2021 (Gonçalves Freitas Maestri) Tuco-Tucos

Thales Renato Ochotorena de Freitas
Gislene Lopes Gonçalves

Renan Maestri Editors
Tuco-Tucos
An Evolutionary Approach
to the Diversity of a Neotropical
Subterranean Rodent
Tuco-Tucos
Gislene Lopes Gonçalves • Renan Maestri
Editors
Tuco-Tucos
An Evolutionary Approach to the Diversity
of a Neotropical Subterranean Rodent
Editors
Thales Renato Ochotorena de Freitas Gislene Lopes Gonçalves
Department of Genetics Department of Genetics
Federal University of Rio Grande do Sul Federal University of Rio Grande do Sul
Porto Alegre, Rio Grande do Sul, Brazil Porto Alegre, Rio Grande do Sul, Brazil
Renan Maestri
Department of Ecology
Federal University of Rio Grande do Sul
Porto Alegre, Rio Grande do Sul, Brazil
ISBN 978-3-030-61678-6 ISBN 978-3-030-61679-3 (eBook)

https://doi.org/10.1007/978-3-030-61679-3
© Springer Nature Switzerland AG 2021

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Foreword
The tucotuco (Ctenomys brasiliensis) is a curious small animal, which may be briefly
described as a Gnawer, with the habits of a mole.
Charles Darwin
The Voyage of the Beagle
Chapter III, Maldonado
Such begin Darwin’s comments on tuco-tucos, based on his encounters with

these animals along the southern coast of Uruguay. Now, 175 years after the original
publication of Darwin’s observations, biologists continue to be fascinated by these
charismatic rodents. In part, this interest reflects the somewhat mysterious nature of
tuco-tucos—even in areas where the animals are locally abundant and can at times
be heard calling from all directions, it is often challenging to catch a glimpse of
these largely subterranean mammals. Increasingly, though, interest in tuco-tucos
reflects our growing understanding of the biology of these animals and the unex-
pected wealth of phenotypic and genotypic variation that they represent. Indeed, as
evident from the contents of this volume, studies of tuco-tucos now encompass
analyses of systematic, phylogenetic, morphological, physiological, ecological, and
behavioral diversity, providing important opportunities to examine the evolutionary
processes underlying divergence within Ctenomys and, concomitantly, convergence
between these and other lineages of subterranean rodents.
One attribute of tuco-tucos that makes them both intriguing and at times chal-
lenging to study is the apparently rapid divergence of species within the genus
Ctenomys. As Diego Verzi and colleagues indicate (Chap. 1), while the family
Ctenomyidae appears to date to the Oligocene, the single extant genus Ctenomys
has arisen much more recently, with current species-level diversification dating only
to the early Pleistocene. This rapid burst of speciation has made it difficult to evalu-
ate the evolutionary relationships among species, as molecular genetic analyses
have often failed to reveal much phylogenetic structure, particularly for deeper
nodes within the genus. Indeed, as Guillermo D’Elía and co-authors state (Chap. 2),
phylogenetic analyses of tuco-tucos are in many ways still in their infancy and will
benefit substantially from inclusion of additional data sets (e.g., genomic-level
sequencing) as well as more comprehensive sampling of putative species and
v
vi Foreword
species groups. At the same time, the rapid diversification within Ctenomys creates
exciting opportunities to explore the evolutionary mechanisms underlying specia-
tion in this lineage. The role of chromosomal rearrangements in promoting specia-
tion has been a particular focus for studies of tuco-tucos due to the often pronounced
karyotypic differences among species, including those for which molecular genetic
analyses fail to detect marked differentiation. This theme is examined by Thales de
Freitas (Chap. 3), who concludes that evidence for distinct processes of speciation
(e.g., allopatric, sympatric, chromosomal) varies in relation to the time since diver-
gence among different members of the genus Ctenomys.
Geographically, tuco-tucos are widespread, occurring throughout much of sub-
Amazonian South America. At the level of individual taxa, however, it has long
been thought that allopatry dominates, with only a few examples of sympatry hav-
ing been identified within Ctenomys. To explore how local spatial relationships
among species translate into the genus-level distribution of these animals, Renan
Maestri and Bruce Patterson (Chap. 4) characterize geographic variation in several
attributes of Ctenomys, including patterns of species richness and range size. These
authors report that although species ranges tend to be smaller in Ctenomys, the
exclusivity of these ranges does not differ from that observed in other lineages of
caviomorph rodents, providing no evidence that allopatry is particularly pronounced
among tuco-tucos. Fernando Mapelli and colleagues (Chap. 5) add a genetic com-
ponent to analyses of geographic variation, arguing that landscape features may
impact the demographic processes that shape patterns of genetic differentiation
within and among species of Ctenomys. Their review suggests that landscape-level
genetic variation reflects a baseline pattern of isolation by distance that is modified
by a complex, species-specific interplay between geographic features, environmen-
tal conditions, and demographic parameters.
In terms of their gestalt, it has been suggested that if you have seen one tuco-
tuco, you have seen them all. This quip reflects the general expectation that the
challenges associated with life in underground burrows have acted to constrain mor-
phological and other forms of phenotypic diversification within Ctenomys. As
knowledge of these animals has increased, it has become increasingly apparent that
they are more phenotypically diverse than has been appreciated. Morphologically,
variation is evident for multiple cranial traits, and, as reported by Rodrigo Fornel
and co-authors (Chap. 6), this variation displays geographic but not phylogenetic
signal, suggesting that environmental conditions may play a critical role in shaping
skull structure in these animals. One obvious environmental factor that may contrib-
ute to this variation is the difference in the soils in which the animals live. As
described by Aldo Vassallo and colleagues (Chap. 7), although tuco-tucos rely pri-
marily on their forepaws to dig, they also routinely use their incisors to chew through
obstructions or loosen hard chunks of soil. Accordingly, the structure of both the
forelimbs and the skull may vary with soil type, and, conversely, constraints on the
biomechanics of digging may preclude the animals from occupying particular soils.
Soil may also be an important determinant of the underappreciated variation in pel-
age coloration that occurs within Ctenomys. Using comparisons of overall pelage
color as well as the structure of individual hairs, Gislene Goncalves (Chap. 8) argues
Foreword vii
that differences in coloration among species of tuco-tucos from the Atlantic coasts
of Brazil and Argentina reflect selection imposed by differences in soil color, with
color matching to local substrates serving to protect animals from predation while
active on the surface.
Interactions between individuals and their environments are also central to stud-
ies of the ecology and physiology of Ctenomys. The role of tuco-tucos as ecosystem
engineers is examined by Bruno Kubiak and Daniel Gailano (Chap. 9), who also
consider the effects of habitat parameters on species’ distributions as well as spatial
and social relationships among conspecifics. Although relevant data are lacking for
many species, the emerging picture is one of greater than expected ecological and
behavioral variation within the genus. One critical aspect of a species’ ecology is its
diet, which can affect not only where animals occur on the landscape but also how
they acquire energy and nutrients, thereby providing a particularly direct link
between external conditions and intrinsic processes. Although all tuco-tucos are
herbivorous, surprisingly few detailed studies of the animals’ diets have been con-
ducted. As Carla Lopes (Chap. 10) reports, the growing use of DNA sequencing of
fecal samples to characterize diets is creating new opportunities to examine dietary
variation within and among members of the genus Ctenomys, including the role of
diet partitioning in shaping the few examples of sympatry that have been reported
for these animals. Maria Sol Fanjul and colleagues (Chap. 11) explore the inner
workings of tuco-tucos in greater detail, revealing how differences in habitat condi-
tions as well as differences in how individuals use their habitats contribute to adap-
tively important variation in multiple physiological systems, including processing
of sensory information, response to external stressors, and regulation of both water
and energy balance. Extrinsically generated differences in physiology may be medi-
ated by variation in individual phenotypes (e.g., sex, reproductive status), thereby
adding an additional layer of complexity to efforts in understanding how external
conditions shape the internal biology of tuco-tucos. In the final chapter of the vol-
ume, Cristina Matzenbacher and Juliana da Silva (Chap. 12) take a more applied
approach to interactions between tuco-tucos and their environments by examining
the role of these animals as bioindicators of environmental change, specifically the
introduction of heavy metals and other toxic compounds as a result of human activ-
ity. More generally, this discussion raises the issue of conservation of the genus
Ctenomys, thereby serving to connect the previous chapters to the increasingly
important need to ensure that members of this lineage are protected from an ever-
growing list of threats.
In closing, one theme that resonates throughout this volume is diversity. From
systematic and phylogenetic revisions of Ctenomys to analyses of interactions
between the environment and specific physiological processes, it is clear that stud-
ies of tuco-tucos are revealing new and sometimes unexpected patterns of diversifi-
cation in this relatively young clade of rodents. Coupled with an ever-growing suite
of analytical tools, this diversity creates novel opportunities to examine long-
standing questions regarding the biology of tuco-tucos. For example, efforts to
understand the often marked karyotypic differences among otherwise closely related
species should benefit from the use of genomic tools to identify the specific portions
viii Foreword
of the genome that are impacted by chromosomal rearrangements. Similarly, as our

ability to characterize the genomic architecture of specific phenotypic traits
increases, we will be better able to examine the genetic bases for adaptive traits such
as the specialized morphological features associated with digging. More generally,
the expanding catalog of diversity within Ctenomys means that members of this
genus are increasingly recognized as important models for research on a wide range
of evolutionary topics, including studies that explore the effects of pathogen com-
munities on immunological function or the role of ecological factors in generating
interspecific differences in social systems. All told, our growing understanding of
the biology of tuco-tucos suggests that the “curious small animals” that intrigued
Darwin will continue to play a central role in biological research for years to come.
Berkeley, USA Eileen Lacey

Introduction
This book examines the biology of tuco-tucos (Ctenomys) from an evolutionary

perspective. Ctenomys is a remarkable lineage of subterranean rodents widely dis-
tributed over the southern half of South America. It exhibits various adaptations for
living underground—mostly solitarily, but in some species also in social groups.
Such a peculiar lifestyle has long attracted the attention of scientists, including
Charles Darwin. In 1832, during the voyage of the Beagle, Darwin had a memorable
experience with tuco-tucos when he stayed in Maldonado, Uruguay, which he reg-
istered in his diary (published in 1839).
The next century of scientific studies of Ctenomys was mostly limited to species
descriptions obtained on European expeditions to South America. Beginning in
1950, a wealth of knowledge on physiology, ecology, genetics, morphology, paleon-
tology, and taxonomy has been documented in scientific journals, as well as in many
theses and dissertations dedicated to this intriguing group. Most studies have docu-
mented local or regional patterns shown by tuco-tucos; however, global or compre-
hensive synopses are still needed. We seek to partly fill this gap by inviting
investigators that have worked for years both in field and laboratory with extinct and
extant tuco-tuco species to review major evolutionary topics and frame these essays
with the breadth of current understanding. We hope that the combination of exten-
sive reviews and original information on tuco-tucos, produced by numerous authors,
will stimulate future studies.
Among the subterranean rodents, the tuco-tucos (Ctenomys) stand along with the
Mediterranean mole rats (Spalax), North American pocket gophers (Thomomys and
Geomys), and species of Bathyergidae in Africa as the major lineages well-known
from long-standing studies. If one considers that tuco-tucos are endemic to the
Neotropics—where funding for basic research is limited—the status of knowledge
reached for Ctenomys is remarkable, resulting from the efforts and passion of many
individuals.
With some frequency—before we decide for this book project—we used to ask
colleagues during annual scientific meetings if they wonder what maintains the pas-
sion for tuco-tucos across distinct generations of scientists (since Darwin) and, par-
ticularly, what makes Ctenomys an exciting group to be studied. In general, people
ix
x Introduction
mentioned that when tuco-tucos are carefully observed in the field, enormous varia-
tion is found, such as in their behavior, pelage, skull, and digging ability. The facili-
ties of working with this group, as in the capture of specimens in the field and the
possibility of keeping them in captivity, and locating their populations and tracking
them in time and space for long years, together with the underlying variation in a
rodent that seems uniform, were considered, in a large degree, the triggers for main-
taining the passion for tuco-tucos. While compiling background and putting them
into context, it became clear—at least explicit—why tuco-tucos are fascinating
from an evolutionary perspective, and we felt motivated to organize this volume.
Considering that the articles on Ctenomys started from the 1950s of the last cen-
tury, we believe that there is currently sufficient data spread over various disciplines
and well-established lines of research that, after 70 years, should be put in a book
trying to make a synthesis of what already exists. We should mention previous
books that included Ctenomys, starting with Evolution of Subterranean Mammals at
the Organismal and Molecular Levels, edited by Eviatar Nevo and Oswaldo A. Reig,
which included a whole chapter about the genus from an evolutionary point of view
(Reig et al. 1990). The genus also appeared on books that featured subterranean
rodents such as Life Underground: The biology of subterranean rodents (2000),
authored by Eileen Lacey, James L. Patton, and Guy N. Cameron, and more recently,
in 2010, the book Subterranean Rodents: News from Underground, by Sabine
Gegall, Hynek Burda, and Cristian E. Schleich. Given how much the scientific com-
munity has learned about tuco-tucos since Reig et al. (1990), we believe it is time
for Ctenomys to have their own book.
Species and local populations of tuco-tucos are the most interesting South
American mammals for studying mechanisms underlying speciation. Basically, two
principal interconnected aspects drive interest in tuco-tucos: chromosomes and spe-
cies diversity. Ctenomys form one of the most karyotypically diverse clades known
in mammals, with chromosomal diploid numbers ranging from 10 to 70 (Cook et al.
1990; Gallardo 1991; Reig et al. 1992; Ortells 1995). In addition, 65 species are
recognized for the genus (Teta and D’Elia 2020, Chap. 2, this volume), more than
any other group of subterranean rodents (Reig et al. 1990; Lessa and Cook 1998;
Castillo et al. 2005; Woods and Kilpatrick 2005). Since Ctenomys appeared in the
late Pliocene, their extant diversity was achieved by remarkable flurry of speciation
events (Verzi et al. 2010; Parada et al. 2011). The age of the genus is quite recent,
estimated at ca. 5 Ma according to molecular evidence (Parada et al. 2011; Upham
and Patterson 2015), which agrees with the paleontological records (Verzi 1999,
2002; Verzi et al. 2010; Chap. 1, this volume).
Reig and Kiblisky (1969) were the first to propose that tuco-tucos are a prime
example of chromosomal speciation. Reig et al. (1990) raised the idea, still accepted,
that diversification may have been facilitated by the isolation of small demes that
characterize population structure in most species and extensive chromosomal rear-
rangements (Reig and Kiblisky 1969; Cook et al. 1990; Gallardo 1991; Ortells
1995). In fact, the high intra- and interspecific chromosomal polymorphisms—once
suggested as the main factor responsible for fast speciation of Ctenomys (Ortells
1995)—do not seem to be directly responsible for its species richness. Thus, rather
Introduction xi
than a triggering speciation in tuco-tucos, chromosomal rearrangements speed up

the process by modulating gene flow rate in certain genome regions (Torgasheva
et al. 2017), suggesting an even more complex scenario for cladogenesis.
The collection of chapters in this book articulates research views that are dis-
seminated across major subjects as paleontology, systematics, evolutionary ecol-
ogy, and genetics. To address such a broad range of topics from the perspective of a
single mammal genus is unusual and transcends good-study-model reasoning. Such
excess might be based on subjective aspects of tuco-tucos since it is a charismatic
animal, which typically touches human feelings, including those from contributors
and readers.
We are most grateful to the authors for their willingness to join us and make this
book happen. Many thanks are also due to Eileen Lacey for writing the foreword
and offering editorial suggestions, as well as to Bruce D. Patterson for helpful com-
ments on our Introduction. We thank Springer Nature Publisher for bringing our
project in the current form, particularly Luciana Christante de Mello, Vignesh
Viswanathan, and Nolan Mallaigh, for their editorial assistance.
Porto Alegre, Rio Grande do Sul, Brazil Thales Renato Ochotorena de Freitas
Renan Maestri
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(Ctenomys; Rodentia, Ctenomyidae): Contrasting mitochondrial and nuclear intron sequences.
J Mammal 86:170–179
Cook JA, Anderson S, Yates TL (1990) Notes on Bolivian Mammals 6: the Genus Ctenomys
(Rodentia, Ctenomyidae) in the Highlands. Am Mus Novit 2980:1–27
Gallardo MH (1991) Karyotypic evolution in Ctenomys (Rodentia, Ctenomyidae). J
Mammal 72:1–21
Lessa EP, Cook JA (1998) The molecular phylogenetics of tuco-tucos (genus Ctenomys, Rodentia:
Octodontidae) suggests an early burst of speciation. Mol Phylogenet Evol 9:88–99
Ortells MO (1995) Phylogenetic analysis of G-banded karyotypes among the South American sub-
terranean rodents of the genus Ctenomys (Caviomorpha: Octodontidae), with special reference
to chromosomal evolution and speciation. Biol J Linnean Soc 54:43–70
Parada A, D’Elia G, Bidau CJ, Lessa EP (2011) Species groups and the evolutionary diversification
of tuco-tucos, genus Ctenomys (Rodentia: Ctenomyidae). J Mammal 92: 671–682
Reig OA, Kiblisky P (1969) Chromosome multiformity in the genus Ctenomys (Rodentia,
Octodontidae). Chromosoma 28:211–244
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Contents
Part I Evolution of Ctenomys

1 The History of Ctenomys in the Fossil Record: A Young
Radiation of an Ancient Family�� 3
Diego H. Verzi, Nahuel A. De Santi, A. Itatí Olivares,
Cecilia C. Morgan, and Alicia Álvarez
2 A Short Overview of the Systematics of Ctenomys: Species
Limits and Phylogenetic Relationships�� 17
Guillermo D’Elía, Pablo Teta, and Enrique P. Lessa
3 Speciation Within the Genus Ctenomys: An Attempt to Find
Models �� 43
Part II Geographic Patterns

4 Geographical and Macroecological Patterns of Tuco-Tucos�� 69
Renan Maestri and Bruce D. Patterson
5 Phylogeography and Landscape Genetics in the Subterranean
Rodents of the Genus Ctenomys�� 83
Fernando Javier Mapelli, Ailin Austrich, Marcelo Javier Kittlein,
and Matías Sebastián Mora
Part III Organismal Biology

6 Skull Shape and Size Diversification in the Genus Ctenomys
(Rodentia: Ctenomyidae)�� 113
Rodrigo Fornel, Renan Maestri, Pedro Cordeiro-Estrela,
and Thales Renato Ochotorena de Freitas
xiii
xiv Contents
7 Biomechanics and Strategies of Digging�� 141

Aldo I. Vassallo, Federico Becerra, Alejandra I. Echeverría,
Guido N. Buezas, Alcira O. Díaz, M. Victoria Longo,
and Mariana Cohen
8 Adaptive Pelage Coloration in Ctenomys �� 167
Part IV Environmental Relationships

9 Environmental and Ecological Features of the Genus
Ctenomys �� 193
Daniel Galiano and Bruno Busnello Kubiak
10 The Diet of Ctenomyids �� 213
Carla Martins Lopes
11 Ecological Physiology and Behavior in the Genus Ctenomys�� 221
María Sol Fanjul, Ana Paula Cutrera, Facundo Luna,
Cristian E. Schleich, Valentina Brachetta, C. Daniel Antenucci,
and Roxana R. Zenuto
12 Effects of Environmental Pollution on the Conservation
of Ctenomys �� 249
Cristina A. Matzenbacher and Juliana da Silva
Index�� 265
Contributors
Alicia Álvarez Instituto de Ecoregiones Andinas, CONICET, Jujuy, Argentina

C. Daniel Antenucci Grupo ‘Ecología Fisiológica y del Comportamiento’,
Instituto de Investigaciones Marinas y Costeras, Universidad Nacional de Mar del
Plata, Consejo Nacional de Investigaciones Científicas y Técnicas, Mar del Plata,
Argentina
Ailin Austrich Departamento de Biología, Facultad de Ciencias Exactas y
Naturales, Universidad Nacional de Mar del Plata, Instituto de Investigaciones
Marinas y Costeras (IIMyC), CONICET – UNMdP, Mar del Plata, Buenos Aires,
Argentina
Federico Becerra Grupo Morfología Funcional y Comportamiento, Instituto de
Investigaciones Marinas y Costeras (IIMyC, UNMDP-CONICET), Universidad
Nacional de Mar del Plata (UNMdP), Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET), Mar del Plata, Buenos Aires, Argentina
Valentina Brachetta Grupo ‘Ecología Fisiológica y del Comportamiento’,
Argentina
Guido N. Buezas Grupo Morfología Funcional y Comportamiento, Instituto de
Mariana Cohen Grupo Histología e Histoquímica, Instituto de Investigaciones
Marinas y Costeras (IIMyC, UNMDP-CONICET), Universidad Nacional de Mar
del Plata (UNMdP), Consejo Nacional de Investigaciones Científicas y Técnicas
(CONICET), Mar del Plata, Buenos Aires, Argentina
xv
xvi Contributors
Pedro Cordeiro-Estrela Departamento de Sistemática e Ecologia, Centro de

Ciências Exatas e da Natureza – Campus I, Universidade Federal da Paraíba, Jardim
Universitário s/n, João Pessoa, PB, Brazil
Ana Paula Cutrera Grupo ‘Ecología Fisiológica y del Comportamiento’, Instituto
de Investigaciones Marinas y Costeras, Universidad Nacional de Mar del Plata,
Consejo Nacional de Investigaciones Científicas y Técnicas, Mar del Plata, Argentina
Juliana da Silva Universidade LaSalle – UNILASALLE and Universidade
Luterana do Brasil - ULBRA, Canoas, RS, Brazil
Thales Renato Ochotorena de Freitas Department of Genetics, Federal University
of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
Nahuel A. De Santi Sección Mastozoología, CONICET, Museo de La Plata, La
Plata, Argentina
Guillermo D’Elía Instituto de Ciencias Ambientales y Evolutivas, Universidad
Austral de Chile, Valdivia, Chile
Alcira O. Díaz Grupo Histología e Histoquímica, Instituto de Investigaciones
Alejandra I. Echeverría Grupo Morfología Funcional y Comportamiento,
Instituto de Investigaciones Marinas y Costeras (IIMyC, UNMDP-CONICET),
Universidad Nacional de Mar del Plata (UNMdP), Consejo Nacional de
Investigaciones Científicas y Técnicas (CONICET), Mar del Plata, Buenos Aires,
Argentina
María Sol Fanjul Grupo ‘Ecología Fisiológica y del Comportamiento’, Instituto
Rodrigo Fornel Programa de Pós-Graduação em Ecologia, Departamento de
Ciências Biológicas, Universidade Regional Integrada do Alto Uruguai e das
Missões – Campus de Erechim, Erechim, RS, Brazil
Daniel Galiano Laboratório de Zoologia, Universidade Federal da Fronteira Sul,
Realeza, Brazil
Gislene Lopes Gonçalves Departamento de Genética, Universidade Federal do
Rio Grande do Sul, Porto Alegre, RS, Brazil
Departamento de Recursos Ambientales, Facultad de Ciencias Agronómicas,
Universidad de Tarapacá, Arica, Chile
Marcelo Javier Kittlein Departamento de Biología, Facultad de Ciencias Exactas
y Naturales, Universidad Nacional de Mar del Plata, Instituto de Investigaciones
Argentina
Contributors xvii
Bruno Busnello Kubiak Programa de Pós-Graduação em Genética e Biologia

Molecular, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
Enrique P. Lessa Departamento de Ecología y Evolución, Facultad de Ciencias,
Universidad de la República, Montevideo, Uruguay
M. Victoria Longo Grupo Histología e Histoquímica, Instituto de Investigaciones
Carla Martins Lopes Departamento de Biodiversidade e Centro de Aquicultura,
Instituto de Biociências, Universidade Estadual Paulista (UNESP), Rio Claro,
SP, Brazil
Facundo Luna Grupo ‘Ecología Fisiológica y del Comportamiento’, Instituto de
Investigaciones Marinas y Costeras, Universidad Nacional de Mar del Plata,
Renan Maestri Departamento de Ecologia, Universidade Federal do Rio Grande
do Sul, Porto Alegre, RS, Brazil
Fernando Javier Mapelli División Mastozoología, Museo Argentino de Ciencias
Naturales “Bernardino Rivadavia”, Buenos Aires, Ciudad Autónoma de Buenos
Aires, Argentina
Cristina A. Matzenbacher Programa de Pós-Graduação em Genética e Biologia
Molecular, Universidade Federal do Rio Grande do Sul - UFRGS, Porto
Alegre, Brazil
Matías Sebastián Mora Departamento de Biología, Facultad de Ciencias Exactas
y Naturales, Universidad Nacional de Mar del Plata, Instituto de Investigaciones
Argentina
Cecilia C. Morgan Sección Mastozoología, CONICET, Museo de La Plata, La
Plata, Argentina
A. Itatí Olivares Sección Mastozoología, CONICET, Museo de La Plata, La Plata,
Argentina
Bruce D. Patterson Negaunee Integrative Research Center, Field Museum of
Natural History, Chicago, IL, USA
Cristian E. Schleich Grupo ‘Ecología Fisiológica y del Comportamiento’,
Argentina
Pablo Teta División Mastozoología, Museo Argentino de Ciencias Naturales
“Bernardino Rivadavia”, Buenos Aires, Argentina
xviii Contributors
Aldo I. Vassallo Grupo Morfología Funcional y Comportamiento, Instituto de

Diego H. Verzi Sección Mastozoología, CONICET, Museo de La Plata, La Plata,
Argentina
Roxana R. Zenuto Grupo ‘Ecología Fisiológica y del Comportamiento’, Instituto
About the Editors
Thales Renato Ochotorena de Freitas has been a full professor in the Department
of Genetics, Institute of Biosciences, Federal University of Rio Grande do Sul
(UFRGS), since 1985. He earned his Ph.D. in genetics and molecular biology
(1990) from UFRGS. He has been a visiting scholar at the Museum of Vertebrate
Zoology, University of California-Berkeley (1996–1997), and visiting researcher at
Laboratoire d'Écologie Alpine, University of Grenoble (2013–2014), where he
worked on cutting-edge genomic tools. Dr. Freitas is currently investigating varia-
tion at population and species levels in Ctenomys, particularly from groups on the
border of the Amazon Forest and coastal plain, using cytogenetic and molecular
genetics. Dr. Freitas serves as director of the Graduate Program in Genetics and
Molecular Biology at UFRGS and was the founder president of the Brazilian
Society of Mastozoology (SBMZ). He teaches classical genetics and conservation
genetics to undergraduate and graduate students.
Gislene Lopes Gonçalves has been a research collaborator in the Department of

Genetics, Federal University of Rio Grande do Sul (UFRGS), Brazil, and at the
Universidad de Tarapacá, Chile, since 2014. She earned her Ph.D. in genetics and
molecular biology (2011) from UFRGS, where she completed her postdoctoral
studies in zoology (2012–2016). She has been a visiting researcher at the Museum
of Comparative Zoology, Harvard University (2009–2010), where she worked on
the genetic basis of hair pigmentation in rodents with Professor Hopi Hoekstra. Dr.
Gonçalves is currently interested in editorial works and collaborates with numerous
research groups in the scope of evolutionary genetics.
Renan Maestri has been an assistant professor in the Department of Ecology,

Institute of Biosciences, Federal University of Rio Grande do Sul (UFRGS), since
2018. He is also a research associate at The Field Museum of Natural History,
Chicago, IL, USA, since 2020. He earned his Ph.D. in ecology (2017) from UFRGS,
with a period in The Field Museum, and completed his postdoctoral studies in the
Graduate Program in Animal Biology (2017–2018), UFRGS. Dr. Maestri teaches
and advises students in the field of ecology and evolution, and coordinates the
xix
xx About the Editors
Laboratory of Ecomorphology and Macroevolution at the Department of Ecology,

UFRGS. He is interested in the areas of evolutionary biology, ecology, and bioge-
ography, with an emphasis on morphological evolution, macroevolution, and mac-
roecology of complex phenotypes.
Part I
Evolution of Ctenomys
Chapter 1
The History of Ctenomys in the Fossil
Record: A Young Radiation of an Ancient
Family
Diego H. Verzi, Nahuel A. De Santi, A. Itatí Olivares, Cecilia C. Morgan,

and Alicia Álvarez
1.1 Introduction
Ctenomyidae is a clade of South American hystricomorph rodents with a peculiar

evolutionary history characterized by: strong morphological differentiation, i.e.,
modernization that took place in the late Miocene; extinction of lineages during the
Plio-Pleistocene, which led to Ctenomys being the only representative of the clade
in the living fauna; and an extremely high rate of speciation of the latter genus,
which is unmatched among caviomorphs (Reig et al. 1990; Lessa et al. 2008; Verzi
2008; Verzi et al. 2014, 2016; Álvarez et al. 2017, 2020). The stage of morphologi-
cal differentiation is defined by the acquisition of a unique dental morphology,
which persists in living species (Reig 1970). Because of its uniqueness, the appear-
ance of this dental morphology has dictated the recognition of ctenomyids in the
fossil record (Wood 1955; Reig et al. 1990; Arnal and Vucetich 2015). In addition,
the skeletal morphology of modern ctenomyids diversified in adjustment to life
underground. Because of their unequivocal recognition, as well as their appealing
adaptive diversification, these modern representatives have attracted the attention of
paleontologists almost exclusively; the corpus of information produced, primarily
systematic and paleobiological, has provided knowledge on the boundaries of spe-
cialization explored by at least part of the clade throughout its history (Reig and
Quintana 1992; Casinos et al. 1993; Quintana 1994; Fernández et al. 2000; Vieytes
et al. 2007; Lessa et al. 2008; Verzi 2008; Morgan and Verzi 2011).
With regard to the other major contribution of fossils, i.e., the estimation of the
time of origin and extinction of lineages and clades, this is an issue that remains still
D. H. Verzi (*) · N. A. De Santi · A. I. Olivares · C. C. Morgan

Sección Mastozoología, CONICET, Museo de La Plata, La Plata, Argentina
e-mail: dverzi@fcnym.unlp.edu.ar; ndesanti@fcnym.unlp.edu.ar;
iolivares@fcnym.unlp.edu.ar; cmorgan@fcnym.unlp.edu.ar
A. Álvarez
Instituto de Ecoregiones Andinas, CONICET, Jujuy, Argentina
© Springer Nature Switzerland AG 2021 3

T. R. O. de. Freitas et al. (eds.), Tuco-Tucos,
https://doi.org/10.1007/978-3-030-61679-3_1
4 D. H. Verzi et al.
partially unresolved and unclear for ctenomyids. This stems not only from the need
for more, and more exhaustive, phylogenetic analyses that include extinct species
but also from the dissimilar current interpretations regarding the evolutionary mean-
ing of these extinct taxa. Fossils provide estimations of the divergence times of
clades as raw information, and subsequently through the calibration of molecular
clocks (Benton and Donoghue 2007; Ronquist et al. 2016). Consequently, they play
a central role in the analysis of evolutionary patterns, models, and rates. Nevertheless,
reliable age estimations require hypotheses regarding the correspondence of fossils
to the different evolutionary stages of a clade: origin, modernization, and establish-
ment of the crown group (Hennig 1965). As previously mentioned, the paleonto-
logical studies of ctenomyids have been essentially focused on the modern species;
however, in this context of analysis, these have little to contribute to the knowledge
of the origin of the clade, or even, depending on their phylogenetic position, of the
origin and diversification of the crown group. Thus, achieving an understanding of
the evolutionary pattern of this family, including the times and rates of taxonomic
and morphological diversification of living species (e.g., Álvarez et al. 2017;
Caraballo and Rossi 2018), still requires a more accurate interpretation of the fos-
sil record.
In this chapter, we offer a brief review of the history of the family Ctenomyidae
such as it can be interpreted through its fossil record. In addition to describing major
characteristics of this history, we provide a critical assessment of the potential con-
tribution of available information to the estimation of divergence times through the
phylogeny of the family, with emphasis on its single living representative, Ctenomys.
1.2 tem Ctenomyids and the Understanding

S
of Ctenomyid Origin
The family Ctenomyidae has been traditionally recognized by the rootless molars
with exceptionally simplified occlusal surfaces that characterize its late Miocene to
Recent representatives (Simpson 1945; Wood 1955; Reig et al. 1990; Vucetich et al.
1999; Arnal and Vucetich 2015). Alternatively, Verzi (1999) proposed an octodon-
toid with conservative rootless molars with lophids and flexids, the lower late
Miocene †Chasichimys, as potential ancestor of the modern ctenomyids (see also
Verzi et al. 2004a). Later phylogenetic analyses supported the position of
†Chasichimys, the related †Chasicomys (late Miocene), and the older †Sallamys
(late Oligocene), †Willidewu, and †Protadelphomys (early Miocene) as stem cteno-
myids (Fig. 1.1; Verzi et al. 2014, 2016). However, this unorthodox phylogenetic
hypothesis is far from consensus. With the exception of †Chasicomys (see Pascual
1967), these genera were initially assigned to Echimyidae (e.g., Simpson 1945;
Wood 1955; Wood and Patterson 1959; Patterson and Pascual 1968; Patterson and
Wood 1982; Vucetich and Verzi 1991), a family whose living representatives main-
tain rooted molars with conservative morphologies (Verzi et al. 2016, Fig. 1.1).
Phylogenetic analyses based essentially on dental characters have supported the
1 The History of Ctenomys in the Fossil Record: A Young Radiation… 5
Fig. 1.1 Strict consensus of eight most parsimonious trees resulting from parsimony analysis of
combined morphological and molecular data. Divergence times for species of the crown group is
according to a Bayesian tip-dating analysis by De Santi et al. (unpubl. results)
inclusion of †Protadelphomys within Echimyidae and have recovered †Sallamys as

a stem Octodontoidea (Carvalho and Salles 2004; Arnal and Vucetich 2015; Boivin
et al. 2019).
These dissimilar results regarding the affinities of these early octodontoids are
due, at least in part, to different interpretations of dental characters for different spe-
cies samplings, an issue that still needs critical revision. In a recent meta-analysis of
phylogenetic reconstructions, Sansom et al. (2017) showed that dental data are gen-
erally less reliable than osteological data as indicators of phylogenetic history. In
this sense, the preserved cranial remains of †Protadelphomys possess at least two
informative traits in the orbital and auditory regions that are shared with modern and
living ctenomyids; in addition, this genus does not share with the Echimyidae any
key synapomorphies of the auditory region that are diagnostic of this latter family
(Verzi et al. 2014, Fig. 6; Verzi et al. 2016, Fig. 9).
In any case, even when accepting †Sallamys, †Protadelphomys, and †Willidewu
as stem ctenomyids, their phylogenetic position is less strongly supported than that
of the late Miocene-Pleistocene modern representatives (see Verzi et al. 2014). In
this sense, the abovementioned and best-known concept of Ctenomyidae, restricted
to the species with rootless simplified molars, is undoubtedly more stable. This
concept of Ctenomyidae represents an apomorphy-based clade, defined by the
acquisition of rootless crescent-shaped molars that took place in the late Miocene
(Figs. 1.1 and 1.2). Such apomorphy-based clade comprises late Miocene to
Fig. 1.2 Chart showing categories of clades and related concepts after de Queiroz (2007, Fig. 1.2).
Grey branches represent lineages lacking extant descendants (side branches); black branches rep-
resent lineages with extant descendants. The apomorphy clade is represented as corresponding to
stage t2 by assuming that the marked apomorphy is that which defines the beginning of this stage
Pleistocene stem representatives and the living Ctenomys (Fig. 1.1). Differentiation
of lineages and genera among these modern ctenomyids would have resulted from
the acquisition of disparate adaptations to digging and life underground (Reig and
Quintana 1992). Four cohesive lineages are recognized: †Eucelophorus (early
Pliocene-middle Pleistocene), †Xenodontomys-†Actenomys (late Miocene-late
Pliocene), †Praectenomys (Pliocene), and †Ctenomys (late Pliocene-Recent). The
†Xenodontomys-†Actenomys lineage would have had fossorial habits, while
Ctenomys and †Eucelophorus independently acquired craniodental specializations
for subterranean life (definition of fossorial and subterranean habits follows Lessa
et al. 2008); †Praectenomys would have been at least fossorial (Verzi 2008; Verzi
et al. 2010).
Beyond their different support or stability, the previously mentioned alternative
definitions of Ctenomyidae are conceptually different and represent different times
of the history of the clade. Three successive stages can be recognized in the evolu-
tionary history of any clade with living representatives, referred to as t1, t2, and t3
by Hennig (1965: Fig. 1.4): t1, the time of its origin by divergence from the most
closely related clade with living representatives; t2, its time of morphological dif-
ferentiation or modernization by the acquisition of the apomorphy or apomorphies
that characterize its extant members; and t3, the time of the origin of the last com-
mon ancestor of the living representatives. The nested clades that result from each
of these points of origin are defined as a total clade, apomorphy clade, and crown
clade, respectively (Fig. 1.2; de Queiroz 2007). A total clade comprises the crown
clade and its corresponding stem group. The stem group is by definition paraphy-
letic and includes both extinct species that are directly ancestral to the crown, i.e.,
those belonging to the stem lineage, and those that are not directly ancestral, i.e.,
side branches.
In this context, modern ctenomyids with derived molars represent Hennig’s stage
t2 (Fig. 1.1). Hennig (1965: 114) pointed out that the delimitation of the stage of
morphological differentiation, t2, depends on subjective criteria concerning the
interpretation of the emergence of particular “types” or “Baupläne”. We consider
that this stage is related to change within lineages, and although its delimitation may
imply subjectivity, it can yield important evolutionary information on
environmentally-driven morphological changes (Verzi et al. 2014, 2015). Beyond
this, even though many of the fossils at this stage of morphological differentiation
may be stem representatives, as occurs in ctenomyids (Fig. 1.1), they do not provide
relevant contributions to the interpretation of the origin of the total clade within
which they are nested. The practice of interpreting the origin of clades from the first
appearances of the main diagnostic characters shared with extant representatives
should be assumed as an operational restriction. As pointed out by de Queiroz
(2007: 968), the origins of total clades have to do with lineage splitting rather than
with character state transformations. Consequently, for an apomorphy to be present
in the earliest members of a total clade, that apomorphy would have to have arisen
and become fixed simultaneously with the lineage-splitting event in which the clade
originated. Because of the nature of evolutionary processes and hierarchies involved
in that lineage-splitting event, the latter is not to be expected. As a result, early stem
members share few “non-key” apomorphies with their corresponding crown-group

(Steiper and Young 2008). In any case, the difficulty of separating the earliest repre-
sentatives of two diverging extant clades does not negate the validity of the splitting
point as the origin of the resulting clades (Briggs and Fortey 2005: 100). Thus,
efforts focused on the recognition of plesiomorphic early stem lineages and side
branches are indispensable to interpret the deep history of a surviving clade.
Here we lend more support to the idea of applying the name Ctenomyidae to the
total clade (see below). †Sallamys, recorded in the late Oligocene of Bolivia and
Peru, is its earliest stem representative (Fig. 1.1; Patterson and Wood 1982; Shockey
et al. 2009). Recently, Pérez et al. (2019) transferred the species †Sallamys quispea
from the late Oligocene of Peru to the genus †Migraveramus; we consider that the
molar morphology of †S. quispea is comparable to that of the type species †Sallamys
pascuali albeit less abbreviated (Shockey et al. 2009, Fig. 6), and therefore suggest
that the former species should remain to be assigned to †Sallamys.
The proposals of paleontological ages younger than 10 My for this family (e.g.,
Reig 1989; Reig et al. 1990; Vucetich et al. 1999; Arnal and Vucetich 2015; Vucetich
et al. 2015) should be reinterpreted as associated to the beginning of the moderniza-
tion stage, t2 (Fig. 1.1). According to biochronological data, the earliest species
corresponding to this stage, †Xenodontomys simpsoni, is approximately 6 My old.
1.3 The Genus Ctenomys
Information on the early history of the lineage that leads to Ctenomys is fragmentary
and unclear. The available data hinder a temporal assignment more precise than the
entire Pliocene for the divergence of this lineage from the sister genus †Praectenomys
(see review of the age of Umala Formation in Cione and Tonni 1996). An unpub-
lished mandibular fragment affine to Ctenomys, but with only a slight reduction of
m3, was recently found in the early Pliocene of western Argentina (Verzi unpub-
lished). Although no phylogenetic analyses have yet been made, this new fossil
would represent an intermediate step in the acquisition of the apomorphies that
characterize the Ctenomys lineage, being closer to the latter than to the one cur-
rently considered as sister genus, †Praectenomys.
Thus, while Ctenomys is a morphologically cohesive genus in the living fauna
(Reig et al. 1990; Vassallo and Mora 2007), its boundaries become less evident
when the variation of the oldest related extinct species is considered. By application
of an adaptation-rooted criterion, which involves an assessment of both the mono-
phyly and the adaptive profiles to delimit genera in the fossil record (Wood and
Collard 1999; Cela-Conde and Ayala 2003), the species †Ctenomys uquiensis (late
Pliocene) and †Paractenomys chapalmalensis (lower early Pleistocene) have been
considered as the earliest members of the genus Ctenomys (Fig. 1.3; Verzi 2008;
Verzi et al. 2010). Although part of their traits are undoubtedly plesiomorphic with
respect to living species (Verzi 2002; Morgan and Verzi 2006, 2011), functionally
significant specializations and the conserved allometry of their masseteric
Fig. 1.3 Mandibles and skulls of some extinct species of Ctenomys mentioned in the text. Left
mandible of: (a). †C. uquiensis MLP 96-II-29-1 (holotype); (b). †C. chapalmalensis MMP 1622-M
(right reversed); (c). †C. dasseni PVL 739 (holotype); (d). †C. kraglievichi MMP M-429 (right
reversed); (e). †C. viarapaensis MLP 2966. Ventral view of skull of: (f). †C. chapalmalensis MMP
481-S; (g). †C. dasseni (holotype of †C. intermedius MACN 1849); (h). †C. kraglievichi MSC MS
20–1; (i). †C. viarapaensis MLP 2935 (holotype). MACN, Museo Argentino de Ciencias Naturales,
Buenos Aires, Argentina; MLP, Museo de La Plata, Argentina; MMP, Museo de Ciencias Naturales
de Mar del Plata, Argentina; MSC, Museo de Ciencias Naturales de Santa Clara, Argentina; PVL:
Colección Paleontología Vertebrados, Instituto Miguel Lillo, San Miguel de Tucumán, Argentina
morphology, in the comparative context of the modern ctenomyids, support the

inclusion of these species within the genus (Verzi 2008; Verzi et al. 2010).
The genus Ctenomys thus delimited represents an apomorphy clade with a mini-
mum age, given by †C. uquiensis, close to 3.5 My (Fig. 1.1). However, given that
these species are ancestral to the crown clade (Verzi 2008; Verzi et al. 2010; De
Santi et al. 2020), this estimation marks a maximum constraint (softbound) on the
age of the crown (Benton and Donoghue 2007). Ongoing phylogenetic analyses (De
Santi et al. 2020; unpublished results) that include the most complete fossil materi-
als of extinct species accepted as valid suggest that the crown clade Ctenomys is
Fig. 1.4 Geographic distribution of the extinct species of Ctenomys accepted as valid. Open
square, Necochea locality (see text)
younger than previously considered; this is supported both by evidence from the
fossil record and by a calibrated tree obtained through Bayesian analysis (Fig. 1.1
and Table 1.1). Even species from the early and middle Pleistocene of central
Argentina (Figs. 1.1, 1.3, and 1.4), markedly younger than †C. uquiensis and
†C. chapalmalensis, are stem representatives according to the phylogenetic hypoth-
eses obtained.
An alternative stance could be that the name Ctenomys be restricted to the crown
clade (see de Queiroz 2007), in which case other genera should be erected for the
stem species. If such a definition were adopted, the content of the clade (as of any
other crown clade) would depend on extinction (see Budd and Mann 2020). In fact,
the decoupling between the origin (t1) and the rise of the crown group (t3) in any
clade results from extinction (Verzi et al. 2016). Although extinction within the
variation of Ctenomys remains to be studied, species and populations of this genus
are vulnerable to this phenomenon on account of some of their distinctive
Table 1.1 Estimated ages (in My) of origin of the crown clade Ctenomys and the total clade
Ctenomyidae (i.e., Ctenomyidae/Octodontidae divergence). Values from this study are fossil-
based estimates of minimum ages; value from De Santi et al. (unpubl.) is from a Bayesian analysis
using fossil constraints after the tree in Fig. 1.1
Ctenomys Octodontidae/Ctenomyidae
This study * ~ 0.78 ** ~ 26.25
De Santi et al. (unpubl.) 1.3 –
Caraballo and Rossi (2018) 5.88 (4.32–7.54) 21.35 (15.59–26.69)
Álvarez et al. (2017) 11.13 25.96
Upham and Patterson (2015) 6.0 (4.6–7.6) 18.9 (15.7–22.1)
Upham and Patterson (2015) 4.3 (2.2–7.4) 19.1 (14.3–23.5)
Parada et al. (2011) 9.2 (6.4–12.6) 17.9 (13.5–23.0)
Castillo et al. (2005) 3.7, 1.3 –
Lessa and Cook (1998) 5.1 (3.3–6.9), 1.4, 1.1 –
*†Ctenomys dasseni (Soibelzon et al. 2009); **†Sallamys quispea (Shockey et al. 2009)
ecological features, such as patchy distribution, limited vagility, and small effective
numbers (Reig et al. 1990). In support of the latter, a marked turnover in the diver-
sity of Ctenomys that involved extinction is detected along a 500 kyr Pleistocene
stratigraphic sequence (ca. 1 Ma to 0.5 Ma) in the locality of Necochea, in east-
central Argentina (Fig. 1.4; Bidegain et al. 2005). In addition, the recently described
†C. viarapaensis from the Holocene of central Argentina is abundant in the fossil
record for ca. 7 kyr until its (at least local) extinction at 0.36 kyr BP (De Santi et al.
2020). This longevity pattern is quite different from that of species of other modern
ctenomyid genera (and even other small mammals, see Prothero 2014, Table 1.1)
whose duration is over a million years (Verzi et al. 2015, Fig. 5.3).
In addition, the application of a name to a crown clade that contains extinct spe-
cies also entails a decision regarding the hierarchy of the clade being delimited. In
the case of Fig. 1.2, two sister crown clades, B and C, could alternatively be recog-
nized within the indicated crown clade A.
Accordingly, defining Ctenomys based on the apomorphy clade is more stable,
given that it is not dependent on extinction.
Beyond which definition of Ctenomys is adopted, it is clear that this genus
reached its unusually high current taxonomic and ecological diversification, as well
as its widespread distribution and considerable morphofunctional disparity (Lacey
and Wieczorek 2003; Bidau 2015; Freitas 2016; Borges et al. 2017; Álvarez et al.
2017; Morgan et al. 2017), in a surprisingly short lapse. The oldest representative of
the crown clade Ctenomys, †C. dasseni, is about 0.78 Ma old according to biochro-
nology and magnetostratigraphy (level D of Punta Hermengo locality, central
Argentina; Soibelzon et al. 2009). Remarkably, the three Pleistocene species recov-
ered as members of the crown group, i.e., †C. dasseni, †C. subassentiens, and
†C. kraglievichi, were clustered into the earliest diverged clade known as frater
group (Parada et al. 2011; Bidau 2015), which currently inhabits the Bolivian-
Paraguayan Chaco and Andean zones. This supports the previous interpretation of
the record of †C. kraglievichi as representing the irruption of a member of this clade
into the pampean region (currently dominated by the mendocinus group), associated
to an important middle Pleistocene warm pulse (near 0.5 Ma; Verzi et al. 2004b).
1.4 Final Considerations
Like the other families of South American hystricomorph rodents (Upham and
Patterson 2015; Álvarez et al. 2017), Ctenomyidae has a deep evolutionary history,
which is evidenced in the fossil record of this family since the late Oligocene. These
earliest fossils, closer to the origin of the clade, and the first (middle Pleistocene)
members of the crown clade are the ones that contribute relevant temporal informa-
tion for the calibrations of molecular phylogenies. The temporally extended stem-
group evidences strong extinction, and includes even middle Pleistocene species of
the only surviving genus, Ctenomys. The history of the strikingly diverse crown
clade which includes at least 69 extant species (Freitas 2016) as well as a few extinct
species is, on the contrary, surprisingly short. Although this could be influenced by
biases inherent to the fossil record, it is nevertheless significant that most Pleistocene
species included in the analyses are recovered as members of the stem group. This
entails a new perspective regarding the timing of taxonomic, ecological, and mor-
phofunctional diversification of extant Ctenomys, which should be taken into
account in future evolutionary analyses of the genus.
Acknowledgments We are especially grateful to the editors, T Freitas, G Lopes Gonçalves, and
R Maestri, for the invitation to contribute to this volume. For access to materials under their care,
we thank P Teta, A Martinelli, M Ezcurra, L Chornogubsky (Museo Argentino de Ciencias
Naturales, Argentina), P Ortiz, M Díaz (Facultad de Ciencias Naturales e Instituto Miguel Lillo,
Argentina), S Bogan (Fundación de Historia Natural Azara, Argentina), M Pérez, E Ruigómez
(Museo Paleontológico Egidio Feruglio), M Rosi (IADIZA, CONICET, Argentina), J Oliveira
(Museu Nacional, Universidade Federal do Rio de Janeiro, Brasil), D Romero, M Taglioretti, F
Scaglia, †A Dondas (Museo de Ciencias Naturales de Mar del Plata, Argentina), P Straccia (Museo
de Ciencias Naturales de Santa Clara, Argentina), J Vargas Mattos (Colección Boliviana de Fauna,
Bolivia), E Tonni, and M Reguero (Museo de La Plata, Argentina). This research was supported by
Agencia Nacional de Promoción Científica y Tecnológica PICT 2016-2881.
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Chapter 2
A Short Overview of the Systematics
of Ctenomys: Species Limits
and Phylogenetic Relationships
Guillermo D’Elía, Pablo Teta, and Enrique P. Lessa
2.1 Introduction
Ctenomys Blainville, 1826 is one of the most species-rich genera of Mammalia; the
current count indicates that the genus has 64 living species (see below). Tuco-tuco
species have mostly allopatric distributions, with some of them having very
restricted geographical ranges (Bidau 2015) and in some cases being only known
from their type localities (Teta and D’Elía 2019). The genus displays one the broad-
est range of chromosomic variation (2n = 10 to 2n = 70; Cook et al. 1990; Novello
and Lessa 1986) of any mammal genus. As such, Ctenomys has long attracted the
attention of evolutionary biologists aimed to characterize its diversity and to under-
stand the drivers of such impressive radiation. These studies have been hampered,
however, by a poor understanding of species boundaries and the species phyloge-
netic relationships.
In recent year several studies have advanced our knowledge of distinct aspects of
the evolutionary biology and ecology of Ctenomys (e.g., Martínez and Bidau 2016;
Morgan et al. 2017; Tomasco et al. 2019; Kubiak et al. 2020); this book is indeed a
more than welcome proof of these advances. Notwithstanding, regarding the evolu-
tionary history of the genus several unclear areas remain. At the base of this sce-
nario lays the fact that the taxonomic status of several populations and nominal
forms is still dubious, either because they have not been evaluated (e.g., those of
several unsampled geographic areas) or results are inconclusive. As such, the
G. D’Elía (*)
Instituto de Ciencias Ambientales y Evolutivas, Universidad Austral de Chile, Valdivia, Chile
P. Teta
División Mastozoología, Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”,
Buenos Aires, Argentina
E. P. Lessa
Departamento de Ecología y Evolución, Facultad de Ciencias, Universidad de la República,
Montevideo, Uruguay

https://doi.org/10.1007/978-3-030-61679-3_2
18 G. D’Elía et al.
taxonomy in Ctenomys is still much unstable (see the species accounts in Bidau
2015). In turn, this scenario hampers the understanding of species phylogenetic
relationships and secondarily all studies that need a phylogeny as input (e.g., studies
of historical biogeography or morphological evolution).
Even though several fossil species are known (e.g., Rusconi 1931; Verzi et al.
2004; De Santi et al. 2020), we focused on living forms and structured the core of
this review into two main sections. The first one deals with species boundaries while
the second one focus on phylogenetic relationships among species of tuco-tucos.
Both sections, after a short summary on the historical development of the knowl-
edge, focus on its current state; emphasis is made on what we known, but also on
gray areas that need to be the focus of future research. We close our literature review
in June 2020.
2.2 Taxonomy
The taxonomic history of Ctenomys is long and complex. In a statement that has
been cited several times, Sage et al. (1986) claimed that “The current taxonomy of
Ctenomys is in a state of general chaos.” As would be commented below several
issues contribute to this view, including some taxonomic practices that should be
avoided (see below), the general scarcity of study samples, as well as the intrinsic
biological complexity of this genus. For instance, it has been widely acknowledged
that the taxonomy of Ctenomys is challenged by the remarkable morphological
homogeneity, both external and cranial, that exists among species. However, De
Santi et al. (2020) shown that morphological differences among species and groups
of species can be uncovered when cranial or dental characters are assessed in detail.
Along the same lines, linear and geometric morphometric analyses have been able
to differentiate species, although differences tend to blur if multiple species are
considered simultaneously (Tiranti et al. 2005; Freitas 2005). Some species exhibit
large chromosomal variation (e.g., 45 distinct karyotypes are known for C. minutus
Nehring, 1887; see Lopes et al. 2013) that, if not considered altogether, may mis-
lead species delimitation. Another fact that challenges the establishment of species
boundaries is that, commonly, there is incongruence among the variation pattern of
distinct data sets (e.g., the large and well-structured chromosomic variation of
C. pearsoni Lessa and Langguth, 1983 contrasting with the patterns of variation of
the mitochondrial genome and the skull morphology; Tomasco and Lessa 2007;
D’Anatro and D’Elía 2011). These challenges are slowly starting to be overcome
with work that incorporates distinct evidence lines and analytical tools.
What follows does not intend to be an exhaustive review of the taxonomy of
Ctenomys (for that we refer the reader to the species accounts written by Bidau
2015; although below we pose some departures); rather, by commenting some study
cases, we highlight distinct aspects of the taxonomic history and practice of
Ctenomys. We aim to give our vision of the current taxonomic knowledge of
Ctenomys, including the identification of some areas in need of additional research,
2 A Short Overview of the Systematics of Ctenomys: Species Limits… 19
as well as to advocate for the abandonment of some practices that have unnecessar-
ily contributed to complicate tuco-tuco taxonomy.
2.2.1 Currently Considered Distinct Species
The first species of Ctenomys to be described was C. brasiliensis Blainville, 1826.

At the same time, this was the first mention of Ctenomys; as such, this was one of
the first named genera of Neotropical rodents. In the remaining of the nineteenth
century, 10 other species of tuco-tucos were described (Table 2.1); of these, four
were described in 1848. An important increase in species description occurred dur-
ing the first three decades of the twentieth century. Between 1903 and 1926, 28
species, currently considered distinct, were described; Oldfield Thomas described
24 of these species (23 by himself and the other with Jane St. Leger). After that,
there was a large period of over five decades (1927–1980) in which only five species
were described. In addition, during this period, the number of recognized species
suffered a marked reduction (e.g., Cabrera 1961 recognized only 26 species within
this genus), with several taxa being uncritically included into the synonymy of oth-
ers, and usually retained as subspecies (e.g., azarae Thomas, 1903; bergi Thomas,
1902; fochi Thomas, 1919; haigi Thomas, 1919; juris Thomas, 1920; latro Thomas,
1918; lentulus, Thomas, 1919; occultus Thomas, 1920; pundtii Nehring, 1900;
recessus Thomas, 1912, and tucumanus Thomas, 1900, were regarded as synonyms
of mendocinus Philippi, 1869). The imprint of this classification is still present in
some species groups, such as is the case of C. frater Thomas, 1902 (see below).
Over the next two decades (1981–2001), a new period of intense species description
occurred, where 12 new species were named and described. Most of these species
were authored by Julio R. Contreras. During the period 2002–2011, no new species
was described. Finally, from 2012 up today (September 24, 2020) eight new species
have been erected. As such, the cumulative count of species still shows a steady
increase in known species richness (Fig. 2.1).
As expected from the dates of description, most species of Ctenomys were only
described based on morphologic (mostly qualitative) differences (see a discussion
of the general trend for rodents in D’Elía et al. 2019). The first species of Ctenomys
whose description included chromosomic evidence was C. coyahiquensis by Kelt
and Gallardo in 1994. As this form is now regarded as a synonym of C. sericeus
J. A. Allen, 1903 (see Teta and D’Elía 2020), the first description incorporating
chromosomic data of what we consider to be a distinct species is from one year later
and corresponds to that of C. osvaldoreigi Contreras, 1995. Although several candi-
date species have been identified based on the analysis of DNA sequences (e.g.,
some Bolivian forms included in the analysis of Lessa and Cook 1998; some
Patagonian forms in the study of Parada et al. 2011), the first species description
incorporating analysis of DNA sequences dates from less than a decade ago, corre-
sponding to that of C. ibicuensis Freitas, Fernandes, Fornel, and Roratto, 2012.
Table 2.1 List of the 64 living species of Ctenomys recognized in this study
Species
Species Sperm type group
1 Ctenomys andersoni Gardner, Salazar-Bravo and Cook, 2014 boliviensis
2 Ctenomys argentinus Contreras and Berry, 1982 symmetric tucumanus
3 Ctenomys australis Rusconi, 1934 asymmetric mendocinus
4 Ctenomys bergi Thomas, 1902 asymmetric mendocinus*
5 Ctenomys bicolor Miranda-Ribeiro, 1914 boliviensis
6 Ctenomys bidaui Teta and D’Elía, 2020 asymmetric magellanicus
7 Ctenomys boliviensis Waterhouse, 1848 symmetric boliviensis
8 Ctenomys bonettoi Contreras and Berry, 1982 asymmetric mendocinus*
9 Ctenomys brasiliensis Blainville, 1826
10 Ctenomys coludo Thomas, 1920
11 Ctenomys conoveri Osgood, 1946 asymmetric frater
12 Ctenomys contrerasi Teta and D’Elía, 2020 asymmetric magellanicus
13 Ctenomys dorbigny Contreras and Contreras, 1984 torquatus
14 Ctenomys dorsalis Thomas, 1900 boliviensis
15 Ctenomys emilianus Thomas and St. Leger, 1926
16 Ctenomys erikacuellarae Gardner, Salazar-Bravo and Cook, boliviensis
2014
17 Ctenomys famosus Thomas, 1920 mendocinus
18 Ctenomys flamarioni Travi, 1981 asymmetric mendocinus
19 Ctenomys fochi Thomas, 1919
20 Ctenomys fodax Thomas, 1910 asymmetric magellanicus
21 Ctenomys frater Thomas, 1902 symmetric frater
22 Ctenomys fulvus Phillippi, 1860 symmetric opimus
23 Ctenomys haigi Thomas, 1919 asymmetric magellanicus
24 Ctenomys ibicuensis Freitas, Fernandes, Fornel and Roratto, torquatus
2012
25 Ctenomys johannis Thomas, 1921
26 Ctenomys juris Thomas, 1920 asymmetric tucumanus*
27 Ctenomys knighti Thomas, 1919
28 Ctenomys lami Freitas, 2001 torquatus
29 Ctenomys latro Thomas, 1918 tucumanus
30 Ctenomys lessai Gardner, Salazar-Bravo and Cook, 2014 frater
31 Ctenomys leucodon Waterhouse, 1848 no group
32 Ctenomys lewisi Thomas, 1926 symmetric frater
33 Ctenomys magellanicus Bennet, 1836 asymmetric magellanicus
34 Ctenomys maulinus Phillippi, 1872 asymmetric no group
35 Ctenomys mendocinus Phillippi, 1869 asymmetric mendocinus
36 Ctenomys minutus Nehring, 1887 symmetric torquatus
37 Ctenomys nattereri Wagner, 1848
38 Ctenomys occultus Thomas, 1920 asymmetric tucumanus
39 Ctenomys opimus Wagner, 1848 symmetric opimus
(continued)
Table 2.1 (continued)

Species
Species Sperm type group
40 Ctenomys osvaldoreigi Contreras, 1995 asymmetric
41 Ctenomys paraguayensis Contreras, 2000
42 Ctenomys pearsoni Lessa and Langguth, 1983 symmetric torquatus
43 Ctenomys perrensi Thomas, 1896 symmetric torquatus
44 Ctenomys peruanus Sanborn and Pearson, 1947
45 Ctenomys pilarensis Contreras, 1993 asymmetric tucumanus*
46 Ctenomys pontifex Thomas, 1918
47 Ctenomys pundti Nehring, 1900 symmetric talarum
48 Ctenomys rionegrensis Langguth and Abella, 1970 asymmetric mendocinus
49 Ctenomys roigi Contreras, 1988 symmetric torquatus
50 Ctenomys rondoni Miranda-Ribeiro, 1914
51 Ctenomys saltarius Thomas, 1912 opimus
52 Ctenomys scagliai Contreras, 1999 symmetric opimus
53 Ctenomys sericeus Allen, 1903 asymmetric magellanicus
54 Ctenomys sociabilis Peason and Christie, 1985 asymmetric no group
55 Ctenomys steinbachi Thomas, 1907 symmetric boliviensis
56 Ctenomys talarum Thomas, 1898 symmetric talarum
57 Ctenomys thalesi Teta and D’Elía, 2020 asymmetric magellanicus
58 Ctenomys torquatus Lichtenstein, 1830 symmetric torquatus
59 Ctenomys tuconax Thomas, 1925 asymmetric tucumanus*
60 Ctenomys tucumanus Thomas, 1900 symmetric tucumanus
61 Ctenomys tulduco Thomas, 1921
62 Ctenomys validus Contreras, Roig and Suzarte, 1977
63 Ctenomys viperinus Thomas, 1926
64 Ctenomys yatesi Gardner, Salazar-Bravo and Cook, 2014 boliviensis
For each species, if information is available, we indicate sperm type and species group as defined
by Parada et al. (2011). An asterick (*) indicates that species group allocation is inferred from the
topology presented by Mascheratti et al. (2000)
After almost two centuries of taxonomic studies centered in Ctenomys, here we

recognize 64 living species of tuco-tucos (Table 2.1). In the last review of Ctenomys
presented by Claudio Bidau (2015) 65 (not 64 as stated in page 820) living species
were listed for the genus. The list of Bidau and ours present several departures
(Table 2.2). Bidau (2015) listed three informal forms that are not nomenclatorially
available; wse omit them here for that reason. Bidau (2015) explicitly stated in the
text (and by using quotation marks) that two of those forms were not available, but
chose to list them as a form of recognizing their distinction at the species level as
well as the need to formalize them. The first of these two is the Bolivian form
C. “mariafarelli” described by Azurdy (2005); our assessment of the literature sug-
gest us that C. “mariafarelli” seems to represent the same lineage of species level
represented by the recently described taxon C. erikacuellarae Gardner, Salazar-
Bravo and Cook, 2014; as such, if it would have been properly described, C.
Fig. 2.1 Number of currently considered distinct living species of Ctenomys proposed between
the years 1826 and May 2020 (triangles). Species accumulated in the same interval of time (circles)
“mariafarelli” would have priority over C. erikacuellarae. Bidau (2015) also

included in his list C. “yolandae”; a form from Santa Fe Province in Argentina,
which was enunciated by Contreras and Berry (1984) in a meeting abstract. Available
evidence, including it being the unique known species with a complex asymmetric
sperm type (Vitullo et al. 1988) and showing a distinctive karyotype of 2n = 50,
FN = 67, 70, 78 (Ortells et al. 1990; Bidau et al. 2005), indicates “yolandae” in fact
represents a distinct species of tuco-tucos; as such, its formal description is needed.
Lastly, Bidau (2015) listed C. “rosendopascuali” Contreras, 1995 (without quota-
tion marks in Bidau 2015), as a distinct species, although raising doubts regarding
its availability. Here we considered that this name is unavailable (see below) and as
such, we exclude it from the species list. As a form to emphasize the need to for-
mally describe species (see below), we have omitted from our list nonavailable
binomens even if available evidence suggests they represent distinct lineages of
species level.
Seven species included in our list (i.e., C. andersoni Gardner, Salazar-Bravo and
Cook, 2014; C. bidaui Teta and D’Elía, 2020; C. contrerasi Teta and D’Elía, 2020;
C. erikacuellarae Gardner, Salazar-Bravo, and Cook, 2014; C. lessai Gardner,
Salazar-Bravo, and Cook, 2014; C. thalesi Teta and D’Elía, 2020; and C. yatesi
Gardner, Salazar-Bravo, and Cook, 2014) were described after the review of Bidau
(2015) entered the publication process. In addition, the forms C. colburni J. A. Allen,
1903, C. coyahiquensis, and C. goodfellowi Thomas, 1921 considered as distinct
species by Bidau (2015) were later synonymized, respectively, under C. magellani-
cus Bennet, 1836, C. sericeus, and C. boliviensis Waterhouse, 1848 based on mor-
phologic, karyotypic, and molecular data (Teta et al. 2020; Teta and D’Elía 2020;
Gardner et al. 2014).
Table 2.2 Departures in the list of distinct living species of Ctenomys presented in this study (see
Table 2.1) from the list of Bidau (2015), the last published taxonomic catalog of Ctenomys
Species Bidau (2015) This work
Ctenomys Signaled as a distinct species Nnot considered because the name is not
“mariafarelli” with a nonavailable name. available (it probably represents the same
species than C. erikacuellarae)
Ctenomys Signaled as a distinct species Not considered because the name is not
“yolandae” with a non-available name. available even when it may be distinct
Ctenomys andersoni Distinct (described after Bidau 2015
entered the press)
Ctenomys bidaui Distinct (described after Bidau 2015)
Ctenomys colburni Distinct Synonym of C. magellanicus
Ctenomys contrerasi Distinct (described after Bidau 2015)
Ctenomys Distinct Synonym of C. sericeus
coyahiquensis
Ctenomys distinct (described after Bidau 2015
erikacuellarae entered the press)
Ctenomys Distinct Synonym of C. boliviensis
goodfellowi
Ctenomys lessai Distinct (described after Bidau 2015
entered the press)
Ctenomys Signaled as a distinct species not considered because the name is not
“rosendopascuali” although expressing doubts available
on its availability
Ctenomys thalesi Distinct (described after Bidau 2015)
Ctenomys yatesi Distinct (described after Bidau 2015
entered the press)
Ctenomys azarae Distinct Synonym of C. mendocinus
Ctenomys porteousi Distinct Synonym of C. mendocinus
Finally, our list differs from that of Bidau (2015) in that two of the species listed
by him as distinct are here synonymized in what constitutes the single nomenclato-
rial act of this contribution. Here we consider C. azarae and C. porteuosi Thomas,
1919 as subjective junior synonyms of C. mendocinus. Both azarae and porteuosi
are forms of central Argentina that were described by Oldfield Thomas during the
first quarter of the twentieth century. Both forms together with C. australis Rusconi,
1934, C. flamarioni Travi, 1981, C. mendocinus, and C. rionegrensis Langguth and
Abella, 1970 are part of the C. mendocinus species group (Massarini et al. 1991;
D’Elía et al. 1999; Parada et al. 2011). The distinction of azarae and porteousi from
C. mendocinus has been largely questioned in the literature (e.g., Massarini et al.
1991: 141). The three taxa present the same karyotype polymorphism of 2n = 46,
47, and 48, as well as patterns of G-bands (Massarini et al. 1991, 1998); in addition,
C. mendocinus also displays a cytotype of 2n = 50 (e.g., Parada et al. 2012).
Multivariate analyses of skull measurements show that neither azarae nor porteousi
differentiate from C. mendocinus, as the three nominal forms have completely over-
lapping morphospaces (Massarini and Freitas 2005; Fornel et al. 2018). Finally,
genealogical analysis of mitochondrial DNA sequences, including sequences of

specimens collected at the three type localities, shows that variants recovered from
specimens of azarae and porteousi mix with variants of C. mendocinus (Mapelli
et al. 2017). The lack of monophyly of the three nominal forms goes beyond what
one expects for a gene tree not recovering the species tree (as it seems to be the case
of C. australis in the same gene tree where its variants form a clade nested within
the variation of C. mendocinus); variants accommodate in three main clades that are
not allopatric, including the fact that mitochondrial clades overlap in different com-
binations at same localities (even when almost half of the sampled localities have
only one or two sampled specimens and no locality has more than five sequenced
specimens). In addition, the divergence observed between clade pairs is relatively
low (up to 1.5% for a fragment of 899 bp of the control region plus 402 bp of the cyt
b gene). As such, for the moment, no emerging property (e.g., morphological or
karyotypic diagnosibility, monophyly) supports the distinction at the species level
of C. azarae or C. porteusi. Then, as here delimited, C. mendocinus is a species with
a large distribution in central Argentina including localities in the provinces of
Buenos Aires, Córdoba, La Pampa, and San Luis.
We close this section noting that, so far, no study of species delimitation of
Ctenomys has used a genomic approach neither the multispecies coalescent model.
As data acquisition is getting easier and cheaper, we expect that in the next few
years the current taxonomic scheme be further tested with these approaches (see
comment in Lessa et al. 2014). In this regard, it would be of much interest to see
how robust are current inferences that, regarding molecular data are mostly, if not
exclusively, based on mitochondrial DNA sequences. Another relevant aspect is that
large geographic areas still remain unexplored, a scenario that should be overcome
with further specimen collection. To visualize the importance of this fact, it is rele-
vant to note that several candidate species have been proposed (see below). Similarly,
the distinction of some nominal forms has been called into question, while the dis-
tinction of a large fraction of currently considered distinct species has not been
adequately evaluated (see below). As such, given these precedents and that currently
several research groups in distinct countries are working on the taxonomy of
Ctenomys, we anticipate that our list of living species would soon be outdated.
2.2.2 Candidate Species
As it happens when any group that is taxonomically assessed, distinct candidate

species of Ctenomys have been identified. As the practice dictates, the distinction of
these forms should be properly evaluated with additional studies, and if proven to be
distinct, we advocate for formally describe them. For example, Teta and D’Elía
(2020) assessed the distinction of some candidate species previously identified on
the base on DNA sequences and karyology by Bidau et al. (2003) and Parada et al.
(2011), and after corroborating their distinction using an integrative taxonomic
approach, named and described C. bidaui, C. contrerasi, and C. thalesi.
Candidate species of Ctenomys have been identified based on distinct sources of

evidence. Next, we illustrate some cases of candidate species involving Argentinean,
Brazilian, and Chilean tuco-tucos, which differ in their complexity and in the evi-
dence used to identify them.
A series of populations from pre-Andean areas of the Araucania Región, Chile,
display a distinct cytotype (2n = 28; FN = 48) from nearby populations of C. mau-
linus brunneus Osgood, 1943 (2n = 26, FN = 50; Gallardo 1979). This difference
prompted Gallardo (1979) to suggest 2n = 28 populations represent an undescribed
species and referred to them as C. sp. No published study has further assessed the
taxonomic status of these populations (but see Aguilar 1988). Similarly, Leipnitz
et al. 2020) identified, on the basis of mitochondrial DNA sequences, two lineages
in the Brazilian states of Mato Grosso and Rondônia, hypothesizing that they are
lineages of species level. Authors referred to these lineages as C. sp. “xingu” and
C. sp. “central” awaiting new analyses to test their distinction.
Another example where candidate species have been identified pertains to popu-
lations of the Corrientes group, an assemblage of populations from Corrientes prov-
ince, Argentina, which is part of the C. torquatus species group (Parada et al. 2011)
and exhibit large chromosomic variation, ranging from 2n = 41 to 2n = 70 (e.g.,
Ortells et al. 1990; Giménez et al. 2002). Three nominal forms, C. dorbignyi
Contreras and Contreras, 1984; C. perrensi Thomas, 1896, and C. roigi Contreras,
1988 are associated with the Corrientes group and are considered as representing
distinct species (e.g., Bidau 2015; Teta et al. 2018; Table 2.1); however, the biologi-
cal boundaries, and then also the geographic distribution, of these species, in par-
ticular of C. dorbignyi and C. perrensi, have been long discussed. The taxonomic
literature is relatively vast, at least when compared to that available for tuco-tucos
from most of the other areas and complexes (the exception would be those well-
studied taxa from southern Uruguay and Rio Grande do Sul, Brazil). Mitochondrial
DNA sequences analyses fail to recover C. dorbignyi and C. perrensi as monophy-
letic, while roigi was found as monophyletic and nested within perrensi (Giménez
et al. 2002; Mirol et al. 2010; Buschiazzo et al. 2018; Caraballo and Rossi 2018a;
see also Gómez Fernández et al. 2012). In addition, there is a lack of correspon-
dence between cytotypes and haplogroups (e.g., Giménez et al. 2002; Caraballo and
Rossi 2018a), blurring the delineation of lineages of species level. This situation is
accentuated by the fact that, surprisingly given that these populations have been
extensively studied since the decade of 1980, there is not a comprehensive assess-
ment of the morphological variation of populations of the Corrientes group.
Available morphologic information is limited to that presented on the species
descriptions, which were based on quantitative assessments of small sample sizes,
and to a single quantitative analysis conducted by Contreras and Scolaro (1986)
centered on four groups of populations then assigned to C. dorbignyi (one of these
groups is more related to C. perrensi than to C. dorbignyi; Gimenez et al. 2002).
Notwithstanding, Giménez et al. (2002) suggested the existence of two candidate
species, which were labeled as Ctenomys sp. α and Ctenomys sp. β; both candidate
species encompass populations assigned to C. perrensi (those of C. sp. β were the
ones earlier assigned to C. dorbignyi by Contreras and Scolaro 1986). This
suggestion was reinforced by the results of Caraballo et al. (2012). In addition,

Caraballo and Rossi (2018a) identified another candidate species, termed
“Sarandicito,” encompassed by an isolated population in southern Corrientes that
has been previously considered as part of C. dorbignyi. We expect that this changing
and complex scenario starts to settle down when patterns of morphological variation
are thoroughly assessed and nuclear gene DNA sequences are incorporated in the
analysis of this complex group of tuco-tucos.
The proposition of candidate species is a natural outcome of taxonomic work.
We advocate conducting species validation analysis to test the distinction of the
candidate species here reviewed as well as the other presented in the literature
(including some for which if proven distinct, names may be available; e.g., C. len-
tulus; see Teta et al. 2020). Then, if results of the validation analyses show that they
in fact constitute distinct species, we advocate to formally describe them to, among
other reasons, avoid situations as the ones commented below. Informal names have
no regulation; as such, the literature shows that they vary from study to study (e.g.,
a lineage first referred as C. sp. α and later as Iberá), at the time that the same deno-
tation (e.g., C. sp. 1) can be applied to distinct candidate species. These facts ham-
per effective communication and should be avoided.
2.2.3 Unavailable Names
A common issue in the literature of Ctenomys is the extensive usage of nonavailable

names to refer to putative lineages of species level. These cases, which includes that
of the already commented “yolandae,” depart from the traditional way of labeling
candidates species (e.g., sp. α; sp. “xingu”) in that putative distinct species are iden-
tified with binomials (C. “yolandae”), which did not accomplish the provisions of
The International Code of Zoological Nomenclature (ICZN 1999) and as such, are
unavailable. Most commonly, these names have been introduced in papers without
selecting a holotype and providing a diagnosis or in meeting abstracts. One of these
names is “eremofilus,” (also referred as C. eremophilus or C. eremicus), which was
advanced by Contreras and Roig (1975) for populations from the Ñacuñán Biosphere
Reserve, Mendoza, Argentina; this name was used regularly in the literature until
Parada et al. (2012) showed that those populations belong to C. mendocinus. As
such, “eremofilus,” besides not being available, does not represent a distinct species,
differing this way from the case of “yolandae” (see above), at the time that resem-
bling that of C. “chasiquensis.” Perhaps the most extensively used informal name in
the literature of Ctenomys is C. “chasiquensis,” mentioned for the first time in a
meeting abstract by Contreras, Manceñido and Ripa Alsina (1970) and mentioned
by Contreras and Maceiras (1970) as a subspecies of C. azarae (C. a. “chasiquen-
sis”) to refer to tuco-tuco populations from the area of the Chasicó Lagoon, south-
western Buenos Aires Province, Argentina. The name, now at the species level,
keeps been used up today (e.g., Mora et al. 2016; Mapelli et al. 2019). However, in
addition to have never been formalized, to be best of our knowledge no evidence

supports the distinction of “chasiquensis” as a distinct species. On the contrary,
karyotypic data (Massarini et al. 1991) and analysis of mitochondrial DNA
sequences (Mapelli et al. 2017; see also Mora et al. 2016) strongly indicates that
populations referred to as “chasiquensis” belong to C. mendocinus. It seems that the
still ongoing mentions to C. “chasiquensis” are caused by some sort of taxonomic
inertia. Here we made a call to no longer use this name because it does not satisfy
nomenclatorial rules and there is no need to do it since populations referred to it
belong to C. mendocinus.
There are other unavailable names, all of them coined by Contreras and collabo-
rators (e.g., C. “alvaromonesi,” C. “avellanedae,” C. “felixi,” C. “nevoi,” C. “para-
milloensis”; see Contreras and Bidau 1999: Bidau et al. 2003), that fortunately have
not gained momentum on the literature. Other names, such as beltzeri, marthae,
monesi, and reigi, described as subspecies of C. minutus by Contreras and Contreras
(1984) in a meeting abstract are also unavailable; however, these names were con-
sidered as synonyms of C. minutus by Woods and Kilpatrick (2005). We highlight
the relevance of complying with the provisions of International Code of Zoological
Nomenclature (ICZN 1999), in particular, with the designating of a holotype. Name
bearing type specimens constitute the only way to unambiguously link taxonomic
names with the lineages to which they apply (see D’Elía 2012 for additional
discussion).
Regarding type specimens, we call attention to an issue that seems to have passed
unnoticed in the taxonomic literature of Ctenomys. It is the fact that Contreras,
when describing C. argentinus Contreras and Berry, 1982, C. bonettoi Contreras
and Berry, 1982, and C. d’orbigny (also the nonavailable C. “yolandae”) designed
two specimens as “holotypes” for each of these species. The Code establishes that
the holotype is a single specimen; as such, the two specimens listed as holotypes of
a given species should be considered as syntypes forming part of a type series (Art.
72), for which one can be selected as a lectotype to serve as the name-bearing type
(Art. 74; see also Art. 72.2).
We close this section mentioning an issue raised by Bidau (2015: 866) regarding
the possibility that the name C. “rosendopascuali” be unavailable, given that it is
not clear if the 1995 issue of Nótulas Faunísticas, were the species description
appears, was in fact published or not. Yolanda Davis communicated one of us (PT)
that that issue was probably not published. In addition, we note that in Contreras
and Bidau (1999), C. “rosendopascuali” is indicated on page 4 without authorship
and as in press 1999, while on page 11 is mentioned with the authorship of Contreras
1999. Recently, Agnolin et al. (2020) stated that in a personal communication to the
first author, J.R. Contreras mentioned that in fact the description of C. “rosendopas-
cuali” was never published. As such, we have not included “rosendopascuali” in the
list of currently distinct and available species.
2.2.4 Synonyms
About nine species of Ctenomys have taxonomic names in their synonymy. Some of
these names are traditionally recognized as subspecies (e.g., C. maulinus brunneus),
although in most, if not all, of these cases, the distinction of these putative distinct
lineages of subspecies level has not been assessed with contemporaneous
approaches. For example, the forms barbarus Thomas, 1921; budini Thomas, 1913;
mordosus Thomas, 1926; sylvanus Thomas, 1919; and utibilis Thomas, 1921,
together with the nominotypical frater Thomas, 1902 are recognized as subspecies
of C. frater, an arrangement that goes back to Cabrera (1961; see also Bidau 2015)
and that since has not been assessed. However, without providing new data, Galliari
et al. (1996) treated barbarus, budini, and sylvanus as distinct species; a similar
scheme was presented by Wood and Kilpatrick (2005) who listed three distinct spe-
cies within this complex: budini (including barbarus as a subspecies), frater (includ-
ing mordosus as a subspecies), and sylvanus (including utibilis as subspecies). The
degree of understanding of the variation within and among frater and associated
names is a good example of the taxonomic knowledge of most species of Ctenomys
with names associated in their synonymy: the distinction of the nominal forms has
been, if so, seldom evaluated and their ranking has shifted over taxonomic history
without necessarily providing evidence supporting changes. We expect this review
to call the attention over these cases and prompted colleagues to study them.
In addition, the status of several currently considered distinct species is dubious,
either because their description was based on minor differences shown by a small
series of specimens and their distinction was not later evaluated or because there is
evidence questioning their distinctiveness. Several species fall in the first category.
For instance, C. tulduco, which was described by Thomas (1921) and is known from
the surroundings of its type locality in San Juan Province, Argentina remains with
unknown karyotype and sperm type (Bidau 2015) and has not been included in any
taxonomic nor phylogenetic study. However, Cabrera (1961) regarded it as a sub-
species of C. fulvus Philippi, 1860. Similarly, the status of C. validus Contreras,
Roig, and Suzarte, 1977, a species from central-northern Mendoza with unknown
karyotype and sperm type, is dubious. When it was described, Contreras et al.
(1997) did not compared it with the nearby and widely distributed C. mendocinus.
However, Rossi et al. (2002), on the basis of a morphometric analysis and unpub-
lished allozimic data (page 281) of Gallardo and collaborators, questioned the dis-
tinction of C. validus with respect to C. mendocinus.
As an example of the species whose distinction has been questioned in the litera-
ture, we mention one involving the type species of the genus, C. brasiliensis.
Fernandes et al. (2012) raised the possibility that C. brasiliensis traditionally
regarded as from Minas Gerais, Brazil, an area where no tuco-tuco is known, be a
senior synonym of C. pearsoni, a form known from southern Uruguay and nearby
areas of Argentina. This hypothesis has its base on a reinterpretation of the place-
ment of the type locality of C. brasiliensis as to Minas, Uruguay. Morphometrically
the type specimen of C. brasiliensis falls in the morphospace of C. pearsoni
(Fernandes et al. 2012) and is close to that a C. torquatus, a species closely related
to C. pearsoni; the known distribution of the later does not reach Minas, but it is no
far from there. As in similar cases pending of resolution, comparisons of DNA
sequences from putative topotypes and the type specimen of C. brasiliensis would
allow clarify this issue. As the area of Minas is quite accessible and the type of
C. brasiliensis is preserved as skin and skull, such a study seems feasible.
2.2.5 Final Remarks of the Taxonomy Section
As it is clear from the cases reviewed above the taxonomy of Ctenomys is far from
been stable; Cabrera (1961) recognized 26 tuco-tuco species, Honacki et al. (1982)
recognized 33, Woods (1993) recognized 38 species, Woods and Kirlpatrick (2005)
listed 60 species, Bidau (2015) listed 65, while here we recognized 64 (with several
departures from those 65 recognized by Bidau). This variation in numbers and con-
tents not only reflects the discovery of new forms of species-level; it is also due to
distinct considerations on the distinction of some populations, as well as distinct
operational criteria adopted on a given list (e.g., to include or not names that are
nomenclatorially unavailable, even if the evidence supports the hypothesis of them
representing distinct species-level lineages).
We close this section on Taxonomy remarking that studies based on the integra-
tion of chromosomal, molecular, and morphological evidence are still scarce (e.g.,
Freitas et al. 2012; Gardner et al. 2014; Teta and D’Elía 2020) and that the sampling
density for data types is, in general, uneven (e.g., species well characterized at the
chromosomic level limits to those of Rio Grande do Sul, Brazil). Similarly, molecu-
lar evidence, even when several specimens have been analyzed in some studies,
limits for the most part to mitochondrial DNA sequences. Moreover, in general, the
geographic coverage of the taxonomic studies is narrow (e.g., the species that we
just described are known from a handful of localities; Teta and D’Elía 2020). Given
these facts, at the time that the taxonomic status of several populations and nominal
forms is dubious, we anticipate that several departures from our list will start to
accumulate in the near future.
2.3 Phylogenetic Relationships
Even though several studies have been published since the early 1990s, it can be
said that tuco-tuco phylogenetic studies are still in their infancy. For instance, only
one study, with a reduced taxonomic sampling, has incorporated nuclear DNA
sequences; all other molecular studies analyze mitochondrial DNA sequences. The
two analyses incorporating morphological evidence have also incomplete taxo-
nomic sampling. In fact, none of the published studies have nearly complete species
sampling.
2.3.1 Early Phylogenetic Analyses
Three strictly phylogenetic analyses of species of Ctenomys were published before

the incorporation of DNA sequences; they included only Bolivian or Argentinean
species. The first one was part of a coevolution study and analyzed qualitative mor-
phological features and karyotypic characters of six Bolivian species (Gardner
1991). Cook and Yates (1994), based on allelic variation at 21 allozyme loci,
assessed relationships among seven species and an at the time undescribed form
from Bolivia (currently recognized as C. yatesi). The third study is that of Ortells
(1995) where, based on G-band patterns, relationships among 11 species and five
karyomorphs from Argentina were assessed.
2.3.2 DNA Sequence-Based Studies
During the last years of the decade of the 1990s, phylogenetic analyses of species of
Ctenomys incorporated DNA sequences as evidence. With a single exception, these
studies are based on sequences of the mitochondrial DNA cytochrome b (cytb)
gene. The single study incorporating nuclear DNA sequences is the one conducted
by Castillo and collaborators (2005) that, in addition to cytb sequences, included
sequences of two nuclear genes, namely the 4th intron of the rhodopsin gene and the
2nd intron of vimentin. This study, which included ca. 20 species, corroborated
some groups found in previous mitochondrial topologies but did not provide resolu-
tion at the base of the tree. It was not expanded in further studies; therefore what we
know about the phylogenetic relationships of species of Ctenomys is basically based
on cytb variation. This is certainly a main limitation of our current knowledge; we
expect that this scenario improves in the next few years with the adoption of a mul-
tilocus or even genomic approach and the use of an exhaustive taxonomic coverage.
Five cytb-based phylogenetics studies of tuco-tucos were published two decades
ago (Cook and Lessa 1998; Lessa and Cook 1998; D’Elía et al. 1999; Mascheratti
et al. 2000; Slamovits et al. 2001), starting the current era of phylogenetic studies of
Ctenomys. The first and last studies present phylogenies that were inferred to assess
the diversification rate of Ctenomys and the evolution of satellite DNA, respectively.
The other three studies were motivated with a more traditional systematic aim, the
clarification of species relationships. The study of Lessa and Cook (1998) includes
Argentinean and Bolivian species; that of D’Elía et al. (1999) builds over the taxo-
nomic sampling of Lessa and Cook (1998) adding species from Brazil, Chile, and
Uruguay. Still, both studies covered a relatively small fraction of the species diver-
sity. The study of Mascheratti et al. (2000) has a broader taxonomic sampling (ca.
28 species), including for the first time a Paraguayan species and additional species
from Argentina, but has a much-reduced character sampling (ca. less than 400 bp vs.
the complete gene in the other studies). This may be the reason behind why subse-
quent phylogenetic studies did not include sequences gathered by Mascheratti et al.
(2000); as such, the phylogenetic position of some species (e.g., C. bergi, C. bon-
netoi, C. pilarensis Contreras, 1993) has not been further assessed after Mascheratti
et al. (2000).
Early in this decade, Parada et al. (2011) published the results of a phylogenetic
analysis that remains the reference topology of the phylogenetic relationships
among species of Ctenomys. Considering that this study was published 10 years
ago, it is clear that not much progress has been achieved since then. Subsequent
studies mostly built on the matrix of Parada et al. (2011) adding sequences of previ-
ously not included species (i.e., ibicuensis, Freitas et al. 2012; bicolor Miranda-
Ribeiro, 1914: Leipnitz et al. 2020, see also Stolz et al. 2013; dorsalis Thomas,
1900: Londoño-Gaviria et al. 2018; famosus Thomas, 1920: Sánchez et al. 2018).
Notwithstanding, some species (e.g., johanis Thomas, 1921; juris Thomas, 1920;
osvaldoreigi; pilarensis; paraguayensis Contreras, 2000; tulduco; validus) remain
without being incorporated in any phylogenetic analysis. As follows from what has
been said, the comments we present below are mostly built on the results of the
study of Parada et al. (2011).
2.3.3 Species Relationships and Species Groups
Most species of Ctenomys fall in eight main clades that Parada et al. (2011) referred
as to species groups and are named using the oldest species name of each group.
These groups are the boliviensis, frater, magellanicus, mendocinus, opimus, tala-
rum, torquatus, and tucumanus species groups. In Table 2.1, we provide the group
affiliation of the species here recognized. The richness of the species groups is rela-
tively homogeneous, ranging between 2 and 8 species. Except for the magellanicus
and torquatus groups, the other species groups were previously identified, with
some differences in composition, and referred with other names (mostly referring to
their geographic distribution), by Contreras and Bidau (1999; see Table 2.1 in
Parada et al. 2011). As after Parada et al. (2011), no gene other than cytb has been
used to assess the phylogenetic relationships of Ctenomys, and the contents of spe-
cies groups have been stable. Modifications only pertain to the inclusion of new
species into already defined groups, as new sequences become available. For
instance, C. famosus was shown to be a member of the mendocinus species group
(Sánchez et al. 2018), and the magellanicus species group was expanded to include
the recently described species C. bidaui, C. contrerasi, and C. thalesi (Teta and
D’Elía 2020).
Four species, C. leucodon Waterhouse, 1848; C. maulinus Philippi, 1872;
C. sociabilis Pearson and Christie, 1985; and C. tuconax Thomas, 1925 were not
recovered as members of any species group by Parada et al. (2011) as there are not
closely related to any other species of the genus. However, given the topology por-
trayed in Mascheratti et al. (2000), C. tucounax, C. juris, and C. pilarensis would
belong to the tucumanus species group. In addition, from that topology, it follows
that C. bergi and C. bonettoi belong to the mendocinus group. However, several
species still remain with unclear affiliation as they have not been included in any
published phylogenetic analysis (Table 2.1). While species groups are relatively
well supported (the exception is the torquatus species group), relationships among
them are poorly supported; the exception being the clade formed by the mendocinus
and talarum groups. This lack of resolution among species groups extends to the
base of the radiation of Ctenomys.
A somewhat surprising, although not strongly supported, result of the phyloge-
netic analysis is that the basal dichotomy of the crown group of Ctenomys sets
C. sociabilis as sister to a clade formed by all other species included in that study
(Parada et al. 2011). The position of C. sociabilis as sister to all other species of the
genus was corroborated by the studies of Londoño-Gaviria et al. (2018), who used
a distinct sequence of C. sociabilis than the one used by Parada et al. (2011), and
Sanchez et al. (2018), as well as in the Maximum Likelihood analysis of Leipnitz
et al. (2020); there is a problem with the placement of the root of the gene tree por-
trayed in Fig. 3, where C. sociabilis appears as sister to the octodontids and not to
the other species of Ctenomys). Other studies have recovered another basal dichot-
omy. The position of C. sociabilis as sister to the other tuco-tucos is not corrobo-
rated in the Bayesian analysis of Leipnitz et al. (2020) and in the studies of Roratto
et al. (2015) and Caraballo and Rossi (2018b); in these topologies, the frater species
group is sister to the clade formed by all other species of Ctenomys, where C. socia-
bilis appears sister to C. tuconax. However, these relationships are not significantly
supported. In sum, relationships at the base of Ctenomys are better portrayed as a
polytomy involving several species groups, as well as some lineages formed by
single species (e.g., C. sociabilis). This basal polytomy has been classically regarded
as resulting from an explosive radiation (e.g., Castillo et al. 2005).
Available topologies need to be further tested with a broader taxonomic sam-
pling that includes those species missing from current datasets as well as with the
incorporation of other characters, in particular nuclear DNA sequences. In this
sense, the study of Castillo et al. (2005) may be regarded as a starting point.
However, we note that the two loci employed by Castillo et al. (2005) are relatively
short for current sequencing technologies (i.e., more characters could be obtained
with the same effort) and that they have not been used in other studies of relation-
ships among caviomorphs (e.g., Upham and Patterson 2015). In addition, a genome-
wide sampling approach to tackle tuco-tuco phylogenetics seems feasible now
(Lessa et al. 2014). Similarly, the inclusion of morphologic characters is much
needed; in this sense, we highlight the analysis recently presented by De Santi et al.
(2020), in which patterns of morphological and molecular variation were integrated.
In this line, the analysis of morphologic variation, in a phylogenetic context, is
much needed to diagnose the recovered species groups.
2.3.4 Subgenera of Ctenomys
Based on trenchant characters, two subgenera have been nominated on the basis of
species of Ctenomys. Thomas (1916) erected Haptomys to contain C. leucodon,
highlighting its procumbency and unpigmented incisors. Subsequently, Osgood
(1946) described C. conoveri under the subgenus Chacomys, remarking in its over-
all large size and distinctly grooved incisors. Cabrera (1961), following Osgood
(1946), only recognized Ctenomys and Chacomys. The phylogenetic analyses
reviewed above lend no support for classification including three subgenera (i.e.,
Chacomys, Ctenomys, and Haptomys). Meanwhile, a scheme of two subgenera with
Chacomys and Ctenomys is weakly supported in some studies if Chacomys is
expanded to encompass the frater species group; we note, however, that Chacomys
would have to be re-diagnosed. In light of available information, we think that it is
adequate to maintain a scheme of one genus without subgenera.
2.3.5 Dating
Four studies, using a dense taxonomic sampling, have dated the radiation of
Ctenomys (Parada et al. 2011; Roratto et al. 2015; Caraballo and Rossi 2018b;
Leipnitz et al. 2020). These studies have in common that they are based on cytb
gene sequences but differ on several implementation aspects, most notably in cali-
bration points (Table 2.3). Estimates presented by Parada et al. (2011) are older than
those of the other studies, and their confidence intervals do not overlap. Those of
Roratto et al. (2015), Leipnitz et al. (2020), and Caraballo and Rossi (2018b) are
more in line among them, although the estimate for the stem age of Ctenomys in the
latter study is the oldest of the four studies. The oldest known fossils of Ctenomys,
gathered at the Uquía Formation in Jujuy, Argentina, have an age of 3.5 Ma (Reguero
et al. 2007; Verzi et al. 2010); this age falls within the confidence interval of
(2.38–5.14) of the younger molecular estimate that corresponds to that of Leipnitz
et al. (2020). As such, this estimate can be discarded as it is contradicted by the fos-
sil record. The remaining three studies imply a ghost lineage for Ctenomys of ca.
2.1–5.7 Ma (0.8–9.1 Ma if confidence intervals are considered). Additional fossil
evidence, as well as sequencing of new loci, will help resolve these uncertainties.
The study of Cook and Lessa (1998; see also Castillo et al. 2005) focused on
diversification rates of Ctenomys and with a reduced taxonomic sampling, found
that the diversification of Ctenomys decreases toward the present. In a broader phy-
logenetic context, Upham et al. (2019) found that Ctenomys is one of the mammal
lineages with an accelerated speciation rate.
Table 2.3 Selected results of four dating analyses of Ctenomys. Mean estimated ages (Mya) are
indicated with 95% confidence intervals in parenthesis
Parada et al. Roratto et al. Caraballo and Rossi Leipnitz et al.
(2011) (2015) (2018b) (2020)
Gene Cytb Cytb Cytb Cytb
Number of calibration 1* 3** 7*** 2****
points#
Stem Ctenomys 17.97 11.17 21.35 10.98
(13.5–23.0) (10.20–12.13) (15.59-26.69) (9.08–12.83)
Crown Ctenomys 9.22 5.64 5.88 3.71
(6.4–12.6) (4.38–6.98) (4.32–7.54) (2.38–5.14)
All Ctenomys minus 7.74 – N/A N/A
sociabilis (5.7–10.2)
Median crown species 1.44–4.97 – – 0.52–2.18
groups
N/A implies that the given node was not recovered in the phylogenetic analysis (see text for
details). A dash (–) indicates information not provided in the paper
#
for details of the implementation of each calibration point (e.g., type of distribution) as well as the
base of each point see the original publications
*
MRCA for Caviomorpha: 28.5–37
**
Origin Caviomorpha: 34 (31–37); Ctenomyidae-Octodontidae: 10.65 (9.8–11.5); origin
Ctenomyidae: 5 (3.3–7.5)
***
Crown Octodontoidea: 27.2–41.0; crown Echimyidae 20.5–24.2; crown Abrocomidae:
2.0–4.0 Myr; crown Octodontinae: 6.8–9.0 Myr; the clade form by Aconaemys and Spalacopus:
5.0–9.0; the clade form by Octomys, Tympanoctomys (including Pipanacoctomys): 2.0–4.0; Crown
Ctenomys: 3.5–6.0 Myr. A second dating analysis with 4 calibration points was also conducted;
results derived from the 4 and 7 calibration point datasets are congruent in terms of overlapping
95% credibility intervals
****
Ctenomyidae-Octodontidae: 10.65 (9.8–11.5); Ctenomys: 5.0 (3.5–6.5)
2.3.6 Trait Evolution
Studies about the evolution of distinct traits of Ctenomys are scarce. Several men-
tions are made in the literature regarding the direction of chromosomal rearrange-
ments, but no study has assessed chromosome evolution at a large scale and with an
explicit phylogenetic context.
Verzi and Olivares (2006), using a qualitative and quantitative approach, recog-
nized two morphotypes within Ctenomys, for which they used the names of “C. dor-
bigny morphotype” and “C. fulvus morphotype.” These two groups differ in the size
and shape of the postglenoid fossa, auditory meatus, and condyloid and postcondy-
loid processes of the mandible. Verzi and Olivares (2006) included in the first mor-
photype the species C. dorbignyi, C. leucodon, C. lewisi, C. pearsoni, C. tucumanus,
and two undescribed forms (C. sp. “bellavista”, C. sp. “perucho”), while in the
second they included C. fulvus, C. australis, C. azarae (here regarded as a synonym
of C. mendocinus), C. opimus, C. maulinus, C. steinbachi, C. talarum, and another
undescribed form (C. sp. “trapalco”). Despite the fact that several species have not
been assessed on regard to these features and that none of these groups is monophy-
letic, it is of interest to note that each of the groups defined by Parada et al. (2011)
include only members of one of the morphotypes described by Verzi and
Olivares (2006).
Penial morphology was studied by Balbontin et al. (1996), who described three
main types of phallus within Ctenomys. One morphotype, referred to as “spike bear-
ing species”, is present in C. australis, C. azarae (= C. mendocinus), C. porteousi (=
C. mendocinus), C. rionegrensis, and C. talarum. The second morphotype called
“spiny-bulb bearing species,” encompasses C. dorbigny, C. pearsoni, C. perrensi,
C. roigi, and some other populations from Corrientes, Argentina. Finally, the third
morphotype is exclusively known from C. “yolandae” and shows both spikes and
spiny bulbs. Broadly viewed, “spike bearing species” correspond to the mendocinus
and talarum species groups, which are sister to each other, while those referred to
as “spiny-bulb bearing species” correspond to the torquatus species group (sensu
Parada et al. 2011).
Species of Ctenomys display three sperm morphotypes: simple symmetric, sim-
ple asymmetric, and complex asymmetric. Most species are characterized by one of
the first two types, whereas the third one is known only from C. “yolandae” (Feito
and Gallardo 1982; Vitullo et al. 1988; Vitullo and Cook 1991). The sperm type of
several species remains unknown (see Table 2.1). The asymmetric morphotype was
first described by Feito and Barros (1982) and presents a paddle-like head with a
postacrosomic process at the base of the head opposite the insertion of the flagel-
lum. This morphotype is not found in other mammals (Vitullo et al. 1988). The
symmetric morphotype lacks the postacrosomic process, while the complex asym-
metric presents two of these processes. Reconstruction of the evolution of sperm
morphs using parsimony agrees with the hypothesis that the symmetric morph is the
ancestral character state (Vitullo et al. 1988; Vitullo and Cook 1991); however, the
asymmetric sperm may have evolved more than once in the history of Ctenomys
(Lessa and Cook 1998; D’Elía et al. 1999). As such, the polyphyly of the asymmet-
ric sperm species falsifies the evolutionary scheme suggested by Feito and Gallardo
(1982), Vitullo et al. (1988), and Vitullo and Cook (1991), who, without formally
postulating the reciprocal monophyly, proposed two major lineages of Ctenomys
based on these sperm types.
We expect those upcoming denser and more robust phylogenies to provide the
framework for the study of the evolution of these and other traits within Ctenomys.
Chromosomal evolution, which is strikingly large and has played a significant role
in the study of the evolutionary biology of Ctenomys (e.g., Gallardo 1991; Lopes
et al. 2013), is probably the most outstanding of these character sets. The evolution
of sociality is a more recent but significant area of great interest (e.g., Lacey
2004, MacManes and Lacey 2012; Tomasco et al. 2019) to include in a phylogenetic
framework.
2.3.7 Final Remarks on Phylogenetic Relationships
Much remains to be learned about the phylogenetic relationships of the species of

Ctenomys, and the same is true about the timing of their diversification. We expect
that in the upcoming years this scenario will change. Perhaps paradoxically, one of
the major challenges still is to secure samples of all recognized species, including
those obtained at or near type localities. Collaborative efforts among research
groups seem to be the most promising way to secure a dense taxonomic sampling
and associated tissue availability for the genus. Meanwhile, a dense molecular char-
acter sampling, even one at the genomic scale, nowadays, appears within reach.
Integrating molecular and morphological characters presents additional challenges
(but see De Santi et al. 2020). All of these difficulties trace back to the need to
assemble adequate specimen series for each species. In this regard, we close this
chapter by making a call to our colleagues on the need to keep collecting voucher
specimens of Ctenomys, an essential step toward the improvement of our under-
standing of its evolutionary history.
Acknowledgments We express our gratitude to the editors of this book for their invitation to
participate. Similarly, we thank all colleagues and students with whom we have exchanged ideas
on the systematics of tuco-tucos over several years. Finally, we would like to recognize the effort
of those who keep collecting specimens in the field and to those working in the collections that
maintain these specimens available for further studies.
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Chapter 3
Speciation Within the Genus Ctenomys:
An Attempt to Find Models
3.1 Introduction
Speciation is a process that results in the emergence of new species, an evolutionary

force opposed to extinction that causes species to disappear. As a result, there is a
natural balance between these two forces that maintain biodiversity. These pro-
cesses, speciation and extinction, cause the biodiversity on Earth to be changed or
renewed over time.
Although the speciation processes are complex, two major classes can be identi-
fied: allopatric and sympatric processes. An allopatric process results from the
emergence of extrinsic factors, like a geographical barrier that separates populations
of the same species, which, throughout time and due to isolation, turn into two dif-
ferent species (Mayr 1942). On the other hand, sympatric speciation, initially pro-
posed by Darwin (1859) and totally refuted by Mayr (1963), is due to the intrinsic
differentiation of a particular species within the same area. Additionally, the sym-
patric process causes mechanisms of reproductive isolation to develop between sis-
ter species leading to differentiation. Noteworthy, a very important condition is that
both forms have never undergone an allopatric process (Li et al. 2016).
Chromosomal speciation has been discussed for many years throughout chapters
of classic books and articles as, for example, White (1978), King (1993), Rieseberg
(2001), and Dobigny et al. (2017), as well as the role of chromosomal rearrange-
ment in this process. Thus, rearrangements such as the Robertsonian translocations
have the role of changing the organization of chromosomes within the nucleus,
increasing or decreasing the number of chromosomes. Inversions do not change the
chromosome number but they change the organization of chromosomes even more
because segments within a chromosomal arm are inverted (paracentric inversion).
T. R. O. de Freitas (*)
Department of Genetics, Federal University of Rio Grande do Sul,
Porto Alegre, Rio Grande do Sul, Brazil
e-mail: thales.freitas@ufrgs.br

https://doi.org/10.1007/978-3-030-61679-3_3
44 T. R. O. de Freitas
Also, the inverted segment can contain the centromere and thus involve the two
chromosomal arms (pericentric inversion).
Hybrid zones in which the hybrids survive beyond the F1 generation, crossing
each other and with their parental types, can form a population with a wide variety
of recombinant types (Harrison 1993). Thus, a hybrid zone favors the introgression
of alleles, which are incorporated from one taxon to the other (Harrison 1993). This
exchange of genes leads to the disappearance of parental differences and, conse-
quently, to the appearance of recombinant species (Barton 2001). Many factors lead
to introgressions, such as selective pressure and consequent fitness due to the viabil-
ity of the hybrid, differentiated dispersion between males and females, and choice
of partners. Importantly, these are the factors that determine the size and direction
of the introgression (Petit and Excoffier 2009).
The genus Ctenomys Blainville, 1826 comprises approximately 65 living species
(Bidau 2015; Freitas 2016; D’Elía et al. 2020; Chap. 3 of this volume), sharing
some common characteristics, such as small populations distributed in fragments,
which are associated with low adult dispersion rates that lead to patterns of low
genetic variation within populations and high genetic divergence between popula-
tions (Busch et al. 2000; Wlasiuk et al. 2003). Additionally, habitat discontinuities,
with numerous barriers to species dispersion, are also responsible for restricting
individuals to their native areas. Such conditions can result in increased intraspecific
competition within populations and, consequently, in inbreeding (Galiano et al.
2016). These characteristics also favor the setting of new chromosomal rearrange-
ments, which is considered important for the diversification of ctenomids (Reig
et al. 1990; Lessa and Cook 1998; Lacey et al. 2000; Freitas 2006; Parada et al. 2011).
The polytypic genus Ctenomys was initially divided into groups of species based
on biogeographic, morphological, and cytogenetic data. Contreras and Bidau (1999)
classified 34 species and divided them into nine groups: Bolivian-Mato Grosso (4
species), Bolivian-Paraguayan (3 species), Patagonian (4 species), Mendocinus (5
species), Oriental (3 species), Chaco (6 species), ancestral (2 species), Corrientes (3
species), Chilean spp. (2 species), and uncertain position (2 species). Parada et al.
(2011) used molecular markers by sequencing the mtDNA Cyt-b gene to analyze 39
species and divided them phylogenetically into eight groups: Boliviensis (4 spe-
cies), Mendocinus (5 species), Torquatus (6 species), Talarum (2 species), Opimus
(4 species), Magellanicus (6 species), Tucumanus (4 species), and frater (4 species).
Afterward, three phylogenetic trees were determined, including 54 species, (Freitas
et al. 2012) and 50 species with 10 species from Bolivia (Gardner et al. 2014). More
recently, a third phylogenetic tree included species from the Midwest of Brazil
(Leipnitz et al. 2020).
The speciation processes in Ctenomys have been always associated with the
Allopatric model because the data showed that each species displayed a geographic
distribution isolated from the other, the only exception being the sympatric process
found between Ctenomys australis and Ctenomys talarum (Reig et al. 1990). In this
chapter, we discuss results that provide evidences about speciation models in
Ctenomys. First, examples of allopatric speciation in four species of Ctenomys are
presented: Ctenomys australis and Ctenomys flamarioni, which inhabit separated
3 Speciation Within the Genus Ctenomys: An Attempt to Find Models 45
regions in South America, and Ctenomys minutus and Ctenomys lami that share the
coastal plain of Rio Grande do Sul, the southernmost state of Brazil. Then, chromo-
somal speciation in Ctenomys is discussed, with an emphasis in C. lami, while
C. minutus is presented as an example of probable sympatric speciation. Furthermore,
the role of hybrid zones, both intra and interspecies, in speciation, are discussed.
Finally, we report other differentiation processes that have occurred or seem to be
starting.
3.2 Allopatric Speciation Model
Two examples related to differentiation events that can be classified as allopatric

processes are found in Ctenomys. The first involves two species:” C. flamarioni,
which currently occupies the coast of southern Brazil, and C. australis, which
inhabits part of the coast of Argentina. The second example involves two other spe-
cies: C. lami and C. minutus, both living on the coastal plain of Rio Grande do Sul,
in the South of Brazil.
3.2.1 C. flamarioni and C. australis
Both species have the same number of chromosomes 2n = 48, with a varying num-
ber of chromosomal arms (FN). The CBG-band pattern is similar between the two
species with heterochromatic blocks in the small chromosome arms. The GBG-
band pattern is the same in both species, which means that they have homologous
chromosomes (Freitas 1994; Massarini and Freitas 2005). These characteristics led
Freitas (1994) to consider C. flamarioni as belonging to the group of species called
Mendocinus, which is formed by C. mendocinus, C. azarae, C. australis, C. porte-
ousi, and C. rionegrensis (Massarini et al. 1991). At the same time, both species
have the same asymmetric sperm shape (Freitas 1995b). However, the skull mor-
phology is different, due to C. australis being larger than C. flamarioni (Massarini
and Freitas 2005; Fornel et al. 2018). Phylogeographic patterns have been described
for both species by different authors. Mora et al. (2006) found 25 different haplo-
types in the geographical distribution of C. australis. Nevertheless, C. flamarioni
has only nine different haplotypes (Fernández-Stolz 2006). Ecologically, both spe-
cies live in the first line of dunes, C. australis, in Necochea (Argentina), and C. fla-
marioni, on the coastal plain of Rio Grande do Sul (Brazil).
These data suggest that C. australis and C. flamarioni were initially a single spe-
cies that occupied a more extensive coastline than the current one, about 18,000 years
ago (Massarini and Freitas 2005). Moreover, Mertens et al. (in preparation), through
ecological niche modeling, found that in the Last Glacial Maximum (22,000 years
ago), both species were distributed in a zone of sympatry that occupied the region
that today is Uruguay. The rise of the sea and its advancement over the coastal plain
of South America resulted in the Rio de La Plata, which ended up being a barrier
that separated the populations of Argentina and southern Brazil. These data lead us
to recognize a process of allopatric speciation between C. flamarioni and C. australis.
3.2.2 C. minutus and C. lami
These two species also show an allopatric speciation process. Both have a parallel
parapatric distribution in the coastal plain of Rio Grande do Sul (Freitas 1995a).
C. lami occupies the first line of dunes in the coastal plain, a region that originated
in the Plio-Pleistocene, while C. minutus occurs in the second line of dunes, which
originated in the Pleistocene.
Both C. lami and C. minutus belong to the Torquatus group, which is formed by
ten species. Ctenomys roigi, Ctenomys perrensis, and Ctenomys argentinus occurs
in the Entre Rios region, in Argentina. Separated from these species by the Uruguay
river, we found C. pearsoni, in Uruguay, C. torquatus, in Uruguay and southern
Brazil, and C. ibicuiensis, only in the south of Rio Grande do Sul, near Uruguay.
Finally, inhabiting the coastal plain of Rio Grande do Sul, we found C. minutus and
C. lami. The intriguing question is how C. minutus and C. lami arrived in the coastal
plain, which is separated by the Patos Lagoon from the other regions where all the
other species of the Torquatus group inhabit by the Patos Lagoon.
The analysis of the separation process between C. minutus and C. lami starts
with understanding the taxonomic confusion about these species. C. minutus was
described by Nehring (1887), who recorded the species in a type of vegetation
known as “fields.” in a location called Taquara do Mundo Novo, in Rio Grande do
Sul. Later, Nehring (1900) reported that the animals were found near the beach of
Tramandaí, on the banks of the Tramandaí river (29° 53′S; 50° 17′W). Reig et al.
(1966) and Reig and Kiblisky (1969), aiming to determine the karyotypes of
Ctenomys species, described until the 1960s at their respective type-locality, failed
to find specimens of C. minutus in the place now known as Taquara. However, they
found a population in the locality of Santo Antônio da Patrulha, in Rio Grande do
Sul, and considered this location as the new type-locality of C. minutus. However,
reports about cytogenetics (Freitas 1994, 1997) and geographic distribution (Freitas
1995a) of C. minutus suggests that the specimens collected by Reig et al. (1966)
were, in fact, a separate taxon, the new species described as C. lami (Freitas 2001).
Both C. minutus and C. lami have separate, but parallel, geographic distributions,
and different chromosomal numbers: C. minutus presents 2n = 42 to 50 (Freitas
1997; Freygang et al. 2004) and C. lami, 2n = 54 to 58 (Freitas 2001). Chromosomally,
when comparing the karyotypes 2n = 46a of C. minutus and 2n = 54 of C. lami, both
species are different due to four Robertsonian rearrangements (Freitas 2001). The
species are separated by swamp areas and are isolated, which probably led to the
beginning of a differentiation, which is still occurring. They are, therefore, two spe-
cies that are still in a process of differentiation. Nonetheless, at the same time, due
to the anthropization of the region, both species are in contact in a hybrid zone,
which will be described later in this chapter.
3.3 Sympatric Speciation Model
As mentioned before, sympatric speciation is characterized by the differentiation of

a particular species due to some mechanism of reproductive isolation that leads to
the appearance of separate species. Herein, we describe findings in C. minutus that
suggest that this species is undergoing a sympatric speciation process.
3.3.1 C. minutus
Before describing the sympatric speciation that is likely to be occurring in C. minu-

tus, it is important to notice the complexity of the facts that occur in this species
involving its different karyotypes and their relationship with molecular markers,
skull morphology, and the evolution of the coastal plain in southern Brazil.
This species has a linear geographic distribution in the coastal plain of southern
Brazil, about 500 km long in a south-north direction (Freitas 1995a). In this direc-
tion, C. minutus occupies the sandy fields of the coastal plain and then occupies the
first line of coastal dunes parallel to the sandy fields until the extreme north of its
distribution. In this area, C. minutus is currently undergoing an evolutionary process
involving variation in the number of chromosomes, high mtDNA variation, and
morphological differences in the skull. Throughout its distribution, C. minutus has
a chromosomal variability of 2n = 42, 46a and 46b, 48a and 48b, 50a, and 50b
(Freitas 1997; Freygang et al. 2004). This variation involves eight pairs of acrocen-
tric chromosomes of the karyotype 2n = 50a (#2, #16, #17, #19, #20, #22, #23, and
#24). The centric fusions 20 + 17 and 23 + 19 form the karyotype 2n = 46a; the same
fusions and the fission of pair #2 in 2p and 2q form the karyotype 2n = 48a; fusions
20 + 17, 23 + 19, in tandem fusion 24 + 16 and the centric fusion of this new seg-
ment with #24 forming a chromosome, result in the karyotype 2n = 42; the centric
fusion 20 + 17, the 2p pericentric inversion, and the in-tandem fusion 24 + 16 gener-
ate the karyotype 2n = 46b; centric fusions 20 + 17, 23 + 19, and pericentric inver-
sion 2p form the karyotype 2n = 48b; finally, fusion 20 + 17 and the pericentric
inversion 2p form the karyotype 2n = 50b (Freitas 1997; Freygang et al. 2004).
Other karyotypes were found as 2n = 49a, and 49b, 47a and 47b, 45 and 43 are
products of hybrid zones, which will be described later.
The evolution of chromosome numbers by rearrangements as Robertsonian
translocations and in tandem, described by Freitas (1997) and Freygang et al.
(2004), can be related to the evolution of the coastal plain in the South of Brazil due
to the outflow of rivers which formed the channels that subdivided it. Based on the
geological data from Weschenfelder et al. (2014), it appears that two different
cut-and-fill events in Patos Lagoon are related with the formation of channels dur-
ing the Pleistocene and Holocene, which would explain the different karyotypes.
Firstly, the 2n = 50 karyotype was separated by Araranguá river generating 2n = 50a
and 2n = 50b (Fig. 3.1a). The channels of the Camaquã and Jacuí rivers that emerged
during the Pleistocene are likely to have separated the karyotypes 2n = 50b, 2n = 42
and 2n = 48a, respectively. The 2n = 48a was separated from 2n = 46a by another
Fig. 3.1 Evolution of the chromosome numbers along the coastal plain of Rio Grande do Sul
related to the geological history of the region with the geographic distribution of diploid numbers.
(a) Separation between 2n = 50 (ancestral) by the Araranguá river in two new karyotypes, 2n = 50a
and 2n = 50b. (b) During the Pleistocene, new karyotypes appeared: 2n = 46a, separated by the
Araranguá river from 2n = 50a, and separated from 2n = 48a by another undetermined channel.
The Jacuí river separated 2n = 48a from 2n = 42, and the Camaquã river separated 2n = 42 from
2n = 50b. (c) In the Pleistocene-Holocene, new channels emerged from the Camaquã and Jacuí
rivers and separated two new diploid numbers, 2n = 46b and 2n = 48b. (d) Nowadays, the channels
have disappeared and hybrid zones between different karyotypes are formed. The Araranguá river
separates 2n = 46a from 2n = 50a, 2n = 49a, and 2n = 48c
probable channel, which was separated from 2n = 50a by the Araranguá river
(Fig. 3.1b). Then, in the late Holocene, the same rivers formed new channels, and
these separated 2n = 50b, 2n = 48b, 2n = 46b, 2n = 42, 2n = 48a, 2n = 46a, and
2n = 50a (Fig. 3.1c). More recently, those river mouths became paleochannels, and
contact zones started to be formed between the different diploid numbers (Fig. 3.1d).
The Araranguá river is still a barrier that separates 2n = 46a from 2n = 50a, 2n = 49a,
and 2n = 48c.
Recently, the mtDNA sequences were analyzed with the same individuals from
the same populations studied by Freitas (1997) and Freygang et al. (2004), and 52
haplotypes were found, forming six haplogroups, which were named as “Norte,”
“Litoral,” “Lagoa dos Barros,” “Mostardas,” “Tavares,” and “Sul” (Lopes et al.
2013). Firstly, we analyze the relationship between the haplotypes network with the
diploid numbers of C. minutus. The haplogroup “Norte” has 10 haplotypes that are
in animals with karyotypes 2n = 50a (7 haplotypes), 2n = 49a (2 haplotypes), and
2n = 48c (2 haplotypes). All karyotypes share one haplotype, and another haplotype
is shared by individuals with 2n = 50a and 2n = 49a. As for the haplogroup “Litoral,”
this group has 10 haplotypes, all occurring in individuals with 2n = 46a. The hap-
logroup “Lagoa dos Barros” has 14 haplotypes, six of which are found in animals
with 2n = 46a, 10 in 2n = 48a, and three in 2n = 47a. One haplotype is shared
between 2n = 46a and 2n = 48a, and two others are shared between 2n = 47a and
2n = 48a. Another haplotype is shared by 2n = 46a, 2n = 47a, and 2n = 48a. The
haplogroup “Mostardas” has 10 haplotypes, seven of them occur in 2n = 48a, while
other two occur in 2n = 42. One haplotype is shared by 2n = 42 and 2n = 48a. The
karyotype 2n = 42 shares one haplotype with the hybrids formed between 2n = 42
and 2n = 48a. As for the haplogroup “Tavares,” this group has seven haplotypes.
Three haplotypes are found in hybrids between 2n = 42 and 2n = 46b. One haplo-
type is found only in individuals with 46b, and another in hybrids with 2n = 47b.
One haplotype is shared by 2n = 46b, 2n = 47b, and hybrids between 2n = 42 and
46b, and another haplotype is shared by the same hybrids and 2n = 42. Finally, the
haplogroup “Sul” has three haplotypes occurring in 2n = 50b, 2n = 49b, and
2n = 48b, respectively. There is one shared haplotype between 2n = 50b and
2n = 49b.
A second analysis reveals the relationships between the haplotypes and the
parental karyotypes within their geographic distribution and the coastal plain geo-
logical evolution. These relationships are summarized in Fig. 3.2, which presents
the diploid numbers of C. minutus and the respective haplotypes.
The parental types 2n = 50a and 2n = 48c have the haplotypes H52 to H47 and
H45, respectively. The 2n = 46a karyotype has two groups of haplotypes: individu-
als inhabiting the first line of dunes present haplotypes H44 to H35, while those
inhabiting on the fields have haplotypes H34 to H31. Individuals with 2n = 48a have
haplotypes H30 to H15 and H13. The haplotype H13 is shared with the karyotype
2n = 42, which has also haplotypes H14, H12, and H11. The karyotype 2n = 46b
present two haplotypes: H6 and H5, while 2n = 48b has only H3, and 2n = 50a, H2
and H1. Interestingly, with the exception of H13 that is shared between 2n = 42 and
2n = 48a, there are no shared haplotypes. At the same time, we can observe that
Fig. 3.2 Geographic distribution of Ctenomys minutus parental karyotypes with the respective
haplotypes. In the Pleistocene/Holocene the coastal plain was divided by river mouths, which are
shown as dashed lines separating the inhabiting areas of different karyotypic forms
channels of the Camaquã and Jacuí rivers, the undetermined channel, and the
Araranguá river separated parental diploid numbers and haplotypes. This is a con-
sequence of the geological evolution of the coastal plain in the South of Brazil dur-
ing the Pleistocene and Holocene, where old channels divided the area, resulting in
the chromosomal differentiation.
A final analysis allows us to understand the relation between the karyotypes and
the haplotypes found along the coastal plain, as a consequence of the geological
evolution (Fig. 3.3). At the north of the geographic distribution, the karyotype
2n = 50a has haplotypes H52 to H46, while 2n = 49a shows H47 and H46, and
2n = 48c has H46 and H45. Haplotypes H46 and H47 occur in individuals having
any of the three karyotypes. The karyotype 2n = 47a shares the haplotype H27 with
its parental karyotype 2n = 46a, and the haplotypes H30 and H28 with the parental
2n = 48a. Individuals from the hybrid zone between 2n = 48a and 2n = 42
(2n = 43–47) have only haplotype H11, which is shared with the parental 2n = 42.
The hybrid zone between 2n = 42 and 2n = 46b (2n = 43–45) has the haplotypes H9,
H8, and H4. The 2n = 47b has the haplotypes H7 and H4, sharing the latter with
individuals from the hybrid zone 2n = 42 and 2n = 46b. Finally, in the south, the
karyotype 2n = 49b has the haplotype H2, which is also in 2n = 50b.
Figure 3.3 shows also the hybridization zones between the C. minutus karyo-
types, where one can notice the haplotypes being shared between different diploid
numbers. It is also observed that the sharing occurs between 2n = 50a, 2n = 48c, and
2n = 49a, between 2n = 46a, 2n = 48a, and 2n = 47a, and between 2n = 50b and
2n = 49b. The haplotype H4 is present only among the hybrids of 2n = 42 and
2n = 46b (2n = 43, 2n = 44, 2n = 45) as well as in 2n = 47b.
Fig. 3.3 Geographic distribution of the several Ctenomys minutus karyotypes and hybrid forms
with the respective haplotypes. In the Holocene, the river mouths transformed to paleochannels,
and contact zones between the parental types were formed. The hybrid zones are shown as gray
areas delimited by dashed lines
C. minutus is the most studied among the species that occur in southern Brazil,
and shows the same pattern in three aspects: the karyotypes and the haplogroups
share the same North-South distribution, and the same occurs with the shape of the
skull in the analysis of geometric morphometry (Fornel et al. 2010). It should be
noticed that the different diploid numbers are not considered as intrinsic barriers to
each other, because when the river mouths and lagoon exit to the sea disappeared,
contact zones started to develop, and sharing started to occur. Again, these facts sug-
gest that chromosomal differences are due to geographical barriers, and so different
karyotypes occurred due to the isolation of populations and genetic drift.
Animals with diploid number 2n = 46a inhabit both the first line of dunes and the
sandy fields of the coastal plain of Rio Grande do Sul (Fig. 3.4) and show an inter-
esting feature regarding the shape of the skull. Freitas (2005), analyzing the skulls
of the animals with 2n = 46a from parapatric populations, found that the skulls of
those animals that occur in the dunes are different from the skulls of the animals that
occur in the sandy fields. This difference indicates that C. minutus uses different
strategies for tunneling, sometimes digging with paws, sometimes digging with
teeth, this being a response to soil hardness (Kubiak et al. 2018). So, the strategies
are not exclusively evolutionary, being probably a consequence of the harder soil
found in sandy fields, which are more difficult to excavate (Kubiak et al. 2018).
These facts suggest two evolutionary alternatives related to the mode of excava-
tion of individuals in populations that occupy different habitats: (i) the presence of
directional selection or (ii) strong phenotypic plasticity in the morphology (Kubiak
Fig. 3.4 The environment of the coastal plain of the South of Brazil, with dunes and sandy fields.
Ctenomys minutus inhabiting the dunes live in large tunnels and show different microsatellite
alleles and haplotypes H35 to H44. Those inhabiting sandy fields live in small tunnels and show
different microsatellite alleles and haplotypes H25 to H34
et al. 2018). Another interesting feature is the size of the home-range of individuals
from two populations of C. minutus that inhabit these different habitats. Nineteen
adult animals were tracked by radio, and the size of each individual’s home-range
was estimated using a 2 x 2-meter grid per cell (GCs) and minimum convex polygon
(MCP) methods. The results from both methods show that the home-range of
C. minutus differs between the two habitats, with an average size 1.75-times greater
for individuals who inhabit the dunes. This difference in the size of the home-range
between habitats is probably associated with differences in resource availability
(plant biomass) or soil conditions (Kubiak et al. 2017). All of these data lead to sug-
gest a differentiation in the populations of fields and sandy and coastal dunes.
Additionally, Lopes et al. (2013) analyzed 14 microsatellite DNA loci and verified
through Bayesian clustering and specimen assignments for the 12 clusters identified
by structure that a change occurs from the sandy fields to the dunes. The authors
also reported that the haplotypes 35, 36, 37, 38, 39, and 40 were found in individu-
als living on dunes, while haplotypes 27, 29, 30, 31, 32, 33, and 34 were found on
the sandy fields.
The current data about behavior ecology, morphology, mtDNA, and microsatel-
lite data, showing genetic differences between dunes- and sandy fields-inhabiting
animals have the same karyotype 2n = 46a, is a primary indication of a sympatric
speciation process in C. minutus. The works carried out with the underground rodent
Spalax galili species shows, with more powerful data (Rad-seq and microRNA), a
similar behavior and a strong indication that a sympatric process of differentiation

occurs between the two super-species (Hadid et al. 2013; Kexin et al. 2016; Li
et al. 2016).
3.4 Chromosomal Speciation Model
Chromosomal speciation is based on the assumption that the fixation of one or more
chromosomal rearrangements in a population favors speciation. For speciation to
occur after rearrangement, the heterozygote has hybrid sterility with reduced fertil-
ity, which can be caused by non-disjunctions, which consequently produces incom-
plete numbers of chromosomes forming unbalanced gametes (White 1978;
King 1993).
For the genus Ctenomys with chromosomal multiformity (2n = 10–70), there is a
classic idea that the chromosomal speciation model (Reig and Kiblisky 1969, Reig
et al. 1990) is responsible for the appearance of the various species that constitute
the genus. Such an idea is because the Ctenomys species meet the conditions
expected for this to occur. The occurrence of chromosomal rearrangements in het-
erozygotes in small isolated demes could generate new karyotypes by genetic drift,
which would be tested by selection and low gene flow. The genus is known as hav-
ing the highest chromosomal variation among mammals (2n = 10 to 70) and, among
rodents, to have the species with the lowest diploid number, for example, C. steiba-
chi has 2n = 10, while C. argentinus and C. pearsoni show 2n = 70. In addition,
there are species with large chromosomal variation like C. pearsoni (2n = 56, 64,
and 70); C. boliviensis (2n = 42–46), C. rionegrensis (2n = 48, 50, 56, and 58);
C. minutus (2n = 42–50), C. lami (2n = 54–58), C. talarum (2n = 44–48), and
C. perrensis (2n = 50, 54, 56, and 58) (Novello and Lessa 1986; Cook et al. 1990;
Reig et al. 1992; Freitas 1997; Massarini et al. 2002; and Freitas 2007).
In the following, we describe a chromosomal speciation process that is likely to
be occurring in Ctenomys, in the coastal plain of Rio Grande do Sul.
3.4.1 C. lami
Among Ctenomys species, C. lami has one of the smallest geographical distribu-
tions described. The species inhabits a sandy terrain that was formed at the begin-
ning of the Pleistocene, in the coastal plain of Southern Brazil. The distribution
region of this species is only 78 km long and 12 km wide, but Freitas (2007) found
a chromosomal variation in which the diploid numbers (2n = 54, 55a and 55b, 56a
and 56b, 57, and 58) associated with the autosomal fundamental numbers (FNs = 74
to 84) form 26 different karyotypes. Such a variation in the diploid numbers is due
to Robertsonian-type fusion and centric fission rearrangements involving pairs 1
and 2, while the variation in the FNs was due to pericentric inversions that occur in
several pairs in different karyotypes. Considering the variation found in pairs 1 and
2, 2n = 58 has eight acrocentric chromosomes, which, by the G-band pattern, are the
respective short and long arms of pairs 1 and 2 of the form 2n = 54. Between these
two configurations, one finds heterozygotes for both pair 1 and pair 2 (2n = 55a and
2n = 55b) and homozygotes for four acrocentric chromosomes and a metacentric
pair (2n = 56a and 2n = 56b). There is also 2n = 57, which is heterozygous for pair
1 and has six acrocentric chromosomes. This chromosomal variation is distributed
in four population blocks. In a sequence from southwest to northeast, one finds
block A, which shows 2n = 54, 55a and 56a; block B, 2n = 57 and 58; block C,
2n = 54 and 55a, and block D, 2n = 56b and 55b (Freitas 2001).
One can hypothesize as to the origin of the chromosomal variation in decreasing
the number of chromosomes from 2n = 58 to 2n = 54 due to Robertsonian centric
fusion rearrangements. An alternative hypothesis is that the basic karyotypes, i.e.,
2n = 54, 58, and 56b, that inhabit the blocks form two contact zones: the first,
between blocks A and C, generates the hybrids 2n = 55a and 55b; and the second
between block B and D generates the hybrid form 2n = 57. While there is this chro-
mosomal difference between blocks A, B, C, and D, molecular markers of mito-
chondrial DNA show that there is a genetic flow between blocks A, B, and C, and
still between C and D. This sharing of haplotypes are observed between the popula-
tion blocks: the haplotypes 2, 4, and 7 are surprisingly shared by the diploid num-
bers 2n = 54 (block A) and 2n = 58 (block B); the haplotype 5 is present in blocks
A (2n = 54), B (2n = 58), and C (2n = 54a), while the haplotype 15 is shared by
blocks C (2n = 54) and D (2n = 56b) (Fig. 3.5).
The sharing of haplotypes between 2n = 54 (block A) and 2n = 58 (block B)
shows that there is a crossing system between them, which would result in the dou-
ble heterozygote 2n = 56 for pairs 1 and 2, which was never found. The meiosis of
2n = 56 would result in the formation of two trivalent chromosomes, and for the
crossing system to be successful, there must be a normal disjunction between these
chromosomes, that is, one metacentric for one pole and two acrocentric chromo-
somes for the other pole of the cell (Fig. 3.5). Research on meiosis has always
focused on explaining how chromosomes are segregated when they form a trivalent
due to a Robertsonian translocation. In the 1980s, several articles showed us the
pairing of the trivalent through silver staining of the synaptonemal complex
(Bogdanov et al. 1986; Hale and Greenbaum 1988). Currently, molecular cytoge-
netics has shown more evidently how these processes occur in heterozygotes. The
alternative pairing of chromosomal rearrangements causes changes in the frequen-
cies of rearrangements and recombination, not being the cause of chromosomal
differentiation. This strengthens the idea that, despite the large chromosomal vari-
ability, this may not be the direct cause of speciation, but a consequence of it. If this
were the case, C. lami would not hybridize with C. minutus, and therefore, there
would be no hybrid zone between 2n = 46a and 2n = 56 and the formation of F1, F2,
and F3 hybrids (Gava and Freitas 2003).
Lopes and Freitas (2012) did not find genetic structure associated with the four
different karyotype blocks separately, or even with the chromosomal rearrange-
ments found in this species. Their findings suggest that the karyotype blocks
Fig. 3.5 Relationship between haplotypes and diploid numbers in Ctenomys lami population
blocks. Four haplotypes are shared by different diploid numbers. The mating of individuals with
different diploid numbers generates double heterozygotes in relation to pairs 1 and 2
described by Freitas (2007) proved to be inconsistent for mtDNA and microsatellite

data, and that chromosomal rearrangements probably do not prevent reproduction
among individuals with distinct karyotypes. Thus, they do not act as reproductive
barriers. The fixation of new chromosomal rearrangements seems to be frequent in
species of the genus Ctenomys and is generally not followed by sterility, reduction
in fitness, or negative heterosis of heterozygous carriers (Tomasco and Lessa 2007).
There are some examples of chromosomal polymorphic ctenomids that do not show
genetic differentiation among karyotypic populations. For example, the Corrientes

group includes species that are genetically very similar to each other, despite their
high degree of karyotype differentiation (2n = 41-2n = 70 and FN = 76, 78, 80, 84,
and 86) (Giménez et al. 2002; Mirol et al. 2010, Caraballo et al. 2012, Caraballo and
Rossi 2017). For C. pearsoni, all populations and karyomorphs studied were poly-
phyletic in their mtDNA (Tomasco and Lessa 2007). Furthermore, the species
C. torquatus and C. minutus, the latter being sister species of C. lami, also do not
share a pattern of genetic and karyotype variation (Fernandes et al. 2009; Lopes
et al. 2013; Lopes and Freitas 2012).
The available information on chromosomal variability, mitochondrial DNA con-
trol region and subunit I sequences of the cytochrome c oxidase I, and 14 microsat-
ellite loci suggests that the genetic structure of this species follows a pattern of
isolation by distance and a clinal genetic variation within the step-stone population
model. These results did not indicate a genetic structure associated with distinct
karyotypes. However, mitochondrial and nuclear molecular markers have demon-
strated the existence of two demes, which are not entirely isolated but are probably
reinforced by a geographical barrier (Lopes and Freitas 2012).
Fornel et al. (2018) tested whether there was an association between chromo-
somal polymorphism and variation in the skull shape and size in two groups of
Ctenomys species from the groups: Torquatus and Mendocinus, showing high
(2n = 42–70) and low (2n = 46–48) chromosomal variations, respectively. The
hypothesis was based that chromosomal rearrangements in small populations, as in
Ctenomys, produce reproductive isolation and allows independent population diver-
sification. The authors analyzed the variation in shape and size of the skull and jaw
using a geometric morphometric approach, with univariate and multivariate statisti-
cal analysis in 12 species of the Mendocinus and Torquatus. A total of 763 adult
skulls were used in the dorsal, ventral, and lateral views, and 515 jaws in the lateral
view, and 93 landmarks in four views. Importantly, no greater phenotypic variation
was found in the Torquatus group than in the Mendocinus group. These results
rejected the hypothesis of an association between chromosomal polymorphism and
variation in the shape and size of the skull. In addition, the Torquatus group also
presented phenotypic variation equal to that of the Mendocinus group. Thus, the
chromosomal variation is not related to the morphological evolution of the skull but
probably due to the heterogeneity of the habitat associated with biomechanical
restrictions and other factors such as geography, phylogeny, and demography
(Fornel et al. 2018).
3.5 Hybrid Zones
Hybrid zones are places where two different forms co-exist and originate hybrids.
The forms may differ according to molecular, chromosomal, morphological, physi-
ological, or behavioral markers. Using one or several markers at the same time, it is
possible to assess the level of hybridization that is occurring within or between spe-
cies. Hybrid zones have two important roles in the evolutionary process: they
increase genetic diversity or trigger an introgressive process, with genes being

exchanged between populations. Nevertheless, a hybrid zone can homogenize pop-
ulations (Barton 2001).
The occurrence of hybrid zones in Ctenomys is still not entirely known. There are
65 species (Teta and D’Elía 2020) described for the genus, and we can hypothesize
that there are hybrid zones both between and within species that have not been
found yet.
So far, few hybrid zones were described between species. A hybrid zone between
C. minutus and C. lami was described by Gava and Freitas (2003), and others
between C. flamarioni and C. minutus reported by Kubiak et al. (2020). What draws
the attention of these two cases is their difference in phylogenetic terms. In the first
case, two closely related sister species are involved. In the second one, both species
are very distant phylogenetically (Parada et al. 2011) and belong to two groups:
C. flamarioni is part of the Mendocinus group, while C. minutus belongs to the
Torquatus group.
3.6 Intraspecific Hybrid Zones
Regarding hybrid zones within species, C. minutus is the one with the largest num-
ber of contacts already described for the genus. These contacts have caused the
great chromosomal variability previously described. In the geographic distribution
of C. minutus, from north to south, we find 2n = 50a, 48a, and 49c, but the Araranguá
river isolates these three diploid numbers. Then, it is possible to observe 2n = 46a
and 2n = 48a distributed parapatrically in the coastal plain, forming a hybrid zone
where the hybrid 2n = 47a occurs. This hybrid shows a stable chromosomal poly-
morphism in a dynamic and a narrow hybrid zone between chromosomal races
(Gava and Freitas 2002; Gava and Freitas 2004).
Gava and Freitas (2002) studied 132 specimens of C. minutus from 22 sites in
that contact zone and adjacent areas, where seven polymorphic sites were identified.
A single chromosomal rearrangement is involved in the observed variation, which
served as a Mendelian diallelic character in a Hardy-Weinberg analysis. Additionally,
the clustering of polymorphic populations revealed that the mating system is ran-
dom among the genotypes. Moreover, the observed frequency of the metacentric
chromosome follows a clinal variation. Importantly, the hybrid zone is located along
an ecotone, which could imply that exogenous selection is important for its
maintenance.
With the same populations, the same hybrid zone was evaluated with six micro-
satellite loci in 107 specimens (Gava and Freitas 2003). Of the 56 alleles discovered
in the study, 39.2% are exclusive to alternative cytotypes or contact populations.
The clinal variation is not obvious because there are no fixed microsatellite differ-
ences between divergent chromosomal populations, but the variation in the Hai 2
locus is gradual in the zone. Local populations are highly differentiated and struc-
tured (Gava and Freitas 2003).
Castilho et al. (2012) found variation in microsatellite loci and in chromosomal

polymorphisms of a parental population with 2n = 42 and 2n = 48a and hybridized
populations of Ctenomys minutus. The cytogenetic analysis and genetic variations
of six microsatellite loci were included in 101 specimens with 2n/AN = 42/74 and
48a/76 and animals of contact zone. The cytogenetic analysis found 26 different
karyotypes in 50 individuals from the hybrid population in an area of 7 km2. Of the
26 karyotypes, only 14% had a parental configuration, and none had the expected 2n
and AN combination for an F1 hybrid. The remaining karyotypes were alternative
hybrid forms, with 2n ranging from 42 to 46, and AN from 68 to 80. These results
suggest that chromosomal rearrangements are of little importance in establishing
reproductive barriers for this species (Castilho et al. 2012). All of these data indicate
that there is no effective barrier between the diploid numbers nor gene flow between
them (Gava and Freitas 2003; Castilho et al. 2012).
Introgressions exist between different chromosomal numbers in the hybrid zone
of 2n = 46a with 2n = 48a, where the microsatellite alleles occur in both parental
diploid numbers. The same is true for the haplotypes H26, H27, and H28. A similar
process was found in the hybrid zone between 2n = 42 and 2n = 48a, where Castilho
et al. (2012) observed the exchange of several microsatellite alleles between these
diploid numbers. Introgression also occurs with the H13 haplotype in the same
hybrid zone.
3.7 Interspecific Hybrid Zones
Contact zones between species in the South of Brazil were described in Ctenomys,
one between C. minutus and C. lami and the most recently found between C. fla-
marioni and C. minutus.
The first interspecific contact zone was reported by Gava and Freitas (2003) and
Ximenez (2009) between divergent populations of Ctenomys minutus (2n = 48a)
distributed parapatrically with C. lami (2n = 56b). In the western region of Barros
Lake, 21 different karyotypes were found between the parents 2n = 48a and
2n = 56b. They were recorded in a sample of 26 specimens: 2n = 48a, NA = 76;
2n = 49, NA = 74, 76; 2n = 50, NA = 74, 76; 2n = 51, NA = 76, 77, 78, and 80;
2n = 52, NA = 79; 2n = 53, NA = 74, 80; 2n = 54, NA = 78, 80; 2n = 55, NA = 76;
2n = 56, NA = 78, 80. It is worth noting the wide chromosomal variability through-
out an area of about 2 km2. Moreover, at the same time, only one individual was
found with 2n = 52, NA = 79, which corresponds to the F1 hybrid; so, the other 20
karyotypes are from F2 onward. The variation is a result of Robertsonian mecha-
nisms of chromosomal evolution and in tandem fusions. Importantly, polymor-
phisms have been considered as the result of a secondary contact between
populations after divergence in allopatry. As described earlier in this chapter, the
geomorphological evolution of the coastal plain provides clues to the existence of
past geographical barriers that modulated the populations of Ctenomys, during the
Holocene.
The second interspecific contact zone in southern Brazil is between C. minutus

and C. flamarioni, and this is the second case of sympatry found for the genus. The
first one was described between C. australis and C. talarum in the coastal dunes in
southern Argentina, (Malizia and Busch 1991). The sympatry between these two
species is very complex due to the interaction between them. Kubiak et al. (2015)
investigated the vegetation structure, plant biomass, and soil hardness selected by
these two species when distributed in sympatry and allopatry. These species show
segregation in their selection of microhabitats, differing in relation to soil hardness,
biomass, and vegetation cover. C. flamarioni and C. minutus show habitat segrega-
tion in the area where they occur in sympatry. Nevertheless, C. flamarioni shows a
distinction in habitat selection when it occurs in allopatry and sympatry, while
C. minutus selected the same habitat characteristics in both conditions. A possible
explanation for the observed pattern is that these species have acquired different
adaptations over time, which allows them to explore different resources and, thus,
avoid competitive interactions (Kubiak et al. 2015).
Species with similar ecological requirements coexisting in the same geographic
region are likely to competitively exclude each other or, alternatively, can coexist.
For this analysis, two methodological approaches were used (ecological niche mod-
eling [ENM] and geometric morphometry) to test two hypotheses: (i) if, given their
behavioral, morphological, and ecological similarities, one species competitively
excludes the other or (ii) if the character changes allow their coexistence in two
places where species are known to occur in sympatry. The results of the ENM-based
approach did not provide evidence of competitive exclusion. However, geometric
morphometric analyses showed the change in the skull size of C. minutus.
Interestingly, this result suggests that C. minutus can exclude C. flamarioni from
areas with softer soils and greater food availability (Kubiak et al. 2017).
While there is interaction between these two species, regardless of their separate
spaces, different morphologies, completely different karyotypes, divergence in evo-
lutionary history, and form of sperm, they are still capable of generating natural
hybrids. Also, this ability to generate natural hybrids demonstrated that females of
both species are capable of generating hybrids with males of the two species. In
addition, the specific chromosome probes isolated from Ctenomys flamarioni
showed chromosomes of each species in the hybrid karyotype, and the homeology
relationship between the chromosomal arms of both species. Moreover, microsatel-
lites also showed the hybridization process at the population level, where through
population assignment, individuals of both parental species were shown, as well as
hybrids. Furthermore, it is worth mentioning that mtDNA analyses allowed to detect
that hybrids have mtDNA from one species or from the other, showing that the
crosses are clearly bidirectional (Kubiak et al. 2020).
3.8 Other Processes
A very recent differentiation was found in the Midwest region of Brazil with some
species lineages. Species from Argentina, Uruguay, Bolivia, and southern Brazil are
well studied at the phylogenetic level. However, the taxonomic status of the species
that inhabit the Mid-western and Northern Brazil remains scarcely known. For the
three species of Ctenomys that occur in these two regions (C. nattereri, Wagner
1848; C. bicolor, De Miranda-Ribeiro 1914; and C. rondoni, De Miranda-Ribeiro
1914), it can only be found one article for each, and a formal description of these
taxa is nonexistent (De Miranda-Ribeiro 1914). Additionally, the geographical dis-
tribution of these species is also unknown in the Brazilian states of Acre, Rondônia,
and Mato Grosso.
Ctenomys bicolor, an endemic species from the northwest of Brazil, is present in
southern Amazonia, specifically at the edge of the Amazon ecoregion. The type was
collected in 1912 by zoologist Alipio Miranda Ribeiro, during the Roosevelt-
Rondon scientific expeditions (Avila-Pires 1968) in a remote area of the Amazon
region, and the specimen described is deposited in the National Museum of Rio de
Janeiro, under the collection I.D. MNRJ-2052. Afterward, the description of this
individual was published 2 years later (De Miranda-Ribeiro 1914), with no indica-
tion of the type locality; but, Bidau and Avila-Pires (2009) redefined it as in the state
of Rondônia, in the locality of Pimenta Bueno. Stolz et al. (2013) confirmed the
location based on a sample of 10 animals. That report determined 2n = 40, NF = 68,
their species status through phylogenetic analysis with 820 bp of Cyt-b, and the
skull was analyzed with geometric morphometry.
Noteworthy, these findings showed a new behavior for Ctenomys. These popula-
tions were found living inside the forest and feeding from tree roots, contradicting
the existing knowledge that practically all species would live in open formations
and feeding on grasses.
It can be hypothesized that the invasion of the Amazonian group in the forest area
seems to have occurred at a time (before 850 thousand years ago, in the quaternary)
when large extensions of savannah existed where today the forest is located (Haffer
and Prance 2002). Such an ancestor, common to the three species, in a more recent
period of high temperatures and humidity, was constrained by the advancement of
forest expansion.
In this changing environment, individuals that could take advantage of the forest
environment to survive were selected and differentiated themselves into a new mor-
photype, adapted to life and food on the soil of the forests. Leipnitz et al. (2020)
studied the samples from the mid-west and north of Brazil, with the intent to ana-
lyze these populations. The authors built a phylogeny based on maximum-likelihood
and Bayesian inference methods with haplotypes of the Ctenomys cytochrome b
gene and with haplotypes representative of the genus Ctenomys. Also, they evalu-
ated skull morphology using geometric morphometric analysis to assess whether
the skull morphology findings agree with the observed phylogenetic patterns. The
results showed the occurrence of two species, namely, Ctenomys bicolor, in the state
of Rondônia, and Ctenomys nattereri, in Mato Grosso and Bolivia. Additionally, the
results revealed two lineages of Ctenomys that are distinct from Ctenomys bicolor
and C. nattereri, which they called Ctenomys sp. B Xingu and Ctenomys sp. B
Central. Importantly, both species and lineages share a more recent ancestor com-
mon with C. boliviensis and are part of the species group known as boliviensis.
Named as Corrientes group, three species, C. roigi, C. perrensis, and C. dorbig-
nyi, are tuco-tucos that inhabit the Iberá region. This area has sandy soils under the
influence of the second largest wetland in South America, located in the northeast of
Argentina, in the province of Corrientes. The phylogeny of these species was stud-
ied under cytogenetic point of view by Ortells and Barrantes (1994). These species
are characterized as a “species complex” with one of the largest chromosomal vari-
ability within the genus. To analyze the evolutionary relationships in the Corrientes
group and its chromosomal variability, the partial sequences of three mitochondrial
markers were obtained by Caraballo et al. (2012, Caraballo and Rossi 2017). These
authors found that the Corrientes group is monophyletic, and they divided the com-
plex into three main clades that grouped the related karyomorphs. Also, in Argentina
there is another group of species, belonging to the Mendocinus group, that shows
interesting evolutive conditions (see Chap. 5).
3.9 Final Comments
Considering the existing 65 species of Ctenomys (Teta and D’Elía 2020), we found
that little is known about the evolutionary patterns and processes that are still shap-
ing this genus. Analyzing the literature, we notice that only about 15 species were
studied from an integrative point of view, that is, chromosomal, morphological, and
molecularly. In this way, we only have the evolutionary panorama of these 15 spe-
cies, i.e., the evolutionary response to the region where these species live. Notably,
this process is observed in the Pampa region, coastal plain of Southern Brazil, the
coast of Uruguay, the region of Entre Rios, in Argentina, and Midwest region of
Brazil. In these regions, evolution is occurring in real time but at different evolution-
ary phases. Species such as C. perrensis, C. roigi, and C. dorbignyi live in the Iberá
region in Argentina, in a sandy area that is influenced by the water regime of the
second largest wetland in South America, and they are undergoing a recent process
of differentiation, although it is not possible (yet) to see a certain evolutionary pat-
tern. Furthermore, the species of Mato Grosso, Brazil, C. bicolor, C. rondoni, and
C. nattereri are undergoing a similar process, with low chromosomal or morpho-
logical variability, also in relation to molecular markers. Moreover, these species do
not have a well-defined pattern, because the process is very recent; however, it is
possible to know that all the variation originated from a colonization of the region
by one or more species came from the Bolivian region. In the coastal plain of south-
ern Brazil, we have three species, C. flamarioni, C. minutus, and C. lami, that are in
an “older” process of differentiation and, thus, we can recognize patterns of chro-
mosomal variation, morphology, and molecular markers. However, hybrid zones
between and within species indicate that they are still in a long process of evolution.
Similarly, the same can be observed in C. pearsoni, on the Uruguayan coast. On the
other hand, C. torquatus exhibits a much more defined pattern, as if it had already
found homeostasis in relation to morphology, chromosomes, and molecular markers.
Acknowledgments I am deeply grateful to my undergraduate, MSc, and PhD students whose

works provided much of the results that I used to write this chapter and the collaborators I had
throughout the years studying Ctenomys. I also thank Carla Freitas for suggestions and reviewing
the text and Raquel Freitas for help with figures and final text revision. Thanks also to Gislene
Gonçalves for valuable discussions. This study was financed in part by Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq), Fundação do Amparo à Pesquisa do Estado do
Rio Grande do Sul (FAPERGS), and Coordenação do Aperfeiçoamento de Pessoal de Nível
Superior (CAPES) – Finance Code 001. I also acknowledge the support from the Graduate
Programs PPGBM, PPGBAN, and PPGEcologia-UFRGS.
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Trabalho de Conclusão, Ciências Biológicas-UFRGS
Part II
Geographic Patterns
Chapter 4
Geographical and Macroecological
Patterns of Tuco-Tucos
Renan Maestri and Bruce D. Patterson
4.1 Introduction
Tuco-tuco is the vernacular name given to subterranean rodents of the genus

Ctenomys, which are widely distributed over the southern half of South America.
Species are distributed from Peru and the Brazilian state of Rondônia in the north
(11°S) to Tierra del Fuego in the south, and from the Atlantic to the west coast
(Bidau 2015; de Freitas 2016). Within those geographic limits, 65–70 species are
currently recognized (mammaldiversity.org; D’Elía et al. 2020). Even more impres-
sively, all of this diversity arose over the last 5 Ma, indicating explosive speciation
compared to other genera of Caviomorpha (Upham and Patterson 2015). Rapid spe-
ciation and geographic colonization gave rise to a large number of allopatric species
(Bidau 2015; de Freitas 2016), with only a few known cases of syntopy (Galiano
and Kubiak 2020). Contiguous allopatry with little overlap between species has
been attributed to the subterranean habits and behavior (Nevo 1979), but a formal
comparison of the exclusivity in the geographic ranges of tuco-tucos is lacking. In
addition, overall patterns in species and range size distribution – a legacy of their
speciation and colonization processes – remain to be explored.
Patterns in the distribution of species richness, geographic range size, and body
size have been common themes in the mammalian literature (Ruggiero and
Kitzberger 2004; Smith and Lyons 2011; Lyons et al. 2019). Macroecological anal-
yses of those characteristics helped to identify general patterns and uncover some of
R. Maestri (*)
Department of Ecology, Federal University of Rio Grande do Sul, Porto Alegre,
Rio Grande do Sul, Brazil
e-mail: renan.maestri@ufrgs.br
B. D. Patterson
Negaunee Integrative Research Center, Field Museum of Natural History, Chicago, IL, USA
e-mail: bpatterson@fieldmuseum.org

https://doi.org/10.1007/978-3-030-61679-3_4
70 R. Maestri and B. D. Patterson
the underlying processes behind mammalian evolution (Brown and Maurer 1989;
Stevens 1989; Brown et al. 1993; Smith et al. 2008). For example, geographical pat-
terns such as Rapport’s rule – the tendency of geographic range size to be smaller at
lower latitudes than at higher latitudes – and Bergmann’s rule – the tendency of
body size to be bigger at higher than at lower latitudes – were first described using
mammals (Bergmann 1847; Rapoport 1982; Stevens 1989; Blackburn et al. 1999).
To date, only a few macroecological investigations of species’ range and/or body
size distribution patterns have been focused on tuco-tucos. Among them, Medina
et al. (2007) found that tuco-tuco body size follows the converse of Bergmann’s
rule, Bidau et al. (2011) found increases in relative tail length with increases in lati-
tude (the converse of Allen’s Rule), and Caraballo et al. (2020) explored the overlap
of species range maps with protected areas.
This chapter will explore spatial patterns in the distribution of (i) species diver-
sity, (ii) geographic range size, and (iii) body size of tuco-tucos in the hope of offer-
ing some light for future investigations on Ctenomys biogeography. The chapter is
organized in three main sections devoted to exhibit the general spatial patterns on
each theme (i, ii, and iii above) and to compare the geographic range size of tuco-
tucos with other caviomorphs.
4.2 Patterns of Species Distribution
Species of tuco-tucos are unevenly distributed in the southern half of South America.
Most species occur in the northern portions of Argentina (Bidau 2015; de Freitas
2016). The pattern of species richness for Ctenomys species is shown in Fig. 4.1.
The range maps of each species (Bidau 2015) were overlaid in a gridded map a
1°× 1° scale to generate the map.
The hotspot of species richness is located between 23° and 29° degrees S lati-
tude, where the range maps of 7–10 species overlap. Outside of this hotspot, between
1 and 6 range maps overlap over the remaining tuco-tuco distribution. Most regions
have only one or two species. It is important to note that an overlap in range maps
should not be interpreted as if the species live in syntopy within those geographic
limits. Tuco-tucos have predominantly allopatric distributions with only three or
four cases of syntopy currently documented (see Galiano and Kubiak 2020),
although more studies investigating the spatial relationships of species living in
close proximity are urgently needed. However, range maps are useful to visualize
broad richness patterns that may steer inferences of large-scale spatial and temporal
processes shaping the biogeographical history of tuco-tucos.
A weak linear relationship was found in the correlation of species richness with
latitude (r = 0.13), longitude (r = −0.04), and elevation (r = 0.17), suggesting an
absence of clear richness gradients along these three axes (Fig. 4.2). The most range
overlaps for tuco-tucos coincide with intermediate latitude, longitude, and eleva-
tion values.
4 Geographical and Macroecological Patterns of Tuco-Tucos 71
Fig. 4.1 Species richness of tuco-tucos across South America on a 1°× 1° scale. Range maps of
each species are shown as grey polygons
4.3 Patterns of Geographic Range Size Distribution
Most species of tuco-tuco have small geographic range sizes (Fig. 4.3), a frequency
distribution that resembles the all-mammalian pattern (Brown et al. 1996; Gaston
1998). However, compared to other families of caviomorphs (Patton et al. 2015;
Maestri & Patterson 2016), the tuco-tucos (sole living members of the family
Ctenomyidae) have one of the smallest geographic range sizes (Fig. 4.4), with the
median of the geographic range size virtually identical to members of its sister
Fig. 4.2 Scatterplot depicting the relationship among species richness, elevation, latitude, and
longitude. Each dot corresponds to a 1°× 1° cell. Geographical variables composed of the three
main axes while richness is shown with colors
Fig. 4.3 Histograms of (a) geographic range size (km2) and (b) log of geographic range size (km2)
Fig. 4.4 Boxplot representing variation in geographic range size of species from different cavio-
morph families. Boxes represent the third and first quartiles, plus the median (bold line), and
whiskers represent upper and lower limits; dots indicate outliers
family Octodontidae, and trailing only the Abrocomidae – a poorly known family of
rodents with few extant species (Patton and Emmons 2015). Moreover, as can be
seen from Fig. 4.4, species of tuco-tucos vary little in geographic range size when
compared to species in other families, which may suggest constraints on the size of
the range around some modal value.
Examining patterns of range overlap, a surprising result was found in the com-
parisons of species-rich genera of caviomorphs (Fig. 4.5). Ctenomys species are
commonly perceived as being strongly allopatric, with species essentially replacing
one another across the landscape with little overlap. We calculated geographic range
exclusivity as the proportion of a species range that does not overlap with any other
species of its own genus. Remarkably, variation in geographic range exclusivity of
65 species of Ctenomys is statistically indistinguishable from that shown by
Dasyprocta (Dasyproctidae, n = 10), Proechimys (Echimyidae, n = 22), and
Coendou (Erethizontidae, n = 14) (Fig. 4.5). Only Coendou and Dasyprocta show
significant differences in range exclusivity (F = 3.02; P = 0.03), probably driven by
the strong degree of contiguous allopatry among species of agoutis.
The geographic range size (log) of tuco-tucos has no obvious relationship with
the mean latitudinal point of each species (R2 = 0.01; P > 0.05) (Fig. 4.6Aa),
Fig. 4.5 Boxplot representing variation in the proportion of geographic range size exclusivity –
the proportion of a species’ range that does not overlap with any other species of its genus. Four
species-rich genus of caviomorph are compared. Boxes represent the third and first quartiles, plus
the median (bold line), and upper and lower limits; dots represent outliers
suggesting the absence of Rapoport’s rule at this interspecific scale. Geographic

range size (log) was also linearly uncorrelated with mean elevation (R2 = 0.005;
P > 0.05) and species’ log body size (R2 = 0.02; P > 0.05) (Fig. 4.6b, c). The rela-
tionship between geographic range size and body size takes the approximate form
of a triangle, where species with large body sizes and small geographic range sizes
are lacking, similar to the general mammalian pattern (Smith et al. 2008; Lyons
et al. 2019). Such a triangular envelope was hypothesized to result from the higher
extinction rates of large-sized species; their low densities over a restricted area of
geographic occurrence result in vulnerably small population sizes (Brown and
Maurer 1989; Diniz-Filho et al. 2005; Tomiya 2013).
Nevertheless, at an assemblage scale, a positive relationship between average
geographic range size (average of range size for all species occurring on each 1 × 1°
cell) and average cell latitude (S) is apparent (R2 = 0.19; P < 0.001) (Fig. 4.7a). This
positive relationship is in accordance with the predicted by Rapoport’s rule (Stevens
1989), suggesting the prevalence of small range-sized species at lower latitudes,
even though the relationship between richness and latitude is weak. No trend was
found for the relationship between average geographic range size and mean cell
elevation (R2 = 0.001; P > 0.05), and a weakly negative relationship was found
between average geographic range size and average body size across cells (R2 = 0.05;
P < 0.01) (Fig. 4.7).
Fig. 4.6 Scatterplot

depicting the relationship
of log geographic range
size (km2) with (a) latitude,
(b) elevation, and (c) body
size – log of skull centroid
size. Each dot represents
a species
Fig. 4.7 Scatterplot

depicting the relationship
of average geographic
range size by cell (km2)
with (a) average latitude,
(b) average elevation, and
(c) average body size by
cell – log of skull centroid
size. Each dot represents a
1°× 1° cell
4.4 Patterns of Body Size Distribution
Species’ body size distribution is right skewed (Fig. 4.8), which is a common pat-
tern in many groups of organisms (Brown et al. 1993). In this chapter, the skull
centroid size was used as a proxy for body size. Skull centroid size was measured
through geometric morphometric techniques applied on 1359 specimens of tuco-
tucos (see Fornel et al. 2020 for a complete methodological description). Centroid
size measures usually correlate well with body length and weight across rodents,
moreover, these results are in accordance with those of Medina et al. (2007) that
used tuco-tucos body weight and length.
A negative relationship was observed between species body size and their aver-
age S latitude (R2 = 0.17; P < 0.002) (Fig. 4.9a). This pattern of larger sizes closer
to the equator (the antithesis of Bergmann’s Rule) was previously reported by
Medina et al. (2007) using body length and weight as a dependent variable. The
subterranean habits of tuco-tucos, which decouple them from air temperature, and
increased resource availability (NPP) at lower latitudes (Blackburn et al. 1999;
Blackburn and Hawkins 2004; Alhajeri et al. 2020) may jointly explain the relation-
ship between body size and latitude in tuco-tucos (Medina et al. 2007). Moreover, it
has been suggested that soil properties are correlated to humidity which in turn is
correlated with latitude (Medina et al. 2007). Soil excavation is an important part of
Fig. 4.8 Histogram representing variation of species’ body size – log of skull centroid size as in
Fornel et al. 2020
Fig. 4.9 Scatterplot depicting the relationship of species’ body size (log of skull centroid size)
with (a) latitude and (b) elevation. Each dot represents a species
Fig. 4.10 Scatterplot depicting the relationship of average body size by cell (log of skull centroid
size) with (a) average latitude and (b) average elevation. Each dot represents a 1°× 1° cell
tuco-tuco’s life habits and shapes their morphological evolution (Vassallo et al.
2020). For example, species inhabiting harder soils tend to be smaller than species
inhabiting soft soils (Borges et al. 2017); if soil hardness is shown to be correlated
with latitude this might also explain the tendency of larger species to occur at lower
latitudes.
There is also a negative relationship between average body size (average of all
species occurring within each cell) and latitude of the cells (R2 = 0.13; P < 0.001)
(Fig. 4.10a). The same pattern of increasing cell-averaged body size at lower lati-
tudes was found for all mammals (Rodríguez et al. 2008) and for sigmodontine
rodents (Maestri et al. 2016) in South America. That various groups of mammals
exhibit the same pattern may decrease the cogency of hypotheses of body-size vari-
ation in tuco-tucos based on their subterranean habits.
At both the interspecific and the assemblage levels, there was little relationship
between body size and elevation (R2 = 0.02, P > 0.05; and R2 = 0.03, P < 0.001)
(Figs. 4.9b and 4.10b).
4.5 Concluding Remarks
We have shown general patterns in the distribution of species diversity, range size,
and body size of Ctenomys. The center of richness for tuco-tucos lies between 23°
and 29° degrees S latitude in northern Argentina, a region that is intermediate in
elevation, longitude, and latitude compared to the entire geographical distribution of
tuco-tucos. The center of richness may be also the center of initial diversification for
tuco-tucos because it harbors one of the oldest fossils of Ctenomys, C. uquiensis
(Verzi et al. 2020). This hypothesis should be tested with phylogenetic and paleon-
tological data, and if supported would provide a striking historical explanation for
the tuco-tucos’ richness gradient. Patterns in the distribution of species’ richness
and range size confirmed that tuco-tucos have mostly allopatric distributions and
species of Ctenomys have small range sizes when compared to other caviomorphs.
However, when tuco-tucos are compared with other species-rich genera of cavio-
morphs in terms of the exclusivity of their geographic ranges, they are unexcep-
tional. The conventional wisdom that suggested tuco-tucos have more exclusive
ranges than other mammals is fallacious. Moreover, environmental correlates of the
latitude may influence the geographic range size of Ctenomys species, judging from
the assemblage-level occurrence of Rapoport’s rule. Body size patterns confirmed
that tuco-tuco species tend to have larger body size in lower latitudes, and this ten-
dency for Ctenomys to defy Bergmann’s Rule was evident at the assemblage level
as well. Future investigations that include phylogenetic information to frame mac-
roecological and biogeographical associations may provide a historical perspective
to existing patterns, and help to understand the processes generating such patterns.
Acknowledgments We thank Claudio Bidau, in memory, for generating the geographic range
maps used here. RM was supported by UFRGS, CAPES, and CNPq (150391/2017-0), BP was
supported by FMNH.
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Chapter 5
Phylogeography and Landscape Genetics
in the Subterranean Rodents of the Genus
Ctenomys
Fernando Javier Mapelli, Ailin Austrich, Marcelo Javier Kittlein,

and Matías Sebastián Mora
5.1 n Overview of Phylogeography

A
and Landscape Genetics
During the last two decades, since the publication of “Phylogeography: The History
and Formation of Species” by John C. Avise (2000), studies of population genetics
at broader time scales gave rise to so-called phylogeography. This discipline estab-
lishes that most species in nature exhibit some degree of genetic structure linked to
geography (Avise 2000; Richards et al. 2007). A species’ phylogeographic structure
is the expected result of the interaction between demographic and genealogical pro-
cesses, in line with the dynamics of the Earth’s processes (geological or climatic;
Hare 2001; Lessa et al. 2003). Phylogeographic studies have made possible to test
biogeographic hypotheses, describe demographic and evolutionary processes that
led to the formation of discrete evolutionary units, and also fostered inferences
about the processes that determined the origin, distribution, and maintenance of
biodiversity (Avise 2000); essential information for conservation and taxonomy.
Alongside, recent and accelerating fragmentation and degradation of natural
environments advanced the development of methodological tools in landscape
genetics. This discipline directly associate landscape characteristics and anthropic
disturbances with parameters that quantify population genetic diversity. In particu-
lar, landscape genetics is a relatively new discipline that focuses on how landscape
characteristics (e.g., topography, suitable habitats vs. unsuitable habitats, barriers
F. J. Mapelli (*)
División Mastozoología, Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”,
Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina
A. Austrich · M. J. Kittlein · M. S. Mora
Departamento de Biología, Facultad de Ciencias Exactas y Naturales, Universidad Nacional
de Mar del Plata, Instituto de Investigaciones Marinas y Costeras (IIMyC), CONICET –
UNMdP, Mar del Plata, Buenos Aires, Argentina
e-mail: kittlein@mdp.edu.ar; msmora@mdp.edu.ar

https://doi.org/10.1007/978-3-030-61679-3_5
84 F. J. Mapelli et al.
such as rivers and mountains, corridors, etc.), and environmental variables (e.g.,
temperature, humidity, and precipitations) mold gene flow. In accordance with its
objectives, this discipline has involved smaller spatial and temporal scales and the
use of highly variable molecular markers (e.g., multi-locus approaches with micro-
satellites or SNPs). In this way, phylogeography and landscape genetics have served
to identify evolutionarily independent units in natural populations providing back-
ground for decisions about the conservation and viability of species.
5.2 hy Study Underground Rodents as Models

W
from Population Genetic Approaches?
Tuco-tucos inhabit all regions of Southern South America, including Bolivia, Brazil,
Peru, Uruguay, Paraguay, Chile, and Argentina; with 68 species within the genus
(that is 45% of all species of subterranean rodents; Lacey 2000; Bidau 2015; Freitas
2016; Teta and D’Elia 2020; Teta et al. 2020; see also D’Elia et al. Chap. 3 in this
volume). These animals are found in a multiplicity of habitats, from the Andes
Mountains to the coastal dunes of the Atlantic and from the humid steppes of the
Pampas to the dry deserts of the Chaco (Mapelli et al. 2017; Torgashevaa et al. 2017;
Kubiak et al. 2020; Teta and D’Elia 2020; Teta et al. 2020). The genus Ctenomys is
probable one of the most extreme cases of high chromosomal diversity (with diploid
chromosome numbers ranging from 10 to 70; Reig and Kiblisky 1969; Freitas and
Lessa 1984; Reig et al. 1990; Freitas 1995; Ortells 1995; Fernandes et al. 2009) and
has experienced a rapid and relatively recent radiation during the Middle/Late
Pleistocene, which resulted in the outburst of the many contemporary species (Reig
et al. 1990; Lessa and Cook 1998; Cook and Lessa 1998; Slamovits et al. 2001;
Reguero et al. 2007; Verzi et al. 2010, 2014; Parada et al. 2011).
Tuco-tucos presents a set of life history traits that position them as excellent
models for the use of population genetic approaches (Reig et al. 1990; Busch et al.
2000; Wlasiuk et al. 2003; Lopes and Freitas 2012; Lopes et al. 2013; Esperandio
et al. 2019). First, tuco-tucos have a high specificity in habitat use; the high cost of
burrowing activities determines that they only occupy environments with sandy, fri-
able, and permeable soils (Busch et al. 2000; Luna and Antinuchi 2007). These soil
types are frequently patchily distributed, fragmented, and inserted in a landscape
matrix with multiple hostile environments for the occupation for tuco-tucos (water-
bodies, flooded areas with marsh vegetation, rocks, dense forests or grasslands,
human impacted areas, etc.), which determines that populations are also commonly
fragmented.
Tuco-tucos are mostly confined to their burrows, have a strong phylopatric
behavior, and move little aboveground as compared to surface-dwelling rodents
(Zenuto and Busch 1995, 1998; Busch et al. 2000; Lacey et al. 2000; Lessa 2000;
Cutrera et al. 2005). Moreover, tuco-tucos are highly territorial and it has been gen-
erally assumed that once they establish their territories, after natal dispersion, they
make few additional dispersal movements (Zenuto and Busch 1998; Busch et al.
2000; Cutrera et al. 2005). Most individuals taken by aerial predators (e.g., owls)
5 Phylogeography and Landscape Genetics in the Subterranean Rodents of the Genus… 85
consist of subadult individuals leaving, presumably, their natal burrows (Vassallo

et al. 1994; Kittlein et al. 2001).
Low population densities, habitat specialization, and low dispersal abilities are
aspects that greatly impact on the scales at which genetic differences in tuco-tucos
are exhibited (Mora and Mapelli 2010; Mora et al. 2010). Low population densities
could arise, in part, because tuco-tucos tend to be solitary, with each adult preserv-
ing its own exclusive-use territory (Busch et al. 2000; Zenuto and Busch 1995,
1998; Cutrera et al. 2005). This mode of life results in small population units,
sparsely distributed, with low genetic variation and high inter-population diver-
gence brought about by genetic drift and low levels of gene flow (Busch et al. 2000;
Lacey 2000, Cutrera et al. 2005; Fernández-Stolz et al. 2007; Mora et al. 2006,
2007, 2010; Cutrera and Mora 2017; Mapelli et al. 2017). A particular combination
of behavioral and eco-physiological characteristics determines that, even at small
spatial scale (at very few kilometers), tuco-tucos show high levels of genetic struc-
ture with strong phylogeographic patterns (Cutrera et al. 2006, 2010; Mora et al.
2006, 2007, 2013; Mapelli et al. 2012a).
High habitat specialization of tuco-tucos has conditioned that population demo-
graphic trajectories were strongly affected by changes in the extent of the environ-
ments that they occupy (Mora et al. 2016). In this regard, historical variations in the
extension and connectivity of their habitats have strongly impacted the evolution
and diversification of populations (Mora et al. 2006; Mirol et al. 2010; Roratto et al.
2014; Caraballo and Rossi 2017; Mapelli et al. 2012a, 2017). Thus, patterns of
genetic variation at the geographical level have allowed to infer the evolutionary
dynamics of the populations and, by extension, the dynamics of the environments
they occupy.
5.3 Patterns of Isolation by Distance
Limited dispersal in Ctenomys species has important consequences on the spatial

distribution of genetic variation (Lessa 2000; Fernández-Stolz et al. 2007; Lopes
et al. 2013). If dispersal distances are small, a pattern of spatial autocorrelation
emerges in the distribution of genetic variation: individuals that are close to each
other are likely to be more related and therefore genetically more similar than indi-
viduals that are spaced farther apart (Mora et al. 2010). Something similar occurs at
larger spatial scales, where the limited dispersal results in relatively higher values of
gene flow between neighboring populations, which finally translate into a positive
relationship between genetic and geographic distances. Consequently, a balance
between the loss of genetic variation due to genetic drift and its gain because of the
immigration of individuals is established. This equilibrium between these evolu-
tionary forces determines that the spatial distribution of genetic variation in the
species conforms to an isolation by distance (IBD) pattern, where the genetic dis-
similarity among populations is proportional to the distance among them. The
genetic population structure of many species (particularly those that have lower
dispersion rates and are widely distributed) is characterized by an IBD pattern, and
it has been frequently observed in nature (Vekemans and Hardy 2004).
Considering the life history traits of tuco-tucos (e.g., low dispersal capabilities,
small population sizes), it is expected that the spatial distribution of the genetic
variation will fit to an IBD pattern. However, there are several studies on population
genetics in Ctenomys showing the absence of an IBD pattern or illustrating conflict-
ing information between different regions of the genome. In this regard, mutation
rate on different molecular markers must be considered in the analyses.
Phylogeographic studies in Ctenomys were primarily based on mitochondrial
DNA (mtDNA) sequence analyses, whereas population genetic approaches at most
recent ecological time scales have analyzed the nuclear DNA variation at microsat-
ellite loci. Different DNA fragments of the genome have different rates of mutation,
with mtDNA rates being from 3–5 times lower than those reported for microsatellite
loci. These differences in mutation rates among different molecular markers have
evident implications in how the genetic variation is recovered in natural popula-
tions. Particularly, it is expected that molecular markers with slower mutation rates
require more time to reach a new equilibrium between genetic drift and migration
after a demographic change event. Thus, for an IBD pattern to arise, it is necessary
that the balance between gene flow and genetic drift be sustained for enough time.
Because of differences in their effective population sizes and mutational rates, it is
expected that historical demographic events affect mitochondrial genetic variation
more strongly than variation at microsatellite loci. Several phylogeographic studies
in Ctenomys show that mtDNA still reflects the historical demographic processes
(population expansions or contractions) that occurred during the Pleistocene and
Holocene (see the next section in this chapter); consequently, the spatial distribution
of genetic variation does not fit to an IBD pattern. For instance, some species of
tuco-tucos such as C. rionegrensis, C. australis, C. magellanicus, and C. torquatus
have experienced recent range expansions (Wlasiuk et al. 2003; Mora et al. 2006;
Gonçalves and Freitas 2009; Fasanella et al. 2013; Roratto et al. 2014) and genetic
estimates of gene flow using mitochondrial DNA are higher therefore, than current
levels, concealing the IBD pattern.
Following Slatkin (1993), D’Elía et al. (1998) suggested that the current distribu-
tion of C. rionegrensis in Uruguay is the result of a recent range expansion, suggest-
ing that genetic estimates of gene flow should be higher than current levels. Wlasiuk
et al. (2003) proposed a scenario in which C. rionegrensis expanded in the recent
past from a more limited geographic range and has subsequently differentiated in
near isolation, with genetic drift most likely playing a major role in overall genetic
differentiation (see also D’Anatro et al. 2011). The recent range expansion in this
species would hide the IBD pattern using mitochondrial and nuclear molecular
markers. Mora et al. (2006) in C. australis, Fasanella et al. (2013) in C. magellani-
cus, and Roratto et al. (2014) in C. torquatus also showed that genetic differentia-
tion was not consistent with a simple IBD pattern when mtDNA was considered,
possibly evidencing a lack of equilibrium between gene flow and local genetic drift.
Like C. rionegrensis, these species experienced a recent demographic expansion as
shown by their pattern of mtDNA variation.
On the other hand, several species of Ctenomys have shown patterns implying
historical IBD, emphasizing the idea that populations have remained long enough in
demographic equilibrium between drift and migration (see Mora et al. 2007, 2013,
2016; Tomasco and Lessa 2007; Lopes and Freitas 2012; Lopez et al. 2013). Two
examples using mtDNA are reported in Mora et al. (2016) and Lopes and Freitas
(2012), in C. chasiquensis and C. lami, respectively. These authors considered the
entire distributional ranges of these species and suggested moderate to high popula-
tion genetic structure at a regional level accompanied by IBD patterns. Equilibrium
between genetic drift and gene flow at larger spatial scales has also been reported in
other species of ctenomyids that are almost linearly distributed (C. talarum, in Mora
et al. 2007, 2013; C. pearsoni, in Tomasco and Lessa 2007; C. minutus, in Lopes
2011 and Lopes et al. 2013). In these examples, the narrow distribution of the spe-
cies limits dispersal and gene flow to a dominant spatial direction as compared to
what is possible in a widespread and two-dimensional distributional range. As was
suggested by Mora et al. (2006), one-dimensional spatial systems usually have a
reduced variance in geographical genetic attributes, as compared to two-dimensional
arrangements, increasing the chance to detect such IBD pattern (see also Slatkin and
Barton 1989 for a more comprehensive discussion).
There is a strong relationship between the mutational rate of molecular markers,
the spatial arrangements of populations (i.e., one-dimensionally vs two-
dimensionally distributed species) and the recovery rate of genetic variation due to
random mutational processes in natural populations following demographic changes
(e.g., bottlenecks and/or population expansions). This means that species that have
undergone recent demographic expansions (accompanied or not by expansion in
their geographical ranges) are more likely to depict IBD patterns in their most vari-
able DNA portions of their genomes (e.g., microsatellite loci in C. australis and
C. porteousi; see Austrich et al. 2020 and Mapelli et al. 2012b, respectively).
Using microsatellite loci, several studies in population genetics of tuco-tucos
have tried to assess the relative importance of geographic distances among popula-
tions and those landscape elements that potentially could mold gene flow patterns.
For some species, distance appears to be the main factor structuring genetic varia-
tion among different populations (Mapelli et al. 2012b; Austrich et al. 2020). In
other cases, landscape features that impose potential barriers to gene flow or are
associated to suitable habitat conditions for the occurrence of a species are more
important than distance among populations (Wlasiuk et al. 2003; Mora et al. 2010,
2017). Thus, close populations can diverge more quickly if there is a strong barrier
to the movement of individuals, whereas populations located in areas of more con-
tinuous habitat can exchange more migrants, promoting the increase of genetic
similarity among them. On the other hand, the divergence rate among populations
can also be variable, hindering the establishment of an IBD pattern. Basically, dif-
ferentiation among populations is a consequence of genetic drift and this process is
directly dependent upon population size. Populations of smaller sizes are subject to
stronger effects of genetic drift, which leads to a faster fixation of allelic variants. It
is thus expected that these populations have reduced genetic variability in compari-
son to larger and more stable populations. For instance, the lack of an IBD pattern
in C. chasiquensis denote that differentiation was not related to geographical dis-

tances, but to the landscape or habitat characteristics where the populations were
sampled (see Mora et al. 2017). In this species, habitat fragmentation (both anthropic
and natural) has caused the isolation of some populations in the eastern area of its
distribution, with the consequent accentuation of genetic drift. This pattern could
explain, at least in recent times, the lack of equilibrium between gene flow and
genetic drift across the entire distribution of the species. A similar pattern was
observed in C. rionegrensis, where genetic drift due to population isolation has been
strong at recent times, minimizing the effects of geographical distances. In these
examples, the lack of an IBD pattern seems to indicate that population divergence is
not related to geographical distances but rather to landscape characteristics.
5.4 Phylogeographic Patterns
As most species of tuco-tucos are endemics, their complete distributional ranges

were covered in several studies, disclosing the factors that have brought about the
observed phylogeographic patterns. In general, these studies show a close associa-
tion between population size variations and historical changes in habitat availabil-
ity; suggesting that diversification patterns both among and within species, were
closely related to historical habitat connectivity (Mora et al. 2006, 2007, 2013,
2016; Tomasco and Lessa 2007; Mirol et al. 2010; Gómez Fernández et al. 2012;
Mapelli et al. 2012a, 2017; Lopes et al. 2013; Roratto et al. 2014; Cutrera and
Mora 2017).
The glacial-interglacial cycles of the Late Quaternary produced extensive distur-
bances not only in different biogeographic areas throughout South America, but
also in many regions of the world (Rabassa et al. 2000, 2011). In South America,
these changes largely conditioned the availability and connectivity of suitable habi-
tats for tuco-tucos, profoundly affecting their phylogeographic patterns (Chan et al.
2005; Mapelli et al. 2012a, 2017; Tammone et al. 2018a, 2018b). In many species
of Ctenomys, demographic expansions and contractions thus seem to have mainly
responded to landscape changes associated with climatic perturbations occurred in
the Late Pleistocene and Holocene (Tammone et al. 2018a, 2018b). These changes
have led to the formation of sandy accumulations, in the form of coastal and conti-
nental dunes, which have allowed the permanence or range expansions of the popu-
lations of many species of tuco-tucos (Mora et al. 2006, 2007). In this regard, not
only the species in Andean regions have altered their distributional ranges as a result
of the glacial-interglacial cycles (Gutiérrez-Tapia and Palma 2016), but species dis-
tributed in many areas distant from the Andes, such as the South American Pampas
and coastal areas, have also been largely affected (Tonni et al. 1999; Mora et al.
2006, 2007, 2016; Mapelli et al. 2012a).
Expansion of sandy areas affected not only the demography and evolution of
tuco-tucos populations, but also impacted on the diversification patterns between
species (Mapelli et al. 2017). Caraballo and Rossi (2018) used phylogeographic
approaches to understand the diversification of the “torquatus” group, a set of
closely phylogenetically related species of tuco-tucos distributed along Uruguay,

eastern Argentina and southern Brazil (C. torquatus, C. lami, C. minutus, C. ibi-
cuiensis, C. pearsoni) and the “Corrientes” group (tuco-tucos distributed around
and between the Iberá marshes in Corrientes province, Argentina, whose taxonomy
is not yet well established). These authors suggest that the geographic expansion of
these lineages and the colonization of the Argentine areas occurred during the most
arid stages of the Pleistocene when sandy soils expanded throughout the region
(between 500,000-1,000,000 ybp) and the effectiveness of the Uruguay River as a
barrier would have diminished considerably.
Mapelli et al. (2017) studied the “mendocinus” species complex (or “mendoci-
nus” group; D’Elía et al. 1998, 1999; Wlasiuk et al. 2003; Massarini and Freitas
2005; Stolz 2006; Fernández-Stolz 2006; Fernández-Stolz et al. 2007; Parada et al.
2011; Mora et al. 2016) in central Argentina and suggest that the dispersion of the
group was associated with the driest stages of the Late Quaternary. The rapid clado-
genesis experienced by the “mendocinus” group was associated with the formation
of large sandy areas during the Late Pleistocene. Paleoclimate estimates indicate
that this period was the driest and coldest time for the last glacial-interglacial cycle
(Iriondo and Kröhling 1995). These climatic conditions generated an increase in the
extension of arid environments and may have permitted a more continuous distribu-
tion of tuco-tucos of C. mendocinus species complex throughout the Pampas in
Central Argentina (Mapelli et al. 2017). On the other hand, the interglacial periods
were characterized by increased temperature and humidity (Quattrocchio et al.
2008). Suitable environments for these tuco-tucos retracted during humid periods
and expanded during arid pulses (Mapelli et al. 2017). Thus, during periods of habi-
tat retraction and lower overall population sizes, the combined effects of natural
selection and genetic drift would have favored the divergence among lineages. In
this form, the divergence among different lineages of the “mendocinus” species
group may have taken place during humid periods, when climate conditions resulted
in environmental discontinuities that isolated populations (Mapelli et al. 2017).
At the Holocene-Pleistocene boundary (about 12,000–9000 ybp) an important
increase in temperature and humidity occurred in the area of the current distribu-
tional range of “mendocinus” group (Tonni et al. 1999). These climatic changes, in
conjunction with a rise of sea level and the flooding of the riverbeds favored the
spread of inland grasslands, increasing plant cover (Quattrocchio et al. 2008) and
decreasing the extent of habitat available for these tuco-tucos. The easternmost
range of this area have been studied in detail from phylogeographic perspectives
and the results obtained highlight the strong imprint of the environmental change
during the Pleistocene-Holocene boundary on the population demography of these
species.
Three of the species belonging to the “mendocinus” group currently occupy rem-
nant and fragmented habitats. Ctenomys rionegrensis is restricted to very fragmen-
tary sandy areas associated with sedimentary deposits formed by the Rio Negro,
Uruguay (D’Elía et al. 1998; Wlasiuk et al. 2003; Kittlein and Gaggiotti 2008;
D’Anatro et al. 2011). C. porteousi is restricted to sandy soils associated with defla-
tion basins in the Lagunas Encadenadas del Oeste, Argentina (Mapelli and Kittlein
2009; Mapelli et al. 2012a, 2012b) and C. chasiquensis is restricted to a series of

sandy paleo valleys in southern Buenos Aires and La Pampa provinces, Argentina
(Mora et al. 2016, 2017). Their geographical patterns of mitochondrial genetic vari-
ation show local populations evolving in relative isolation, with a strong effect of
genetic drift at local level and low gene flow between populations (Wlasiuk et al.
2003; Mapelli et al. 2012a; Mora et al. 2016). However, it is interesting to highlight
that the differences between the phylogeographic patterns of these species are very
subtle and are related to the history of the environments they currently occupy.
C. rionegrensis shows very low genetic variation by locality, with very few haplo-
types shared among populations. This suggests a strong effect of genetic drift
locally, and low values of gene flow between localities; as expected for animals with
low vagility evolving in relatively isolated populations (Wlasiuk et al. 2003). C. por-
teousi also shows strong effects of genetic drift and low levels of gene flow between
populations (Mapelli et al. 2012a); but the effects do not seem to be as marked as in
C. rionegrensis. C. porteousi, however, showed a greater number of shared haplo-
types among populations, suggesting a less marked effect of genetic drift. Population
differentiation seems not to have been generated by a combination of unique haplo-
types as in C. rionegrensis, but by distinctive assortments of shared haplotypes. This
pattern suggests a greater connectivity between populations in the past, with strong
subsequent effects of genetic drift at local level.
The phylogeographic pattern of C. chasiquensis was similar to that observed in
C. porteousi, but with higher past connectivity between populations and greater
genetic variation by locality (Mora et al. 2016). In addition, populations located
towards the East of the range of C. chasiquensis (a more fragmented area with
higher intrusions of grasslands) presented evidence of higher past isolation as com-
pared with populations in the western range of the species (Mora et al. 2016, 2017).
Historical demographic reconstructions for C. porteousi (Mapelli et al. 2012a) and
C. chasiquensis (Mora et al. 2016) show that their population dynamics were
strongly impacted by environmental changes at the Holocene-Pleistocene bound-
ary; in both species there was a significant reduction in population sizes associated
with these historical events. Accordingly, the loss and fragmentation of their natural
habitats would have had a profound impact on the spatial distribution of mitochon-
drial genetic variation, reducing connectivity between populations and increasing
the effects of genetic drift.
On the other hand, C. australis (sand-dune tuco-tuco), another species included
in the “mendocinus” group that occupies the sand dunes along the Atlantic coast of
Buenos Aires province, Argentina, presents a completely different phylogeographic
pattern to that observed in its sister species (Mora et al. 2006, 2010; Cutrera and
Mora 2017). This endemic and endangered subterranean rodent presents an ances-
tral haplotype distributed in high frequency throughout the entire range of its distri-
bution, which indicates a marked process of population growth associated with a
notable geographic population expansion (see Fig. 5.1). The coastal sand dunes
occupied by this species are a relatively recent habitat originated during the sea-
level fluctuations produced by oceanic regressions and transgressions in the Middle
Holocene (6500–3000 ybp; Isla 1998; Isla et al. 2001). C. australis is an extreme
Fig. 5.1 Examples of haplotype networks depicting the contrasting patterns in the spatial distribu-
tion of genetic variation of mitochondrial DNA in two species of tuco-tuco from Argentina and
Brazil: (a) Ctenomys australis (modified from Mora et al. 2006), (b) C. minutus (modified from
Lopes et al. 2013). Circles correspond to different mtDNA haplotypes, and their size is propor-
tional to their frequency in the samples. Colors of the circles designate their spatial location (local-
ity or group of localities) on the maps (a and b), and lines connecting the circles represent the most
likely genealogical relationships between haplotypes (the crossed marks represent the number of
mutational steps among haplotypes). Light green polygons on the map represent suitable habitat
for the species. Different localities in C. minutus have different groups of haplotypes, which
diverge by several mutational steps. This pattern is indicative that populations have occupied the
area in demographic equilibrium long enough to establish an isolation by distance (IBD) pattern.
In contrast, C. australis shows an ancestral haplotype with much higher frequency than the other
haplotypes in the network and occurring in all localities. The other haplotypes diverge from the
ancestral haplotype in one or only a few mutational steps and occur only in some localities. This
pattern is indicative of a recent demographic expansion, possibly accompanied by a geographical
range expansion
specialist to this type of habitat and shows strong associations with the unstable
conditions of the sand-dune system (Malizia et al. 1991; Zenuto and Busch 1995,
1998; Mora et al. 2006). It can be assumed that its populations are at least as old as
coastal dunes in the region since no fossilized remains of this species have been
found outside its current distributional range (Contreras and Reig 1965). Mora et al.
(2006) propose a recent occurrence of C. australis in the Quaternary coastal dune
system; but whether it extended its distribution to the coastal dunes with the forma-
tion of this novel habitat or derived from an ancestral form coming from another
geographic area remains unknown. The phylogeographic pattern in C. australis
would thus reflect the rapid colonization and expansion along the recently origi-
nated coastal dune systems. Again, historical changes in habitat availability seem to
have molded, in some extent, the spatial distribution of mitochondrial genetic varia-
tion in this species.
Ctenomys talarum (the Talas tuco-tuco) occurs in the Pampean region of
Argentina, and unlike the species of the “mendocinus” group, this species occupies
environments with higher plant cover (Vassallo 1998; Mora et al. 2007, 2013; Luna
and Antinuchi 2007). Its distribution is mainly coastal in Buenos Aires province, but
it is also present inland in areas with a highly fragmented habitat matrix (Mora et al.
2013). Similar to the species of the “mendocinus” group, their populations seem to
be strongly affected by the environmental changes occurred during the Pleistocene-
Holocene boundary. The phylogeographic pattern recovered for C. talarum through-
out its distribution range tells contrasting histories for different groups of populations.
Some populations appear to be characterized by demographic stability and no sig-
nificant departures from neutrality, but others show departures from strict neutrality,
with signals of a recent demographic expansion (Mora et al. 2013). The progressive
increase in humidity and temperature during the Pleistocene-Holocene boundary
and, the consequent associated increase in plant cover, apparently initiated the frag-
mentation of inland populations, but not to such high levels as those affecting the
species of the “mendocinus” group which have more restrictive requirements
regarding the occurrence of sandy soils. In C. talarum, several inland populations in
Buenos Aires province seem to have become extinct recently (Quintana 2004),
while others persist in a highly fragmented matrix, associated to stream sand banks
and inland areas with paleo-dunes (Mora et al. 2007, 2013). Their patterns of mito-
chondrial variation resemble that reported for C. rionegrensis: very low genetic
variability by location and almost no haplotypes shared between locations, suggest-
ing that they have remained in relative isolation (Mora et al. 2013). Genetic diver-
sity and differentiation of coastal populations tell a different story. At present, these
populations occupy sand-dune environments that originated as a result of sea-level
fluctuations during the Holocene. The patterns of genetic variation in these areas
indicate population expansions, evidently associated with the colonization of these
novel environments of recent formation (Mora et al. 2007, 2013).
Interestingly, estimates of the main demographic changes observed in several
Ctenomys species from the central Argentina seem to be temporarily coincident. It
seems that while some species adapted to environments with low plant cover under-
went population retractions (e.g., C. porteousi and C. chasiquensis; Mapelli et al.
2012a; Mora et al. 2016), other species adapted to environments with higher plant
cover (e.g., C. talarum) remained demographically stable in some regions, and
underwent population expansions in others (Mora et al. 2007, 2013). This shows
some differences in the way environmental change affected the demography of each
species.
The Patagonian tuco-tucos Ctenomys sociabilis and Ctenomys haigi offer other
interesting example of differential demographical responses to concomitant changes
in the environment. C. sociabilis is highly endemic to the upper valley of the Limay
River (Chan et al. 2005; Chan and Hadly 2011; Tammone et al. 2018a, 2018b);
while C. haigi is widely distributed in the eastern Limay Valley and adjacent areas
of Río Negro province, Argentina (Lacey et al. 1997, 1998). Chan et al. (2005) and
Tamnone et al. (2018a, b) quantified the mitochondrial genetic variation in current
and fossil samples and observed important evidence of a marked reduction in the
genetic variability of C. sociabilis, but not in C. haigi, during the last 12,000 years.
The reduction in genetic variability observed in C. sociabilis is of such magnitude
that its populations are currently characterized by a single haplotype throughout its
entire distributional range (see Fig. 5.2). Differential demographic responses
observed between these species could have been influenced by species-specific
attributes such as the documented differences in habitat specialization. C. sociabilis
Fig. 5.2 Relationship between haplotype and nucleotide diversity for mitochondrial DNA in some
representative species of Ctenomys. Haplotype diversity is a measure of how many haplotypes are
in the gene pool of a population or species, while nucleotide diversity is a measure of how different
these alleles are from each other. In this figure, species that underwent recent population expan-
sions present low values of nucleotide diversity and high values of haplotype diversity. Only those
studies that include the entire or almost complete distribution of a species are given: C. australis
(Mora et al. 2006), C. chasiquensis (Mora et al. 2016), C. lami (Lopes and Freitas 2012), C. magel-
lanicus (Fasanella et al. 2013), C. minutus (Lopes et al. 2013), C. porteousi (Mapelli et al. 2012a),
C. sociabilis (Chan et al. 2005), C. talarum (Mora et al. 2013) and C. torquatus (Roratto et al. 2014)
inhabits a more restricted subset of habitats compared to the ecologically more gen-
eralized C. haigi (Lacey et al. 1997, 1998; Lacey and Wieczorek 2004). During the
Middle-Holocene, the area occupied by these species was spatially and temporally
dynamic and this variability might have contributed to the decline of C. sociabilis
but not of C. haigi at the study sites (Chan et al. 2005; Tammone et al. 2018a,
2018b). Consequently, the greater habitat specialization of C. sociabilis would have
made this species more sensitive than C. haigi to different environmental changes
(Tammone et al. 2018a, 2018b).
The Late Quaternary environmental changes have also had significant effects on
the historical demography of Ctenomys magellanicus (Fasanella et al. 2013), the
southernmost species of this genus. The species is fragmentarily distributed both in
the steppe and in the forest ecotone of the Isla Grande de Tierra del Fuego in the
southern extreme of Argentina and Chile, although there is fossil evidence that the
species previously occupied the final extreme of continental Patagonia in Santa
Cruz Province, Argentina (Pardiñas 2013). The phylogeographic pattern recovered
for the species suggests a recent history of demographic expansion with a recent
colonization of the northern area of its distribution. Possibly one or a few haplo-
types of the southern distribution have colonized the northern area during the
Pleistocene when rivers had low-flow regimes (Fasanella et al. 2013). These authors
have proposed this species might have lived at a Pleistocene refuge during the
adverse environmental conditions in Tierra del Fuego (see also Rabassa et al. 2000,
2011). At the same time that the northern populations disappeared from the island,
they also were extinct from the continental areas of Argentina in the other side of the
Straits of Magellan. Subsequently, when environmental conditions led to the expan-
sion of the steppe environments, C. magellanicus recolonized the northern sector of
the Isla Grande de Tierra del Fuego. It is likely that this recolonization occurred
after the Island split-up from the continent, so that the Strait of Magellan prevented
the recolonization of continental Patagonia. Interestingly, there is no present-day
migration occurring between the two areas where tuco-tucos are currently present in
Isla Grande de Tierra del Fuego, suggesting an appreciable effect of rivers and
streams as potential barriers to gene flow (Fasanella et al. 2013).
The phylogeographic pattern observed in C. pearsoni also appears to be associ-
ated with the Holocene environmental imprint that resulted in variations of sea lev-
els in a highly dynamic landscape (Tomasco and Lessa 2007). This species mainly
inhabits the coastal plains of southern Uruguay by the Río de la Plata and the
Atlantic Ocean (Tomasco and Lessa 2007; Caraballo et al. 2016). Mitochondrial
genetic variation in C. pearsoni adjusts to an IBD pattern, suggesting that this spe-
cies conserved a stable regime of differentiation by genetic drift under limited gene
flow for a long term. Additionally, C. pearsoni shows no signs of possible depar-
tures from neutrality; in fact, this regional stability in its historical demography
might have been retained in spite of Holocene marine transgressions and other envi-
ronmental and climatic perturbations that affected the distribution of the species
(Tomasco and Lessa 2007). Locally, however, populations may have shifted their
distributions in response to sea-level changes, especially on the coast in the north-
west area of Uruguay, where it occupies systems of coastal dunes of very recent
formation (Tomasco and Lessa 2007). Possibly, more relaxed restrictions associated
to the type of habitat that this species occupy (not certainly restricted to the first strip
of coastal dunes as C. australis and C. flamarioni; see Mora et al. 2006 and
Fernández-Stolz et al. 2007, respectively) have prevented local extinctions or dras-
tic population reductions on the Uruguayan coast.
Several phylogeographic studies have also been carried out in species of
Ctenomys from southern Brazil at different spatial scales (El Jundi and Freitas 2004;
Stolz 2006; Fernández-Stolz et al. 2007; Gonçalves and Freitas 2009; Gonçalves
et al. 2012; Lopes and Freitas 2012; Lopes et al. 2013). For instance, C. torquatus
(a species with one of the widest geographical ranges, occurring in the lowland
grasslands of southern Brazil and northern Uruguay; Freitas 1995; Fernandes et al.
2009) shows a phylogeographic pattern explained by the habitat availability for the
species and the historical patterns of colonization of this area. Mitochondrial genetic
variation shows a process of population expansion and a recent colonization of the
peripheral areas of its global distribution (Fig. 5.2). This population expansion has
been associated with greater environmental availability during the Late Pleistocene
(Lopes and Freitas 2012; Roratto et al. 2014). Conditions of greater aridity prevail-
ing during this period would have allowed a greater expansion of the grassland
environments with a concomitant reduction of gallery forests along the rivers; which
would have allowed the population expansion of C. torquatus and the colonization
of novel areas (Roratto et al. 2014). The formation of coastal dune systems during
the Holocene would also have allowed the expansion of this species into these areas.
Demographic reconstructions of populations occupying these environments support
this hypothesis and indicate that colonization and expansion in these areas would
date back to the last 5000 years, when the most recent sand-dune system was formed
(Tomazelli et al. 2000; Roratto et al. 2014).
C. lami and C. minutus, two very related species, occur in areas close to C. tor-
quatus, but their phylogeographic patterns are somewhat contrasting: these two spe-
cies are characterized by IBD patterns and their populations do not appear to have
been so affected by the climatic oscillations of the Late Quaternary (El Jundi and
Freitas 2004). C. lami is highly endemic with a very restricted distribution; this spe-
cies only occupy an area of 936 km2 (Lopes and Freitas 2012). This zone consists of
sandy fields formed by fluctuations of the sea level in the beginning of the
Pleistocene, approximately 400,000 years ago (Tomazelli et al. 2000). A clinal pat-
tern of genetic variation could be observed in C. lami, since the haplotypes are com-
monly shared between geographically close localities. The absence of any evidence
of population expansion and the fact that populations adjust to an IBD pattern sug-
gests demographic stability and low effects of Late Quaternary environmental
changes on their populations.
A similar pattern can be observed in C. minutus; this species has a coastal distri-
bution in the Southeastern of Brazil, occupying sand-dune systems formed during
the marine transgressions and regressions in the Pleistocene and Holocene (Lopes
2011; Lopes et al. 2013). Despite the marked environmental dynamics of the region,
C. minutus shows demographic stability and one of the strongest phylogeographic
breaks within the genus: in general, most haplotypes are separated by several
mutational steps, and were limited to single area (see Fig. 5.1). This pattern of spa-
tial distribution of genetic variation suggests that populations of this species have
remained stable for a considerable period and their demographic trajectories have
been little affected by environmental changes (Lopes et al. 2013). These latter
authors used Bayesian approaches and showed that the effective population sizes
were mostly constant, with only some minor sudden retractions observed from the
last 13,000 ybp (see also Lopes 2011).
Phylogeographic approaches have also been applied for the study of the species
of the “Corrientes” group distributed in the northeast of Argentina (Mirol et al.
2010; Caraballo et al. 2012; Gómez Fernández et al. 2012). The “Corrientes” group
represent a typical case of uncertainty on species boundaries; including three named
species (C. roigi, C. perrensi, and C. dorbignyi), and several other populations
whose taxonomical status has not been determined yet (Giménez et al. 2002; Mirol
et al. 2010; Caraballo and Rossi 2017). The diversification of the “Corrientes” group
also seems to be due to the historical availability of the habitat; although in this case
habitat availability is not seem to be associated with the glacial and interglacial
cycles but to variations in the hydrological regimen in the region. Tuco-tucos from
this group occupy sandy soils located in elevated sites inside a large wetland, influ-
enced by the effects of the Paraná River and the Iberá Wetland (Giménez et al. 2002;
Mirol et al. 2010). The flooded systems typically observed in this extensive wetland
create a highly dynamic and unstable environment for the presence of tuco-tucos.
This is because water-level fluctuates depending on rainfall and the soil composi-
tion as well as underground rivers flow. Thus, the wetlands changes in time and
space, altering the availability of suitable habitat for tuco-tucos, connecting or iso-
lating populations at different times and in different areas (Mirol et al. 2010; Gómez
Fernández et al. 2016). Although very few haplotypes in this group of species are
shared between locations, they appear closely related, differing in few mutational
steps. At the same time there is no clear geographical structure and there is evidence
of an important historical gene flow between some sites (Caraballo et al. 2012;
Gómez Fernández et al. 2012). The highly dynamic hydrological regime seems to
be responsible for temporal connection and disconnection among populations,
affecting the patterns of genetic differentiation at regional level.
In summary, some studies in Ctenomys have shown concordant phylogeographic
patterns across species. There are very few cases (e.g., C. minutus, C. lami, C. pear-
soni; Lopes and Freitas 2012; Lopes et al. 2013; Tomasco and Lessa 2007) in which
species of tuco-tucos have remained stable sufficiently long to permit a demo-
graphic stability. These species adjusts to an IBD pattern, suggesting that the popu-
lations have remained stable long enough time to have achieved a balance between
genetic drift and migration. On the contrary, many species of Ctenomys have suf-
fered recent demographic processes (population growth or decrease) that have pro-
foundly impacted the distribution of genetic variation. Most species (C. rionegrensis,
C. australis, C. porteousi, C. chasiquensis, C. talarum, C. torquatus) show strong
demographic signals associated with historical variations in the extend of the natu-
ral habitats. The glacial-interglacial cycles of the Late Quaternary have strongly
impacted the distribution and demography of these tuco-tucos, particularly the
environmental change occurred in the Pleistocene-Holocene boundary, both for

their magnitude and temporal proximity. These climatic and environmental pro-
cesses have left deep traces not only in the distribution of mitochondrial genetic
variation, but also in nuclear DNA (see Cutrera and Mora 2017).
5.5 Spatial Scales of Population Genetic Structure
During the last three decades, researchers have used diverse genetics tools to define
population boundaries. In this regard, the use of highly variable molecular markers
in studies of assigning individuals to populations has revolutionized population
genetics. SNPs and microsatellite loci have been particularly efficient in elucidating
population boundaries at fine spatial scales and to define significant evolutionary
units in hundreds of species (Lacey 2000; Helyar et al. 2011).
The use of microsatellite loci (Lacey et al. 1999; Lacey 2001; Roratto et al. 2011)
in addition to statistical Bayesian approaches have allowed to infer putative genetic
populations and assign individuals to them in several Ctenomys species. These
approaches have permitted to understand, to some extent, the factors and processes
shaping the population structure and spatial distribution of genetic diversity in this
genus (Wlasiuk et al. 2003; Gava and Freitas 2004; El Jundi and Freitas 2004;
Fernández-Stolz et al. 2007; Gonçalves and Freitas 2009; Mirol et al. 2010; Mora
et al. 2010; Gómez Fernández et al. 2012; Lopes and Freitas 2012; Mapelli et al.
2012b; Fasanella et al. 2013; Lopes et al. 2013; Mora et al. 2017; Esperandio et al.
2019). In general, these studies describe the tuco-tucos as species very prone to be
spatially structured. High habitat specificity, a phylopatric behavior and low disper-
sal capabilities of tuco-tucos determine that gene flow occurs most probably within
the same local population. Consequently, gene flow between nearby localities is low
and strongly conditioned by the connectivity among populations, which leads to
high interpopulation divergence.
But not only the distances between localities determine the population connec-
tivity in Ctenomys. Several studies (Mora et al. 2010; Lopes and Freitas 2012;
Mapelli et al. 2012b; Lopes et al. 2013; Mora et al. 2017; Esperandio et al. 2019)
have shown that habitat discontinuities strongly condition gene flow patterns
between populations, so numerous landscape elements can become important barri-
ers to population connectivity (in addition to geographical distances between popu-
lations). Species of tuco-tucos inhabit more fragmented habitats have shown the
strongest patterns of population structure than species distributed in landscapes with
continuous and more conserved habitats (Mapelli et al. 2012b, 2020; Mora et al.
2017). Similarly, within the same species, populations located in areas with low
habitat connectivity show greater divergence relative to areas where the habitat of
tuco-tucos is less fragmented (see Mora et al. 2017; Mapelli et al. 2020). This can
also be illustrated in several studies, where populations occupy fragmented habitats
(both by natural and anthropic action) and where sampling locations only 10–15 km
away from each other constitute independent genetic units (Wlasiuk et al. 2003 in
C. rionegrensis; Gonçalves and Freitas 2009 in C. torquatus; Mapelli et al. 2012b in

C. porteousi). The case of C. rionegrensis is paradigmatic since populations located
at these distances seem to evolve in almost complete isolation with extremely
reduced gene flow (Wlasiuk et al. 2003; D’Anatro et al. 2011). In species occurring
in a more continuous habitat the genetic population units are defined at larger spatial
scales. This can be observed in C. lami (Lopes and Freitas 2012), in C. minutus
(Castilho et al. 2012; Lopes et al. 2013), and in C. chasiquensis (Mora et al. 2017).
All these studies show how patterns of population genetic structure are strongly
conditioned by the landscape configuration.
Another aspect to consider is how genetic variation recovers after an event of
demographic change. Because population bottlenecks often accompany coloniza-
tion events that occur when a species expands its range, peripheral populations fre-
quently have lower genetic diversity than central populations. Greater effective
population sizes in central populations than in peripheral populations suggest also
an important role of genetic drift after colonization (Swaegers et al. 2013). In this
regard, these patterns are recurrent in tuco-tucos: greater genetic divergence among
the populations located in peripheral areas relative to those populations from the
core area, and lower genetic diversity in the peripheral populations.
Several authors have shown how populations of tuco-tucos found in the periph-
eral areas are characterized by low genetic diversity, and in some cases tend to
establish independent genetic units (Gómez Fernández et al. 2012; Lopes and
Freitas 2012; Mapelli et al. 2012b; Lopes et al. 2013; Mora et al. 2017; Austrich
et al. 2020). As was suggested previously, this pattern could be explained by the
greater effect of genetic drift in peripheral populations (recurrent founder events
lead to greater fluctuations in population sizes; Mapelli et al. 2012b). Some extreme
cases of this scenario can be observed in C. rionegrensis (Wlasiuk et al. 2003), in
C. australis (Austrich et al. 2020), and in the species of the “Corrientes” group
(Gómez Fernández et al. 2012, 2016), where the most isolated populations com-
pletely lack genetic variability in several microsatellite loci. Another example is
C. magellanicus, a species that expanded its range into the northernmost areas of
Isla Grande de Tierra del Fuego after the Pleistocene glaciation, and populations in
these relatively recently colonized areas have considerably lower genetic diversity
than populations within the areas of former glacial refuge (Fasanella et al. 2013).
In general, tuco-tucos are structured following a classical metapopulation model
(Hanski and Ovaskainen 2000; Hanski and Gaggiotti 2004), with local populations
exchanging individuals depending on the habitat connectivity among sites. In this
way, landscape characteristics and distances among populations have a significant
effect in limiting the movement of individuals and structuring the genetic variation
of tuco-tucos (Mora et al. 2017), perhaps to a greater extent than social factors (e.g.,
encounter of couples during reproduction, maintenance of home-range, etc.). In
such a context, several species have shown that some minor landscape features such
as changes in soil texture and porosity, watercourses, roads, afforestations, and
some other barriers appear to play a significant role in limiting the dispersion and
gene flow (Wlasiuk et al. 2003; Mapelli et al. 2012b; Mora et al. 2010, 2017;
Esperandio et al. 2019). In these studies, the spatial distribution of genetic clusters
Fig. 5.3 Population genetic structure in Ctenomys lami along its complete distributional range
(modified from Lopes and Freitas 2012). Circles on the map correspond to sampling sites and their
colors denote membership to the one of the main genetic clusters identified using microsatellite
markers. The figure shows how the spatial distribution of genetic clusters was shaped by major
habitat discontinuities. Connections between Pachecos and Taurus Swamps divide the main
genetic clusters; there are other four genetic clusters confirmed by only one or two neighboring
localities. These small populations are in peripheral areas of the species’ distributional range and
have less connectivity, thus subject to greater effects of genetic drift. Consequently, they diverged
faster than populations in the central areas of the species distribution
appears to be limited by the availably of the suitable habitat for the species. Here,
the main landscape discontinuities generally coincide with the boundaries between
populations (see Fig. 5.3; Lopes and Freitas 2012, Lopes et al. 2013). Although
sample locations distanced by 20–40 km can constitute a single genetic population
in areas of greater habitat connectivity, in those areas with greater fragmentation the
population genetic structure usually is given at smaller spatial scales (see Mora
et al. 2010).
5.6 andscape Genetics: Which Factors Structure

L
the Genetic Variation?
Landscape genetics is a novel discipline focuses on how geographical and environ-

mental features structure the genetic variation on populations (Manel et al. 2003,
2005). Although still incipient, it has allowed to analyze jointly population genetic
and geospatial data derived from satellite images and several types of thematic maps
(altitude, vegetation, degree of anthropic impact, etc.) in order to understand how
the landscape configuration affect the connectivity among populations (Storfer et al.
2007; Waits et al. 2016; Bradburd et al. 2018).
Even though there are few approaches that have used tools derived from land-
scape genetics in tuco-tucos, these studies show some common factors as respon-
sible in structuring populations at the genetic level. As mentioned above, these
studies revealed that populations located in areas with greater habitat fragmentation
are more genetically differentiated, indicating that habitat discontinuities strongly

restrict gene flow patterns in Ctenomys (Mapelli et al. 2012b; Mora et al. 2017;
Austrich et al. 2020). The landscape effects on the population structure at fine spa-
tial scale can be inferred from the studies of Mora et al. (2010) in C. australis and
Esperandio et al. (2019) in C. minutus. Studying the dispersion patterns in C. aus-
tralis and considering a sampling design at small geographic scales (< 4 km), Mora
et al. (2010) showed how habitat discontinuities have had more relevance in struc-
turing the genetic variation at microsatellite loci than geographical distances among
sampling sites. Esperandio et al. (2019) evaluated the degree of genetic differentia-
tion between pairs of sampling sites distanced to approximately 1 km, one pair
divided by a road, and the other with no potential barrier between them. In this study
is demonstrated, although incipient, an effect of the road restricting the gene flow
between sampling units.
Kittlein and Gaggiotti (2008) using a landscape genetic approach and combining
geospatial and genetic data (information of microsatellite loci extracted from
Wlasiuk et al. 2003) assessed the potential causes that have promoted the genetic
differentiation among populations in C. rionegrensis. These authors showed how
sampling locations at lower altitudes exhibited signs of having experienced strong
genetic drift. Thus, elevation of sampling locations strongly conditions the genetic
differentiation, leading to a faster divergence of the populations located at lower
sites. Despite populations of C. rionegrensis are distributed in rather flat areas,
lower areas could be relatively more affected by flood from nearby rivers than popu-
lations at relatively higher elevations. Under this scenario, floods would reduce
habitat availability and, consequently, population sizes in areas of low altitude.
Thus, populations located in areas of low altitude will be subject to greater effects
of genetic drift and, therefore, would diverge more quickly (Kittlein and Gaggiotti
2008; see also D’Anatro et al. 2011).
Later, Mapelli et al. (2012b, in C. porteousi) and Mora et al. (2017, in C. cha-
siquensis) derived landscape variables using satellite images, elevation, and soil
maps, and analyzed the effect of these features on the population genetic structure.
Among several environmental variables analyzed in C. porteousi, the main land-
scape factor that has affected the population differentiation was the geographic
location that the local populations occupy relative to the most suitable habitats for
this species. Populations located in peripheral areas, where the suitable habitat of
tuco-tucos is distributed more fragmentary, showed greater genetic differentiation,
denoting strong effects of gene drift in the recent past (Mapelli et al. 2012b). The
results obtained in C. chasiquensis show something similar to C. porteousi (Mora
et al. 2017). This species is distributed in a series of paleo-valleys that were filled by
wind sediments (Mora et al. 2016); so their habitat is then basically linear, thinning
toward the East, as the paleo-valleys enter the Pampas Grassland. As a result, popu-
lations located further East showed a greater degree of genetic differentiation. Thus,
again, it is observed that the degree to which local populations are structured is a
consequence of the position that they occupy in relation to the distribution of the
most suitable habitat for the species. Consequently, it is expected that populations
occupying areas of lower environmental availability have suffered stronger effects
of gene drift and have diverged more quickly. Interestingly, the degree of vegetation
cover was a strong determinant of genetic differentiation among populations of
C. chasiquensis. These tuco-tucos are notoriously affected by vegetation cover,
showing very little tolerance to soils with a high percentage of vegetation.
Consequently, Mora et al. (2017) showed that genetic differentiation in C. cha-
siquensis was more pronounced in those areas where the vegetation cover was
higher. This result highlights the importance of habitat quality, and not just its quan-
tity, on the pattern of population structure in Ctenomys. Thus, it is expected that
areas of lower habitat quality will sustain population of smaller size, increasing the
rate of genetic drift and favoring a faster differentiation.
At a larger spatial scale, Gómez Fernández et al. (2016) showed in the “Corrientes
species complex” of Ctenomys how the distribution of genetic variation among lin-
eages of this group was primarily shaped by the habitat distribution. Populations of
these tuco-tucos are distributed on sandy soils where its permanence depends of the
historical variations of the course of Parana River and the subsequent environmental
evolution of the region. Moreover to the distance among localities, the main deter-
minant of population differentiation was also related to the occurrence of the most
suitable habitats of these species. Thus, sites with greater habitat availability sup-
port populations of larger sizes which retain greater genetic variability (see also
Mirol et al. 2010).
Although the species studied by Mapelli et al. (2012b), Gómez Fernández et al.
(2016) and Mora et al. (2017) inhabit an anthropically fragmented landscape and,
their studies included landscape metrics that describe this fragmentation, this pro-
cess does not seem to have much effect on the pattern of population-genetic struc-
ture in Ctenomys. All these studies covered the entire distributional range of the
species under study, and therefore they only captured the most important factors that
have somehow affected their population genetic structure. At these spatial scales (>
15 km), the most suitable habitat for the occupation of the species seems to be the
main factor limiting the population genetic structure. At lower spatial scales, how-
ever, the effects of anthropic activities begin to become more evident. Mapelli et al.
(2020) studied how different landscape elements have affected the population
genetic structure in an area of approximately 150 km2 occupied by populations
belonging to the “Corrientes” group. Using the observed genetic data, these authors
applied a novel tool and assessed the cost that different landscape elements offer to
gene flow. The habitat occupied by these species sits within a landscape matrix
dominated by water bodies and flood-prone habitats. Consequently, the altitude was
the main landscape factor that conditioned the genetic differentiation among locali-
ties, with low and flooded areas presenting very high friction values to the move-
ment of the tuco-tucos. It should be noted, however, the second cause of
differentiation between sampling locations was related to the anthropic impacts in
the area. Only the forested areas (mainly with exotic tree species such as Pinus and
Eucalyptus destined to commercial forestry) older than 20 years have strongly
impacted the population genetic structure of tuco-tucos, limiting the gene flow
between among localities. This is the first study that documents the negative effects
of anthropic habitat fragmentation on the genetic connectivity among populations

of tuco-tucos. Due to the negative effects of the anthropic activities on the tuco-tuco
populations reported in the last decades, understanding how the landscape and envi-
ronmental features have molded the genetic variability in natural populations will
be very significant for the development of proper management and conserva-
tion plans.
5.7 What About Conservation Genetics?
The inferences of dispersal patterns and population genetic structure provides

essential knowledge for the conservation of threatened species (Hanski and
Gaggiotti 2004; Manel et al. 2005). Consequently, a crucial objective of conserva-
tion genetics is not only to recognize the overall genetic structure, diversity, and
connectivity between populations, but also to understand the factors that shape such
patterns (Apodaca et al. 2012). In this form, assessing the magnitude of these threat-
ening processes is critical in conservation management and has become a priority in
studies of conservation biology (Ciofi et al. 1999).
As was suggested by Waples and Gaggiotti (2006), the management of endan-
gered species requires the identification of units that behave independently in terms
of population dynamics. One of the means to study the population dynamics of spe-
cies and delimit spatially discrete units in highly fragmented landscapes is to quan-
tify the population connectivity and gene flow based on inferences about migration
rates (Hanski and Gaggiotti 2004). The movement of individuals (and their linked
genes) will mostly depend on the characteristics of the environment they inhabit,
since a fragmented landscape offers a greater degree of heterogeneity that can
restrict the dispersion (Hanski and Gaggiotti 2004; Crooks and Sanjayan 2006).
Thus, the loss or reduction of genetic variability, as a result of habitat fragmentation,
leads to a decrease in the connectivity and a reduction in effective population sizes
(Hanski and Gaggiotti 2004), increasing the effect of genetic drift and levels of
inbreeding and decreasing the evolutionary potential of the species (Crispo
et al. 2011).
Although some species of Ctenomys have wide distributions over distinct ecore-
gions, many have narrow distributional ranges and occupy severely fragmented
environments, either by natural or anthropogenic causes (Mapelli and Kittlein 2009;
Freitas 2016). One interesting example is the recent described species C. ibicuensis
from sandy soils on the western slopes of the state of Rio Grande do Sul in southern
Brazil (Freitas et al. 2012). Populations of this species have a narrow geographic
distribution in a small area (approximately 500 km2) that has been suffering from
anthropogenic pressure from soybean, pine, and eucalyptus plantations, as well as
desertification. All of these situations have led the researchers to consider C. ibi-
cuiensis as endangered. Other similar cases are C. australis and C. porteousi, which
currently have minor ranges than 500 km2 (Mora et al. 2006; Mapelli and Kittlein
2009). C. australis occupy a restricted geographic range immerse in a highly
fragmented sand dune landscape in the Southeast of Buenos Aires province,

Argentina (Mora et al. 2006; Cutrera and Mora 2017). The habitat of this species is
being continuously fragmented due to the development of coastal towns and cities
oriented to the exploitation of beach tourism, which generates strong pressure on
their populations (Mora et al. 2010). C. porteousi is an endangered rodent with a
very narrow distributional range in central Argentina. Their suitable habitat was
estimated in only 509 km2, which represents less than 10% of its geographical range
(Mapelli and Kittlein 2009, Mapelli et al. 2012b). The habitat of this species is natu-
rally fragmented, but in recent years the degree of fragmentation has notably
increased because of the extraordinary expansion of soybean cultivation in the
region, a very similar situation currently observed in many species of Ctenomys.
The continuous exploitation of natural habitats for agriculture, livestock, for-
estry, and urbanization has a strong negative impact on the natural habitats of many
species of tuco-tucos. Anthropic fragmentation of their natural habitats placed most
Ctenomys species in a highly vulnerable situation. As the recent recategorization of
mammals species from Argentina shows, more than 50% of the total species of the
genus Ctenomys are classified in some risk category (Red list of the mammals of
Argentina; http://cma.sarem.org.ar/).
As was mentioned previously, several population genetic studies showed that
peripheral and isolated populations have low genetic diversity and low effective
population sizes (Lopes and Freitas 2012; Mapelli et al. 2012b; Gómez Fernández
et al. 2012; Lopes et al. 2013; Mora et al. 2017; Austrich et al. 2020). Furthermore,
Mora et al. (2010), Esperandio et al. (2019), and Mapelli et al. (2020) showed that
small discontinuities in the suitable habitat can establish a strong barrier to the
movement of tuco-tucos, and can increase isolation among populations. Thus, these
isolated populations would be more vulnerable to the effects of demographic and
environmental stochasticity and, consequently, more likely to experience local
extinction processes. A significant conclusion of these studies is that habitat frag-
mentation strongly conditions gene flow patterns and population structure in
Ctenomys, even at small spatial scales.
On the other hand, phylogeographic studies have showed how the history and
evolution of the landscapes have intensely conditioned the mode in which genetic
variation has been geographically partitioned (Mora et al. 2006, 2013, 2016; Mapelli
et al. 2012b; Roratto et al. 2014). Thus, these studies illustrate how the population
demography of tuco-tucos was strongly impacted by environmental changes that
have modified the extension and fragmentation of their natural habitats.
In this context, the availability of suitable habitat for tuco-tucos seems to be the
main variable to consider in order to design future conservation plans. Even for
widely distributed species, management plans must be strictly aimed to protect
those environments having suitable habitat characteristics (e.g., sandy and well-
drained soils) for the long-term permanence of tuco-tucos. Due to the high habitat
specificity and low vagility that characterize these species, the main efforts should
be aimed at securing the connectivity and suitability of the environments they
occupy. Several species of Ctenomys (e.g., C. australis, C. flamarioni, and C. minu-
tus) occur exclusively in coastal environments, associated with sand-dune systems
originated during the Late Quaternary. Other species mainly occurring on coastal
dune environments (C. talarum and C. pearsoni) from Argentina and Uruguay are
also found in some fragmentary inland populations (Mora et al. 2013; Tomasco and
Lessa 2007). The occupation of these coastal environments determines linear distri-
butions, so that even punctual anthropic impacts can easily fragment the tuco-tuco
populations.
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Part III
Organismal Biology
Chapter 6
Skull Shape and Size Diversification
in the Genus Ctenomys (Rodentia:
Ctenomyidae)
Rodrigo Fornel, Renan Maestri, Pedro Cordeiro-Estrela,

and Thales Renato Ochotorena de Freitas
6.1 Introduction
The genus Ctenomys (Blainville 1826) is the sole member of the family Ctenomyidae,
the most speciose genus of subterranean rodents. It has a large geographic distribu-
tion with species occurring in South America, from the Andes to the Atlantic coast,
and from southern Peru to Tierra del Fuego in Argentina (Reig et al. 1990). Also
known as tuco-tucos, it has been used as an example of the explosive cladogenesis
that occurred during the Pleistocene and currently comprises more than 70 recog-
nized valid species (Verzi et al. 2010a; Gardner et al. 2014; Bidau 2015; Freitas
2016; Teta and D’Elía 2020). The members of Ctenomys also have high karyotypic
diversity, with diploid numbers varying from 2n = 10 (C. steinbachi) to 2n = 70
(C. dorbignyi and C. pearsoni), which may be the highest rate of chromosomal
R. Fornel (*)
Programa de Pós-Graduação em Ecologia, Departamento de Ciências Biológicas,
Universidade Regional Integrada do Alto Uruguai e das Missões – Campus de Erechim,
Erechim, RS, Brazil
R. Maestri
Departamento de Ecologia, Universidade Federal do Rio Grande do Sul,
Porto Alegre, RS, Brazil
e-mail: renan.maestri@ufrgs.br
P. Cordeiro-Estrela
Departamento de Sistemática e Ecologia, Centro de Ciências Exatas e da Natureza – Campus
I, Universidade Federal da Paraíba, Jardim Universitário s/n, João Pessoa, PB, Brazil
e-mail: estrela@dse.ufpb.br
T. R. O. de Freitas
Department of Genetics, Federal University of Rio Grande do Sul, Porto Alegre,
Rio Grande do Su, Brazil
e-mail: thales.freitas@ufrgs.br

https://doi.org/10.1007/978-3-030-61679-3_6
114 R. Fornel et al.
evolution among mammals (Reig and Kiblisky 1969; Reig et al. 1990; Bidau et al.
1996; Cook and Lessa 1998; Cook and Salazar-Bravo 2004). The high speciation
rate and rapid evolution of morphological and cytogenetic characteristics in
Ctenomys have been explained by their patchy distribution, limited vagility, territo-
riality, small effective numbers, and high karyotypic polymorphism, which are also
probably associated with their subterranean lifestyle and environmental changes at
the end of the Pliocene (Reig et al. 1990).
The species also vary greatly in size (from the small C. pundti with 100 g of body
weight and 220 mm of total length to the large C. conoveri with 1100 g and 430 mm),
color (from pale yellow-grayish to black), and in the angle of incisor procumbency
(Reig et al. 1990; Vassallo 1998; Mora et al. 2003). Many species of Ctenomys have
been characterized as scratch (claws) and chisel-tooth (incisors) diggers to build
their tunnel systems; they are considered to be an interesting model to investigate
functional morphological adaptations to the subterranean niche (Vassallo 1998;
Lessa et al. 2008; Steiner-Souza et al. 2010; Morgan and Álvarez 2013). They pos-
sess reduced tails, strongly built anterior limbs, and comb-like hairy fringes along
the manus, from which they gained the name Cteno (= comb) mys (= mice). They
also show reduced ear pavilion, eyes and ears located on top of the head, and labial
located posteriorly to the incisors, allowing chisel-tooth digging without ingesting
soil. All of these characteristics show a high degree of fossoriality where the skull
integrates different selective pressure to this habit.
Among several morphological traits that play important roles in a vertebrate, the
skull is a very important anatomical structure because it houses the brain and sense
organs, and it has regions of origin and insertion of muscles (Hanken and Thorogood
1993). Moreover, for most mammalian taxa, well-preserved crania and mandibles
can be found in scientific collections. Many studies have explored the morphologi-
cal skull variations in Ctenomys, at both intraspecific and interspecific levels (i.e.,
Vassallo 1998; Marinho and Freitas 2000; Mora et al. 2003; Freitas 2005; D’Anatro
and Lessa 2006; Verzi and Olivares 2006; Fernandes et al. 2009; Fornel et al. 2010;
Fernandes et al. 2012; Borges et al. 2017; Fornel et al. 2018; Kubiak et al. 2018). At
the intraspecific level, a large amount of variation has been detected between allo-
patric and parapatric populations and among chromosomal groups (Fernandes et al.
2009; Fornel et al. 2010; Kubiak et al. 2018). Interestingly, this amount is often of
the same order of magnitude as interspecific differences. Large-scale interspecific
studies are few. Among them, in a study including 23 species of Ctenomys, Mora
et al. (2003) showed significant morphological differences in skull size and in the
angle of incisor procumbency, which is very likely an adaptation to digging. In the
same fashion, a study of 24 species by Borges et al. (2017) found differences in bite
force among Ctenomys resulting from different excavation strategies. However, a
broad view of the morphological variations in the genus is still lacking (Lessa
et al. 2008).
In recent decades, studies based on chromosomal polymorphism, biogeography,
morphological similarity, and molecular data have contributed to the formulation of
groupings of species within the genus Ctenomys. Six species groups have been pro-
posed: mendocinus, Boliviano-Matogrossense, Boliviano-Paraguaio, Chaco,
6 Skull Shape and Size Diversification in the Genus Ctenomys… 115
opimus-fulvus, and pundti-talarum (Massarini et al. 1991; Freitas 1994; Lessa and
Cook 1998; Contreras and Bidau 1999; D’Elía et al. 1999; Mascheretti et al. 2000;
Slamovits et al. 2001; Castillo et al. 2005; Tiranti et al. 2005). Nonetheless, Parada
et al. (2011), based on molecular phylogenetic analysis, confirmed the six species
groups as clades suggested two additional clades; thus, they renamed the eight
groups with the oldest species in each group. Henceforth, we use these groups: (1)
boliviensis, (2) frater, (3) opimus, (4) tucumanus, (5) torquatus, (6) talarum, (7)
mendocinus, and (8) magellanicus (Parada et al. 2011). Freitas et al. (2012) described
a new species C. ibicuiensis; based on cytochrome-b divergence, they added it and
C. dorbignyi to the torquatus group. The geographic distribution of each Ctenomys
clade and type localities of the species may be viewed in Fig. 6.1 based on data from
Woods and Kilpatrick (2005), Freitas et al. (2012) and Gardner et al. (2014).
Fig. 6.1 Map of type localities (circles) of 65 Ctenomys species based on data from Woods and
Kilpatrick (2005), Freitas et al. (2012), and Gardner et al. (2014). The meaning of abbreviations
are scientific names given in the proper figure. Dashed lines and numbers indicate the eight main
Ctenomys clades proposed by Parada et al. (2011)
Given the rapid evolution in the explosive cladogenesis of tuco-tucos in the sub-
terranean niche (Reig et al. 1990; Castillo et al. 2005) and the lack of information
about its large-scale morphological diversification, we conducted the most compre-
hensive survey of tuco-tucos morphological variation by including 49 species of the
genus. We explored skull morphological evolution in Ctenomys and the morpho-
logical relationship among main clades to understand how morphological diversifi-
cation occurs within subterranean radiation. The aims of this study were as follows:
(1) to understand the morphological relationships among Ctenomys species and to
test the hypothesis that the eight groups proposed for Ctenomys (Parada et al. 2011)
based in a neutral molecular marker are supported by a quantitative analysis of the
morphology of the cranium and the mandible; and (2) to test if the changes in skull
shape are related to changes in size (i.e., allometry) and geographical distribution.
6.2 Sample and Data Collections
Skulls of 1359 adult specimens of 49 living Ctenomys species were examined

(Table 6.1), along with a sample of 820 mandibles of Ctenomys species. We reduced
the number of mandibles due to damaged structures that precluded landmark digita-
tion. The skulls and mandibles analyzed are housed in the following museums and
scientific collections: 1) Departamento de Genética, Universidade Federal do Rio
Grande do Sul, Porto Alegre, Brazil (UFRGS); 2) Museo Nacional de Historia
Natural y Antropología, Montevideo, Uruguay (MUNHINA); 3) Museo Argentino
de Ciencias Naturales “Bernardino Rivadavia”, Buenos Aires, Argentina (MACN);
4) Museo de La Plata, La Plata, Argentina (MLP); 5) Museo Municipal de Ciencias
Naturales “Lorenzo Scaglia”, Mar del Plata, Argentina (MMP); 6) Museum of
Vertebrate Zoology, University of California, Berkeley, USA (MVZ); 7) American
Museum of Natural History, New York, USA (AMNH); and 8) Field Museum of
Natural History, Chicago, USA (FMNH).
6.3 Geometric Morphometric Analysis
Each cranium was photographed in the dorsal, ventral, and left lateral views of the
skull and on the left lateral side of the mandible using a digital camera with 3.1
megapixels (2048 × 1536) of resolution with a macro function at the same focal
distance (15 cm). The software tpsUtil 1.40 (Rohlf 2008) was used to manage skull
image files. We used two-dimensional landmarks proposed by Fornel et al. (2010):
29 landmarks for the dorsal, 30 for the ventral, and 21 for the left lateral views of
the cranium, and 13 landmarks for the left lateral view of the mandible (Fig. 6.2; SI
1). Anatomical landmarks were digitized for each specimen using tpsDig version
Table 6.1 Skull sample sizes of 49 species examined from the genus Ctenomys
Species Clade N Species Clade N
C. argentinus Tucumanus 3 C. magellanicus Magellanicus 23
C. australis Mendocinus 35 C. maulinus – 34
C. azarae Mendocinus 32 C. mendocinus Mendocinus 24
C. bicolor – 1 C. minutus Torquatus 197
C. boliviensis Boliviensis 59 C. nattereri Boliviensis 1
C. bonettoi – 2 C. occultus Tucumanus 6
C. budini – 1 C. opimus Opimus 80
C. coludo – 2 C. pearsoni Torquatus 77
C. conoveri Frater 4 C. perrensi Torquatus 9
C. Coulburni Magellanicus 30 C. peruanus – 14
C. coyhaiquensis Magellanicus 1 C. porteousi Mendocinus 30
C. dorbignyi Torquatus 13 C. pundti Talarum 5
C. dorsalis – 6 C. rionegrensis Mendocinus 2
C. flamarioni Mendocinus 34 C. roigi Torquatus 7
C. fochi – 3 C. scagliai Opimus 1
C. fodax Magellanicus 1 C. sericeus Magellanicus 2
C. frater Frater 11 C. sociabilis – 15
C. fulvus Opimus 26 C. steinbachi Boliviensis 12
C. haigi Magellanicus 74 C. sylvanus – 6
C. ibicuiensis Torquatus 16 C. talarum Talarum 76
C. knighti – 2 C. torquatus Torquatus 222
C. lami Torquatus 89 C. tuconax – 17
C. latro Tucumanus 8 C. tucumanus Tucumanus 23
C. leucodon – 8 C. yolandae – 2
C. lewisi Frater 13
Total 1359
1.40 software (Rohlf 2004), and all landmarks were taken by the same person (R.F.).
Coordinates were superimposed using a generalized Procrustes analysis (GPA)
algorithm (Dryden and Mardia 1998). GPA removes differences unrelated to the
shape: scale, position, and orientation (Rohlf and Slice 1990; Rohlf and Marcus
1993; Bookstein 1996a, b; Adams et al. 2004; Adams et al. 2013). We symmetrized
both sides (left and right) of the landmarks in the dorsal and ventral views of the
skull to avoid redundancy, and only the symmetric part of the variation was ana-
lyzed (Kent and Mardia 2001; Klingenberg et al. 2002; Evin et al. 2008). The size
of each skull was estimated using its centroid size, the square root of the sum of the
squares of the distances of each landmark from the centroid (mean of all coordi-
nates) of the configuration (Bookstein 1991).
Fig. 6.2 Morphological landmark locations on skull of Ctenomys. a) dorsal, b) ventral, and c)
lateral views of the cranium, and d) lateral view of the mandible (Fornel et al. 2010)
6.4 Shape and Size Analysis
For skull size, we summed the skull centroid size (logarithm transformed) for each
view of the skull (dorsal, ventral, and lateral) to generate one value for cranium size.
Centroid size variation among species was visualized through boxplots. The size
difference was tested between sexes, clades, and species with a three-way ANOVA
(nested: sex*clades: species). For multiple comparisons, we used Tukey’s test.
Shape differences between sexes, among groups, and among species, as well as
their interactions, were tested through multivariate analysis of variance (MANOVA).
The Bonferroni correction for multiple comparisons was applied when needed to
adjust the significance level (Wright 1992). Due to the small sample size of man-
dibles, we used them solely in sexual dimorphism and group comparisons, not for
species analysis.
For skull shape, an exploratory analysis was carried with principal components
analysis (PCA) using the variance-covariance matrix of generalized least-squares
superimposition residuals. Principal components (PCs) of the covariance matrix of
superimposition residuals were used as new shape variables to reduce the dimen-
sionality of the data set as well as to work on independent variables (Baylac and
Friess 2005; Evin et al. 2008). To choose the number of PCs to be included in the
linear discriminant analysis (LDA), we computed correct classification percentages
with each combination of PCs (Baylac and Friess 2005). We selected the subset of
PCs giving the highest overall correct classification percentage. We used a leave-
one-out cross-validation procedure that allowed an unbiased estimate of classifica-
tion percentages (Ripley 1996; Baylac and Friess 2005). Cross-validation was used
to evaluate the performance of classification by LDA. LDA explored differences in
shapes between species. We performed an LDA on PCs in combined with and with-
out the sum of the logarithms of dorsal, ventral, and lateral centroid sizes (Cordeiro-
Estrela et al. 2006).
Mahalanobis distances were used to compute trees with a neighbor-joining algo-
rithm to visualize the morphological relationships among groups and among spe-
cies, with and without size information. The visualization of shape differences for
the skull views were obtained through multivariate regression of shape variables on
discriminant axes and the consensus configuration.
6.5 Geographical Structure of Skull Shape
We tested the association between skull morphology and geographical distribution

of Ctenomys species following Fornel et al. (2010). We used a morphological dis-
tance matrix based on Mahalanobis distances, calculated from skull morphometric
data, and a geographic distance matrix based on geodesic distances of each species
calculated with the software Geographic Distance Matrix Generator, version 1.2.3
(Ersts 2009). The correlation between the two distance matrices was tested with an
RV coefficient randomization test (Heo and Gabriel 1997), using 10,000 random
permutations as implemented in the ade4 library. We also tested for the degree of
association of cranial shape with spatial distribution with a two-block partial least-
squares analysis (2B-PLS) between shape data and geographical coordinates of cen-
troids of specimens for each species as implemented in the geomorph library.
Finally, we tested for global spatial autocorrelation of centroid size and mean shape
of species at different spatial scales with a Moran’s I correlogram for size and by the
centered Mantel static for shape (Bjørnstad et al. 1999). The number of distance
classes was chosen according to Legendre and Legendre (1998).
For all statistical analyses and to generate the graphics, we used the R language
and environment for statistical computing version 2.14.1 for Windows (R
Development Core Team, http://www.R-project.org), as well as the following librar-
ies: MASS (Venables and Ripley 2002), ape version 1.8–2 (Paradis et al. 2004), stats
(R Development Core Team 2009), ade4 (Dray and Dufour 2007), and geomorph
(Adams and Otarola-Castillo 2013). Geometric morphometric procedures were car-
ried out with the Rmorph package: a geometric and multivariate morphometrics
library for R (Baylac 2008).
6.6 Variation in Cranium and Mandible Size
The centroid size of the cranium and mandible was significantly different between
sexes (generally males are larger than females), among clades, and among species
(Table 6.2). The interaction term between species and sex indicates that sexual
dimorphism varies significantly among species of the genus Ctenomys (Table 6.2).
The variance of the three factors tested, represented by mean squares value, shows
that most of the variance in skull and mandible size is found among the eight clades,
then among species and finally between sexes (Table 6.2).
Table 6.2 ANOVA of logarithms of centroid size (log CS) of the craniums (dorsal, ventral, and
lateral views pooled = three views) and mandibles of Ctenomys between sexes and among clades,
species, and the sex-versus-species interaction (n.s., not significant)
Sincranium Mean of squares d.f. F P
SKULL log CS 3 views
Sex 5.68 1 190.304 < 0.001
Clade 60.54 7 289.742 < 0.001
Species 32.09 25 42.999 < 0.001
Sex × species 0.73 20 1.223 n.s.
MANDIBLE log CS
Sex 0.55 1 110.38 < 0.001
Clade 7.29 7 206.51 < 0.001
Species 4.34 17 50.63 < 0.001
Sex × species 0.11 17 1.34 < 0.001
Among species, we found a large variation in centroid size, with five significant
different groupings for Tukey’s test (P < 0.05) (Fig. 6.3a). A partial superimposition
only among few species by the size distribution is unimodal with extreme values for
C. conoveri with a skull about 2.4 times larger than the smallest species, C. latro
(Fig. 6.3a).
Among the eight Ctenomys clades, we also found five significant different group-
ings for skull centroid size (Fig. 6.3b). The bolivienesis and frater clades have the
larger centroid size, and the magellanicus clade is the smaller one. There are large
variations in skull centroid size within some clades, such as in frater, mendocinus,
Fig. 6.3 Skull size variation in genus Ctenomys, with boxplots of the sum of dorsal, ventral, and
lateral log centroid sizes. (a) Among species in increasing order, with sample size larger than two
skulls for each species. (b) Skull size variation within and among the eight Ctenomys clades.
Different letters above boxes represent significant differences among groups (Tukey’s test). NS,
nonsignificant pairwise comparison at the 5% level
and magellanicus. We found a smaller variation among species within boliviensis,

opimus, talarum, torquatus, and tucumanus clades (Fig. 6.3b).
6.7 Variation in Cranium and Mandible Shape
The MANOVA showed a highly significant sexual dimorphism in shape for the
dorsal, ventral, and lateral views of the cranium, as well as for the mandible
(Table 6.3). The interaction between sex and clade effects was also significant for
shape for all views (Table 6.3). The interaction between sex and species was signifi-
cant just for dorsal and ventral views of the skull (Table 6.3).
For clades, Table 6.3 shows the significant difference in skull and mandible
shape. For MANOVA comparisons between different effects, the highest F value
was found for groups. Using the F values as a differentiation index, there is three
times more differentiation among clades than among species or between sexes,
except for the mandible (1.2 times). The pairwise MANOVA among the eight
Table 6.3 MANOVA of cranium and mandible shapes of Ctenomys between sexes and among
clades, and species (n.s., not significant)
Skull (Dorsal) Wilks’λ d.f. F P
Sex 0.678 1 26.276 < 0.001
Clade 0.001 7 82.292 < 0.001
Species 0.007 25 12.926 < 0.001
Sex × clade 0.762 7 2.2 < 0.001
Sex × species 0.641 20 1.264 < 0.001
Skull (ventral) Wilks’ λ d.f. F P
Sex 0.651 1 29.81 < 0.001
Clade 0.002 7 78.854 < 0.001
Species 0.01 25 11.854 < 0.001
Sex × clade 0.816 7 1.64 < 0.001
Sex × species 0.67 20 1.132 < 0.05
Skull (lateral) Wilks’ λ d.f. F P
Sex 0.734 1 20.058 < 0.001
Clade 0.003 7 71.524 < 0.001
Species 0.009 25 12.01 < 0.001
Sex × clade 0.842 7 1.38 < 0.01
Sex × species 0.687 20 1.059 n.s.
Mandible Wilks’ λ d.f. F P
Sex 0.641 1 20.65 < 0.001
Clade 0.028 7 24.911 < 0.001
Species 0.033 17 8.5 < 0.001
Sex × clade 0.706 7 1.881 < 0.001
Sex × species 0.601 17 1.13 n.s.
Ctenomys clades using three views of the skull pooled shows that all comparisons
were significant (Table 6.4), as well as for the mandible (Table 6.5). Moreover, the
percentage of correct classification using LDA shows higher values for the tor-
quatus group for three views of the skull (100% pooled and separated) and smaller
values for the tucumanus group for the mandible (56%) (Table 6.6). The morpho-
logical structure with the higher percentage of correct classification on average was
the dorsal view of the skull (91.07%), whereas the mandible had a smaller percent-
age (73.86%) (Table 6.6).
The PCA did not show a clear ordination in the skull or mandible shape scores
for different axes. However, in the discriminant analysis for the eight Ctenomys
groups, we found an ordination in the two first Canonical Variate (CV) axes. For the
dorsal view of the skull, CV1 shows an ordination from east to west and CV2 shows
a north-to-south ordination, with boliviensis having positive scores and magellani-
cus having negative ones (Fig. 6.4a). These two axes explain 57.9% of the variation.
In CV1, the negative scores are represented by a proportionally enlarged auditory
meatus and the positive scores by a proportionally elongated rostrum (Fig. 6.4c).
The negative scores on CV2 represent a proportionally shorter and narrow rostrum
and the positive scores represent a proportionally enlarged zygomatic arch
(Fig. 6.4b). For the ventral view of the skull, CV1 showed a small ordination west
to east, and CV2 showed a small ordination north to south (Fig. 6.4e). These two
axes explain 54.5% of the variation. In CV1, the positive scores are represented by
a proportionally enlarged tympanic bullae (Fig. 6.4g). CV2 showed negative scores
with a narrower skull compared with positive scores (Fig. 6.4f). For the lateral view
of the skull, the CV1 showed east-to-west ordination, and CV2 showed north-to-
south ordination (Fig. 6.5a). These two axes explain 59.4% of the variation. In CV1,
the negative scores are represented by a proportionally flat skull and enlarged tym-
panic bullae, whereas the positive scores show a deep rostrum (Fig. 6.5c). CV2
showed negative scores with a proportionally enlarged tympanic bulla, whereas the
positive scores showed a deep and elongated rostrum and enlarged zygomatic arch
(Fig. 6.5b). For the mandible lateral view, the two first CVs explain 50.7% of the
variation, and there is no clear ordination in these data (Fig. 6.5e). CV1 showed
negative scores for proportionally shorter coronoid processes and elongated
Table 6.4 Pairwise MANOVA of cranium shape. Summary F values and significance among
eight Ctenomys clades (results for pooled dorsal, ventral, and lateral datasets)
boliviensis frater opimus tucumanus torquats talarum mendocinus
Frater 28.71* –
Opimus 124.39* 45.34* –
Tucumanus 77.36* 24.09* 38.09* –
Torquatus 277.92* 105.82* 195.99* 84.63* –
Talarum 113.33* 30.39* 25.79* 23.01* 68.45* –
Mendocinus 149.33* 73.51* 35.79* 46.93* 214.37* 13.77* –
Magellanicus 247.59* 87.81* 77.61* 5.46* 212.92* 28.98* 33.87*
P < 0.001; after Bonferroni correction
*
Table 6.5 Pairwise MANOVA of mandible shape. Summary of F values and significance among
eight Ctenomys clades
boliviensis frater opimus tucumanus torquats talarum mendocinus
Frater 12.13* –
Opimus 13.88* 18.04* –
Tucumanus 9.22* 9.13* 19.43* –
Torquatus 18.74* 26.02* 57.36* 10.26* –
Talarum 18.25* 12.54* 26.31* 12.08* 29.63* –
Mendocinus 15.06* 11.18* 19.35* 17.23* 61.42* 8.87* –
Magellanicus 12.79* 9.77* 12.11* 15.31* 33.81* 16.24* 19.29*
P < 0.001; after Bonferroni correction
*
Table 6.6 Percentage of correct classification from linear discriminant analysis (LDA) of shape
of dorsal, ventral, and lateral views of the cranium of the three pooled datasets and for the lateral
view of the mandible for Ctenomys clades
Clade SkullDorsall SkulllVentral Skulllateral Skull3 views Mandible
1- boliviensis 94.44 97.22 100 97.22 66.66
2- frater 92.86 92.85 82.14 89.28 69.23
3- opimus 86.92 85.98 90.65 87.85 83.33
4- tucumanus 95 90 95 92.5 56
5- torquatus 100 100 100 100 93.29
6- talarum 74.63 77.61 77.61 76.11 70.21
7- mendocinus 87.84 83.11 83.78 86.48 82.05
8- magellanicus 96.87 96.09 85.15 96.09 70.12
Average 91.07 90.35 89.29 90.69 73.86
condylar processes relative to the positive scores (Fig. 6.5g). For CV2, the main
difference is a proportionally deep mandibular body negative score (Fig. 6.5f). The
phenogram for Mahalanobis distances for clades shows a geographic structure for
the skull (Figs. 6.4d, h, and 6.5d) but not for the mandible (Fig. 6.5h). For the skull,
there is a clear pattern with frater and boliviensis clades in one side of the tree (north
of the geographic distribution) and mendocinus and magellanicus in the other side
of the tree (south of the geographic distribution) (Figs. 6.4d, h and 6.5d).
As previously mentioned, we found significant differences in shape among all
clades for all morphological structures (Tables 6.4 and 6.5). Figure 6.6 presents the
consensus shape of each clade for each view of the cranium and also for the lateral
view of the mandible. To facilitate the interpretation, the clades are arranged from
the north (boliviensis) to the south (magellanicus) of the distribution, and the arrows
indicate the main shape differences. Basically, for skull shape, the northern shapes
are more robust, with larger rostrum and larger zygomatic arches, whereas the
southern shapes are more gracile, with delicate rostrum and jugal with thinner bones
than northern groups (Fig. 6.6). For the mandible, despite significant differences
among all clades, visualizing these differences is less obvious than for the skull
shape (Fig. 6.6).
Fig. 6.4 Discriminant analysis for eight clades of Ctenomys for dorsal and ventral views of the
cranium. (a) Scatter plot of canonical variate axes (CV1 and CV2) for dorsal view of the skull. (b)
Skull shape variation for second canonical variate axis (CV2). (c) Skull shape variation for first
canonical variate axis (CV1). (d) Phenogram of Mahalanobis distance for dorsal view of the skull.
(e) Scatter plot of canonical variate axes (CV1 and CV2) for ventral view of the skull with cranium.
(f) Skull shape variation for second canonical axis (CV2). (g) Skull shape variation for first canoni-
cal axis (CV1). (h) Phenogram of Mahalanobis distance for the ventral view of the skull. Symbols
represent group mean and ellipses represent 95% confidence interval. The percentage of variance
explained by each axis is given in parenthesis. In grids of shape variation for each canonical axis,
solid lines indicate positive scores and dashed lines indicate negative scores
Fig. 6.5 Discriminant analysis for eight clades of Ctenomys for lateral view of the cranium and
lateral view of the mandible. (a) Scatter plot of the canonical variate axes (CV1 and CV2) for lat-
eral view of the skull. (b) Skull shape variation for second canonical axis (CV2). (c) Skull shape
variation for first canonical axis (CV1). (d) Phenogram of Mahalanobis distance for lateral view of
the skull. (e) Scatter plot of the canonical variate axes (CV1 and CV2) for the mandible. (f)
Mandible shape variation for second canonical axis (CV2). (g) Mandible shape variation for first
canonical axis (CV1). (h) Phenogram of Mahalanobis distance for the lateral view of the mandible.
Symbols represent mean plot and ellipses represent 95% confidence interval. The percentage of
variance explained by each axis is given in parenthesis. In grids of shape variation for each canoni-
cal axis, solid lines indicate positive scores and dashed lines the negative ones
Fig. 6.6 Consensus configuration of landmarks (mean shape) for the eight main groups of
Ctenomys for dorsal, ventral, and lateral views of the cranium, and lateral view of the mandible.
Consensus configuration for all groups are given in dashed lines. The arrows indicate the main
differences among groups. See the text for more detailed description
Species-level comparisons indicated highly significant differences among

Ctenomys species for skull and mandible shapes (Table 6.3). The percentage of cor-
rect classifications by the LDA function for each species with skull sample size
equal to or superior to three individuals (total of 37 species) is given in Table 6.7.
The majority of species (33 of 37 species) were correctly classified, with percent-
ages higher than 75% for shape for three views together. Nineteen species were
Table 6.7 Percentage of correct classification from discriminant analysis of shape for dorsal,
ventral, and lateral views of the cranium, individually, combined, and with size plus shape (form)
for Ctenomys species. All species with sample size equal or superior to three individuals
Species Dorsal Ventral Lateral 3-views shape 3-views form
C. argentinus 100 66.66 66.66 66.66 100
C. australis 100 94.28 91.42 100 100
C. azarae 84.37 93.75 93.75 90.62 96.87
C. boliviensis 96.61 98.3 100 96.61 96.61
C. conoveri 100 100 100 100 100
C. Coulburni 93.33 86.66 80 93.33 93.33
C. dorbignyi 100 76.92 100 100 92.3
C. dorsalis 100 100 100 100 100
C. flamarioni 100 100 100 100 100
C. fochi 66.66 100 33.33 66.66 66.66
C. frater 90.9 90.9 81.81 90.9 90.9
C. fulvus 79.92 38.46 76.92 80.76 80.76
C. haigi 90.54 87.83 83.78 90.54 95.94
C. ibicuiensis 96.66 86.95 95.65 95.65 97.66
C. lami 86.51 82.02 79.77 85.39 84.26
C. latro 100 100 75 100 100
C. leucodon 100 100 100 100 100
C. lewisi 100 92.3 100 100 100
C. magellanicus 91.3 100 91.3 91.3 100
C. maulinus 91.17 94.12 91.17 91.17 91.17
C. mendocinus 70.83 45.83 45.83 70.83 66.66
C. minutus 91.37 90.35 91.87 92.89 94.41
C. occultus 100 100 66.66 100 100
C. opimus 93.75 95 97.5 95 97.5
C. pearsoni 84.41 93.5 93.5 84.41 87.01
C. perrensi 88.88 100 88.88 88.88 88.88
C. peruanus 100 100 100 100 100
C. porteousi 60 76.66 66.66 70 80
C. Pundit 80 40 80 80 80
C. roigi 100 57.14 85.71 100 100
C. sociabilis 100 100 100 100 100
C. steinbachi 100 100 100 100 100
C. sylvanus 100 83.33 100 100 100
C. talarum 86.84 82.89 82.89 86.84 86.84
C. torquatus 96.84 94.59 94.59 97.29 96.84
C. tuconax 94.12 100 88.23 100 100
C. tucumanus 91.3 78.26 91.3 91.3 91.3
Average 91.85 87.21 86.62 91.7 93.28
correctly classified in at least one view of the skull with 100%. Of these 19 species,
7 reached 100% of classification for all views of the skull. The worst classification
was for C. Fochi, with 33.33% for the lateral view of the skull. The size contribution
to form (shape plus size) increases correct classification on average, with 1.58% in
reclassification (Table 6.7).
The skull shape phenogram for 49 Ctenomys species shows two phonetic groups
highly congruent with the phylogenetic topology (the torquatus and boliviensis
groups) and one group partially congruent (the tucumanus group; Fig. 6.7a). For the
tree using shape plus size (form), the groups of torquatus, boliviensis, and tucuma-
nus were congruent with the phylogenetic hypothesis; opimus, mendocinus, and
magellanicus groups were partially congruent (Fig. 6.7b). In both trees, the largest
branches were found for C. conoveri, which appear closer to C. peruanus. Moreover,
in the two phenograms, C. sociabilis is closer to the mendocinus group species
(C. australis and C. flamarioni) (Fig. 6.7).
6.8 Geographical Structure of Cranial Shape
For the association between variations in skull morphology and geographic dis-
tances among species populations, the RV test showed significant correlation for the
dorsal (r = 0.45, P < 0.001), ventral (r = 0.37, P < 0.001), and lateral views of the
skull (r = 0.25, P < 0.05). Two block partial least squares also showed that shape
covaries significantly with latitude and longitude for dorsal (r = 0.82, P = 0.001),
ventral (r = 0.67, P = 0.002), and lateral views of the skull (r = 0.695, P < 0.001).
Autocorrelograms showed no spatial autocorrelation for size. Shape showed signifi-
cant spatial autocorrelation at different spatial scales, especially at a 245-km inter-
val (dorsal P = 0.012 at a 245-km interval, P = 0.02 at a 1073-km interval; ventral
P = 0.014 at a 245-km interval, P = 0.009 at a 1528-km interval and P = 0.02 at a
3835-km interval; lateral P = 0.037 at a 245-km interval).
6.9 Morphological Variation in Ctenomys: An Overview
The morphological variation of skull shape in Ctenomys is spatially structured,

which suggests homogeneity of the subterranean niche across geographical space.
Especially at a 245-km scale, the strong spatially structured populations (migration/
mating patterns) are mainly responsible for the cranial evolution in Ctenomys. The
mechanisms of geographic isolation coupled with the spatial gradient of variation in
the subterranean niche are likely to be the more important selective agents in shap-
ing the ctenomyid cranium.
We found a high variation in size within Ctenomys groups, except the torquatus
group (Fig. 6.3a and b) as observed in Fornel et al. (2018). Apparently, size shows a
similar range of variation within groups but is constrained to intermediate values in
Fig. 6.7 Neighbor-joining trees of Mahalanobis distances among 49 Ctenomys species for dorsal,
ventral, and lateral views of the skull pooled. (a) Phenogram for Ctenomys skull shape. (b)
Phenogram for skull form (shape plus size). The solid keys indicate monophyletic groups congru-
ent with phylogenetic hypothesis, and dashed keys indicate partially congruent groups with phylo-
genetic hypothesis
the torquatus group. C. conoveri is the single species with extreme values that fall
outside the range of other species. This variation within clades in skull size could
result from adaptive radiation with size like a line of least evolutionary resistance
(Marroig and Cheverud 2005). Medina et al. (2007) found that body size follows the
converse Bergmann’s rule at the interspecific level in Ctenomys. These authors sug-
gested that this pattern could be related to seasonality, ambient energy, primary
productivity, or predation pressure. Our data show significant differences in skull
size; apparently, this variation does not follow a clear latitudinal pattern. In general,
cranium size by clades was ordered from north to south seem to decrease in size
(Fig. 6.3b).
The Mahalanobis trees from the different views, except of the mandible, show
that morphology follows a geographic structure. The boliviensis and frater groups
at one extreme and the magellanicus group are on a north-south gradient (Figs. 6.4
and 6.5). These results are congruent with an isolation-by-distance pattern proposed
for Ctenomys at the intraspecific level with molecular data (Mora et al. 2006, 2007).
Moreover, our results show a large difference between northern and southern and
between eastern and western species of Ctenomys. We found a gradient from north
to south with more robust species in the north and more delicate species in the
south—a skull morphological cline for shape but not completely for size. This pat-
tern of isolation-by-distance in skull morphometric data was also was observed at
the intraspecific level in C. minutus (Fornel et al. 2010). The boliviensis and frater
groups in the north of Ctenomys distribution have a skull with strong and enlarged
zygomatic arches with long processes and a deep and enlarged rostrum (the “robust”
shape). In contrast, the magellanicus group have a more delicate skull with a thin
zygomatic arch and a narrow rostrum (the “gracile” shape) (Fig. 6.6).
The phenogram for cranium shape among Ctenomys groups (Figs. 6.4 and 6.5)
showed a more geographic signal than mandible shape. The skull is more complex
than mandible in genetic and anatomical complexity, as well as a number of devel-
opmental modules (Atchley and Hall 1991; Caumul and Polly 2005). Therefore, we
suggest that mandible shape in Ctenomys is a more functionally and developmen-
tally constrained structure than the skull.
6.10 ongruence Between Morphological

C
and Molecular Data
One question of this study was whether morphological phenograms are congruent
with phylogenetic trees from molecular data. The answer is partly that the skull
morphology trees are not completely congruent with molecular phylogeny
(Fig. 6.7a, b). The only completely congruent groups were torquatus, boliviensis,
and tucumanus. Moreover, our results showed that C. dorbignyi is associated with
the torquatus group, as also found by Mascheretti et al. (2000) and Freitas et al.
(2012), who both used molecular data. Therefore, we propose the inclusion of
C. dorbignyi in the torquatus group as well as C. ibicuiensis, as suggested by Freitas

et al. (2012), based on molecular and skull morphological similarities.
The tucumanus clade (C. argentinus, C. latro, C. occultus, and C. tucumanus) is
partially congruent for shape data (Fig. 6.7a) and congruent for form data (Fig. 6.7b).
The magellanicus group is only partially congruent because C. magellanicus is not
associated with this group’s other species. We propose that C. coulburni be inte-
grated into the magellanicus group because the skull form and geographical distri-
bution are very similar to the species from this clade. The opimus and talarum
groups are not congruent. The mendocinus clade is not congruent, but there is a
close association between C. flamarioni and C. australis (Fig. 6.7b). Moreover, the
strong relationship between C. flamarioni and C. australis was already described by
Massarini and Freitas (2005).
The most singular Ctenomys skull belongs to C. conoveri. Besides the strong
morphological difference observed in the phenograms, C. conoveri is associated
with C. peruanus (Fig. 6.7a and b). This high morphological divergence in
C. conoveri was noted by Osgood (1946), who proposed a new subgenus Chacomys
to accommodate only C. conoveri species (Lessa and Cook 1998). The subgenus
Haptomys was proposed by Thomas (1916) to comprise only C. leucodon, which
differs from other tuco-tucos because the incisors are extremely proodont. Our
results for skull morphology do not support the Haptomys group because, in the
phenograms, C. leucodon was associated with other groups.
We found a significant congruence at the interspecific level and support for some
groups. Cardini (2003) enumerated several factors that might explain the lack of
correspondence between phenotypic and phylogenetic trees, such as sampling error,
retention of plesiomorphic traits, genetic drift, morphological convergence, and
misrepresentation of the phylogenetic analysis. This last factor occurs in Ctenomys,
which lacks a complete gene tree representing all species of the genus (Lessa and
Cook 1998; Mascheretti et al. 2000).
6.11 Size Versus Shape
We found a weak but significant correlation between the size and shape of the skull.
The percentages of correct classification for species increased only 1.58% on aver-
age when size was added. The skull size is highly variable within groups (the tor-
quatus group is an exception). Thus, the relationship between size and shape in the
genus Ctenomys seems considerable in the phylogenetic context, and the interspe-
cific allometries are not negligible. The robust shape in the north and the gracile
shape in the southern Ctenomys species are not completely related to skull size. Our
results support the observations made by Verzi et al. (2010b) for postnatal ontoge-
netic series for five Ctenomys species, which suggested that interspecific skull shape
differences found in Ctenomys are not associated with differences in size alone. Our
results suggest that skull size shows a large variation within groups while skull
shape shows a large variation among Ctenomys groups. Therefore, the skull shape
is more similar within groups, but the size is more variable within groups.
6.12 Morphological Diversification
In the genus Ctenomys, Cook and Lessa (1998) and Parada et al. (2011) found an
increase in the diversification rate at the base of Ctenomys clade. These authors sug-
gested an increase in diversification of approximately ~3 mya. However, Rowe et al.
(2011), using multilocus molecular phylogenetic, suggested that the genus Rattus,
despite rapid diversification, displayed little ecomorphological divergence among
species and did not fit the model of adaptive radiation.
Nevertheless, we found a remarkable variation in skull form in genus Ctenomys,
and our results support a great morphological diversification in shape and size.
However, the cause of this diversification remains unclear (Parada et al. 2011).
Despite this, our data show that Ctenomys varies in body size, coat color, karyotype,
sperm morphology, angle of incisor procumbency, and skull form.
The mandible shape was less variable than cranium. This characteristic is consis-
tent with conserved morphology by biomechanical constraints. For skull shape, we
have low support for an ecological speciation (Gavrilets and Losos 2009). Despite
Ctenomys occupying large latitudinal and longitudinal gradients and several envi-
ronments, their life history in burrow systems buffers above-ground conditions
(Medina et al. 2007). However, we found a continuum in variation in skull shape
among the eight main groups, a strong signal of geographical variation, and support
for genetic drift as being the more likely cause generating the observed pattern.
Simultaneously, the selection for underground life (digging, communicating, etc.) is
likely strong and should drive constraints on cranial shape. This pattern suggests
that the adaptive landscape of Ctenomys is holey (sensu Gavrilets 2003), with low
fitness genotypes not fit for underground life and highly fit genotypes that form
continuously within the underground niche. This scenario suggests a model of geo-
graphical parapatric speciation without strong and/or obvious barriers to gene flow.
Current evidence suggests this model to be likely. Reproductive isolation has been
investigated in sister species by Lopes and Freitas (2012) and Freitas (2006), show-
ing the presence of hybrid zones at the intraspecific and interspecific levels between
sister taxa Ctenomys lami and C. minutus. Few other hybrids have been detected
either because population/species differentiation is rapid as predicted analytically
(Gavrilets 2003) or because of limited taxon sampling. Genetic differentiation com-
patible with isolation by distance model has been found in most species analyzed
except for C. australis (Mora et al. 2010), coupled with the fine-scale geographic
structure for C. talarum (Mora et al. 2007), C. rionegrensis (Wlasiuk et al. 2003),
C. minutus (Lopes and Freitas 2012), C. flamarioni (Fernandez-Stolz et al. 2007),
C. pearsoni (Tomasco and Lessa 2007), C. torquatus (Roratto et al. 2015), and even
C. lami (Lopes and Freitas 2012) with a distribution area of 78 × 12 km. Isolation
by distance differentiation signal in the skull was also found in C. minutus (Fornel
et al. 2010), C. rionegrensis (D’Anatro and Lessa 2006), and likely in C. torquatus
(Fernandes et al. 2009). Ecological differentiation has been rarely tackled within the
niche of tuco-tucos but metagenomic results indicate differentiation in diet between
distantly related sympatric/parapatric species or between populations (Lopes et al.
2015), despite significant differences in skull morphology between habitats (Fornel
et al. 2010).
In summary, we found remarkable skull shape variation in Ctenomys forming a
continuum with a strong geographical signal and little evidence of distinct selec-
tion regimes between species, which agrees with patterns observed by Lessa et al.
(2008), indicating that morphological adaptations in ctenomyids are more variable
and complex than supposed anteriorly. In this interspecific scenario, parapatric spe-
ciation in a holey adaptive landscape is likely. This skull shape variation could be
the result of small populations, limited dispersal, and weak selection in Ctenomys.
Acknowledgements We are very grateful to Fabiano A. Fernandes, Daniza Molina-Schiller, and

Gisele S. Rebelato for their help with photographing skulls. We thank Michel Baylac for the
Rmorph package. Thanks for all curators and collection managers that provided access to Ctenomys
specimens: Enrique González (MUNHINA), Olga B. Vacaro and Esperança A. Varela (MACN),
Diego H. Verzi and A. Itatí Olivares (MLP), A. Damián Romero (MMP), James L. Patton, Eileen
A. Lacey, and Christopher Conroy (MVZ), Eileen Westwig (AMNH), and Bruce D. Patterson
(FMNH). This work was supported by Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES);
Fundação de Amparo à Pesquisa do Rio Grande do Sul (FAPERGS); Departamento de Genética –
UFRGS; and Projeto Tuco-tuco. P. C-E. was supported by the CNPq/CAPES PROTAX Program
for Taxonomy. R. F. was supported for this project by a doctoral fellowship from Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq) [grant proc. no. 142953/2005-9].
Supporting Information
SI 1.
Definitions of morphological landmarks with numbers and localizations for each
view of the cranium and lower jaw of Ctenomys (represented in Fig. 6.2). The same
definitions were used by Fornel et al. (2010).
Dorsal View of the Cranium
1, anterior tip of the suture between premaxillas; 2–3, anterolateral extremity of

incisor alveolus; 4, anterior extremity of the suture between nasals; 5–6, anterior-
most point of the suture between nasal and premaxilla; 7–8, anteriormost point of
the root of zygomatic arch; 9, suture between nasals and frontals; 10–11, anterolat-
eral extremity of lacrimal bone; 12–13, point of least width between frontals; 14–15,
tip of extremity of superior jugal process; 16–17, anterolateral extremity of suture
between frontal and squamosal; 18, suture between frontals and parietals; 19–20, tip
of posterior process of jugal; 21–22, anterolateral extremity of suture between pari-
etal and squamosal; 23–24, anterior tip of external auditory meatus; 25–26, point of
maximum curvature on mastoid apophysis; 27, posteriormost point of occipital
along the midsagittal plane; 28–29, lateral extremity of suture between jugal and
squamosal.
Ventral View of the Cranium
1, anterior tip of suture between premaxillas; 2–3, anterolateral extremity of incisor

alveolus; 4–5, lateral edge of incisive foramen in suture between premaxilla and
maxilla; 6–7, anteriormost point of root of zygomatic arch; 8–9, anteriormost point
of orbit in inferior zygomatic root;10–11, anteriormost point of premolar alveolus;
12–13, posterior extremity of III molar alveolus; 14, posterior extremity of suture
between palatines; 15–16, anteriormost point of intersection between jugal and
squamosal; 17–18, posteriormost point of pterygoid processes; 19–20, anterior
extremity of tympanic bulla; 21–22, anterior tip of external auditory meatus; 23–24,
posterior extremity of mastoid apophysis; 25–26, posterior extremity of paraoccipi-
tal apophysis; 27, anteriormost point of foramen magnum; 28–29, posterior extrem-
ity of occipital condyle in foramen magnum; 30, posteriormost point of foramen
magnum along midsagittal plane.
Lateral View of the Cranium
1, inferiormost point of incisor alveolus; 2, posteriormost point of incisor alveolus;

3, anteriormost point of premaxilla; 4, anteriormost point of the suture between
nasal and premaxilla; 5, anterior tip of nasal; 6, inferiormost point of suture between
premaxilla and maxilla; 7, suture between premaxilla, maxilla and frontal in supe-
rior zygomatic root; 8, inferiormost point of suture between lacrimal and maxilla; 9,
inferiormost point of infraorbital foramen in inferior zygomatic root; 10, anterior-
most point of premolar alveolus; 11, extremity of inferior jugal process; 12, extrem-
ity of superior jugal process; 13, tip of posterior jugal process; 14, medial point of
suture between parietal and squamosal; 15, posterior extremity of postglenoid fossa;
16, inferior extremity of mastoid apophysis; 17, inferior extremety in suture between
pterygoid and tympanic bulla; 18, anteriormost margin of paraoccipital apophysis;
19, posteriormost margin of paraoccipital apophysis; 20, posterior extremity of
intersection between occipital and tympanic bulla; 21, superior extremity of lamb-
doidal crest.
Lateral View of the Mandible
1, upper extreme anterior border of incisor alveolus; 2, extreme of the diastema

invagination; 3, anterior edge of the premolar alveolus; 4, intersection between
molar alveolus and coronoid process; 5, tip of the coronoid process; 6, maximum of
curvature between the coronoid and condylar processes; 7, anterior edge of the
articular surface of the condylar process; 8, tip of the postcondyloid process; 9,
maximum curvature between condylar and angular processes; 10, tip of the angular
process; 11, intersection between mandibular body and masseteric crest; 12, poste-
rior extremity of the mandibular symphysis; 13, posterior extremity border of inci-
sor alveolus.
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Chapter 7
Biomechanics and Strategies of Digging
Aldo I. Vassallo, Federico Becerra, Alejandra I. Echeverría, Guido N. Buezas,

Alcira O. Díaz, M. Victoria Longo, and Mariana Cohen
7.1 Introduction
For small rodents, running and hiding are the main strategies to protect themselves
from predators, and burrows offer an excellent shelter against most of them
(Reichman and Smith 1990; Andino et al. 2014). Particularly, in arid and semiarid
ecosystems, digging (i.e., to break up and remove the soil) and burrowing (i.e., to
hide in burrows, to construct by tunneling, or to progress by or as if by digging) are
common behaviors in many mammals (Nevo 1999; Whitford and Kay 1999; Lacey
et al. 2000; Cutrera et al. 2006). Numerous terrestrial mammals have evolved fosso-
rial adaptations, and rodents, in particular, have repeatedly diversified into under-
ground habitats (Hopkins 2005; McIntosh and Cox 2019). Across the globe, in all
continents but Australia and Antarctica, at least 250 extant rodent species (38 gen-
era, 6 families – according to the classification applied) spend most of their lives in
self-constructed burrows (Begall et al. 2007). Life underground is achieved through
several distinct methods of burrow excavation. From studies performed in terrari-
ums (Giannoni et al. 1996), as well as in natural soils (Vassallo 1998), it is known
that tuco-tucos break up the soil through the use of both the foreclaws of the manus
(scratch-digging) and the incisors (upper and lower) in a chewing motion
A. I. Vassallo (*) · F. Becerra · A. I. Echeverría · G. N. Buezas

Grupo Morfología Funcional y Comportamiento, Instituto de Investigaciones Marinas y
Costeras (IIMyC, UNMDP-CONICET), Universidad Nacional de Mar del Plata (UNMdP),
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET),
Mar del Plata, Buenos Aires, Argentina
e-mail: avassall@mdp.edu.ar; fbecerra@mdp.edu.ar; aiechever@mdp.edu.ar
A. O. Díaz · M. V. Longo · M. Cohen
Grupo Histología e Histoquímica, Instituto de Investigaciones Marinas y Costeras (IIMyC,
UNMDP-CONICET), Universidad Nacional de Mar del Plata (UNMdP), Consejo Nacional
de Investigaciones Científicas y Técnicas (CONICET),
Mar del Plata, Buenos Aires, Argentina
e-mail: adiaz@mdp.edu.ar; mvlongo@mdp.edu.ar

https://doi.org/10.1007/978-3-030-61679-3_7
142 A. I. Vassallo et al.
(chisel-tooth digging) according to soil requirements. However, the use of these dif-
ferent digging tools varies between species and habitats occupied. Scratch-digging
is the predominant mode of digging among rodents. A final mode of digging, and
the least common among rodents, is head-lift digging (Hopkins 2005), which is
performed through the use of the nose in a spade-like manner, and sometimes
assisted by the lower incisors.
Tuco-tucos are proficient scratch-diggers that dig primarily by means of vigor-
ous scraping movements, which comprise rapid alternating strokes of their forefeet
(e.g., Vassallo 1998). These movements allow them to loosen the soil through the
use of the hands, especially the foreclaws. Recently, Echeverría et al. (2019) found
that the palms of tuco-tucos’ hands show a pad pattern similar to that observed in
other fossorial rodents (all Spalacinae occurring at least in Europe; several bathyer-
gids: Georychus, Cryptomys, and Bathyergus; Pedetes capensis Pedetidae;
Geomyidae and the dipodine Jaculus; for references see Ade and Ziekur 1999). This
pattern is defined by a lack of distinct distal pads accompanied by strongly devel-
oped proximal pads (the hypothenar and thenar pads, this latter has been now rede-
scribed as a false thumb by Echeverría et al. 2019). According to Ade and Ziekur
(1999), this condition indicates that the animals can dig by using their paws in a hoe
or scraper-like manner, since digits II-III-IV-V project ventrally and the flexed palm
forms a kind of scraper or hoe.
According to the requirements of the substrate, tuco-tucos complementarily use
their large and procumbent incisors (i.e., incisors with a protrusion angle greater
than 90°) to assist in loosening and breaking obstacles such as rocks, nodules of
CaCO3 (“tosca”) or hard soil, and fibrous roots (see Ubilla and Altuna 1990; De
Santis et al. 1998; Vassallo 1998; Stein 2000; Becerra et al. 2013). For instance,
Vassallo (1998) found that when confronted with sandy and friable soils, C. talarum
(Los Talas’ tuco-tuco) exclusively use their forelimbs to break down the soil.
Conversely, when confronted with harder and clayey soils, they behave as scratch-
and chisel-tooth diggers, also using the incisors to dig. The digging strategy used is
perhaps related to the external forces that the muscles involved can exert, probably
being greater in those muscles that act during tooth-digging (Tables 7.1, and 7.2).
Although the actual force values at scratch-digging are still unknown, anatomy-
based estimations of forces exerted by selected muscles done at the level of the
foreclaw’s tip and the incisors support this statement (Table 7.2).
The bite force of several Ctenomys species was measured in vivo (Becerra 2015).
For instance, in C. talarum, bite force ranges from 32 to 27 N (in adult males and
females, respectively). Considering that soil hardness of C. talarum’s typical habitat
averages 100 N/cm2, and taking into account the incisor’s cross-section, it was
assessed that the pressure exerted by jaw adductor muscles at the level of the inci-
sors is three times higher than that required for soil penetration (Becerra et al. 2011).
The following sections will introduce the basic biomechanical principles under-
lying force production and transmission during digging. The burrowing system of
tuco-tucos is described, emphasizing its dynamic attributes and dependence on the
characteristics of the environment. Finally, we addressed the functional morphology
7 Biomechanics and Strategies of Digging 143
Table 7.1 Miology of skull and pectoral appendicular regions in Ctenomysa

Structure Muscle Origin Insertion Action
Jaw Masseter superficialis By a round and strong Medial surface of the Jaw adductor
tendon, from a small angle of the mandible,
area on the anteroventral it inserts high up the
surface of the zygomatic deep medial fossa, near
arch and posterior
Masseter profundus From the anteroventral Lateral surface of the
surface of the zygomatic mandible, on the
arch masseteric crest
Posterior masseter It originates from the Lateral surface of the
posteroventral surface of mandible ventral to the
the zygomatic arch condyle, on the
postcondyloid process
Zygomaticomandibularis From the medial surface On the lateral surface of
of the zygomatic arch the mandible
Zygomaticomandibularis Represented by fibers of Lateral surface of the
infraorbitalis the mandible. Along the
zygomaticomandibularis dorsal aspect of the
that arise from the bony masseteric fossa
walls of the infraorbital
foramen. It originates
from the fossa on the
lateral side of the
rostrum, the superior
zygomatic root, and the
medial surface of the
maxillary and jugal
parts of the zygomatic
arch
Temporalis According to Woods On the coronoid
(1972), the muscle is process of the
composed of these three mandible. The main
parts. It originates from part inserts on the tip,
the surface of cranium, the orbital part inserts
may include the mostly on the medial
temporalis fascia and surface, the posterior
the surface of the part inserts on the
cranium superior and lateral surface
posterior to the
zygomatic arch
Pterygoideus Externus. From the edge Externus. Below and
of the lateral pterygoid posterior to the condyle
plate, the surface of the on the medial surface of
alisphenoid bone, and the condyloid process.
the adjacent edge of the Internus. Medial side of
maxillary bone the mandible, on the
Internus. From the dorsal surface of the
margin and deep inside flattened angular
the pterygoid fossa. Two process
parts
(continued)

Shoulder Subscapularis From the entire On the lesser tuberosity Medial rotator
subscapular fossa of the of the humerus of the arm. It
scapula provides
shoulder joint
stability
Rotator Supraspinatus Two parts. From the The muscle parts are External rotation
cuff anterior surface of the moderately fused. The of the shoulder
proximal half of the part from the scapular joint. Shoulder
scapular spine, the spine is more joint stabilizer
ventral surface of the superficial than the
spine in the region of second part, and inserts
the great scapular notch, onto its surface. The
and the surface of the combined parts narrow
septum separating this to a thin tendon which
muscle from the inserts on the
infraspinatus. The dorsocranial surface of
remaining and larger the greater tuberosity of
part is from the the humerus
supraspinous fossa, the
superior border of the
scapula, and the
anteromedial surface of
subscapularis muscle
Infraspinatus From the surface of the The muscle narrows to External rotation
infraspinous fossa, the a tendon and inserts on of the shoulder
dorsal margin of the the greater tuberosity of joint. Shoulder
axillary border under the the humerus. The joint stabilizer
teres major muscle, the muscle passes
vertebral border, and the anteroventrally beneath
spine of the scapula. the spine of the scapula
The part of the muscle (great scapular notch).
from the spine The notch extends
originates on the along the distal
posterior margin of the two-thirds of the length
spine proximal to the of the scapular spine
great scapular notch and
on the ventral surface of
the spine in the region
of the notch
Teres minor From the distal On the distal edge of External rotation
two-fifths of the axillary the greater tuberosity. of the shoulder
border of the scapula, The insertion is via a joint. Adduction
and from the anterior tendon and is distal to and extension of
surface of the and separate from the the shoulder.
well-developed insertion of the When the
aponeurotic envelope. infraspinatus muscle humerus is
The muscle is small, and stabilized,
largely covered by the abducts the
infraspinatus muscle inferior angle of
the scapula.
Shoulder joint
stabilizer.
(continued)

Teres major From the most posterior Distally, on the shaft of Its main action
margin of the vertebral the humerus is flexing the
border and the dorsal shoulder
third of the axillary according to
border of the scapula. anatomical
Some fibers also descriptions.
originate from the Acting alone
fascial surface of the from a position
infraspinatus the with the
subscapularis muscles shoulder
semi-flexed, it
would thus be
expected to raise
the elbow, and
its activity may
relate to initial
paw lift.
However, due to
its insertion
medially on the
humeral neck, it
is possible that
in some
positions of the
humerus it could
exert a force
tending to
medially rotate
the humerus,
and thus
contribute to
medial
movement of the
paw, which
occurs at the
same time and
to which it was
also highly
correlated
Latissimus dorsi From the last five On the medial side of Its action is to
thoracic vertebrae and the humerus below the pull the arm
the lumbodorsal fascia lesser tuberosity. The backward and
muscle joins with the upward
teres major muscle to
insert via a common
tendon
Dorsoepitrochlearis Dorsolateral border of Posteroventral aspect of Forelimb
the m. latissimus dorsi the olecranon extensor
Triceps longus Axilar border of the Posterior surface of the Forelimb
scapula olecranon extensor
(continued)

Triceps medialis Distal four-fifths of the Posterodorsal surface of Forelimb
shaft of the humerus the olecranon. The deep extensor
fibers are continuous
with the anconeus
muscle and insert on
the lateral aspect of the
olecranon
Triceps brachii (=triceps Ventrolateral surface of On the ventrolateral Forelimb
lateralis) the greater tuberosity of surface of the olecranon extensor
the humerus and onto the surface of
the triceps longus
muscle
Biceps brachii The short head, from the The two heads merge Forelimb flexor
tip of the coracoid into one and inserts on
process. Long head, the brachial ridge of the
from the base of the ulna. Distally, inserts
coracoid process and lip secondary on the radius
of the glenoid fosa
Supinator On the proximal 2/3 of Fleshy. On the proximal Forearm
the humerus. From the half of the lateral supinator
surface of the radial surface of the radius
collateral ligament and
from the deep surface of
the distal end of the
lateral epicondyle
Forearm Pronator teres Medial epicondyle of Middle 2/3 of the radius Forearm
the humerus. Fleshy pronator
Palmaris longus Medial epicondyle of Thenar and hypothenar Hand flexor
the humerus pads
Flexor carpi ulnaris Ventral surface of the Base of the pisiform Hand flexor
olecranon and proximal bone
end of the ulna
Flexor carpi radialis Medial epicondyle of On the base of the Hand flexor
the humerus second and third
metacarpals
Extensor carpi radialis Humerus. From the Half of the second and Hand extensor
proximal end of the third metacarpals
lateral epicondylar ridge
Extensor carpi ulnaris Lateral epicondyle of On the unciform bone Hand extensor
the humerus and
proximal half of the
ulna (distal to the
middle of the semilunar
notch)
(continued)
Flexor digitorum Via four heads. The first The tendons join in one Digits flexor
profundus and the second, from the that passes through the
medial epicondyle of the wrist and splits into
humerus; the third from four tendons. They
the proximal 2/3 of the insert onto the
radius; the fourth, from sesamoids of the
the proximal 2/3 of the terminal phalanxs of
ulna digits II to V. From the
tendon of digit II, a side
tendon develops and
passes out onto the
thumb
Sublimis (=flexor Medial epicondyle of Splits in three tendons Digits flexor
digitorum superficialis) the humerus which pass out onto the
base of digits II, III,
and IV. Beyond the
metacarpophalangeal
junction, each tendon
splits into two small
branches which pass
out on either side of the
large tendon of the
flexor digitorum
profundus and insert on
the base of the second
phalanx
Extensor digitorum Lateral epicondyle of Insertion tendon passes Digits extensor
the humerus through the wrist
(between the ulna and
radio epiphyses) and
splits in 4 tendons that
insert on the terminal
phalanges of the second
through fifth digits
Abductor pollicis longus Ulna and radius Point of bifurcation False thumb and
between the thumb and thumb abductor
the thenar pad
Hand Flexores breves profundi Distal carpal Onto the sesamoids at Digits flexors
corresponding to each the
digit metacarpophalangeal
joints of each digit
Abductor pollicis brevis Proximal border of the Onto the radial side of Thumb abductor
radial sesamoid (skeletal the metacarpal of the
support of the false thumb
thumb)
Flexor pollicis brevis Base of the radial Base of the metacarpal Thumb flexor
sesamoid of the thumb
Adductor pollicis brevis Common deep palmar Incorporates to the Digits adductor
ligament muscle complex
connecting the false
thumb, the thumb, and
the digit II
Taken from Woods (1972); Druzinsky et al. (2011); Echeverría et al. (2017, 2019)
a
Table 7.2 Mechanical advantage and forces exerted by selected forelimb and jaw adductor
muscles involved in digging in Ctenomys talarum
Triceps Biceps Masseter Masseter
longus brachii superficialis profundus
Muscle mass (g) 0.78 0.10 0.61 0.60
% muscle mass to body mass 0.53 0.04 0.42 0.41
PCSA (mm2) 27 6 72 94
Li/Lo, mechanical advantage 0.21 0.16 0.20 0.30
Fi, muscle internal force (N) 8.23 1.77 18.03 23.55
Fo, external force (N) at claws 1.73 0.28 3.61 7.06
(triceps, biceps) and incisors
(masseter)
of the so-called digging tools, i.e., incisors, claws, and functionally related struc-
tures, including issues related to its structural strength.
7.2 A Biomechanical Approach to Digging Behavior
Briefly, we will introduce why a biomechanical approach allows us to understand

the tuco-tucos’ digging behavior. Classical mechanics deals with the behavior of
physical entities when they are affected by forces or displacements. Biomechanics,
as a discipline, integrates anatomical attributes and principles of classical mechan-
ics to understand the functioning and adaptations of animals at different levels of
organization, from the cellular level, organs or structures, to the whole organism
(e.g., Vogel 2013). Locomotion and feeding are two of the main functions on which
biomechanics focuses on. Terrestrial locomotion requires the production of forces
to sustain the body by counteracting the force of gravity, as well as to propel the
animal and produce its displacement (Biewener 2003). On the other hand, feeding
requires the production of forces to disaggregate a food item (Ungar 2010). In sub-
terranean rodents, such as Ctenomys, part of the anatomical structures used for the
basic functions of locomotion and feeding (which they possess because they are
terrestrial mammals) were co-opted to produce, in addition, a new function, dig-
ging. These now partially modified structures are called “digging tools,” but it is
important to note that they have not lost their original function. For instance, when
a tuco-tucos moves inside its burrow or moves over the surface to access to aboveg-
round plant parts, it does so according to a typical mammal locomotor pattern (Lobo
Ribeiro 2017). Of course, digging tools involve more anatomical elements and rela-
tionships between them than specifically the incisors and foreclaws. For example,
for the incisors to properly perform as digging tools, it is necessary that the cervical
musculature act stabilizing the head, so that these muscles are well developed in
chisel-tooth digging rodents, as well as the jaw muscles (Hildebrand 1985; Van
Daele et al. 2009).
One of the most important structural modifications concerning digging is the

increase of the mechanical advantage (MA) of the involved muscles. In musculo-
skeletal systems (e.g., limbs; jaw and associated muscles), the mechanical advan-
tage is a measure of the amplification of force exerted by a certain arrangement of
muscles and bones, given by the equation:
MA = Fo / Fi = Li / Lo ; Fo = Li / Lo × Fi.
Where Fo is the resultant external force exerted (or applied) by the system, Fi is
the contributive fraction of the internal force – produced by muscle contraction – to
the Fo (effective force), and Li and Lo are determined by the moving bones that
rotate on a joint, that is, the lever arms. This formalism of classical mechanics,
known as the static equilibrium equation, allows us to analyze the anatomical char-
acters involved in digging in two complementary approaches. On the one hand, it
allows us to interpret the function of a complex feature consisting of different bones,
joints, muscles, and sites of origin and insertion, i.e., understanding the relationship
between structure and function (Fig. 7.1). On the other hand, it allows us to predict
on which anatomical attributes natural selection could act on, leading to adaptations
related to specialized behaviors such as digging and subterranean habits.
7.3 The Burrow System in Tuco-Tucos
For mammals that evolved to live underground, the burrow represents an exoso-
matic structure which constitutes, in fact, a materialization of the relationship
between the individuals and the environment. The nexus established by the tuco-
tuco’s burrow with its habitat is so strong and complex that it is difficult, or even
arbitrary, to establish the boundaries between the species’ phenotype and the sur-
rounding environment. Burrows serve a number of purposes such as thermoregula-
tion (they provide a stable environment), shelter against predators, procurement,
and storage of food, facilitation of social interactions and mating (e.g., Fleming and
Brown 1975; Ellison 1995; Ebensperger and Blumstein 2006; Antinuchi et al. 2007;
Zelová et al. 2010). The tuco-tuco’s burrow structure was well studied by Antinuchi
and Busch (1992). These authors found that tuco-tucos are capable of building
extensive and elaborated tunnel systems with many feeding openings, which are
characterized by a branching structure; i.e., a main axial tunnel (representing 48%
of the total length), a single nest, and a variable number of lateral blind and foraging
tunnels. The burrow openings are of two types: some serve to take out the soil
removed by the animal (product of the maintenance of the burrow or the construc-
tion of new tunnels), which are usually surrounded by loose soil that takes the form
of lobed or fan-shaped mounds, and others are used as an exit to the outside to col-
lect plant material, or eventually interact with other tuco-tucos, for example, during
the excursions made by the males to visit the female’s burrow during the reproduc-
tive season (Zenuto et al. 2002). The studied species by Antinuchi and Busch (1992),
Fig. 7.1 Biomechanical analysis of chisel-tooth and scratch-digging in tuco-tucos. Above: The
mandible is in static equilibrium between two forces acting in opposite directions (third-order lever
system). The force produced by the mandibular adductor musculature (Fi) tends to rotate the jaw
by closing it, while (Fo) is the reaction force that the substrate exerts on the lower incisors which
tends to rotate the jaw in the opposite direction. Below: The forearm (ulna and radius) is in static
equilibrium between two forces acting in the same direction (first-order lever system). The force
produced by the arm extensor musculature (Fi) tends to rotate it by extending the elbow, while (Fo)
is the reaction force that the substrate exerts on the claws which tends to rotate the forearm in the
opposite direction. Each force acts on its respective lever arm, Li and Lo. This static equilibrium
can be thought of as the situation prior to the breakage of the ground either by the incisors (above)
or claws (below), so Fi x Li = Fo x Lo. P represents the joint’s position (pivot). Photographs taken
from a video film of a C. talarum adult male (body mass 150 g) digging in soil typical of its habitat;
above: modified from Becerra et al. (2011). In white, over imposition of mandible (above) and
ulna, humerus, and scapula (below)
C. talarum, lives in well-vegetated grassy and highly variable habitats, ranging from
sand dunes to inland grasslands with sandy and friable to hard, clayey soils (Malizia
et al. 1991). The soil hardness at which this species is found is 15–25 kg/cm2 for
5 cm of penetration (Vassallo 1998). It should be noted that this tuco-tuco species is
not among the largest species of Ctenomys, but despite their small body size
(♀ = 122 g, and ♂ = 154.4 g; Malizia and Busch 1991), it can build tunnel systems
with a burrow area of 8 ± 6 m2, and a burrow length of 14 ± 8 m in average (Antinuchi
and Busch 1992).
Burrow systems are dynamic structures. The animals prolong the galleries daily
toward patches with abundant vegetation, while other sections are abandoned and
obliterated with sediment to prevent the transit of predators (Dentzien-Dias and
Figueiredo 2015). The existence of areas surrounding C. mendocinus burrow sys-
tems showing past signs of burrowing activity and a relatively lower vegetation
cover (Rosi et al. 2000) agrees with this observation. These facts are further evi-
dence that burrow systems are dynamic structures, where grazed areas with partly
depleted food are temporally abandoned, and feeding galleries are extended through
microhabitats with relatively higher food availability, as suggested by Rosi et al.
(2000). This burrowing dynamic allows tuco-tucos to access plant resources distrib-
uted over a relatively large area, by means of short and fleeting excursions to the
surface from several burrow openings. There is evidence of intraspecific differences
in digging dynamics: populations of C. mendocinus that occupy different habitats in
terms of plant abundance and diversity differ in burrow attributes such as the total
length of the burrow and number of gallery bifurcations (Rosi et al. 2000). A radio-
telemetry interspecific study by Cutrera et al. (2010) found that the home-range size
of the sand dune tuco-tuco C. australis was ∼19 times larger than that of Los Talas’
tuco-tuco C. talarum, in spite of C. australis being only 2 to 3 times larger and hav-
ing a BMR 1.3–2.6 higher than C. talarum. These authors suggested that the differ-
ences in the composition of the plant community of the different habitats of these
species could affect the turnover rate of the consumable plants, and ultimately habi-
tat food availability through time, leading the sand dune tuco-tuco’s burrow to cover
greater areas to meet their energy requirements.
In line with this, Kubiak et al. (2017) argued that C. minutus inhabiting sand
dunes probably need longer galleries covering larger areas to access sufficient food
resources because this habitat has relatively less plant biomass than other habitats
occupied by the species. In sum, and contrary to what happens with those species
that use the burrow only as shelter and/or breeding, subterranean rodents carry out
daily routine digging activities to build their burrow in such a way that its structure
and dynamics are in line with the variable attributes of the occupied habitat and the
energy requirements of the species.
Contrary to what one might think in the case of a subterranean rodent, burrowing
does not seem an activity to which tuco-tucos devote much to the daily time budget.
In fact, in C. talarum it has been estimated that the daily lengthening of the burrow
for accessing new patches of vegetation is ~1 m/day (Vassallo 2006), and ~3 m in
larger species such as C. australis (AIV, pers. obs. based on the daily appearance of
new mounds). Based on a digging speed of 1.5–5 m/hour, which varies depending
on the hardness of the soil (Vassallo 1998; Luna and Antinuchi 2007), it can be
inferred that daily lengthening can demand 12–40 net minutes of digging. Pioneering
studies by Vleck (1979) showed that progressing by digging requires energy expen-
diture 360–3400 times greater than moving the same distance aboveground.
However, looking at the daily energy expenditure, digging represents only 12% of
the thermoregulation cost, and 10% of the maintenance cost, as estimated by
Antinuchi et al. (2007). Even daily displacements through the tunnel system (patrol-
ling; access to foraging openings) would consume more energy than digging.
Hence, it is intriguing why a relatively limited activity concerning daily time and
energy budgets is associated with major changes in morphology. These include
those shown by tuco-tucos, especially in its biomechanical and functional morphol-
ogy attributes. Probably, this is partly due to the relatively high forces required to
disaggregate and transport the substrate.
7.4 Functional Morphology of Digging Strategies
7.4.1 Scratch-Digging
The morphological adaptations related to scratch-digging are well-known in mam-

mals, and they are mainly found in the forelimbs. For example, the superstructures
(i.e., bone eminences such as the teres major, deltoid, and olecranon processes, and
the distal, lateral, and medial epicondyles of the humerus) of long bones are strongly
developed in scratch-diggers. One of the main functions of bone superstructures is
to provide an attachment point for tendons and ligaments, which transmit force
from the contracting muscles to the skeleton (Mc Henry and Corruccini 1975; Polly
2007; Lessa et al. 2008). Thus, in scratch-diggers, the more developed bone super-
structures on forelimbs allow an increased strength in flexing the large digits and the
wrist, extending the elbow, flexing the humerus on the scapula, and stabilizing the
shoulder (Hildebrand 1985; see Table 7.1). For example, a study of limb morphol-
ogy and function in caviomorph rodents not including Ctenomys (Elissamburu and
Vizcaıno 2004) showed that, in general terms, caviomorph diggers are characterized
by morphofunctional indices (ratios) that represent higher humeral and ulnar robust-
ness, higher deltoid and epicondylar development, and increased mechanical advan-
tage of elbow extensor muscles. In Ctenomys, Echeverría et al. (2014) investigated
the postnatal ontogeny of limb proportions and functional indices in C. talarum, and
found that during the early development of this species, several morphological traits
that are typically associated with scratch-digging are already present (suggesting
that they are prenatally shaped), and other traits develop progressively. For instance,
when compared with juveniles and adults, young pups show relatively more robust
humeri, ulnae, and femora (being robustness the anteroposterior diameter at diaphy-
sis divided by length), wider humeral epicondyles, longer zeugopodial elements, a
proportionally shorter olecranon, as well as a poorly developed teres major pro-
cesses and an incipient deltoid crest (Echeverría et al. 2014). Bone robustness can
be related to the need to support the body mass during locomotion or of developing
the forces required for more specific functions of the limb, such as digging activity
(Elissamburu and De Santis 2011). Thus, the greater bone robustness observed in
young C. talarum may indicate a possible compensation for lower bone stiffness,
while the wider epicondyles may be associated with improved effective forces in
those muscles that originate onto them (see Table 7.1), compensating the lower
muscular development. Echeverría et al. (2014) also found a gradual increase in the
relative olecranal length (i.e., the index of fossorial ability: IFA = olecranal length/
functional ulnar length), suggesting a gradual enhancement in the scratch-digging
performance due to an improvement in the mechanical advantage of forearm exten-
sors (i.e., m. triceps and m. dorsoepitrochlearis). Middle limb indices, which indi-
cate the extent to which the forelimb is apt for fast movement (brachial index) and
how well the hindlimbs are apt for speed (crural index), is higher in pups than in
juveniles-adults, reflecting relatively more gracile limbs in their middle segments,
which is in accordance with their incipient fossorial ability.
In terms of behavioral development, and in accordance with what was previously
observed at the morphological level by Echeverría et al. (2014), Echeverría et al.
(2015) observed that in C. talarum scratch-digging and burrowing express early
during postnatal ontogeny, particularly, during lactancy age (Fig. 7.2). In this study,
it was found that the digging of a “true burrow” (i.e., a completely closed tunnel of
at least the animal’s body length) clearly preceded the dispersal age, and it hap-
pened around 18 (lactation) and 47 (post-weaning) postnatal days (Fig. 7.2). Also,
it was observed that young pups are capable of loosening the soil using their fore-
claws, and remove the accumulated substrate using their hindfeet as adults do (i.e.,
via an inchworm locomotion in reverse, see Hickman 1985). Prior to weaning age,
young are able to construct simple burrows to shelter, and they dig for the first time
their own burrow in the absence of either a burrowing demonstrator or an early
subterranean environment (i.e., a natal burrow). Thus, in this species, immature dig-
ging behavior gets expressed early during ontogeny and develops progressively.
Taking into account that (a) some morphological adaptations related to scratch-
digging are already present in very young individuals and others develop progres-
sively (Echeverría et al. 2014), (b) subterranean lifestyle is assumed to be highly
Fig. 7.2 Postnatal ontogeny of salient behaviors related to digging in C. talarum. (Modified from
Echeverría 2011)
costly due to the high energetic demand of tunnel extension (Vleck 1979; Luna
et al. 2002), and (c) C. talarum displays a long maternal care period (~2 months;
Zenuto et al. 2002), the early occurrence of scratch-digging and burrow construc-
tion would provide of enough time to reach a proper musculoskeletal and behavioral
development, mainly to deal with energetic and biomechanical demands. Epigenetic
effects of the early biomechanical environment have been documented in several
groups of mammals and, therefore, it is widely accepted that both genetic and epi-
genetic factors determine the final shape and strength of the skeleton (Carter et al.
1998; Nowlan and Prendergast 2005). For example, physical activity may promote
normal development of muscles and bones (e.g., Herring and Lakars 1981), and
patterns of muscle contraction can cause a differential growth in those bone areas
that are closest to peak strains (Young et al. 2009; and references therein). Thus,
functional demands would affect relatively more the position of bone structures
associated with muscle and ligament attachment (e.g., deltoid process) than the
attainment of the fundamental form of the skeleton, as well as the presence of bone
superstructures (e.g., condyles, articular surfaces, tuberosities, grooves) (Murray
1936; Hall 1978).
7.4.2 Tooth-Digging
Ctenomys species have spread throughout a remarkable portion of South America,

having to confront a huge variety of environmental conditions when digging is per-
formed (Bidau 2015). For many of those species, scratch-digging would not be
enough to break down the clayey rocks, roots, and all sorts of obstacles in the soil,
and a less flexible – but still more robust and powerful – apparatus needs to show up
in the scene: the jaws (see, for instance, Vassallo 1998; Lessa et al. 2008; Becerra
et al. 2013). In order to do so, these animals use their procumbent incisors to apply
powerful forces, much more powerful even than the expected ones for a small-to-
medium size caviomorph rodent (Becerra et al. 2011, 2013, 2014; Becerra 2015;
Borges et al. 2016; Fig. 7.3). Even though this strong biting – relative to body size –
seems to have been selected for an underground life (Lacey et al. 2000), some
authors have even proposed that it might also be selected as a key factor for males
success at controlling a certain habitat range and the resources therein (Becerra
et al. 2012a). This fact may be the reason why, even though tuco-tucos inhabit a
wide spectrum of soils, bite force is not directly related to habitat’s mechanical
constrains (Fig. 7.3).
Chisel-tooth digging is a burrowing strategy that involves more than the complex
craniomandibular system – which cannot be exclusively dedicated to it, but it is
shared with feeding needs and the main sensory systems – recruiting also much of
the neck musculature (AIE and FB, pers. obs.). Nevertheless, in this section, we will
focus on the head, especially on the incisors’ properties as digging tools and the
overall biomechanics of biting.
Fig. 7.3 Scatter-plot of residual values of a phylogenetic generalized least squared regression
(PGLS) between the log-transformed bite force and log-transformed body mass – to avoid the
potential distortive effect of either body size or evolutionary affinity – in six Ctenomys species and
four other caviomorph rodents as step-wise out-groups. Phylogeny was built upon cytochrome-b
sequences available on GenBank. Species used with GenBank vouchers in brackets: Ct.cha,
C. “chasiquensis” (MF770015.1); Ct.por, C. porteousi (AF370682.1); Ct.tal, C. talarum
(AF370698.1); Ct.aus, C. australis (AF370697.1); Ct.tuc, C. tuconax (AF370684.1); Ct.hai,
C. haigi (HM777476.1); O.deg., Octodon degus (Fam. Octodontidae; AF007058.1); M.coy,
Myocastor coypus (Fam. Echimyidae; AF422919.1); Ch.lan, Chinchilla lanigera (Fam.
Chinchillidae; JX312692.1); D.pat, Dolichotis patagonum (Fam. Caviidae; GU136724.1). Gray
scale within Ctenomys species (except for C. “chasiquensis”) represents the soil hardness mea-
sured in situ. Taking into account that the phylogeny was constructed at the species level, standard
deviations were rescaled based on the mean values after PGLS. Data from Becerra (2015)
It has been previously observed that tuco-tucos attack most hard obstacles by
anchoring the upper incisors and applying the force at the lower incisors tip, by
repetitive firing movements of the mandible (Vassallo 1998; Becerra et al. 2013).
Therefore, incisors’ resistance toward abrasion is highly important. Justo et al.
(1995), De Santis et al. (2001), and Vieytes et al. (2007) studied the incisors’
schmelzmuster (i.e., enamel pattern) in ctenomyids. They found out that the thicker
external (radial) enamel would be present when a more intense tooth-digging habit
is performed, in order to withstand the concomitant microgrinding on the surface of

incisors from the soil. Nevertheless, while soil content and texture seem to be highly
linked to the external enamel properties (Justo et al. 1995; De Santis et al. 2001),
bite force does not follow the same trend (e.g., C. australis and C. haigi inhabiting
sandy dunes and more compact, clayey soils, respectively; Fig. 7.3). On the inner
side, those authors found that the enamel’s bulk is organized in Hunter-Schreger
bands (decussated prisms in a nearly orthogonal setup), preventing cracks from
propagating as a consequence of surface fracture during tooth-digging. Beyond any
macroscopic property of incisors (e.g., the angle of protrusion from the jaws, bend-
ing, and torsion stress resistance), their microscopic architecture seems to be spe-
cially molded by nature giving tuco-tucos a “high-performance” tool for digging.
From a more holistic (and anatomical) point of view, the amount of force applied
at the incisors’ tip strictly depends on the effective force (i.e., the fraction of the
muscle force being transmitted to the biting point; for a more detailed discussion,
see Becerra et al. 2014) and the mechanical advantage of the jaws adductor muscu-
lature (see the “Biomechanical approach of digging” section). It has been assumed
for quite a long time that the shorter snout exhibited by tuco-tucos would lead to a
shorter out-lever arm and, therefore, to a more advantageous system (Hildebrand
1988; Vassallo and Verzi 2001; Verzi 2002). Nevertheless, Becerra et al. (2014) ana-
lyzed how the biting biomechanics change between tuco-tucos and non-digging,
social, caviomorph rodents, and strikingly showed that the snout shortening is
highly related to a backward displacement of the muscles line of action, keeping the
mechanical advantage fairly similar. In other words, it might be possible that these
overall proportions have been inherited from a rodent generalized developmental
model (i.e., bauplan) already fairly optimized, from which all lesser deviations
radiated.
On a mesoscale, it would be logical to think that the angle at which incisors pro-
trude from the jaws slightly but directly affects the out-lever arm, and inversely do
so to the mechanical advantage and bite force (on equivalent muscle input setups).
Yet, previous studies have found some positive correlation between procumbency
angle and a burrowing lifestyle, beyond phylogenetic or body size differences
(Lessa 1990; Lessa et al. 2008; Becerra et al. 2012b). These results might be indicat-
ing that this “mechanically disadvantageous” condition would be negligible com-
pared to the great improvement of having front-forwarded incisors for better
anchoring to the soil. Within the genus Ctenomys, on the other side, the story seems
to differ in one step further showing no clear pattern of procumbency variation with
regard to skull’s gross morphology or environment (see Mora et al. 2003; Verzi et al.
2010; Echeverría et al. 2017). This fact leads us to consider a putative scenario, at
which tuco-tucos as a group would have already achieved a state of the character
above what is strictly needed for tooth-digging. Therefore, at the highly specialized
ctenomyid bauplan – much more than the generalized caviomorph one – incisors
would protrude at an angle wide enough to withstand the mechanical demands of
soil break down, and deviations from this model would be unrelated to these physi-
cal needs.
On the flip side, and being negligible the above-mentioned differences in propor-
tion of bony elements (i.e., mechanical advantage), differences in bite force should
be a consequence of mere behavioral dissimilarities, or of an uneven degree of
development for the adductor musculature. In this regard, Becerra et al. (2014)
showed that when both superficial and deep masseter muscles and both heads of the
zygomaticomandibularis muscle (sensu Druzinsky et al. 2011; Table 7.1) pull in
concert, they account for over 90% of the bite force. For all of them, except for the
head of the latter coming through the infraorbital foramen (plesiomorphic condition
for all hystricomorph rodents), a significantly greater degree of development was
coincidently shown for tuco-tucos than for other non-subterranean caviomorph
rodents (Becerra et al. 2011, 2013, 2014). A closer look at all jaw adductor muscles
across ctenomyids, based on data from these last references, showed that they do not
depend on body size (all p-values > 0.05). We could, then, assume that the strong
biting that tuco-tucos apply during tooth-digging has not been evolutionarily
achieved by rearranging the skull morphology, but by hypertrophying their muscu-
lature. Likewise, preliminary studies that analyze the masseteric musculature of
C. talarum and Cavia aperea, a non-digging and gregarious caviomorph rodent,
showed an increase in the heterogeneity of fiber types in tuco-tucos (Fig. 7.4), with
higher proportions of fibers capable of generating more powerful forces (Longo
et al. 2017, 2018).
Summarizing, ctenomyid species have evolved associated to an underground
lifestyle, developing some anatomical specializations which help them accomplish
tooth-digging tasks. Although gross head morphology does not enhance their dig-
ging performance, their hypertrophied adductor jaw musculature lets them apply
biting forces stronger than needed to easily break down the soil and obstacles while
burrowing. Moreover, incisors macro- and microstructure allow tuco-tucos not only
to attack the soil at an advantageous angle but also to withstand the high stress and
abrasion produced by specific clayey, silty, or sandy soils. Overall, the fact that
specializations observed within this genus (e.g., bite force, adductor musculature
development, and procumbency angle) clearly exceed the particular needs for tooth-
digging could respond to a highly derived bauplan, or to cover other (behavioral?)
needs; e.g., mating competition. Being that for one or the other, it could also explain
the lack of association between any of those specializations and soil conditions.
7.5 Structural Strength of Bones
When a subterranean rodent like the tuco-tuco digs using the so-called digging
tools, it must exert forces strong enough to break down the soil, which is allowed by
their powerful forelimb and mandibular muscles. In turn, when these forces are
exerted against the substrate, the digging tools receive reaction forces of equal mag-
nitude and opposite direction (i.e., the Newtonian principle of action and reaction,
which also applies to subterranean rodents! See vector Fo in Fig. 7.1). These reac-
tion forces are transmitted to the rest of the body, in particular to the bones, which
Fig. 7.4 Fiber types of superficial masseter muscles in Ctenomys talarum (A, B) and Cavia aperea
(C, D). (A, C) Succinate dehydrogenase activity, indicative of the oxidative capacity of fibers. (B,
D) Periodic acid Schiff, to detect the glycolitic activity of fibers. Different staining intensities
indicate different fiber types. Scale bars: 100 μm
must be able to resist them without structural failure, i.e., without breakage or per-
manent deformation. Since these forces are concentrated in places such as sites of
muscle origin and insertion, sites of contact between bones (e.g., joints), or the tip
of digging tools, this force per unit area which can be expressed as stress and mea-
sured in pascals (i.e., newton per square meter). As an adaptation to these efforts and
their concomitant stresses, not only the claws and incisors but also the limb long
bones and skull of subterranean rodents are relatively more robust than those of
their non-digging species counterparts (Becerra et al. 2012b; Echeverría et al. 2014;
McIntosh and Cox 2016). One method to estimate the stress that these forces pro-
duce is by means of the development of a virtual model (Fig. 7.5A, B) with the same
geometry as real bones, both internally and externally, from axial tomographic
images (Fig. 7.5C) and specific software for image integration (see it in detail in
Buezas et al. 2019). The mechanical properties of bone are assigned to this model,
a procedure known as finite element analysis (FEA; Rayfield 2007). In a second
stage, the model is subjected to in silico experiments, at which the application and
reception of forces are simulated. Ideally, these forces should be consistent with the
real forces that animals exert in nature. To measure these forces, experimental val-
ues are obtained from live animals with force transducers, while biomechanical
estimations are based upon muscle dissections and lever arm measurements (e.g.,
Becerra et al. 2011, 2013, 2014). By using a FEA approach, it was quantified that
the stress to which C. talarum skull is subjected to during tooth-digging can vary in
Fig. 7.5 Structural bone resistance in C. talarum’s skull. Finite element analysis (FEA) showing
the stress to which the cranium (A) and mandible (B) are subjected during incisor biting. The FE
model was loaded with the corresponding jaw adductor muscle forces, which resulted in a bite
force similar to the one measured in live specimens using a force transducer. Areas subjected to
relatively high stresses are towards the red side of the spectrum (von Mises stress >25 MPa). Note
that, within the cranium, these areas spread throughout the posterior portion of the zygomatic arch
(zones 1, 2, 6), dorsal origin of the anterorbital bar (4), dorsal- and ventral-most regions within the
diastema (5, 7), and the lingual side of incisors. In the mandible, areas of high stress mainly spread
throughout the ascending ramus and anterior end of masseteric crest (1, 5). Descriptions, stress
values and safety factors for numbered zones are provided in Table 7.3. Coronal microCT slice at
the level of molar 1 (C), highlighting some features responsible of the architecture-based mechani-
cally enhanced skull in this species (1, thick frontal bones; 2, anterorbital bar; 3, enlarged maxillar
bones; 4, long-rooted molariform teeth; 5, thick mandible; 6, incisor roots moving back to the
ascending ramus). Scale bars: 5 mm.
more than one order of magnitude depending on the anatomical region being con-
sidered (Buezas et al. 2019).
Another question related to structural strength is: what is the relationship between
the (theoretical) maximum stress that the bone can withstand, and the correspond-
ing values achieved during the real performance in nature? A proper way to answer
this question is by calculating the safety factor (SF), a mechanical estimation of how
below the stress at a certain point is from that one at which bone begins to experi-
ence a nonreversible change of shape in response to applied forces (e.g. Currey
2006). Therefore, these values are computed as the quotient between the critical
yield stress and the local von Mises stress. It is shown that stress and SF values are
more heterogeneous within the mandible than on the cranium in C. talarum
(Table 7.3). This is because of the natural variation of mandibular bone thickness:
thick at the diastema and the mandibular corpus, where incisor and molar roots are
hosted (regions 3 and 4, respectively), and relatively thin in the ascending ramus
(regions 2 and 5).
Table 7.3 Anatomical descriptions, von Mises stress values (in MPa), and safety factors (SF) in
14 regions across the mandible and cranium in Ctenomys talarum (see Fig. 7.5), corresponding to
an incisor bite force of 32 N
Cranium Mandible
Stress Stress
Region (MPa) SF Region (MPa) SF
1 Most posterior end of the 84.19 1.94 1 Region between the 66.7 2.4
zygoma coronoid and the condyloid
processes
2 Most dorsal region of the 26.87 6.07 2 Base of the coronoid 21.2 7.7
zygoma process
3 Central region of the 18.21 8.95 3 Dorsal region of the 3.3 49.4
zygoma mandible, at the level of
the premolar
4 Dorsal origin of the 25.72 6.34 4 Dorsal region of the 9.7 16.8
anterorbital bar diastema
5 Frontal region at the level of 19.31 8.44 5 Anterior end of the 52.8 3.1
the diastema’s posterior end masseteric crest
6 Postero-ventral region of the 19.23 8.95 6 Lateral region of the 6.2 26.3
zygoma diastema
7 Posterior end of the 23.73 6.87 7 Antero-ventral region of 6.5 25.1
diastema – anterior end of the diastema
the zygoma
Summarizing, biting forces received at incisors tips during chisel-tooth digging

are directly transmitted to the skull, a structure which provides anchorage and pro-
tection to sensory organs and the central nervous system. Bone distribution and
skull geometry in subterranean rodents like tuco-tucos can have significant effects
on the adequate dissipation of biting forces and stress dampening. Together with the
complexity of the cranial sutures (Buezas et al. 2017), these traits seem to be impor-
tant structural adaptations related to the burrowing behavior.
7.6 Conclusions
As reviewed above, the predominant mode of digging among subterranean rodents

is scratch-digging, followed by chisel-tooth digging when soils or underground
obstacles become tougher to disaggregate or break down. Tuco-tucos – as other
subterranean mammals – are characterized by several physiological, morphological,
and behavioral adaptations related to their subterranean lifestyle (Nevo and Reig
1990; Nevo 1999) and, strikingly, they are among the few subterranean rodents
capable of performing those two digging methods on an efficient manner (see other
remarkable examples in Jarvis and Sale 1971; Hickman and Brown 1973; Lessa and
Thaeler Jr. 1989). This functionally dual capacity – coupled with the highly opti-
mized biomechanics of both musculoskeletal systems – allows tuco-tucos to inhabit
a wide range of habitats and soils, from the Andean Puna above 4000 m to the
coastal sandy dunes of the Atlantic, and from the mesic and humid Pampas to the
dry Chaco and Monte desert (Reig et al. 1990; Bidau 2015; Fig. 7.1). Particularly,
the soil types where they are found include very friable and soft sandy soils; grav-
elly soils; hard, clayey soils, hard soils rich in humus, and with a low percentage of
silt or clay; sandy rock-free soils; well-compacted, humus-rich, or loamy soils; deep
humid soils; black soil but extremely light; compact sandy soils, among others (see,
for example, Table 7.1 in Echeverría et al. 2017). However, in spite of occupying a
wide geographical and environmental range, the ecological niche they occupy and
the behaviors they exhibit are broadly homogeneous (Mora et al. 2003). In general
terms, subterranean rodents classified as scratch-diggers according to their morpho-
logical specializations are often found in sandy and friable soils, whereas chisel-
tooth diggers are found in a broader range of soils (Lessa and Thaeler Jr. 1989).
Thus, species such as C. talarum (both chisel-tooth and scratch-digger) efficiently
performs in both well-compacted, humus-rich, or sandy soils – where the fairly
sympatric C. australis (a mainly scratch-digger) is confined (Vassallo 1998).
Concluding, other factors – besides morphofunctional specializations – such as
physiological adaptations or predation pressure might be constraining the presence
of a particular species in specific soils, and this must be taken into account to explain
habitat preferences in Ctenomys species.
Acknowledgments We thank the editors for the invitation to contribute to this collective volume
on Ctenomys evolution. This study was carried out under the support of the National Council for
Scientific and Technical Research of Argentina (CONICET) (grants PIP 2014–2016 N°
11220130100375 and UE N°0073) and National University of Mar del Plata (grants EXA918/18
and ING516/18). We thank colleagues from various countries and laboratories for the friendly
exchange of knowledge about an underground friend in common, the Tuco-Tuco.
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Chapter 8
Adaptive Pelage Coloration in Ctenomys
8.1 Introduction
In rodents, pelage color tends to resemble background habitat coloration, suggest-

ing an adaptive significance (Sumner 1934; Dice and Blossom 1937; Cott 1940;
Endler 1978; Krupa and Geluso 2000). But how does it work in subterranean lin-
eages, in which individuals expend most of their lifetime in burrowing systems
(Lacey et al. 2000)? Surprisingly, long stand studies in pocket gophers (Thomomys
bottae and Geomys bursarius) and the Israeli subterranean mole rat (Spalax ehren-
bergi) have demonstrated similar patterns to aboveground rodents, i.e., a strong cor-
relation between dorsal pelage and soil coloration (Ingles 1950; Kennerly 1954,
1959; Krupa and Geluso 2000; Heth et al. 1988), presumably reflecting an influence
of selective pressure when they are active on the surface. This concealment color-
ation is also substantiated in Ctenomys (Langguth and Abella 1970; Vassalo et al.
1994), in which pelage color varies continuously, both inter- and intraspecies
(Langguth and Abella 1970; Freitas and Lessa 1984; Wlasiuk et al. 2003; Gonçalves
and Freitas 2009; Gonçalves et al. 2012). Overall, coat coloration ranges from light
to dark brown in tuco-tucos (Fig. 8.1). However, brown with white patterns, grayish,
and melanic phenotypes are also present. Similarly, variation is found in the back-
ground environment, as species are spread throughout South America, including a
variety– and vast areas – of habitats, e.g., pampas of Puna (above 4000 m), high
mountain steppes, low valleys of the west, dunes of the Atlantic coast of the east,
mesic and humid plains, desert or semi-deserts, open areas among subtropical
G. L. Gonçalves (*)
Departamento de Genética, Universidade Federal do Rio Grande do Sul,
Porto Alegre, RS, Brazil
Departamento de Recursos Ambientales, Facultad de Ciencias Agronómicas,
Universidad de Tarapacá, Arica, Chile
e-mail: lopes.goncalves@ufrgs.br

https://doi.org/10.1007/978-3-030-61679-3_8
168 G. L. Gonçalves
Fig. 8.1 Inter- and intraspecific variation in pelage color of Ctenomys. (a) general view of a speci-
mens’ drawer of tuco-tucos from the Museum of Vertebrate Zoology (MVZ), revealing the typical
brown pattern found. (b) C. torquatus; (c) C. yolandae; (d) C. haigi; (e) C. bonettoi; (f) C. roigi;
(g) C. dorbigny; (h) C. magellanicus; (i) C. maulinus; (j) C. sociabilis; (k) C. argentinus; (l)
C. perrensi; (m) C. mendocinus; (n) C. fulvus; (o) C. peruanus; (p) C. opimus. (Photographs
(except b [from G. L. Gonçalves]) by T. R. O. Freitas – courtesy of mammal collection from the
Museum of Vertebrate Zoology, UC Berkeley)
forests, and steppes of Terra del Fuego (Reig et al. 1990; Lacey et al. 2000; Bidau
2015; Freitas 2016).
In particular, two pairs of species that live in the Atlantic coast catches not only
evolutionary biologists but anyone’s eyes for its marked differences in pelage
8 Adaptive Pelage Coloration in Ctenomys 169
associated with habitat background. The first is Ctenomys australis Rusconi, 1934
and Ctenomys talarum Thomas, 1989, occurring in a coastal dune region in south-
ern Buenos Aires province of Argentina (Contreras and Reig 1965; Reig et al. 1990),
and the second is Ctenomys flamarioni Travi, 1981 and Ctenomys minutus Nehring,
1887 (Freitas 1995a, b), which inhabit the southern Brazil coastal plain (Fig. 8.2a).
C. australis and C. flamarioni have blonde coat color (light phenotype) and inhabit
the sandy dunes, whereas C. talarum and C. minutus have brown pelage (dark phe-
notype) and inhabit sandy fields (Fig. 8.2b) that correspond to a continuum of
coastal dunes toward the continent (Freitas 1995a, b; Busch et al. 2000) (Fig. 8.3a);
these two habitats can be distinguished by soil color (Fig. 8.3b) and hardness, and
plant cover (Malizia et al. 1991; Cutrera et al. 2010; Kubiak et al. 2015; Lopes et al.
2015; Kubiak et al. 2018). Phylogenetic relatedness between and within these pair
of species also vary. C. australis, C. talarum, and C. flamarioni belong to the men-
docinus species group, whereas C. minutus are placed in the torquatus species group
(Parada et al. 2011; Chap. 2, this volume). In this context, the repeated phenotypes
might represent convergence to similar habitats, in which ecological function is
potentially cryptic anti-predation behavior (Langguth and Abella 1970; Vassalo
et al. 1994), which has never been explored.
Two studies have investigated pelage variation in Ctenomys from an evolutionary
genetics perspective. First, Wlasiuk et al. (2003) demonstrated that genetic drift
underlies pelage forms in different populations of Ctenomys rionegrensis Langguth
and Abella (1970) that include brown, dark-backed, and melanic phenotypes.
Second, Gonçalves et al. (2012) performed a molecular approach targeting a key
gene-driven of coatcolor – the Melanocortin 1 receptor (MC1R) –, including a wide
range of species with distinct color pelages.
Fig. 8.2 (a) Geographic distribution of Ctenomys flamarioni (FLA), Ctenomys minutus (MIN),
Ctenomys australis (AUS), and Ctenomys talarum (TAL) in the coastal plain of Argentina and
southern Brazil with schematic shades of its pelage. (b) Convergence pattern of light-dark pheno-
types (FLA-MIN and AUS-TAL) inhabiting contiguous habitats of sandy dunes and sandy fields
Fig. 8.3 (a) Habitats of Ctenomys in the coastal system: sandy dunes and sandy fields. (b) Soil
coloration of each species’ habitat. TAL, C. talarum; MIN, C. minutus; AUS, C. australis; FLA,
C. flamarioni
Hair and skin color in rodents are largely determined by the amount, type, and
distribution of melanin packaged in the melanosomes of epidermal cells and hair
follicles (Jackson 1997). Mc1r acts as a pigmentary switch in the production of
melanin: when activated by α-melanocyte stimulating hormone (α-MSH), it signals
the production of black/brown pigment (eumelanin) and in the absence (or inhibi-
tion) of a-MSH, red/yellow pigment (pheomelanin) is synthesized (Jackson 1997).
In mice, Mc1r dominant mutations are often associated with a hyperactive or con-
stitutively active receptor resulting in predominantly black coat color (Jackson et al.
1994), whereas recessive loss-of-function mutations tend to trigger the production
of pheomelanin, which leads to predominantly yellow or red coat color (Robbins
et al. 1993). Similarly, in wild rodents several mutations were identified in Mc1r,
and associated with the adaptive variation, e.g., the rock pocket mice (Chaetodipus
intermedius; Nachman et al. 2003) and the beach mice (Peromyscus maniculatus;
Hoekstra et al. 2006); also, melanism in British gray squirrel (Sciurus carolinensis)
was linked to a 24-bp deletion in Mc1r (McRobie et al. 2009). In tuco-tucos, several
coding substitutions were detected in Mc1r (Gonçalves et al. 2012), but none of
them with plausible link to the phenotypes examined, especially the light pelage of
C. australis and C. flamarioni, or melanic forms of C. rionegrensis and C. tor-
quatus. Additionally, patterns of Mc1r expression were described for dorsal, flank,
and ventral regions, but differences were not found between light and dark pheno-
types; even though, the distinction among body regions was clear (Gonçalves
et al. 2012).
8.2 Pelage Variation: From Genotype to Phenotype
Simple Mc1r mutations of large effect have not contributed to adaptive differences
among species of tuco-tucos, thus the variation in coat-color among Ctenomys sug-
gests that this trait might have a more complex or even polygenic basis. Finding the
genes underlying this variation is probably a daunting task, which will require map-
ping and association studies involving more markers and defined populations. A
suitable candidate gene is the Agouti signaling protein (Agouti), an antagonist of
Mc1r; in mice, a local expression that varies both spatially and temporarily (Bultman
et al. 1992; Siracusa 1994) results in suppression of synthesis of eumelanin and
increased production of phaeomelanin. Agouti is the second most important gene
linked with adaptive pelage color variation in rodents (e.g., beach mice (Steiner
et al. 2007)), which remains to be explored, particularly in the blonde pelages of
tuco-tucos, such as in C. australis, C. flamarioni, and C. mendocinus that also pres-
ent an intraspecific variation of lighter pelage (see Fig. 8.1).
Typically, wild rodents have a pelage pattern of light ventral, which results from
constitutive Agouti expression and associated production of phaeomelanin. In con-
trast, dorsal hairs have a banded pattern (commonly referred to as agouti hair): ter-
minal and subterminal bands and a base. This banding derives from a pulse of
Agouti expression during the intermediary phase of the hair cycle, resulting in the
deposition of phaeomelanin during the middle of hair growth and deposition of
eumelanin at the beginning and end of hair growth (Hoekstra and Nachman 2006).
In the agouti-type pelage distinct variables may be target by the selection, as the
distribution of pigment, i.e., bandwidth, and the density of pigment deposited in it,
resulting in lighter or darker phenotypes. A few studies have dissected the pigment
structure in the hair (e.g., Peromyscus (Linnen et al. 2009); Spalax (Singaravelan
et al. 2010, 2013)) and ultimately inferred its contribution to overall appearance and
convergence as well.
In this chapter, an original study on the pigmentation of Ctenomys is reported
from a morphological perspective, hypothesizing an association of pelage and soil
coloration. The hair pattern and pigment density are characterized in species of
tuco-tucos from the Atlantic Coastal dune system that present repeated adaptive
phenotypes, to test the existence of convergence.
8.3 Quantifying Hair, Pelage, and Soil Coloration
In vertebrates, the visible color spectrum typically ranges from 400 to 700 nm (blue
to red) (Krupa and Geluso 2000). Therefore, it was used to measure the pelage and
soil coloration. A total of 123 specimens of C. talarum (TAL = 20), C. minutus
(MIN = 40), C. australis (AUS = 28), C. flamarioni (FLA = 35) from both field-
caught and taxidermized specimens from scientific collections of the following
institutions were used: Universidade Federal do Rio Grande do Sul (UFRGS),
Universidad Nacional de Mar del Plata (UNMDP), and Museo Municipal de

Ciencias Naturales Lorenzo Scaglia (MCNLS) (Appendix). For each specimen,
three body regions were analyzed: dorsal, flank, and ventral, determined to infer
distinct selective pressures, since there are a differential influence on the individu-
al’s overall coloration (i.e., dorsal and flank are considered more relevant for evolu-
tion (see Linnen et al. 2009; Manceau et al. 2010)).
Quantification was obtained by the pixel densitometry method; the unit is defined
as Gray for the RGB (red, green, and blue) system. Samples (pelage and soil) were
photographed with the Munsell (X-Rite Inc.) universal color card to correct the
value obtained in relation to the standardized black and white estimates, using the
following formula:
Obtained Value - Black

Calculated Color =
White - Black
The photographs were analyzed using the software AxioVision version 4.8 (Carl
Zeiss Microimaging System Inc.); the central area was delimited using the outline
spline tool, and the densitometry values were individually generated for red, green,
and blue pixels. For each sample, three measurements were performed and averaged
for each pixel. Then, the global average of RGB pixels was calculated.
Microscopic slides were prepared for the dorsal, flank, and ventral regions of
each specimen, plucking 10 guard hairs per individual per region. Hairs were rinsed
in 50% ethanol and immersed in colorless enamel under the coverslip. Each slide
was photographed with a Sony® Cybershot DS20 camera attached to the Leica®
M125 stereoscopic microscope using the 0.8X magnification for the whole hair, and
10X for the terminal and subterminal band images. The photographs were analyzed
using the AxioVision, measuring hair width, and terminal and subterminal band-
width. Also, the densitometry values of the pigment deposited in the terminal and
subterminal bands were analyzed, zooming the same region analyzed (largest diam-
eter) for all species.
For habitat characterization, soil samples were collected along an 80 m-transect,
randomly delineated in each habitat. For C. flamarioni and C. minutus sampling was
placed in Xangri-lá (29o47′S; 50o01′W) and Osório (29o31′S; 50o32′W)
Municipalities, in southern Brazil. For C. australis and C. talarum, sampling sites
were located in Necochea Municipality (38o03′S; 57o49′W and 38o02′S; 57o56′W,
respectively), in Argentina. Soil samples were taken from the surface in every 10 m
of transects and stored in 15 ml tubes. Additionally, eight samples were randomly
taken from the burrowing system of each species for sampling comparison of under-
ground vs aboveground. A total of 64 samples were individually placed in Petri
dishes and dehydrated at 58 °C for 24 h. For plant coverage analysis, a specific area
was photographed in each sampling stations of C. minutus and C. flamarioni, using
a 1 m tape measure at the center of the image as a reference, in order to standardize
the area (1 m2). The percentage of plant coverage was estimated using the Braun-
Blanquet method (1932). Previously published data from C. australis and C. tala-
rum were taken from Cutrera et al. (2010).
Normal distribution of variables was tested using the Kolmogorov-Smirnov test,

which is suitable for small sample sizes (Steinskog et al. 2007). Also, the heteroge-
neity of variance was tested with Bartlets test. Most of the data fit in a normal curve;
however, significant heterogeneity variance was found. Thus, the data were treated
as nonparametric. For comparisons in dorsal, flank, and ventral regions for differ-
ences in the distribution (bandwidth) and density (color) of pigment deposited in
hair and pelage, the Kruskal-Wallis nonparametric test was used, followed by
Dunn’s multiple paired comparisons; the p-value (<0.05) was adjusted for multiple
comparisons using the Bonferroni. Also, this test was used to compare microhabitat
characteristics (soil coloration and plant cover) among species. To test the existence
of an association between soil and pelage (dorsal, flank, and ventral) color, a simple
linear regression analysis was used. Statistical analyzes were performed using the
software XLSTAT (Addinsoft). Results of bandwidth/hair width, densitometry anal-
ysis, and substrate color are presented using the box-plots graphical method, includ-
ing minimum and maximum values, mean, first, and third quartiles; other values are
presented as mean (χ) ± standard error (SE).
8.4 Phenotypic Variation: Pigment Distribution and Density
In the dorsal pelage, tuco-tucos have the agouti hair type, presenting the banding
pattern with black and yellow pigments alternately deposited (Fig. 8.4). In the flank
and ventral hairs, the terminal band is absent. FLA has almost no pigment in ventral
hairs; when present, it is composed only by pheomelanin. In the other three species,
hairs from the flank and ventral regions have two-band patterns (subterminal and
base), with pheomelanin in a lower density. Differences in the width of the terminal
and subterminal bands were observed between light and dark phenotypes (Figs. 8.4,
8.5, and 8.6). TAL and MIN have the proportional widest terminal band and the
shortest subterminal band in dorsal hairs; conversely, AUS and FLA present propor-
tionally shortest terminal width and widest subterminal band in such region
(Fig. 8.6). Contrary, the subterminal band in flank and ventral hairs did not vary
significantly (also in proportion) between light and dark phenotypes (Fig. 8.6).
Differences in dorsal pelage coloration were identified among species of tuco-
tucos, also within the light and dark phenotypes (Fig. 8.7): TAL and MIN presented
significantly higher pigment density compared to AUS and FLA. Thus, TAL repre-
sents the darkest phenotype, whereas FLA the lightest. In the flank, the dark pheno-
types significantly differ to the light ones; within phenotypes, differences were
found only for light pelages (Fig. 8.7). In the ventral region, there were no signifi-
cant differences between light and dark phenotypes (TAL, MIN, AUS). However,
FLA showed marked distinction to all other species (Figs. 8.4 and 8.7). Significant
differences in the pigment density within the terminal band were found between
phenotypes (Fig. 8.7): dark species presented lower values compared to light ones.
Similarly, dark phenotypes had distinct values for the subterminal band compared
to the light ones.
Fig. 8.4 Schematic representation of Ctenomys dorsal, flank, and ventral hairs, in scale. TAL,
C. talarum; MIN, C. minutus; AUS, C. australis; FLA, C. flamarioni
These results suggest a remarkable influence of the density deposited in the ter-
minal and subterminal bands on the overall coloration of an individual. Accordingly,
the greatest functionality is supposed for the dorsal region in comparison to the
flank (that is less intense) reinforced by the small variation. Results of pigment
density in the ventral hairs corroborated this hypothesis, since no significant differ-
ences were found for the subterminal band and overall coloration between light and
dark phenotypes (Fig. 8.7). Since the ventral region is relatively less exposed, the
widest range of variation found might result from selective pressure relaxation. To
test this assumption, the variances were estimated in several parameters analyzed
(e.g., terminal and subterminal bandwidth, total hair width, pigment density within
the terminal and subterminal bands in dorsal, flank, and ventral regions); eight of
them presented heterogeneity among species. Not surprisingly, most occurred in
parameters taken from the flank and ventral regions. In the dorsal, the terminal
bandwidth shown the lowest values in light phenotypes. The dark phenotypes
Fig. 8.5 Box-plots representing variability found in the terminal band (a), the subterminal band
(b), and total hair (c) width in the four Ctenomys species: TAL, C. talarum; MIN, C. minutus; AUS,
C. australis; FLA, C. flamarioni, showing the mean and first and third quartiles. Different letters
over the box-plot indicate statistical significance between species, within each body region ana-
lyzed (dorsal, flank, and ventral). The colors indicate the phenotypes (see Fig. 8.2 and inlet sche-
matic legend)
Fig. 8.6 Box-plots

representing variability
found in the proportional
width of the terminal (a)
and the subterminal bands
(b) in the species of
Ctenomys: TAL,
C. talarum; MIN,
C. minutus; AUS,
C. australis; FLA,
C. flamarioni, showing the
mean, and the first and
third quartiles. Different
letters over the box-plot
indicate statistical
significance between
species, within each body
region analyzed (dorsal,
flank, and ventral). The
colors indicate distinct
phenotypes (see Fig. 8.2
and inlet schematic legend)
Fig. 8.7 Box-plots representing variability in hair density for the terminal (a) and subterminal (b)
band and for the pelage (c) in the species of Ctenomys: TAL, C. talarum; MIN, C. minutus; AUS,
C. australis; FLA, C. flamarioni, showing the mean and first and third quartiles. Different letters
on the box-plot indicate statistical significance between species, within each body region analyzed
(dorsal, flank, and ventral). The colors indicate distinct phenotypes (see Fig. 8.2 and inlet sche-
matic legend)
showed 4–10 times the greatest variance compared to light for terminal bandwidth;
therefore, the variation might be constrained in markedly cryptic light phenotypes,
suggesting greater selective pressure (Table 8.1). Contrary, in the densitometry
parameter the subterminal band showed similar variance in light and dark pheno-
types, thus indicating less influence on the overall coloration comparatively to the
terminal bandwidth. Significant differences in soil coloration were found between
the two habitats (Fig. 8.8). No significant differences were observed between sur-
face and burrow soil samples within sandy fields (TAL, P = 0.48; MIN, P = 0.35)
and dunes (AUS, P = 0.06; FLA, P = 0.06), allowing sufficient representativeness
of samples from transects. Similar to dorsal coat color, soil from TAL microhabitat
had a higher density (i.e., lower values), whereas for FLA indicated the lowest den-
sity (i.e., higher values) (Table 8.2).
Additionally, linear regression analysis indicated a strong association (R2 = 0.87;
P < 0.001) of soil with dorsal and flank coat-color (R2 = 0.71; P < 0.001), and mod-
erate association with ventral (R2 = 0.57; P < 0.001), which is mainly influenced by
FLA pelage values in relation to other species (Fig. 8.9). For plant coverage, two
markedly distinct groups of values were recovered, one representing sandy fields
and the other sandy dunes (Table 8.2). Estimates from sandy fields were twice as
high as those in sandy dunes, and did not differ significantly (P > 0.05) between
similar microhabitats (Table 8.2).
8.5 Cryptic Coloration in Ctenomys
Tuco-tucos have a predominantly fossorial habit; though they are also active aboveg-
round, particularly foraging in close to burrows (Comparatore et al. 1991; Busch
et al. 2000). In particular, a high frequency of mobility was described for TAL
(Busch et al. 1989), AUS (Vassalo et al. 1994), and FLA (Fernández-Stolz et al.
2007; Stolz 2006). Contrary to Spalax, in which aboveground exposure is recog-
nized as accidental (Heth 1991), the regular activity in open areas indicates that
predation might be more common in Ctenomys than any other subterranean lineage.
Ctenomids are often preyed on by several vertebrates, for example, burrowing
owl (Athene cunicularia), pampas fox (Pseudalopex gymnocercus), lesser grison
(Galictis cuja), white-eared opossum (Didelphis albiventris), Molina’s hog-nosed
skunk (Conepatus chinga), small hairy armadillo (Chaetophractus vellerosus), and
Neuwied’s lancehead (Bothrops neuwidi) (Busch et al. 2000).
Specifically, TAL and AUS represent 16% and 2%, respectively, of owl prey
items (Athene cunicularia, Asio flammeus, and Tyto alba) (Vassalo et al. 1994), and
such difference is attributed to markedly distinct body sizes (TAL ca. 118 g and
AUS ca. 360 g). Predation in AUS occurred predominantly in subadult individuals,
likely due to constrain of the predator in carrying out the prey (Vassalo et al. 1994).
However, there is no data on the influence of cryptic behavior in these, or any other,
Ctenomys species preventing predation (i.e., differential survival), linking to micro-
habitat selection.
Table 8.1 Analysis of significant variance among Ctenomys talarum (TAL), Ctenomys minutus
(MIN), Ctenomys australis (AUS), and Ctenomys flamarioni (FLA) for different hair and pelage
parameters
Parameter Species Var. χ2calc P
Terminal width – dorsal 45.72 <0.001
TAL 8.24
MIN 26.16
AUS 1.19
FLA 2.83
Total hair width – ventral 31.46 <0.001
TAL 269.22
MIN 528.77
AUS 191.98
FLA 28.29
Subterminal band width – flank 9.10 0.02
TAL 22.25
MIN 28.27
AUS 31.85
FLA 76.89
Subterminal band width – ventral 7.92 0.04
TAL 15.31
MIN 15.41
AUS 4.68
FLA 8.83
Subterminal band densitometry – dorsal 9.38 0.02
TAL 16.85
MIN 57.85
AUS 55.32
FLA 25.74
Subterminal band densitometry – flank 9.10 0.02
TAL 22.25
MIN 28.27
AUS 31.85
FLA 76.89
Subterminal band densitometry – ventral 7.92 0.04
TAL 15.31
MIN 15.41
AUS 4.68
FLA 8.83
Pelage densitometry – flank 16.58 0.001
TAL 211.99
MIN 27.93
AUS 132.68
FLA 123.96
Fig. 8.8 Association of

the color of the soil with an
appearance in the species
of Ctenomys: TAL,
C. talarum; MIN,
C. minutus; AUS,
C. australis, FLA,
C. flamarioni. (a)
Box-plots representation of
the variability found in the
color of the soil (filled
boxes) and dorsal pelage
(non-filled boxes), showing
the mean, and first and
third quartiles. Different
letters on the box-plot
indicate statistical
significance between
species, within a given
analyzed body region. The
colors indicate distinct
phenotypes (see Fig. 8.2
and inlet schematic legend)
Table 8.2 Estimates of mean ± standard error of soil color and plant cover in the microhabitats of
C. talarum (TAL), C. minutus (MIN), C. australis (AUS), and C. flamarioni (FLA)
Sandy fields Sandy dunes Kruskal–Wallis
TAL MIN AUS FLA Kobs P
Soil coloration 58.14 ± 2.00 70.77 ± 1.78 82.03 ± 1.37 111.06 ± 0.85 53.37 < 0.001
Plant cover 57.18 ± 16.12 60.50 ± 12.31 25.31 ± 17.36 30.50 ± 10.72 43.56 < 0.001
Fig. 8.9 Linear regression

of soil color by coat color.
The circles represent
Ctenomys talarum and
Ctenomys flamarioni, and
the triangles Ctenomys
minutus and Ctenomys
australis. The colors
indicate the phenotypes
(see Fig. 8.2)
This study characterizes, for the first time, hair, pelage, and soil coloration in
tuco-tucos. Specifically, data on distribution and density of pigment deposited in the
terminal band of dorsal hairs highlights the biological relevance of such region also
in this lineage. The smallest variances were found in the light phenotypes; therefore,
dorsal coloration of AUS and FLA might be more restricted to vary. Accordingly,
subtle changes in coat-color in these two species might contrast in pale dunes that
present low plant cover (i.e., more exposed area), making them more susceptibility
for predation capture, which remains to be tested. These species have the highest
dispersion rates in the genus (Stolz 2006; Vassalo 1998; Garcias et al. 2018), rein-
forcing the importance of cryptic behavior, assuming intense selective pressure in
this system. In contrast, the widest range of variation was found in TAL and MIN,
which may reflect the complexity of microhabitat with the highest plant cover,
favoring camouflage despite an individual’s overall coloration. Consequently, subtle
changes in coloration of these dark phenotypes are unlikely to have an intense effect
on differential survival. Moreover, a relevant aspect in MIN is the empirical obser-
vation that young individuals (2–3 months old) are lighter in color than adults
(Fonseca 2003).
This corroborates the hypothesis of local adaptation, whose function is to protect
young specimens that, in general, are the main target of predation (Vassalo et al.
1994; Lacey 2000). Although the species pairs occur in allopatry with areas of sym-
patry (Kubiak et al. 2015), they clearly present microhabitat selection, differing in
relation to soil hardness, plant biomass, and plant cover (Vassalo 1998; Cutrera
et al. 2010; Kubiak et al. 2015). AUS has a larger body size (Busch et al. 2000) and
inhabits less resistant soils, whose primary productivity is reduced (Cutrera et al.
2010). Contrary, TAL occurs in rigid soils with dense and diverse plant cover
(Malizia et al. 1991).
Previous studies have shown that the excavation energy cost is similar in these
species, even in different soil types (Luna and Antinuchi 2007). Therefore, energy
expenditure does not seem to be the main factor that might explain soil selection by
TAL and AUS. Similarly, FLA inhabits less resistant sandy soils than MIN, and
excavator activity and soil composition have non-significant differences between
phenotypes (Rebelato 2006; Kubiak et al. 2015). Thus, the association in soil color-
ation with dorsal pelage observed suggests that crypsis is a potential factor influenc-
ing habitat dependence, with coat coloration being a significant variable, prior to
selection by excavation activity and/or soil composition. Accordingly, each species
might be constrained to its corresponding micro habitat due to the disadvantage of
contrast with the background, especially given their high activity aboveground.
Therefore, the similarity of ecological niches occupied by TAL-MIN and AUS-FLA
are shreds of evidence of repeated local adaptation in dynamic habitats (e.g.,
Southern Brazil Coastal Plain; Tomazelli and Villwock 2000), in which population
ecology and demography vary in time and space. In this context, the fixed ecologi-
cal factor responsible for maintaining these local adaptations is potentially differen-
tial survival.
8.6 Convergent Evolution
AUS and FLA belong to the Mendocinus species group, defined by morphological
characteristics (e.g., asymmetric sperm), karyotype (2n = 47–48; similar G and C
band patterns), and molecular data (Castillo et al. 2005; D’Elia et al. 1999; Freitas
1994; Lessa and Cook 1998; Massarini and Freitas 2005; Parada et al. 2011; Chapter
2, this volume). Such similarity of characters has raised questions such as whether
these two species, recognized as phylogenetically close related, share the same most
recent common ancestor (Freitas 1994, 1995a; Fernández-Stolz 2007; Malizia et al.
1991; Mora et al. 2006). Comparative assessment of skull morphology revealed
significant morphological differences (Fornel 2009; Massarini and Freitas 1995,
2005; Travi and Freitas 1984). Then, it was suggested that FLA might have split
from an ancestral form from Argentina, by migration, isolation, and further differ-
entiation of AUS (Freitas 1994; Massarini and Freitas 2005). This migration would
have occurred in the Pleistocene when the coastal plain was under arid conditions,
approximately 100 km wider than at present; thus, the River Plate was not a relevant
geographical barrier (Corrêa et al. 1992). However, current evolutionary analysis of
the Mendocinus group place AUS closer to C. mendocinus (Parada et al. 2011). In
this context, the convergence observed in AUS and FLA in terms of body size and
light coloration might represent repeated evolution in the occupation of coastal
environments instead of the strict retention of an ancestral character (Fig. 8.10).
Interestingly, a pale pelage coloration is found in museum specimens of C. men-
docinus deposited in the Museum of Vertebrate Zoology (see Fig. 8.1). The
Mendocinus species group might have a common genetic background that underlies
the quality and quantity of pigment deposited in the hair, which should be fur-
ther investigated. Since the phenotypic variation of C. mendocinus is intraspecific,
it is a candidate species to explore the hair pattern together with the Agouti gene in
populations of both forms, to understand the genetic basis of this adaptive pheno-
type in Ctenomys.
The increase in the subterminal band (and consequent reduction in the terminal
band) of the hair, as well as the lower pigment density, provide the dilution of the
overall color of the individuals, making them paler (i.e., the blond color of AUS and
Fig. 8.10 Variation of pelage color within the mendocinus species group under a phylogenetic
context (for details see D' Elia et al. Chapter 2, this volume), including Ctenomys australis,
Ctenomys flamarioni, Ctenomys mendocinus and Ctenomys rionegrensis. Phenotypes observed
are: blond (australis and mendocinus), brown (mendocinus and rionegrensis), pale blond (flamari-
oni), dark-backed (rionegrensis) and melanic (rionegrensis)
FLA) (Fig. 8.9). However, there are significant differences at fine-scale between
light phenotypes. AUS has a dark-blond pelage compared to FLA, whose corre-
spondence is directly reflected in its darker soil. Thus, the data generated in this
study indicate convergent mechanisms of crypsis in the same ecological context.
Similarly, the data showed parallel evolutionary trajectories in the generation of
dark phenotypes in TAL and MIN. These species are phylogenetically distant
(Parada et al. 2011), and converge in terms of body size, microhabitat, coat-color,
and soil. Interestingly, mechanisms used to generate dark phenotypes are identical:
increased eumelanin distribution at the tip of the hair (longer terminal width), and
pheomelanin density in the subterminal band. These two small changes generate
potentially advantageous phenotypes on dark soils, in which small variation is
linked to the ability to turn into cryptic in a more complex environment.
8.7 Final Remarks
The determinants of color patterns in animals are still poorly understood, but three
main functions are suggested: intraspecific communication, predator avoidance, and
thermoregulation (Endler 1978). Tuco-tucos have a predominantly solitary habit
and rarely are in direct contact with other individuals of the same species, suggest-
ing that chemical and vocal communication rule the reproductive behavior in these
animals (Francescoli 1999; Zenuto et al. 2004). Thus, it is assumed that coloration
has little significant involvement in communication. Conversely, the results of this
study suggest that pelage phenotypes of TAL, MIN, AUS, and FLA have an evolu-
tionary significance of predator evasion, possibly also contributing to better thermo-
regulation (see Cutrera and Antinuchi 2004). The function of cripsis is reinforced by
the differences in coat color in each of the four species, converging in parallel to two
groups: light and dark phenotypes. Also, additional support comes from the strong
association between Ctenomys dorsal pelage and soil coloration. Differences in
plant cover of the four habitats corroborate this hypothesis, as they also show varia-
tion at the macroecological level, contributing to a fine-tuning of unique local adap-
tation of each species. Thus, the data allow to propose that natural selection may be
the main evolutionary factor responsible for convergence in tuco-tucos. The exis-
tence of specific areas of sympatry in the distributional range of TAL-AUS (Reig
et al. 1990; Contreras and Reig 1965) and MIN-FLA (Freitas 1995a; Kubiak et al.
2015) led to ask how the cryptic phenotypes behave when in contrasting habitat
background, i.e., when opportunistically the dark phenotype occupy the sandy
dunes, and light phenotype the sandy fields. The recent discovery of hybrids between
FLA and MIN (Kubiak et al. 2015, 2020) reinforce the existence of admixture
between phenotypes and habitats. Do the color phenotypes in contrasting soils have
disadvantage comparatively to the cryptic ones? Will the disadvantage, if it exists,
be higher in open habitats than in plant-covered fields? Quantitative studies involv-
ing controlled experiments, particularly using these natural laboratories of sym-
patry, are fundamental to evaluate rates of predation (i.e., prey capture) associated
with cryptic behavior in these species, which will clarify the effect of coloration on
the differential survival of adaptive phenotypes.
Acknowledgments I thank to M.D. Romero (Museo Municipal de Ciencias Naturales Lorenzo

Scaglia) for permission to photograph and collect hair samples of C. australis and C. talarum; to
C. M. Lopes for the preparation of skins of C. minutus, M.S. Mora for the whole support during
fieldwork in Argentina to collect C. australis and C. talarum and their soil samples, T.R.O. Freitas
for taking photographs of Ctenomys pelage in the Museum of Vertebrate Zoology, and
G.R.P. Moreira for suggestions on statistical analysis and fieldwork support. Financial support:
CAPES, CNPq, PPGBM-UFRGS, and FAPERGS (PRONEX 16/2551-0000485-4).
Appendix
Ctenomys specimens used:

C. talarum: UNMDP4; UNMDP5; UNMDP6; UNMDP7; UNMDP8; UNMDP9;
UNMDP10; UNMDP11; UNMDP12; UNMDP13; UNMDP14; UNMDP15;
UNMDP16; UNMDP17; MCNLS 93-1; MCNLS 93-3; MCNLS 93-2 UNMDP;
MCNLS. C. minutus: TR579; TR639; TR640; TR641; TR642; TR643; TR644;
TR645; TR646; TR647; TR648; TR649; TR650; TR651; TR652; TR653; TR654;
TR655; TR656; TR657; TR1201; TR1202; TR1203; TR1207; TR1212; TR1219;
TR1220; TR1221; TR1222; TR1225; LAMI2; TR1125; TR1126; TR1128; TR1129;
TR1130; TR1132; TR1133; TR1137; TR1231; C. australis: UNMDP 1-1; UNMDP
1-2; UNMDP 1-3; MCNLS 81-1; MCNLS 82-22; MCNLS 82-67; MCNLS 82-68;
MCNLS 82-69; MCNLS 82-71; MCNLS 82-238; MCNLS 82-239; MCNLS
82-240; MCNLS 82-241; MCNLS 82-242; MCNLS 82-243; MCNLS 82-244;
MCNLS 82-245; MCNLS 84-20; MCNLS 84-23; MCNLS I-737; MCNLS I-740;
MCNLS I-1044; MCNLS 1; MCNLS 2; MCNLS 4; UNMDP 37; UNMDP 38;
UNMDP 39, UNMDP; MCNLS; C. flamarioni: PUC278; TR449; TR473; TR474;
TR500; TR1152; TR1153; TR1154; DZRS01; G123; PUC408; TR476; TR478;
TR1272; TRNI1, TRNI2.
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Part IV
Environmental Relationships
Chapter 9
Environmental and Ecological Features
of the Genus Ctenomys
Daniel Galiano and Bruno Busnello Kubiak
9.1 Introduction
Life underground imposes general constraints for most subterranean and fossorial
rodent species. The habitat requirements and ecological characteristics of subterra-
nean influence numerous aspects of their biology, including where individuals live
and how they behave. Subterranean rodents of the genus Ctenomys, commonly
referred to as “tuco-tucos,” are widely distributed throughout South America. There
are currently approximately 65 valid species based on morphological, karyotypic,
and molecular data (D’elia, Teta and Lessa, Chap. 2 this volume). These animals are
typically solitary, have low mobility, and are usually found distributed among small
patches of suitable habitat. They occupy a wide range of habitat types, particularly
in open areas such as grasslands, steppes, deserts, and sand dunes (Lacey et al.
2000), with a few species occurring in forest regions (Gardner et al. 2014; Leipnitz
et al. 2020) (Fig. 9.1). Regional distributions of Ctenomys species vary substantially
with soil and vegetation characteristics and resource availability. Soil features are
known to influence some aspects of their biology (e.g., burrow system characteris-
tics and excavation strategies), with temporal and spatial variation in population
density largely attributed to interactions between limiting environmental factors
combined with species life-history characteristics. As in other fossorial animals,
tuco-tucos can increase local environmental heterogeneity at the landscape level by
aiding in the formation, aeration, and mixing of soils, and may enhance the infiltra-
tion of water into the soil. This modification of vegetation and soil characteristics
can in turn impact herbivore food webs. Tuco-tucos are also a significant food
D. Galiano
Laboratório de Zoologia, Universidade Federal da Fronteira Sul, Realeza, Brazil
B. B. Kubiak (*)
Programa de Pós-Graduação em Genética e Biologia Molecular, Universidade Federal do Rio
Grande do Sul, Porto Alegre, Brazil

https://doi.org/10.1007/978-3-030-61679-3_9
194 D. Galiano and B. B. Kubiak
Fig. 9.1 Mosaic of photographs of Ctenomys habitats, burrows, and mounds. The different types
of habitat are represented, such as grasslands, steppes, sand dunes, and forest areas. Localities: (a)
Viamão, RS, Brazil; (b) Xangri-lá, RS, Brazil; (c) Capão da Canoa, RS, Brazil; (d) Viamão, RS,
Brazil; (e) Manoel Viana, RS, Brazil; (f) Rio Grande, RS, Brazil; (g) Osório, RS, Brazil; (h)
Cáceres, MT, Brazil; (i) Nova Mutum, MT, Brazil; (j) Feliz Natal, MT, Brazil; (k) Feliz Natal, MT,
Brazil; (l) Cáceres, MT, Brazil
source for many avian and mammalian predators. In this chapter, we discuss the
spatial use patterns and general ecological characteristics of species interactions and
social structure in tuco-tucos, as well as their impacts on the local environment and
general aspects of conservation.
9 Environmental and Ecological Features of the Genus Ctenomys 195
9.2 Spatial Utilization and Species Interactions
Tuco-tucos are usually associated with arid or semiarid habitats in open areas such
as deserts, savannas, and dunes (Bidau 2015; Freitas 2016), with few exceptions of
species occurring in forest regions (Gardner et al. 2014; Leipnitz et al. 2020). The
tendency to occupy open habitats may be related to the ecological pressures imposed
by the subterranean lifestyle. The distribution of critical resources, including suit-
able habitat and food supply, is thought to strongly influence the distribution of
small mammal species, including subterranean rodents (Jarvis et al. 1998; Tammone
et al. 2012; Galiano et al. 2014a, b). When resources such as suitable habitat or food
supply are unevenly distributed, habitat use is typically nonrandom, with animals
preferentially occupying patches with sufficient resource availability
(Garshelis 2000).
Tuco-tucos excavate networks of tunnels in which they live and perform most of
their vital activities, making life underground more energetically costly than life on
the surface (Lacey et al. 2000) (see Fig. 9.1g). Above and below ground soil and
vegetation features are important factors for tuco-tuco habitat choice. For example,
Bongiovanni et al. (2019) demonstrated that soil depth is an important habitat fea-
ture for Ctenomys mendocinus activity in the Puna Desert, where climatic condi-
tions are extreme. Individuals from this species tend to select areas with deeper
soils, likely because deeper burrow systems facilitate thermoregulation in cold envi-
ronments (i.e., the amplitude of temperature fluctuation diminishes with increasing
soil depth) (Bennett et al. 1998; Burda et al. 2007). The presence of shrubs also has
a significant positive relationship with C. mendocinus activity on both micro and
macro scales, since shrubs can represent a seasonal food source and provide protec-
tion against aerial predators. Another study by Scheich and Zenuto (2007) demon-
strated that Ctenomys talarum is able to identify soils with richer food supply (e.g.,
grasses) and discriminate quality through olfaction, corroborating the importance of
vegetation as a determining factor in habitat selection for this species.
The hardness and compaction of soils can also be considered a limiting factor for
habitat occupation by tuco-tucos. It is well-documented that excavating and living
in harder soils is more energetically costly than in softer soils (Luna and Antinuchi
2006; Antinuchi et al. 2007). However, different species of tuco-tucos still occupy a
wide range of habitat with respect to soil characteristics. This limitation seems to be
overcome by some species through differential allocation of effort between the inci-
sors and limbs during excavation (Vassallo 1998; Morgan et al. 2017) or variations
in morphological structures linked to bite force (Borges et al. 2017). For example,
Ctenomys minutus populations show variation in humerus shape and skull size
shape among the habitats they occupy. Individuals in sandy field habitats with
harder soils have greater bite force and use their limbs more intensively in excava-
tion than do individuals in sand dunes with softer soils (Kubiak et al. 2018).
Likewise, C. mendocinus exhibits variation in activity depending on habitat soil
characteristics. In the Puna Desert region, soil hardness was not associated with
C. mendocinus activity (Bongiovanni et al. 2019); however, there was a negative
association between soil hardness and C. mendocinus activity in the Monte Desert
region (Albanese et al. 2010). The differences in determining factors for habitat
selection among C. mendoncinus populations might be explained by environmental
characteristics, as the Puna Desert is a more extreme climate with lower resource
availability.
Soil moisture is another factor that can influence habitat selection for Ctenomys
species. Flooded regions were negatively correlated with the selection of mac-
roscale habitats for C. mendoncinus (Bongiovanni et al. 2019) and microscale habi-
tats for C. minutus (Galiano et al. 2016), although the level and duration of tolerance
for higher levels of soil moisture need to be investigated further.
The degree to which habitat features influence the ecology of Ctenomys becomes
evident for species that occupy contrasting habitat types like grasslands and sand
dunes, such as C. minutus in the coastal region of southern Brazil (see Fig. 9.1c) and
C. talarum in the coastal region of Argentina. Both habitat types show clear varia-
tion in soil hardness and resource availability, with sand dunes having softer soils
and lower food availability compared to grasslands (Comparatore et al. 1992;
Malizia et al. 1991; Galiano et al. 2016; Kubiak et al. 2018). C. minutus inhabiting
sand dunes have a home range that is 1.75 times larger than individuals inhabiting
grassland (Kubiak et al. 2017a) and a weaker bite force (Kubiak et al. 2018). In
C. talarum, individuals from Mar de Cobo sand dunes have a comparatively larger
body size than those from Necochea grasslands (Zenuto 1999), but the home range
size does not differ (Cutreta et al. 2010). These species also differ in food types
consumed between habitats (Del Valle et al. 2001, Lopes et al. 2015), which could
be related to changes in plant species composition and subsequent differences in
food availability along their geographical distribution (Lopes et al. 2020, Chap. 10
this volume). Seasonal changes in food availability can also influence Ctenomys
feeding during the year. For example, some studies have shown that when the avail-
ability of grasses is scarce during dry seasons, consumption of shrubs increases
(Madoery 1993; Comparatore et al. 1995; Altuna et al. 1999). In general, the heter-
ogenous nature of the habitat these species occupy and environmental discontinui-
ties along the species distribution range probably strongly influence habitat selection
and resource use for Ctenomys.
Most tuco-tuco species are characterized by allopatric distribution. There are
only three cases of sympatry officially documented to date (Contreras and Reig
1965; Reig et al. 1990; Freitas 1995; Kubiak et al. 2015) (Fig. 9.2). A fourth case
describes the capture of C. dorsalis and C. conoveri at the same time and location in
Colonia Fernheim, Paraguay (Londoño-Gaviria et al. 2019). However, there is no
other information regarding this possible region of sympatry, and this case warrants
further investigation (Londoño-Gaviria et al. 2019). One of the three confirmed
areas of sympatry occur in a portion of the coastal dunes in the Necochea region in
the province of Buenos Aires, Argentina, between C. australis and C. talarum.
These species present well-documented habitat segregation according to soil and
vegetation types (Malizia et al. 1991; Comparatore et al. 1992). This segregation is
Fig. 9.2 Distribution ranges of 65 extant Ctenomys species in South America. Conservation status
is based on IUCN (2020), and sympatric zones are indicated
likely the result of interspecific competition, as demonstrated by Vassallo (1993).

On the other hand, this author also highlighted that competition plays a much
smaller role in sustaining the pattern of microhabitat segregation between species
compared to habitat preference associated with species differences in body size and
color. There are also documented interspecific differences in home range size, with
C. australis having a home range 19 times larger than C. talarum (Cutrera
et al. 2010).
The other two sympatric zones occur in the coastal region of southern Brazil for
the species C. flamarioni and C. minutus (Freitas 1995; Kubiak et al. 2015). One of
these sympatric zones is located in the first line of sand dunes, similar to the sym-
patric zone in Argentina, and covers a length of approximately 15 km. The other
sympatric zone occurs in sandy fields in the southern portion of their distribution
and was only recently described (Kubiak et al. 2015). In the dune region of southern
Brazil, Kubiak et al. (2015) found results similar to those described for the sympat-
ric region of Argentina, where species show segregation in microhabitat according
to differences in soil hardness, plant biomass, and plant cover. Further, the authors
also describe different patterns of habitat selection in allopatric and sympatric popu-
lations of C. flamarioni, whereas C. minutus selected the same habitat characteris-
tics under both conditions. However, no interspecific differences in home range size
were detected, nor were there differences between sympatric or allopatric popula-
tions for this trait (Kubiak et al. 2017b). The authors suggested that co-occurrence
may not influence home range size in these species, perhaps due to environmental
adaptations that facilitate coexistence (e.g., microhabitat segregation and dietary
modifications) (Lopes et al. 2015).
Moreover, Kubiak et al. (2017c) pointed out that these species differ in micro-
habitat selection and morphological characteristics in areas of sympatry: C. fla-
marioni selects different microhabitats while C. minutus does not, and C. minutus
present morphological modifications (i.e., reduction in skull length and body mass)
when occurring in sympatry. This may be a result of temporal segregation of
microhabitats. The displacement of morphological characteristics might reflect
changes resulting from many generations of habitat segregation between C. fla-
marioni and C. minutus in the sympatric area. The plausible effects of both of these
phenomena (i.e., spatial segregation and morphological differentiation) might
point in the same direction, with the co-occurrence of species causing an ecologi-
cal shift in the known zones of sympatry. In this context, interspecific interactions
such as competition and competitive exclusion are likely among the main factors
determining allopatric distribution in subterranean rodents (Miller 1964;
Nevo 1979).
Another intriguing observation near the contact zones of C. flamarioni and
C. minutus was the occurrence of hybrids (Kubiak et al. 2020). These species have
extensive differences in chromosome organization (Freygang et al. 2004; Gava and
Freitas 2003; Massarini and Freitas 2005), phenotype, evolutionary history (Parada
et al. 2011), sperm morphology (Freitas 1995), and genetic characterization (Kubiak
et al. 2020), but still can generate natural hybrids. This hybridization occurs bi-
directionally (females of both species can generate hybrids). The importance of
hybridization in the processes of adaptation and speciation has been rigorously
debated in the literature, and the consensus that hybridization often plays an impor-
tant role in evolution has recently been under debate (Taylor and Larson 2019). In
the case of tuco-tucos, if hybrid individuals such as the ones mentioned above occur
in higher numbers than imagined and possess some capacity to reproduce (either
between hybrids themselves or with parental individuals), then interactions between
tuco-tucos may be more dynamic than expected.
9.3 Social Structure
Few tuco-tuco species have been effectively characterized based on social structure
and ecological features. Most known species to date are solitary, and in solitary spe-
cies, individuals occupy their own burrow system and aggressively defend it.
However, it is relatively common to find young individuals and pups sharing the
tunnel system with their mothers for some time after the reproductive period.
Mothers typically give birth to one to two pups per pregnancy (Pearson 1959; Rosi
et al. 1992, 1996; Malizia and Busch 1991, 1997; Zenuto and Busch 1998; Marinho
and Freitas 2006; Garcias et al. 2018). Some authors have observed that the parental
care period for C. talarum was between 56 and 65 days (Malizia and Busch 1991;
Zenuto et al. 2002) and that individuals live approximately 20–22 months (Busch
et al. 1989; Malizia and Busch 1997). In this context, individuals of C. talarum have
parental care and parent-offspring interactions that correspond to approximately
9.6% of their lifetime. However, the gestation period, extent of parental care, and
number of offspring varies among tuco-tuco species (Pearson 1959; Zenuto 1999).
Male and female tuco-tucos interact at minimum during the reproductive season.
Burrows are usually connected for a brief period to allow courtship and copulation
(Altuna et al. 1999), and it is common to capture animals with injuries possibly
resulting from conflict during mating periods (personal communication; Thales
Freitas). In the solitary species C. talarum, laboratory studies have shown that indi-
viduals travel above ground to search for mates and copulate (frequently the male
travels) (Zenuto et al. 2002). There is also evidence of below-ground access to
females by males for C. minutus over long distances (~70 m) (Kubiak et al. 2017a).
Although most species are solitary, three patterns of sociality have been identi-
fied among tuco-tucos: solitary, facultative sociality, and social (O’Brien et al.
2020). Ctenomys australis, C. flamarioni, C. haigi, C. Minutus, and C. talarum have
a solitary social structure in which each adult lives alone in a tunnel system and
displays minimal or zero spatial overlaps with other adults (Lacey et al. 1998;
Cutrera et al. 2006, 2010; Kubiak et al. 2017a, b). On the other hand, C. sociabilis
has been shown to be social (Lacey et al. 1997), and C. opimus presents an interme-
diate pattern between these two and is considered to be facultatively social (O’brien
et al. 2020). Ctenomys rionegrensis also shows signs of facultative sociality
(Tomasco et al. 2019). The social pattern presented by C. sociabilis differs from
most other species. Social individuals have a high overlap of home range with con-
specific individuals, and burrow systems are routinely occupied by multiple adult
females and, in many cases, a single adult male (Lacey et al. 1997; Lacey and
Wieczorek 2003). In this context, only adult females in a group are kin, and while
females of this species are philopatric, males are not (Lacey and Wieczorek 2003).
The intermediate, or facultatively social pattern, of C. optimus is characterized
by the extensive but incomplete overlapping of home ranges (O’Brien et al. 2020).
Interestingly, C. rionegrensis apparently has sporadic spatial overlap among adults,
documented by both the capture of multiple adults in a single burrow entrance
(Lessa et al. 2005) and overlapping of home range area between females and males
(Tassino et al. 2011). However, individuals of C. rionegrensis do not appear to regu-
larly share the same tunnel gallery (Tassino et al. 2011; Estevan et al. 2016).
According to Lacey and Wieczorek (2003), comparisons of ctenomyids over larger
spatial scales are required, and interactions of subterranean rodents may be more
diverse than previously expected.
9.4 The Influence of Tuco-Tucos on the Environment
Subterranean rodents excavate and inhabit extensive burrow systems, and changes
in plant diversity, abundance, and community composition are typical consequences
of high activity level of these animals (Andersen 1987; Contreras and Gutiérrez
1991; Huntly and Reichman 1994; Malizia et al. 2000; Campos et al. 2001;
Reichman and Seabloom 2002; Kerley et al. 2004; Lara et al. 2007; Hagenah and
Bennett 2013; Galiano et al. 2014a; Miranda et al. 2019). They also alter soil condi-
tions, such as soil granulometry, aeration, and chemical and physical characteristics
(Schauer 1987; Cox and Roig 1986; Borghi et al. 1990; Malizia et al. 2000; Lara
et al. 2007; Šklíba et al. 2009; Hagenah and Bennett 2013). These effects of subter-
ranean rodents on vegetation and soil can arise from burrow dynamics, diet selec-
tion, or foraging behavior (Huntly and Reichman 1994). Because of the great impact
they have on entire ecosystems, including soil, water, and air content, decomposi-
tion processes of plant material, nutrient cycling, and composition of local biota
(Hole 1981), subterranean rodents are regarded as ecosystem engineers (Cameron
2000; Reichman and Seabloom 2002; Reichman 2007). Their actions constitute a
major factor in soil and vegetation dynamics.
Several studies have focused on the effects of Ctenomys on vegetation (Malizia
et al. 2000; Campos et al. 2001; Tort et al. 2004; Lara et al. 2007; Galiano et al.
2014a) and soil conditions (Malizia et al. 2000; Lara et al. 2007; Galiano et al.
2014a). Most of these studies found that Ctenomys species alter vegetation and
nutrient content of soil, although there is some variation in magnitude of these
effects. For example, Galiano et al. (2014a) found significant effects of Ctenomys
minutus activity on vegetation and soil conditions in patches of sandy fields. The
authors observed a 37% reduction in plant biomass areas where individuals of the
species were present versus absent. This reduction is within the range of other her-
bivorous subterranean rodents (e.g., plant biomass reduced by 25–50%; Reichman
and Smith 1985). Additionally, reduction in plant biomass was observed for other
species of Ctenomys. For example, C. mendocinus was associated with a 44%
reduction in plant community biomass in the southern Puna Desert (Lara et al.
2007), while C. talarum was associated with a 31% reduction in biomass in grass-
lands of Buenos Aires Province (Malizia et al. 2000). This reduction could be a
direct result of activities related to feeding and burrow construction.
Albanese et al. (2010) found that grasses were the dominant plant type consumed
by C. mendocinus (79%), and grass leaves were the most representative item among
consumed plant parts (89.5%). Decreased densities of grasses in areas with

Ctenomys were reported by Campos et al. (2001) and Lara et al. (2007). This pattern
suggests an effect of Ctenomys foraging on the abundance of grasses, which may in
turn promote further effects on community dynamics. This modification of vegeta-
tion patterns by Ctenomys species suggests that they may act as keystone species.
Moreover, tuco-tucos are herbivorous predators that can facilitate the propagation
and reproduction of several plant species.
Burrowing activities of tuco-tucos can also affect mycorrhiza. Miranda et al.
(2019) detected changes in the abundance of arbuscular mycorrhizae and dark sep-
tate endophytes as a consequence of burrowing activity by Ctenomys aff. Knighti.
These authors observed that soil patches from burrows contain soil enriched with
organic matter, soil microorganisms that decompose it into nutrients available for
plants, fungal propagules that can establish mutualistic relationships with their
roots, and greater water retention in relation to the surrounding landscape. Soil mix-
ing by burrowing mammals is an important pedogenic process (Johnson 1990) and
increases soil porosity.
During burrowing, subterranean rodents redistribute soil among different hori-
zons which contributes to aeration, irrigation, and fertilization of soils (Reichman
and Smith 1990). According to Malizia et al. (2000), the formation of new mounds
by tuco-tucos (C. talarum) increased the levels of N, P, Na, K, and Mg; however,
higher levels of Ca and pH were found in undisturbed areas. Lara et al. (2007) also
found that the activity of C. mendocinus increased nutrient concentration (N, K, P)
in bare soil compared to bare soil in undisturbed areas, and Galiano et al. (2014a)
observed high concentrations of P and K just below the soil surface where C. minu-
tus were present. Even though the effects on different nutrients vary between stud-
ies, it is well known that tuco-tucos and other subterranean mammals (e.g., pocket
gophers, Geomyidae) modify the distribution of nutrients (Abaturov 1972; Mielke
1977; Grant and Mc Bryer 1981; Hole 1981; Spencer et al. 1985; Inouye et al. 1987;
Reichman and Seabloom 2002) as a result of moving, mixing, or bringing soil to the
surface from lower levels as well as the incorporation of nutrients into the soil.
Moreover, the extensive excavations and their associated impacts might generate a
dynamic mosaic of nutrients and soil conditions that promote diversity and maintain
disturbance-dependent components of plant communities.
These animals have a significant effect on vegetation composition and dynamics
as well as soil properties. While this obviously will affect ecosystem processes such
as decomposition or productivity, the consequence of all the effects that tuco-tucos
have on ecosystems are difficult to predict. In fact, tuco-tucos seem to be an impor-
tant physical force in the ecosystems in which they occur, and their combined eco-
logical effects may be a significant force in the maintenance and conservation of
these ecosystems. To date, few studies have focused on the environmental effects of
tuco-tucos in the Neotropics. While not all effects can be considered positive, the
fact that tuco-tucos are a major source of heterogeneity in the ecosystems they
inhabit is unquestionable.
9.5 Species Conservation
Conservation of small mammals requires knowledge of the genetically and ecologi-

cally meaningful spatial scales at which species respond to habitat modifications,
and effects on small mammals may have cascading effects across the environments
they inhabit (Manning and Edge 2004). The geographical distribution of species is
also an important trait for biodiversity conservation and management policies
because it provides relevant and meaningful information regarding the requirements
for ecological success (Margules and Pressey 2000). As subterranean small mam-
mals, tuco-tucos shows low vagility levels and high levels of population subdivision
across landscapes, and consequently, low genetic variability (Reig et al. 1990;
Lacey et al. 2000). These characteristics together with factors such as habitat frag-
mentation and degradation, urbanization, climatic changes, and limited knowledge
concerning the areas of occurrence and life history of these small mammals enhance
the risk of extinction of species (Gallardo et al. 1996; Freitas et al. 2012; Gómez
Fernández et al. 2016; Caraballo et al. 2020). Additionally, the life history of these
small mammals has contributed to difficulties in developing conservation initiatives
(Fernandes et al. 2007).
Tuco-tucos have been considered agricultural pests for decades because of
their fossorial habits and, consequently, are exposed to risk of predation by
humans (Massoia 1970; Pearson et al. 1968; Freitas et al. 2012). The conservation
status of all Ctenomys species is an important issue to be addressed since most
species have a small geographic range that is restricted to sandy-soil grassland
habitats. Such habitat specificity combined with anthropogenic threats described
above and typical patchy distribution suggests higher vulnerability of tuco-tucos
than presently supposed, and that conservation effort should be based on consis-
tent and detailed studies of habitat occupation (Fernandes et al. 2007; Galiano
et al. 2014b). In this context, the development of habitat conservation plans is
urgently needed for many fragmented landscapes, and the first step in this process
should be to better understand the responses of animal populations to different
scenarios of fragmentation. For example, Mapelli and Kittlein (2009) analyzed
the influence of patch and landscape characteristics on patch occupancy by
Ctenomys porteousi. They observed that species distributions were affected not
only by characteristics of the habitat patches but also by those of the surrounding
landscape matrix. This implies that ongoing modification of the matrix landscape
through anthropogenic activities might have important effects on the conservation
of these species.
The lack of protection for areas occupied by subterranean species is also a major
problem for the conservation of tuco-tucos. Caraballo et al. (2020) studied the over-
lap between protected areas and the 67 extant Ctenomys species, pointing out that
only 34 of these species have significant overlapping distributions with protected
areas. According to the authors, the most concerning species are those at risk (vul-
nerable (VU), endangered (EN), or critical endangered (CR), according to the
International Union for Nature Conservation, IUCN), and nonevaluated ones (i.e.,
not included in the IUCN Red List or data deficient) which have little or no overlap
with protected areas. In other words, half of the extant tuco-tucos correspond to
nonevaluated species, most of which are known only from the type locality or its
surroundings. Under this scenario, we can assume that nonprotected areas have a
unique importance for Ctenomys conservation, and that data deficiency and regional
habitat transformation pose a serious threat to these species.
Regarding the conservation status, 53 out of 65 species (D’elía, Teta and Lessa,
Chap. 2 this volume) were evaluated according to the IUCN Red List. Of these,
approximately 24.5% were considered to be under some degree of threat (one clas-
sified as VU, nine as EN, and three as CR), three (5.7%) are considered “near threat-
ened,” 16 (30.2%) as “least concern,” 21 (39.6%) are classified as “data deficient”
(IUCN 2020), and 12 species were not evaluated (Fig. 9.2). Further, information
regarding population trends is only available for 52% of species. This means that
there is limited knowledge regarding the true conservation status with only basic
information available on population parameters for nearly half of the species. The
conservation status, areas of occurrence, population trends, and major threats for
species are listed in Table 9.1. We note that not all tuco-tuco species are included in
this list.
The current state of knowledge of Ctenomys conservation is lacking, and the lack
of data to guide conservation actions and species management is among the major
roadblocks. In this context, the most recent compilations of the genus (Bidau 2015;
Freitas 2016; Teta and D’Elia 2020) recognize that the number of species will
increase with the combination of additional field collections and revisionary work.
The need to investigate basic ecological information to provide adequate conserva-
tion measures is urgent for species that are not well described. Regardless, the con-
servation of soil and vegetation in regions of occurrence is certainly one of the best
conservation strategies that can be implemented for these species.
Although it is broadly understood that subterranean rodents act as ecological
engineers, obtaining ecological information about tuco-tucos remains challenging
due to the primarily underground lifestyle. Since tuco-tuco populations vary spa-
tially and temporally, the ecology of these animals is also expected to be strongly
influenced by regional factors. Long-term ecological studies are needed to better
understand the interactions between these rodents and their effects on inhabited
ecosystems. Given the high degree of habitat specialization and endemicity of
Ctenomys, regional scenarios of habitat transformation pose a serious threat to spe-
cies viability, and the current lack of knowledge regarding tuco-tuco biology and
ecology could negatively impact population management. Despite many studies
that have utilized various approaches to estimate ecological data for these species,
it remains difficult to corroborate those data with ecological parameters at
broad scales.
Table 9.1 Species, distribution countries, IUCN conservation status, major threats, and population
trends of extant Ctenomys species
IUCN
conservation Population
Species Countriesa statusb Threatsc trends
C. andersoni BOL NE – –
C. argentinus ARG NT AA Decreasing
C. australis ARG EN AA; RCD Decreasing
C. bergi ARG EN AA Decreasing
C. bicolor BRA NE – –
C. bidaui ARG NE – –
C. boliviensis ARG; BOL; LC There are no major threats Stable
BRA; PRY
C. bonettoi ARG EN AA Decreasing
C. brasiliensis URY DD The threats to this species Unknown
are unknown
C. coludo ARG DD There is no information Unknown
available on the threats
C. conoveri BOL; PRY LC There appear to be no Stable
major threats
C. contrerasi ARG NE – –
C. dorbignyi ARG NT BRU Decreasing
C. dorsalis PRY DD AA; RCD Unknown
C. emilianus ARG LC There are no imminent Decreasing
threats
C. BOL NE – –
erikacuellarae
C. famosus ARG DD There is no information Unknown
C. flamarioni BRA EN EPM; HID; RCD Decreasing
C. fochi ARG DD There is no information Unknown
C. fodax ARG DD There is no information Unknown
C. frater BOL LC There do not appear to be Decreasing
any major threats to this
species
C. fulvus CHL DD There is no information Unknown
C. haigi ARG LC There are no known threats Unknown
to this species
C. ibicuiensis BRA DD AA, NSM Unknown
C. johannis ARG DD There is no information Unknown
C. juris ARG DD There is no information Unknown
available on any threats to
this species
(continued)

IUCN
C. knighti ARG DD AA Unknown
C. lami BRA VU AA; RCD Decreasing
C. latro ARG EN AA Decreasing
C. lessai BOL NE – –
C. leucodon BOL; PER LC BRU Stable
C. lewisi BOL LC There appear to be no Stable
major threats to this species
C. magellanicus ARG; CHL LC AA Decreasing
C. maulinus ARG; CHL LC No major threats to this Unknown
species are described
C. mendocinus ARG LC There are no major threats Unknown
known to this species
C. minutus BRA DD AA; RCD Stable
C. nattereri BOL; BRA NE – –
C. occultus ARG EN AA Decreasing
C. opimus ARG; BOL; LC There appear to be no Stable
CHL; PER major threats to this species
C. osvaldoreigi ARG CR AA; NSM Decreasing
C. PRY NE – –
paraguayensis
C. pearsoni URY NT AA; RCD Decreasing
C. perrensis ARG LC There are no known threats Stable
to this species
C. peruanus PER LC There appear to be no Stable
C. pilarensis PRY EN AA; BRU Decreasing
C. pontifex ARG DD There is no information Unknown
this species
C. pundit ARG EN AA Decreasing
C. rionegrensis ARG; URY EN AA Decreasing
C. roigi ARG CR AA Decreasing
C. rondoni BRA NE – –
C. ARG NE – –
rosendopascuali
C. saltarius ARG DD There is no information Unknown
this species
C. scagliai ARG DD There is no information Unknown
this species
(continued)

IUCN
C. sericeus ARG DD There is no information Unknown
this species
C. sociabilis ARG CR AA; BRU Decreasing
C. steinbachi BOL LC There appear to be no Stable
C. talarum ARG LC There are no known threats Unknown
to this species
C. thalesi ARG NE – –
C. torquatus BRA; URY LC AA; EPM Unknown
C. tuconax ARG DD AA Unknown
C. tucumanus ARG DD AA Unknown
C. tulduco ARG DD The threats to this species Unknown
are unknown
C. validus ARG DD There is no information Unknown
available on threats to this
species
C. viperinus ARG DD The threats to this species Unknown
are unknown
C. yatesi BOL NE – –
Data presented here are based on the Caraballo et al. (2020) and IUCN (2020)
aAcronyms (Countries): ARG, BOL, BRA, CHL, PER, PRY, and URY, correspond to Argentina,
Bolivia, Brazil, Chile, Peru, Paraguay, and Uruguay, respectively
bAcronyms (IUCN conservation status): NE, DD, LC, NT, VU, EN, and CR, correspond to Not
evaluated, Data deficient, Least concern, Near threatened, Vulnerable, Endangered, and Critically
endangered, respectively
cAcronyms (Threats): AA, BRU, EPM, HID, NSM, and RCD, correspond to Agriculture and aqua-
culture; Biological resource use, Energy production and mining, Human intrusions and distur-
bance, Natural system modifications, and Residential and commercial development, respectively
Acknowledgments We thank the editors for the invitation to contribute to this collective book on
tuco-tucos. We are grateful to all colleagues and students that we have discussed ideas on the ecol-
ogy of tuco-tucos over the years. We thank Diego A. Caraballo for the invaluable help in sending
us the distribution ranges of Ctenomys. BBK received financial support from Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq; 158250/2018-4). Lastly, we would like to
thank everyone who studies or has studied the ecology of these incredible animals, the tuco-tucos.
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Chapter 10
The Diet of Ctenomyids
Carla Martins Lopes
10.1 An Overview
What does this animal eat? This is one of the first questions that comes out when
someone is introduced to a new species. Researchers are trying to answer this ques-
tion and all other aspects related to the diet of ctenomyids at least since 1989
(Torres-Mura et al. 1989). Assessing the dietary composition of species in their
natural environment is a clue to unravel central questions in ecology and evolution.
Ecological processes such as intra- and interspecific competition, interactions
between predator-prey and herbivore-plant, population fluctuations, and species
geographical distributions can be strongly influenced by the availability of food
resources in the environment and how species use these resources (Begon et al.
2006). All these aspects, ultimately, affect ecosystem functioning and evolutionary
processes.
Ctenomyids are considered strictly herbivorous, generalist, and opportunistic
species able to consume a wide range of plant groups. This behavior has been asso-
ciated with adaptation to high costs of burrowing and low levels of energy available
at the subterranean ecotope (del Valle et al. 2001). The availability of food in the
natural environment is what ultimately drives species selectively (Emlen 1966).
Despite ctenomyids can use a wide variety of plants as a food source, they often
show a preference for the aerial vegetative part of grasses (Fig. 10.1) (Comparatore
et al. 1995; Puig et al. 1999; del Valle et al. 2001; Rosi et al. 2003, 2009). This food
preference has been associated to specific nutritional requirements, palatability, and
lower harvesting and handling times required for feeding these items, which could
minimize the time that individuals expend outside their burrows, diminishing their
predation risk (Puig et al. 1999; Rosi et al. 2003).
C. M. Lopes (*)
Departamento de Biodiversidade e Centro de Aquicultura, Instituto de Biociências,
Universidade Estadual Paulista (UNESP), Rio Claro, SP, Brazil

https://doi.org/10.1007/978-3-030-61679-3_10
214 C. M. Lopes
Fig. 10.1 Specimen of

Ctenomys flamarioni
eating the aerial vegetative
part of grass. (Photograph
by Jose F. B. Stolz)
The feeding behavior of ctenomyids was described as individuals squat on their

haunches when handling their food, hold the plants with one or both forepaws,
between the three central digits and the thenar pad (Fig. 10.1), and shake it up and
down quickly for cleaning. Coprophagy is also a common behavior observed but
animals examine feces before deciding to eat or discard them. The individuals seat
on their hindquarters, bring their head closer to the anus, and take the feces using
lips and incisors. There is almost no feces manipulation with the forepaws. Only on
few occasions animals were observed eating feces collected from the floor. The
ingestion of feces was associated with better assimilation of nutrients and retention
of water (Altuna et al. 1998; Martino et al. 2007).
10.2 Methods for Diet Analysis
Several methodologies have been developed and applied in order to better under-
stand the dietary composition of animals. Some of them provide information about
the ingested or egested items, as observation of foraging behavior and examination
of fecal and gut contents by means of visual inspections, microscopy, or DNA-
based methods. However, not all items ingested are assimilated and incorporated
into the tissue of the consumers. The analysis of stable isotopes, biomarkers, and the
near-infrared reflectance spectroscopy, e.g., can estimate the components that are
10 The Diet of Ctenomyids 215
assimilated by the animal (Johnson et al. 1983; Symondson 2002; Valentini et al.
2009; Pompanon et al. 2012; Nielsen et al. 2018). Each method addresses different
aspects related to the diet of species and must be applied depending on the question
to be further explored. Most of the knowledge about dietary habits and preferences
of ctenomyids is based on the analysis of ingested and egested food items. The
information provided in this chapter are mainly based on these studies.
The direct observation of foraging is one of the simplest methods to unravel the
feeding habits of animals. However, it can be very time-consuming or even impos-
sible to apply for elusive or generalist species living in complex environments, like
herbivorous (Valentini et al. 2009). Cafeteria tests consist of capturing animals in
their natural environment, and keep them at laboratory conditions, where the food
availability is controlled and their feeding behavior can be observed. This approach
provided much knowledge about harvesting and feeding behavior of ctenomyids
and some of their food preferences (Camin and Madoery 1994; Altuna et al. 1998;
del Valle et al. 2001; Martino et al. 2007). However, the controlled conditions in a
laboratory do not recreate the natural environment where these species live, which
may result in major changes in their behavior (Symondson 2002).
The most used method until recent years for unraveling diet preferences of cte-
nomyids was the microhistological analysis of fecal and gut samples. The epidermal
cells of plants have taxon-specific characteristics that can be used for species iden-
tification. The microhistological technique consists of comparing the leaf epiderm
morphology of food particles recovered from fecal and gut samples with those
obtained from plants recovered in the surrounding area of study (Johnson et al.
1983). This technique has provided much valuable knowledge about the diet of
ctenomyids, reviewed below. However, it is a very time-consuming method, prone
to unspecific and context-dependent results, depending on the training of the
observer and the amount and quality of samples (Soininen et al. 2009; Pompanon
et al. 2012).
DNA-based methods have been largely applied in recent years to determine the
diet of species, mainly after the development of the next-generation sequencers.
These methods are particularly useful to assess the diet of generalist species that can
explore a large variety of food resources available in highly diverse environments,
fluid feeders, elusive species, or species for which the other methods cannot be eas-
ily applied or provide reliable results. These methods consist in amplify the DNA
recovered from gut or fecal samples using species-specific or universal primers
(Pompanon et al. 2012; Nielsen et al. 2018). The most recent and promising of these
methods is the DNA metabarcoding approach, which has been successfully used to
describe the diet composition of a wide range of animals, including herbivorous
species (Pompanon et al. 2012). Basically, the total DNA is extracted from the gut
or fecal samples, a small fragment of DNA is amplified using universal primers for
targeting a group of the ingested species of interest. The amplified DNA is sequenced
using the next-generation technology. Finally, a sequence reference database is used
to assign taxonomy to the sequences recovered from fecal and gut samples
(Fig. 10.2). The DNA metabarcoding approach allows the analysis of several sam-
ples in one single experiment, even when these samples are composed by a high
216 C. M. Lopes
Fig. 10.2 Flowchart diagram showing the main steps for assessing the diet composition of species
by means of the metabarcoding approach. The analysis of fecal and gut samples are represented in
yellow. The sequence reference database assembling, for the ingested species, is represented
in green
diversity of food items. The taxonomic resolution of this method depends on how
informative is the fragment of DNA amplified and how complete is the sequence
reference database (Pompanon et al. 2012; Taberlet et al. 2018). The dietary compo-
sition of ctenomyids was assessed only recently using this approach (Lopes et al.
2015, 2020), and is reviewed below.
10.3 What Do Ctenomyids Eat?
Despite the importance of knowing ctenomyids diet, just a few species of the genus
Ctenomys were given appropriate attention in studies of diet composition and
dietary habits. Feeding preferences of C. mendocinus, a species distributed in the
Mendoza Province, Argentina, were better explored than in any other ctenomyid.
The five studies performed to evaluate their diet were based on the microhistologi-
cal technique (Torres-Mura et al. 1989; Madoery 1993; Puig et al. 1999; Rosi et al.
2003) or cafeteria tests (Camin and Madoery 1994). The results showed that C. men-
docinus is generalist, preferring aboveground plant parts than roots (Torres-Mura
et al. 1989; Camin and Madoery 1994; Rosi et al. 2003). They use about 65% of the
plant genera present in their environment as food resource (Puig et al. 1999).
However, a high preference for grasses was observed during the four seasons
(70–94.5% of the diet content). The most eaten genera, depending on the popula-
tion, were Poa, Panicum, Stipa, Setaria, Aristida, and Elymus. Some shrub, forb,
and succulent forms were also recovered as part of their diet (Madoery 1993; Puig
et al. 1999; Rosi et al. 2003). Males and females eat similar proportions of plants
from different categories. However, males have a more varied diet during winter,
and females showed a higher specialization on grasses during spring. These dietary
differences in males and females have been associated with specific nutritional
requirements during the reproductive season, pregnancy, and lactating periods (Puig
et al. 1999). Camin and Madoery (1994) observed that not all plants harvested were
consumed, suggesting that some part of plant material is used for storage and
nesting.
Special attention was given to ecological aspects shaped by the food preferences
of populations of C. talarum and C. australis distributed in sympatry. These species
are allopatric distributed in the grassland dunes of the Province of Buenos Aires,
Argentina, with some populations occurring in sympatry. In their sympatric zone,
C. talarum occupy areas with dense vegetation, compact, and shallow soils, while
C. australis occupy areas with sparse vegetation, sandy, and deep soils (Comparatore
et al. 1995). Moreover, the latter species weights three times less than C. talarum.
Comparatore et al. (1995) observed that populations of both species in the sympat-
ric zone are generalist herbivorous, preferring to consume aerial than subterranean
or reproductive plant parts, and grasses over forbs. del Valle et al. (2001) confirmed
that the diet of C. talarum populations inhabiting Mar del Cobo, in Argentina, is
predominantly composed of the aerial vegetative plant parts of perennial grasses.
Their forage behavior changes seasonally, but they keep their preference for plant
parts with a higher fiber/protein ratio. Despite C. talarum is able to feed selectively,
and males are slightly more selective than females, this species consumes a high
variety of plants present in the grasslands, which are an import source of nutrients.
Rosi et al. (2009) determined the effects of cattle grazed and ungrazed sites on
the diet preferences of a C. eremophylus population inhabiting the arid plain of
Mendoza, Argentina. The results of microhistological analyzes showed that the veg-
etative part of grasses (mainly Panicum and Setaria genera) was the dominant food
source in their diet, followed by low shrubs. Tall shrubs and forbs were observed in
minor proportions. In the grazed area, C. eremophilus showed higher dietary diver-
sity, lower percentage of grasses, and higher consumption of low shrubs and repro-
ductive plant parts than in the ungrazed area, reinforcing the expectation that cattle
grazing force the species to change their dietary habits. During autumn-winter, the
availability of their preferred plants becomes lower. As a response, these rodents
shifted to a lower dietary selectivity, eating a higher variety of plants available
closer to their burrows, as consequence, decreasing their risk of predation.
Most recently, the dietary composition of seven ctenomyids species inhabiting
the southern and midwestern Brazil was assessed using the DNA metabarcoding
approach (Lopes et al. 2015, 2020). One of the central questions in community ecol-
ogy, the competitive exclusion principle, was addressed by Lopes et al. (2015) using
218 C. M. Lopes
C. minutus and C. flamarioni as a model of study. Under this principle, two com-
plete competitor species could not occupy the same habitat under limited resources,
unless one species exclude the other or some kind of niche partition takes place
(Pianka 2011). Ctenomys minutus and C. flamarioni are parapatrically distributed in
the southern Brazilian coastal plain, with a narrow area where they occur in sym-
patry and can compete for food resources. The authors recovered 19 plant families
as part of their diet, 13 for C. minutus, and 10 for C. flamarioni. Despite some dif-
ferences in their diet composition, Poaceae, Araliceae, and Asteraceae were the
most common plants identified in the feces of both species, together with Fabaceae
for C. minutus. These plant families are highly frequent and have high species rich-
ness in the southern Brazilian coastal plain, reinforcing the previous knowledge
about generalist and opportunistic feeding habits of ctenomyids. No remarkable diet
overlapping was observed in the area where these species co-occur. Ctenomys fla-
marioni showed a more homogeneous and less variable diet in the sympatric region
when compared to C. minutus or to other populations of C. flamarioni, suggesting
that some level of dietary partitioning was developed to avoid competition, allowing
these species to occur in sympatry.
Further investigations about the degree of diet specialization and diet overlap
were assessed by Lopes et al. (2020) for ctenomyids inhabiting southern Brazil,
using the DNA metabarcoding approach. Besides C. flamarioni and C. minutus, the
authors evaluated the diet composition of C. lami, a species that inhabit more inter-
nalized dunes of the southern Brazilian coastal plain; C. torquatus and C. ibicuien-
sis, that inhabit lowlands of the State of Rio Grande do Sul; and, specimens sampled
in the hybrid zone between C. minutus and C. lami. The results showed that these
species consume more than 60% of the plant families recovered in soil samples col-
lected around their burrows. Once again, grasses are the preferred food item by all
ctenomyid species analyzed. Despite some particularities observed in the diet of
each species, that are determined by the availability of food resources in the sur-
rounding environment, these ctenomyids showed a high niche overlap in the plant
families and Molecular Taxonomic Units consumed. Authors hypothesized that
these species are ecologically similar and able to use the same range of resources
when available in the environment, because they are allopatrically distributed and
the interspecific competition is not a limiting factor. Lopes et al. (2020) also ana-
lyzed diet preferences of C. bicolor and C. sp., species that inhabit sandy soils in the
States of Rondônia and Mato Grasso, respectively. They are distributed in a patched
environment between fragments of the Amazonian forest and deforested areas occu-
pied by crops. Both species are generalists, using grasses as the main food source.
Plants from crops were absent or represent a small proportion of their diet, contrast-
ing common knowledge that these species frequently use rubber trees and cassava
as a food source (Lopes et al. 2020).
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Chapter 11
Ecological Physiology and Behavior
in the Genus Ctenomys
María Sol Fanjul, Ana Paula Cutrera, Facundo Luna, Cristian E. Schleich,
Valentina Brachetta, C. Daniel Antenucci, and Roxana R. Zenuto
11.1 Introduction
Underground environments are considered among those that have most influenced
the evolution of the organisms that inhabit them. Subterranean animals spend much
of their lives in a moist, dark, poorly ventilated, hypoxic, and hypercapnic environ-
ment, also characterized by low primary productivity (Buffenstein 2000). Many of
the sensory signals present aboveground are limited underground (Francescoli
2000), affecting vital activities such as food searching, reproduction, and territorial
defense. Concomitantly, the underground habitat provides major advantages to its
occupants: environmental stability (e.g., thermal insulation) and protection against
predators (Nevo 1999). Therefore, similar adaptations are expected in underground
mammals due to shared selective pressures. However, this expected evolutionary
convergence is also accompanied by differences in biotic and abiotic factors that
operate at different geographic scales, contributing to the adaptive divergence of
underground mammals (Nevo 1999). Ecological physiology and behavior, as com-
ponents of the biology of subterranean rodents, have been reviewed in several
opportunities. In some of these reviews, the focus was on a particular species of
subterranean rodents, as was the case with naked mole-rats (Sherman et al. 1991),
South African mole-rats (Bennet and Faulkes 2000), and blind mole-rats (Nevo
et al. 2001). Nevo and Reig (1990) put together the first and more comprehensive
Authors María Sol Fanjul, Ana Paula Cutrera and Facundo Luna have equally contributed to
this chapter
M. S. Fanjul · A. P. Cutrera · F. Luna · C. E. Schleich · V. Brachetta · C. D. Antenucci

R. R. Zenuto (*)
Grupo ‘Ecología Fisiológica y del Comportamiento’, Instituto de Investigaciones Marinas y
Costeras, Universidad Nacional de Mar del Plata, Consejo Nacional de Investigaciones
Científicas y Técnicas, Mar del Plata, Argentina
e-mail: msfanjul@mdp.edu.ar; acutrera@mdp.edu.ar; fluna@mdp.edu.ar;
cschleic@mdp.edu.ar; vbrachetta@mdp.edu.ar; antinuch@mdp.edu.ar; rzenuto@mdp.edu.ar

https://doi.org/10.1007/978-3-030-61679-3_11
222 M. S. Fanjul et al.
review covering anatomical, physiological, ecological, and evolutionary studies in

several groups of subterranean rodents. Subsequent efforts (Lacey et al. 2000;
Begall et al. 2007) significantly expanded both the representation of species and the
issues addressed. Studies on ecological physiology and behavior in Ctenomys were
marginally included in the first review (Reig et al. 1990) but were more represented
in later opportunities (Busch et al. 2000; Lacey 2000; Francescoli 2000; Lacey and
Cutrera 2007; Schleich et al. 2007). The interest in understanding the multiplicity of
physiological and behavioral responses associated with the challenges of under-
ground life has grown since then. In this chapter, we aim to review the studies on
physiological and behavioral responses to subterranean environmental conditions
and food resources, as well as those that explore interactions with conspecifics (e.g.,
reproduction and territoriality) and heterospecifics (predators, parasites, and patho-
gens) in the genus Ctenomys. This genus is particularly interesting due to its high
species diversity (63 species, Patton et al. 2015) in which solitary species are domi-
nant while fewer are social (Lacey 2000; Tassino et al. 2011; Tomasco et al. 2019).
These organisms conduct most of their activities within the burrow systems and
hence are subject to many of the selection pressures typical of the underground
environment. However, in many species, food collection is carried out aboveground
(e.g., Ctenomys peruanus, Pearson 1959; Ctenomys opimus, Pearson 1959,
Ctenomys talarum, Busch et al. 2000; Ctenomys australis, Busch et al. 2000;
Ctenomys sociabilis, E.A. Lacey pers. comm.; Ctenomys haigi, E.A. Lacey pers.
comm.; Ctenomys fulvus, Cortés et al. 2000; Ctenomys mendocinus, Puig et al.
1992) and in at least one of them (C. opimus, E.A. Lacey pers. comm.), vegetation
is also consumed on the surface. This aboveground activity may lead to differences
in the manner ctenomyids use sensory cues to assess their environment, how they
cope with stressors both from above and below ground, how they thermoregulate
and maintain energetic and water balance, how they reproduce and establish their
territories, and how they face the challenges of parasite and predator exposure with
respect to more strict subterranean rodents. These particular aspects of the ecologi-
cal physiology and behavior of Ctenomys are reviewed in the following sections.
11.2 Sensory Biology
The sensory capabilities of individuals determine both the mode and extent to which
animals sense changes in their environment and respond to them (Scott 2005). The
sensory biology of the genus Ctenomys has been extensively studied. The first stud-
ies on this topic covered the olfactory and acoustic channels, two senses highly
relevant for a genus that inhabits a dark and monotonous subterranean environment
that limits the transmission of most signals and cues.
Up until now, most of the information regarding the use of olfactory cues in cte-
nomyids comes from studies conducted in C. talarum. Olfactory cues derived from
urine, feces, or anogenital exudations are used by C. talarum to assess individual
identity, reproductive condition, sex, and population of origin of conspecifics. Also,
11 Ecological Physiology and Behavior in the Genus Ctenomys 223
when the participation of the vomeronasal organ in the identification of odor cues is
allowed, tuco-tucos can discriminate the reproductive condition of opposite-sex
conspecifics (Zenuto and Fanjul 2002; Fanjul et al. 2003; Zenuto et al. 2004).
Familiarization by odor cues reduces aggression between partners during courtship
(Zenuto et al. 2007) and interacting males in a territorial context (Zenuto 2010, see
below). In addition, the mating behavior of C. talarum seems to be linked to olfac-
tory signals in mate evaluation and selection (Fanjul and Zenuto 2008a, 2012, 2013;
Fanjul et al. 2018). Altogether, these findings reveal the key role of chemical cues in
the territorial and reproductive biology of this rodent. Odor-based gender discrimi-
nation in males was also reported for C. sociabilis (Schwanz and Lacey 2003).
Moreover, the use of odor cues is not restricted to conspecific interactions.
Individuals of C. talarum use olfaction to orient their digging while foraging, both
detecting substances released by the plants to the soil and discriminating different
plant species (Schleich and Zenuto 2007, 2010). In addition, they also distinguish
odors from predators, thus avoiding them, and choosing to feed in areas where these
cues are not present (Brachetta et al. 2019a).
Vocalizations are an important means of communication in solitary and social
members of Ctenomys. Acoustic signals emitted in territorial, mating, or aggressive
encounters were described in several species (C. peruanus, Pearson 1959; C. haigi,
Pearson and Christie 1985; Ctenomys pearsoni, Francescoli 1999, 2001, 2002, 2011,
2017; C. talarum, Schleich and Busch 2002; C. mendocinus, C. sociabilis Francescoli
and Quirici 2010; Anillaco tuco-tuco, Ctenomys sp., Amaya et al. 2016). Most of the
described vocalizations are within the mid- to low-frequency range, suggesting a
convergent adaptation to the subterranean environment, where only low-frequency
sounds can propagate over long distances. In addition, the acoustic characteristics of
the vocalizations of subterranean rodents were also found to be relatively coincident
with the hearing morphology – particularly the location and density of cochlear
receptors – of the species studied so far (Mason 2004; Schleich and Busch 2004).
Although the subterranean ecotope is assumed to favor acoustic, olfactory, and
tactile senses in detriment of vision, the only existing studies on the latter reveals a
different perspective. Both solitary C. talarum and Ctenomys magellanicus present
normally-developed eyes – in terms of size and functionality – with a significant
proportion of two spectral cone types, indicating that photopic vision has a func-
tional significance in these facultative subterranean rodents. Although active vision
seems to be lost in both species studied, it is believed that the diurnal surface activ-
ity exhibited by these ctenomyids may be responsible for the maintenance of their
reactive vision capabilities, that is, visual surveillance, escape reactions, and preda-
tor detection (Schleich et al. 2010; Vega-Zuniga et al. 2017).
Which sensorial cues are used by subterranean rodents to orient themselves in
the complex and dark subterranean tunnels has deserved much attention. In
Ctenomys, the orientation of the burrows of tuco-tucos (NNW-SSE) suggests that
individuals may use the Earth’s magnetic field as a common heading indicator
(Malewski et al. 2018). However, until today, no further evidence that this genus
relies on the geomagnetic field to orient underground has been obtained (Schleich
and Antinuchi 2004), leaving the question of whether this genus has the sensory
capability to use information from the Earth’s magnetic field still unresolved.
11.2.1 Spatial Cognition
Spatial orientation, or the ability of individuals to learn to find their way through the
environment without getting lost (Vorhess and Williams 2014), has been studied in
different families of subterranean rodents due to the particularly complex structural
characteristics of their habitats. In the genus Ctenomys, the species for which this
type of information is available is C. talarum. Members of this species display a
highly developed capacity to learn and memorize structurally complex labyrinths;
individuals rapidly improved their spatial performance after the initial trials and
were able to memorize a complex maze for a period of between 30 and 60 days after
the learning process (Schleich and Antinuchi 2004; Mastrangelo et al. 2010).
Interestingly, and regardless of sex differences in home-range size, males and
females performed similarly in both tasks (Mastrangelo et al. 2010).
Spatial performance of an animal can influence its ability to perform crucial
activities that depend on proper spatial orientation, such as food searching or mate
localization. In C. talarum, animals exposed to predatory cues or an immune chal-
lenge, similar to what may be triggered by a parasite infection, showed a poorer
navigation capacity (Mastrangelo et al. 2010; Brachetta et al. 2014; Schleich et al.
2015). This clearly indicates that life-threatening or energetically-challenging stim-
uli can negatively impact on spatial memory formation and recall, which play
important roles in cognitive processes in tuco-tucos.
11.3 Stress Response and Individual Condition
Most organisms face stressful situations from different origins (physical, social,
and/or psychological) on a daily basis. The consequent loss of homeostasis depends
on the nature of the stress factor, as well as the magnitude of the response triggered
(Armario 2006). The underground habitat is particularly interesting due to their
distinctive physical and ecological characteristics that condition individual perfor-
mance; e.g., poorly ventilated environments, with low primary productivity, and
deprived of many sensory signals present aboveground (Buffenstein 2000;
Francescoli 2000). To understand how tuco-tucos are affected by different ecologi-
cal and environmental challenges, it is crucial to know about the physiology that
underlies the response to a stressor. In vertebrates, the activity of Hypothalamic-
Pituitary-Adrenal (HPA) axis is stimulated by several harmful stimuli, or stressors,
triggering the secretion of glucocorticoids (GCs) from the adrenal glands. Therefore,
both cortisol and corticosterone are commonly used to assess stress condition; basi-
cally, short- term high GCs levels are associated to the adaptive “fight or flight”
response, while sustained high levels during long-lasting exposure to stressors are
harmful to individuals (Sapolsky et al. 2000; Boonstra 2005). GCs are important
hormones regulating energy balance since they are involved in the mobilization of
energy reserves (Sapolsky et al. 2000) and high levels are considered indicative of
high energetic demands (Boonstra 2005; Mac Ewen and Wingfield 2003; Vera et al.
2018; Mac Dougall-Shackleton et al. 2019). In rodents, corticosterone is the domi-
nant GC, although there is variability within this group. In the genus Ctenomys, only
two species were studied in relation to these hormones, showing clear differences
between them with regard to the dominant GC found. GCs were assessed in C. socia-
bilis using blood and feces matrices (Woodruff et al. 2010, 2013), and higher base-
line levels of corticosterone than cortisol were detected in both free-living and
captive females. Individuals challenged with Adrenocorticotropic (ACTH) hormone
showed an increase in the levels of corticosterone metabolites, confirming that cor-
ticosterone is responsive to stress. Furthermore, corticosterone levels were higher in
free-living than captive females, and challenging conditions in the wild were pro-
posed to explain such differences (Woodruff et al. 2010). Moreover, the physiologi-
cal consequences of group living were explored in C. sociabilis, which live alone or
in groups in nature (Woodruff et al. 2013). Data from captive and free-living indi-
viduals provide evidence that females living alone or in groups differ in their base-
line GCs levels. Lone females showed higher levels of metabolite of GCs, so the
achievement of basic activities such as territory defense and food provision may
have more physiological consequences than social interactions with conspecifics.
On the other hand, studies in C. talarum revealed that both cortisol and corticoste-
rone circulate in the plasma, but females have higher levels of cortisol (Vera et al.
2012). These hormones differ in their seasonal and annual variation patterns in free-
living individuals, suggesting differences in their endogenous regulation and affec-
tation by environmental stimuli (Vera et al. 2011a, 2012, 2013). Cortisol is
responsive to the factors that typically regulate GC concentrations (acute stressors
and ACTH), but corticosterone is not (Vera et al. 2011a, 2012, 2013). Furthermore,
only cortisol levels were affected by sustained low-quality diet and fasting (Vera
et al. 2019). In contrast, angiotensine II – the main biologically-active hormone of
the renin-angiotensin system – stimulated corticosterone, but not cortisol secretion,
denoting its participation in mineral-water balance (Vera et al. 2019). Decreased
levels of both GCs – although more so for corticosterone – and lower negative feed-
back efficacy for corticosterone in captivity, may account for differential responses
to chronic stress conditions (Vera et al. 2019; Dickens and Romero 2013). Overall,
these results reveal that cortisol and corticosterone are not interchangeable hor-
mones and suggest different physiological roles in C. talarum (Vera et al. 2019).
Even though GCs are commonly considered as synonymous of “stress hor-
mones,” the activation of HPA is only one component of the complex stress response
in vertebrates (MacDougall-Shackleton et al. 2019). In this sense, the idea of “stress
profile” emerges as a more complex alternative, where a suite of biological param-
eters are considered instead of a single indicator for evaluating animal condition and
dysregulation (Milot et al. 2014). The evaluation of changes in several indicators of
animal condition, such as the neutrophils: lymphocytes ratio (N/L), body mass,
hematocrit, glucose, triglycerides, accompanied GCs response (mainly cortisol) to

challenging conditions in C. talarum. Decreased levels of glucose, triglycerides,
body mass, and inflammatory response were related to food restriction (Merlo et al.
2016a). In some cases, increases in both cortisol and N/L followed long-lasting
nutritional stress (Vera et al. 2019), while in others, no changes in cortisol levels
were found, although a higher N/L was detected (Schleich et al. 2015; Merlo et al.
2016a). In contrast, increases in cortisol levels without changes in N/L were found
under short-term exposure to stress conditions, such as a 24 h fasting or a brief
immobilization (Vera et al. 2019; Brachetta et al. 2019b). Although linear relation-
ships between GC levels and N/L are frequently proposed (Davis and Maney 2008,
2018), these parameters may be complementary in understanding the stress status of
animals due to their different sensitivities in the responses according to different
stress factors and duration of stimuli (Müller et al. 2011).
11.4 Energetics, Water Balance, and Thermoregulation
One of the most important aspects of the biology of species is the understanding of
how different factors, internal (e.g., digestive capacity) and/or external (e.g., ambi-
ent temperature, water availability, or social interactions) impose limits on their
energy budget (Karasov 1986; Weiner 1992; Withers et al. 2016). As animals can be
understood as open systems to the flow of materials and the energy (Wiegert 1968),
energy balance can be interpreted as the integration of intake, storage, and loss of
energy (Fig. 11.1).
11.4.1 Energy Intake
Animals can increase efficiency in obtaining energy by modifying ingestive and

digestive processes, as well as decreasing the cost of different internal mechanisms
(Karasov 1986). For subterranean species, maintaining efficiency, hence a balanced
energy budget, is expected to be more challenging due to the high cost of living
underground, where food is scarce and/or quality of food is poor (Antenucci et al.
2007). Low energy intake challenges animals to display compensatory mechanisms.
For instance, when C. talarum were fed a low-quality diet in captivity, they con-
sumed more food and devoted more time to feeding activities (Martino et al. 2007;
Perissinotti et al. 2009). In mammals, changes in the morphology of the gastrointes-
tinal tract and the activity of digestive enzymes could represent other digestive strat-
egies to address seasonal variation in food quality/availability. Such changes were
also observed in C. talarum (del Valle and López Mañanes 2008, 2011). Increased
efficiency can also rely on the re-ingestion of fecal items (coprophagy), as observed
in C. pearsoni (Altuna et al. 1998) and C. talarum (Martino et al. 2007; Perissinotti
et al. 2009; Fig. 11.1).
Fig. 11.1 Factors that affect energetic, thermoregulatory, and osmoregulatory variables in
Ctenomys species. The effect – or the lack of effect – of different factors on physiological variables
are indicated by different symbols. Upward and downward arrows indicate increment and decrease,
respectively; x marks indicate no variation; tick marks indicate variation (both increments and
decreases) in relation to developmental stages (growth, reproduction) and time (seasons, days) in
the specific physiological variable; unequal marks indicate differences between species
11.4.2 Basal Energy Expenditure
A balanced energy budget depends on how energy intake is maximized and expen-
diture is minimized. Experimental estimation of the minimum energy expenditure
needed by an individual to maintain homeostasis is the basal metabolic rate (BMR,
Hulbert and Else 2004). In Ctenomys, basal metabolism was evaluated under differ-
ent external conditions in different species. Depending on the species, different
natural or experimentally induced changes in external factors might lead to different
BMR responses (see Luna et al. 2017). Under a low quality diet or high soil hard-
ness, C. talarum decreased their BMR (Perissinotti et al. 2009). However, no effect
of controlled regimes of ambient temperature or seasonal changes in natural ambi-
ent temperatures was observed (Luna et al. 2012; Meroi et al. 2014). In C. aff.
Knighti seasonal energy saving, based on BMR estimation, was detected (Tomotani
et al. 2012; Tachinardi et al. 2017). Interestingly, when interspecific variability of
BMR was evaluated, Luna et al. (2009) observed that body mass, but not biogeo-
graphic factors – such as latitude, ambient temperature, or precipitation – affect
BMR (Fig. 11.1). Similarly, body mass was the main determinant of BMR between
C. talarum and C. australis living in the same area (Busch 1989). Basal metabolism
can also be seen as the minimum heat production at thermoneutrality. Thus, main-
taining a stable body temperature depends on the relationship between heat produc-
tion and heat loss through the animal surface (thermal conductance; Naya et al.
2013; Withers et al. 2016). Even at thermoneutrality, body temperature of C. aff.
Knighti shows daily variations (Tachinardi et al. 2014), which are synchronized to
light-dark cycles by their rhythmic locomotory activity and excursions outside the
burrow (Valentinuzzi et al. 2009; Fig. 11.1).
11.4.3 Thermoregulation and Water Balance
At ambient temperatures below thermoneutrality, an extra source of heat is needed

to maintain stable body temperature. The increase in energy metabolism under this
condition is related to shivering and non-shivering thermogenesis, which are com-
ponents of the total thermogenic capacity (Hohtola 2002; Cannon and Nedergaard
2011). In C. talarum, shivering is the main component of thermogenic capacity and
is not affected if individuals are acclimated to different temperature regimes (Luna
et al. 2012). Interestingly, this pattern is not maintained across Ctenomys species.
Mechanisms of heat production are species-specific, varying from a combination of
shivering and non-shivering thermogenesis (e.g., Ctenomys roigi, Ctenomys por-
teusi, and C. talarum) to a complete use of shivering thermogenesis (e.g., C. austra-
lis and Ctenomys tuconax; Fig. 11.1). Similarly to basal metabolism, biogeographical
factors did not affect the variability of total thermogenic capacity among Ctenomys
species (Luna et al. 2019). At ambient temperatures above thermoneutrality, another
problem arises: the need to lose heat. Individuals can lose heat by direct physical
routes (i.e., conduction, convection, and radiation) and by evaporation. Evaporation
is particularly effective at high temperatures since it does not rely on a thermal gra-
dient (Withers et al. 2016). Evaporative water loss of C. talarum increases above
thermoneutrality and remains low and stable within and below it (Baldo et al. 2015).
The maximal value of evaporation does not change in individuals exposed to con-
trasting humidity acclimation (Baldo et al. 2016). Since the atmosphere of burrows
can restrict evaporation – particularly at high ambient temperatures, due to the high
water vapor content – non-evaporative routes of heat loss are also used (Cutrera and
Antinuchi 2004; Luna and Antenucci 2007b). Evaporation cannot increase indefi-
nitely because it is also a significant route of water loss. Water is an essential com-
ponent of the body, and its balance must be regulated. One important non-evaporative
venue for water loss is through urine excretion. Besides behavioral strategies (see
Bozinovic and Gallardo 2006), mammals can minimize their urinary water loss by
producing concentrated urine (Schmidt-Nielsen 1997). Interestingly, neither
Ctenomys eremophilus (Diaz 2001 in Diaz et al. 2006) nor C. talarum (Baldo and
Antenucci 2019) can concentrate urine. However, under environmental restrictions
(i.e., low water or high salt content of food), C. talarum increased urine osmolarity
(Baldo and Antenucci 2019; Fig. 11.1). Further, as urine concentration capacity
depends on the morphology of the kidney (McNab 2002), medullary thickness can
be used as an index of renal performance. Medullary thickness is only reported for
C. fulvus, C. opimus, and C. eremophilus showing values similar to those found in
mesic surface-dwelling rodent species.
11.4.4 Non-basal Energy Expenditure
Measurement of non-basal metabolism in a wide range of conditions provides infor-

mation about different components of energy budget at different timescales. In gen-
eral, building new tunnels involves an extremely high cost of digging (see Vleck
1979). Soil hardness was proposed to be the main factor explaining the convergent
morpho-physiological features found in phylogenetically unrelated subterranean
species. In both C. talarum and C. australis, hard soils increased the digging meta-
bolic rate (Luna et al. 2002; Luna and Antenucci 2006, 2007a). Also, in C. talarum,
the increment in digging expenditure affects burrow architecture (Antinuchi and
Busch 1992), associated with the energy restriction to construct tunnels with angles
greater than 40° (Luna and Antenucci 2007b). At low ambient temperatures, C. tala-
rum used the heat produced during digging to supply the energy required for ther-
moregulation, but at high temperatures, digging increases the risk of overheating,
which is minimized by conduction to the soil (Luna and Antenucci 2007c; Fig. 11.1).
Thus, digging represents a high physiological cost for Ctenomys species having a
profound impact on the daily and annual energy budget (Antenucci et al. 2007). On
the other hand, reproduction is considered the most energetically costly period of
the life of a female (Tomasi and Horton 1992). Females of C. talarum increase their
energy metabolism during periods of gestation and lactation (Zenuto et al. 2002a).
After birth, pups show an increase in metabolic rate until day 10, when they start to
eat solid food but still cannot maintain stable body temperature. During this critical
period, contact with their mother and siblings is crucial for a normal thermoregula-
tory development (Baldo et al. 2014). After day 10, metabolic rate starts to decrease,
reaching adult’s BMR and body temperature values (Zenuto et al. 2002a; Cutrera
et al. 2003; Fig. 11.1).
From an evolutionary perspective, both internal (e.g., digestive constraints) and
external factors (e.g., ambient temperature) may have determined the balance
between energy gains and losses in Ctenomys. The metabolic scope is defined as the
difference between the lower and the upper limit of energy expenditure (i.e., basal
and maximal metabolism, respectively), and represents the amount of energy avail-
able to the individual for activities over the lower limit of energy required to main-
tain basal physiological mechanisms. In Ctenomys species, the metabolic scope can
be affected by the degree of underground commitment, since it appears to be lower
than in surface-dwelling species (Luna et al. 2015). Thus, restricted metabolic scope
can have a great impact on the species-specific energy budgets, and factors (e.g.,
low O2 content within burrows) that constrain limits in energy expenditure can
probably act independently on different energetic variables, such as basal metabo-

lism or thermogenic capacity (Luna et al. 2009, 2017, 2019).
11.5 I ntraspecific Interactions: Physiology and Behavior

of Territory Defense and Reproduction
11.5.1 Social Behavior and Territoriality
Most Ctenomys species show solitary habits with one individual occupying each bur-
row system, only shared during mating and the maternal care of the young. Sources
of evidence to account for solitary behavior are diverse: minimal or no home-range
overlap, animal capture and visual verifications of no further activities in a given bur-
row, and anecdotal reports (Table 11.1). In most solitary species, both sexes show
aggressive reactions toward conspecifics, but few studies addressed this topic from a
behavioral perspective. In C. talarum, both sexes defend their burrow systems, but
only males utter the typical “tuc-tuc” territorial vocalization warning potential intrud-
ers about the presence of the owner in its territory (Schleich and Busch 2002). Studies
using seminatural enclosures in captivity showed that males engage in aggressive
interactions with other males (Zenuto et al. 2002b). Such aggressive interactions
result in dominance hierarchies, which are also linked to the access to females during
the mating season. Moreover, as mentioned before, territorial aggression is modu-
lated by odor familiarity (i.e., the “dear enemy phenomenon”; Temeles 1994), as a
mechanism mediating territorial behavior in C. talarum (Zenuto 2010). Despite
dominance not being a simple function of aggressiveness, the acquisition and main-
tenance of certain status often require different degrees of aggression. Androgenic
steroid hormones, such as testosterone, mediate aggressive behavior. In the wild,
male C. talarum testosterone levels peaked during the reproductive season but were
also highly variable among individuals, ranging from barely detectable to extremely
high concentrations in comparison to other mammals (Vera et al. 2011b, 2013). This
could be related to their capacity to defend a territory and/or monopolize the access
to females. On the other hand, the role of aggression in habitat segregation was iden-
tified by means of experimental interactions involving two species naturally living in
sympatry, C. talarum and C. australis (Vassallo and Busch 1992).
Considering group living as part of a sociality continuum, few social species
have been identified in the genus Ctenomys so far. The first reported social species
was C. peruanus (Pearson 1959), followed by C. sociabilis (Pearson and Christie
1985). In C. sociabilis, the groups are stable and defined, and individuals are
involved in cooperative tasks such as excavation of tunnels, nest sharing, and off-
spring attendance during the reproductive season (Lacey et al. 1997; Izquierdo and
Lacey 2008). Later, radiotelemetry and genetic studies have found that burrow sys-
tems of C. rionegrensis are not strictly exclusive, and sporadic overlap occurs
among adult residents (Tassino et al. 2011, Tomasco et al. 2019). Also, for C.
Table 11.1 Social and reproductive physiology and behavior in Ctenomys species
11
Ovulation Mating system

type Sexual
Social Territorial Reproductive (Postpartum dimorphism Sexual
Species Climatea behavior defense seasonality estrum, PPE) (sex ratio) selection Parental care References
C. flamarioni Humid Sep to March Male > female Fernández-Stolz et al.
FCS
subtropical (births) (1:2.21) (2007) and Garcias
et al. (2018)
C. minutus Humid SolitaryHR Oct to Dec FCS Male > female Marinho and de
subtropical (1:1.26) Freitas (2006) and
Kubiak et al. (2017)
C. lami Humid SolitaryFC June to Dec FCS (No PPE) Polygyny? El Jundi and de
subtropical (perforated and Male > female Freitas (2004)
pregnant (1:1)
females)
C. torquatus Humid Solitary? Female CO Talice and Laffitte de
subtropical Mosera (1958)
C. pearsoni Humid Solitary FC Yes? June to Oct Induced (No Male > female Altuna et al. (1991,
FCS
subtropical (?) PPE) 1998) and Francescoli
(2011)
Ecological Physiology and Behavior in the Genus Ctenomys
C. opimus High Solitary FC Ago to Feb FCS Pearson (1959) ,

Mountain Facultatively (births) O´Brien et al. (2020)
social HR
C. rionegrensis Humid Solitary/ Jun to Dec FCA Induced? Male > female Tassino and Passos
subtropical SocialHR, GE (2010, Tassino et al.
2011) and Tomasco
et al. (2019)
(continued)
231
232
Ovulation Mating system

type Sexual
Social Territorial Reproductive (Postpartum dimorphism Sexual
Species Climatea behavior defense seasonality estrum, PPE) (sex ratio) selection Parental care References
C. mendocinus Cold arid Solitary Yes FC Ago to Feb FCA (No PPE) Male > female Female CO Puig et al. (1992),
or semiarid (1:1) Rosi et al. (1992,
1996, 2005) and
Camín (1999, 2010)
C. talarum Cool SolitaryHR, CE Yes CE Jun to Dec Induced CE Polygyny CE,GE Female Female Busch et al. (1989),
oceanic (pregnant (PPEE) Male > female sexual exclusively Zenuto et al. (2001,
females)FCA, CO (1:1.63) selection CO 2002a), Fanjul et al.
CE, FC, GE
(2006, 2018), Fanjul
and Zenuto (2008a,
2012, 2017), Zenuto
(1999, 2010) and
Cutrera et al. (2012)
C. australis Cool Solitary HR Yes FC Absent FCS (PPE) Polygyny? Zenuto and Busch
oceanic (pregnant Male > female (1998)
females) (1:2.12)
C. haigi Mountain Solitary HR (1:1.2) Lacey et al. (1998)
cold
semiarid
C. sociabilis Mountain Social HR May to Dec FCS (No PPE) Polygyny?: Communal Lacey et al. (1997),
cold Multi-female female Lacey (2004) and
semiarid groups and careFC, HR Lacey and Wieczorek
single females Female care (2004)
Supraindexes indicate data source: HR home range and polygons estimated by telemetry, FC field capture (A annual survey, S seasonal survey), CO observa-
tional studies in captivity, CE experimental studies in captivity, GE genetic studies,?: no data provided. aKöppen–Geiger climate classification system of
study sites
M. S. Fanjul et al.
opimus (previously reported as a solitary species; Pearson 1959), radiotelemetry

studies revealed their facultative social behavior, as some individuals tend to aggre-
gate during the night (O’Brien et al. 2020).
11.5.2 Seasonality and Regulation of Reproduction
Most ctenomyids are distributed within latitudes characterized by seasonal variation

in climate and food availability (i.e., subtropical, temperate, arid, or mountain
regions, Reig et al. 1990). Therefore, it is expected that reproduction occurs in a
restricted period of time where food and climate conditions are optimum, thus max-
imizing reproductive output. Studies conducted suggest that the timing and length
of reproductive activities are adjusted so that the most energetically-demanding
activities (e.g., gestation and lactation, Zenuto et al. 2002a) are assured (Table 11.1).
Specifically, the reproductive season of some Ctenomys species occurs when food is
abundant (C. talarum, Fanjul et al. 2006), and temperatures are more benevolent
(C. pearsoni, Tassino and Passos 2010). In addition, the length of the reproductive
period – and the potential for a second litter – in C. mendocinus varies depending on
the climatic rigorousness of the habitat (Rosi et al. 1992, 1996). Environmental cues
regulate such timing, contributing to secure these favorable conditions. In this sense,
studies in C. talarum in captivity showed some evidence of female reproductive
responsiveness to photoperiodic cuing (Fanjul and Zenuto 2008b). However, the
relevance of photoperiod in triggering reproduction in C. talarum could be more
important in the wild, where food quantity (and possibly quality) is variable, par-
ticularly at the onset of the reproductive season (Fanjul et al. 2006).
11.5.3 Ovulation and Receptivity
Induced ovulation appears to be the rule among rodents that are solitary, occur at
low population densities and/or inhabit highly seasonal environments (Zarrow and
Clark 1968; Milligan 1982). So far, the only ctenomyid studied in this regard
(C. talarum, Weir 1974) is an induced-ovulator, in which the induction depends on
the amount of copulatory stimulation that the female receives, but not on the sole
male presence or presence of its chemical cues (Fanjul and Zenuto 2008a). Penis
morphology in this genus is characterized by the presence of spines and spikes
which is consistent with the stimulatory requirement to induce ovulation (Balbontín
et al. 1996; Rocha-Barbosa et al. 2013). Even though ovulation in C. talarum is trig-
gered by copulation, female reproductive behavior varies with progesterone and
oestradiol levels, with both hormone levels and vaginal cytology being affected by
male presence (Fanjul and Zenuto 2012).
11.5.4 Courtship and Mating
In solitary species, courtship leads to lower levels of aggression and allows the
assessment of potential partners in relation to their receptivity and mate quality
(C. pearsoni, C. mendocinus, and C. talarum; Altuna et al. 1991; Camín 1999;
Fanjul and Zenuto 2008a). As expected, courtship in ctenomyids involves chemical
and vocal signals (Altuna et al. 1991; Camín 1999; Fanjul and Zenuto 2008a). The
occurrence of multiple intromissions – and possibly multiple ejaculations – observed
in C. talarum (Fanjul and Zenuto 2008a, 2012) and C. pearsoni (Altuna et al. 1991)
during copulation supports the hypothesis of induced ovulation in these species.
11.5.5 Mating Preferences
In mammals, the internal development and lactation impose a differential cost of

reproduction assumed by females. Then, females maximize their reproductive suc-
cess by being more choosy in terms of mate preference (Andersson 1994). Female
mate choice seems to be crucial for C. talarum as we found that is a prerequisite for
mating in captivity (Zenuto et al. 2007). Female familiarization with male odors
affects the outcome of a reproductive encounter (Zenuto et al. 2007), and females
prefer novel than familiar males (Fanjul and Zenuto 2013). Moreover, females pre-
fer dominant males (Fanjul and Zenuto 2017) and those that hold territories even
after they were scent-marked by a competitor (Fanjul et al. 2018). Furthermore, the
Major Histocompatibility Complex (MHC) genes play a key role in mate choice in
C. talarum, since females mate preferentially with heterozygous males and those
that carry specific MHC alleles (Cutrera et al. 2012), thus supporting the idea that
females prefer males with “good genes,” which would increase the chances of
resisting infections for the offspring.
11.5.6 Mating System and Parental Care
Polygyny is the predominant mating system in mammals, which is characterized by

the monopolization and defense of several females by a single male and exclusive
maternal care of the young (Clutton-Brock 1989). The extent of polygyny has been
associated to sexual size dimorphism (males of bigger size) and female-biased oper-
ative sex ratios (Mitani et al. 1996). Considering this indirect evidence, polygyny
seems to be the most frequent mating system among solitary and social ctenomyids
(Table 11.1). Confirmation of this hypothesis in C. talarum comes from molecular
data (Zenuto et al. 1999). Parental care is conducted only by females (Zenuto
et al. 2001) involving feeding of the young as well as thermal protection via direct
contact (C. mendocinus, Camín 2010; C. talarum, Zenuto et al. 2001, 2002a; Cutrera
et al. 2003). For C. sociabilis, telemetry data revealed communal nest attendance by
several females (Izquierdo and Lacey 2008).
11.6 I nterspecific Interactions: Physiology and Behavior

of Predator Avoidance and Defense Against Parasites
and Pathogens
11.6.1 Predator Avoidance
Predators affect prey both directly by killing them and indirectly by affecting their
behavior, foraging patterns, reproduction, and stress physiology, thus affecting their
fitness (Clinchy et al. 2013; Moll et al. 2017). The nonlethal impact of predators
could be even of greater demographic magnitude than that produced by the death of
prey, also involving trans-generational effects (Clinchy et al. 2013). So far, studies
on the physiological and behavioral effects of predatory risk in the genus Ctenomys
are limited to C. talarum. Although the subterranean environment provides protec-
tion against predators, individuals become vulnerable to aerial and terrestrial preda-
tors while dispersing or foraging aboveground (Busch et al. 2000). This species is
often predated by owls, foxes, wildcats (Vassallo et al. 1994; Busch et al. 2000),
dogs, and domestic cats (C.E. Schleich, pers. obs.). Experimental studies show that
both acute and chronic exposure to direct cues indicating the presence of a predator
(immobilization and cat urine) affected spatial performance in this species (Brachetta
et al. 2014). Similarly, tuco-tucos exposed to predator odors generated an anxiety
state and showed avoidance behaviors, even in juveniles prenatally exposed to pred-
atory risk (Brachetta et al. 2015, 2016, 2018). The relationship between behavioral
and physiological responses to stress by predation was experimentally proved.
Moreover, the moderate magnitude found in both responses is proposed to be con-
sistent with a predation pressure buffered by the use of the underground environ-
ment (Brachetta et al. 2019b).
11.6.2 Defense Against Parasites and Pathogens
The study of the interactions between host physiology (i.e., immune function) and
disease ecology (i.e., pathogen prevalence) in a wide range of environments and
animal species allows us to understand the extrinsic and intrinsic factors leading to
immune function variation and disease susceptibility in natural populations (Demas
and Nelson 2012). Variation in immune responsiveness among individuals and spe-
cies may be the result of genetic factors, but also of the interplay among immunity,
demography, and life-history traits in an ecological context (Schoenle et al. 2018).
These factors have been explored in a few Ctenomys species, in an effort to under-
stand the sources of variation in pathogen resistance in this group.
Previous studies in model species have shown that parasite resistance is under
genetic control (see Charbonnel et al. 2006 for a review). More specifically, MHC
genes code for glycoproteins involved in the recognition and binding of foreign
antigens (Klein 1986). The high levels of polymorphism of MHC genes are the
result of pathogen-mediated selection (Doherty and Zinkernagel 1975). This selec-
tive model has been explored in tuco-tucos in relation to variation in social habits
between species (C. haigi and C. sociabilis, Hambuch and Lacey 2002), differences
in demographic traits among populations (C. talarum, Cutrera and Lacey 2006), the
impact of distinct demographic histories (C. australis and C. talarum, Cutrera et al.
2010a; Cutrera and Mora 2017), and patterns of evolution of MHC loci across 18
ctenomyid species (Cutrera and Lacey 2007). Further evidence of selection on
MHC genes came from later studies that showed that (1) MHC allelic and genotypic
variation are associated to parasite resistance and immunocompetence in C. talarum
(Cutrera et al. 2011); (2) the strength of this association may vary among popula-
tions of this species (Cutrera et al. 2014a), and (3) as mentioned before in this
review, female C. talarum choose their mates in relation to their MHC genotype,
among other factors (Cutrera et al. 2012). Together, these studies suggest a role for
parasite-driven selection and female mate choice in maintaining MHC variation,
and hence parasite resistance, in natural populations of C. talarum.
Mounting an immune response is presumed to be costly, and these costs may
mediate the trade-offs between immune function and other costly physiological pro-
cesses. The different outcomes of these trade-offs may also explain the variation in
immune responsiveness (Norris and Evans 2000) among species and individuals.
Therefore, it becomes essential to estimate the magnitude of the immune response
and the costs associated with its activation. One way of doing so is assessing the
increase in oxygen consumption that may be associated with triggering an immune
response (Demas et al. 2012). For C. talarum, the metabolic costs of mounting an
antibody-mediated response (Cutrera et al. 2010b), a local inflammatory response
(Merlo et al. 2014), and an acute-phase innate response (Cutrera, Luna, and Zenuto,
unpublished data) to artificial antigens have been estimated directly using respirom-
etry in captivity, suggesting that costs are variable among the different arms of
immunity of tuco-tucos, with the antibody-mediated response being the most costly
response to activate and maintain, followed by the acute-phase response.
Surprisingly, the local inflammatory response was not associated with a significant
increase in oxygen consumption (Merlo et al. 2014; Fig. 11.1). Another way of
exploring the costs of immunity is to do so indirectly, by assessing if the magnitude
of an immune response is negatively affected by other physiological processes
occurring at the same time (i.e., growth, reproduction, see Sheldon and Verhulst
1996; Lochmiller and Deerenberg 2000). In C. talarum, possible trade-offs between
mounting a local inflammatory response and several energetically-demanding pro-
cesses, such as growth (Cutrera et al. 2014b), reproduction (Merlo et al. 2014), and
mounting a simultaneous humoral response (Merlo et al. 2019) were assessed.
Further, the effects of diet (Merlo et al. 2016a), parasitism (Merlo et al. 2016b), and
body condition (Merlo et al. 2018) on the magnitude of the local inflammatory
response were also explored in C. talarum, suggesting that additional costs, besides
the energetic, maybe mediating the trade-offs between immune function and other
energetically demanding activities of tuco-tucos. The studies conducted so far show
a generally low immune responsiveness of the different arms of the immune system
of C. talarum (innate and adaptive responses, both induced and constitutive) com-
pared with other vertebrate species. This pattern of low immune responsiveness
coupled with the low parasite richness found in C. talarum – which is presumed to
be a consequence of their restricted mobility, high territoriality, and spatial isolation
(Rossin and Malizia 2002) suggests an important role of the underground habitat in
the evolution of immune strategies in this group of subterranean rodents.
11.7 Conclusions and Prospects
Early studies on physiology and behavior in members of the genus Ctenomys date
from the 1970s with the contributions of Wise et al. (1972) and Weir (1974) report-
ing glucose regulation and reproductive behavior in captive C. talarum. Later,
Busch (1987, 1989) performed her studies about physiological adaptations to under-
ground conditions in C. talarum and C. australis, particularly assessing hematologi-
cal traits that allow tuco-tucos to cope with the low oxygen and high CO2 that
characterize the burrow atmosphere, as well as their thermoregulatory capacity and
basal metabolic rate. In addition, identifying dominance hierarchy between species
as a possible cause of habitat segregation between sympatric species was an impor-
tant first step for behavioral studies in Ctenomys (Vassallo and Busch 1992).
Nowadays, there is a greater suite of studies on physiological and behavioral
responses to diverse biotic and abiotic challenges faced by this group. While the
number of species of tuco-tucos assessed is increasing, they still represent only a
quarter of the diversity of the genus. In addition, some topics have only been
addressed in one species – mostly C. talarum – or in a few species, making it diffi-
cult to identify general response patterns in an effort to synthesize our knowledge.
Thus, to reach a better understanding of the multiple physiological and behavioral
responses associated with living underground, we need to make a greater effort to
include more Ctenomys species into the picture. Field and laboratory studies are
needed for this commitment, even though the secretive habits of these organisms
make it particularly difficult to obtain information in the wild and to develop ade-
quate housing conditions for controlled experiments in captivity. Assessing gluco-
corticoid levels, together with a complete biochemical profile, will allow the
evaluation of changes in energy demands and stress conditions associated with dif-
ferent challenges, both in nature and in captivity.
Despite these difficulties, it is now possible to identify some patterns of physio-
logical and behavioral responses that are in accordance to what is expected for sub-
terranean rodents. One example of this is the role of chemical and acoustic cues in
intraspecific communication, especially in the contexts of territorial and reproduc-
tive behavior. The chemical channel also plays an important role in food searching
as well as in predator avoidance. Further, spatial orientation appears as a critical
skill in individuals living in structured and complex systems such as underground

burrow systems. Energy balance is strongly modulated by restrictions on energy
intake, as well as the reduction of costs associated with the maintenance of homeo-
stasis and energy-demanding activities, such as digging. This energy limitation
clearly affects reproductive seasonality. Finally, patterns of immune function varia-
tion and parasite resistance, at least in C. talarum, seem to have evolved according
to the lower parasite exposure of the subterranean habitat in comparison to the chal-
lenges of the aboveground environment.
On the other hand, some studies suggest that the use of the aboveground environ-
ment during food collection impacts on other traits not closely related to living
underground, such as the development of visual abilities, the relevance of photope-
riod in the regulation of female reproductive activity, and the daily variation in body
temperature synchronized to light periods by the rhythmic activity pattern and sur-
face excursions. Moreover, detected differences in determinants of energy budget
between Ctenomys species may account, at least in part, for different commitments
to below and aboveground activities. Even though burrow systems protect individu-
als from aerial and terrestrial predators, response to predatory cues shows that indi-
viduals suffer predatory risk when exposed aboveground.
Beyond the adaptive convergence and divergence framework proposed by Nevo
(1979), we suggest that future studies on ecological physiology of subterranean
rodents will take into account the pace of life perspective. Ctenomys species may be
considered “slow-living,” given their relatively altricial development – at least for
solitary species –, late acquisition of sexual maturity and longevity. In light of this,
predictions can be made regarding the physiological responses of this group of
rodents. For example, considering immune function, it is expected that tuco-tucos
rely more strongly on the adaptive arm of immune defense, even when it is more
energetically expensive than the innate arm, because the adaptive immunity confers
memory against repeated infections that are more likely to occur in long-lived spe-
cies (Lee 2006). Therefore, integrating the studies of the physiological ecology and
behavior of ctenomyids into the pace of life syndrome (POLS) concept, which
describes the covariation of life-history, physiological (particularly metabolic, hor-
monal and immunological), and behavioral traits or personality, under specific con-
ditions (Stamps 2007), may offer insight into the relationship between the low levels
of energy expenditure of ctenomyids and their slow pace of life.
Acknowledgments We wish to dedicate this chapter to Cristina Busch who initiated the study of
ecology, physiology, and behavior of tuco-tucos in the Universidad Nacional de Mar del Plata. We
are grateful to the editors for inviting us to contribute with this chapter. Our research was supported
by grants from Agencia Nacional de Promoción Científica y Tecnológica, Consejo Nacional de
Investigacion Científica y Tecnológica and Universidad Nacional de Mar del Plata.
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Chapter 12
Effects of Environmental Pollution
on the Conservation of Ctenomys
Cristina A. Matzenbacher and Juliana da Silva
12.1 Introduction
All species on the planet have developed adaptations, in different ways, that pro-
mote enough aptitude to sustain different populations for long periods of time and
environmental changes. This is the result of the interaction between the genome and
the environment. The ability of species or population to adaptations from a polluted
environment is called toxicology evolutionary (Bickham et al. 2000). Currently, the
influence of epigenetic factors on adaptations has been discussed, alone and associ-
ated with mutations or natural selection or genetic drift. Epigenetics involves mei-
otically and mitotically stable alterations in gene expression that are not based on
DNA sequence changes but involve processes that impact the packaging of DNA
(chromatin structure) (Kalisz and Purugganan 2004). Although epigenetic variation
can occur in the absence of genetic variation, genetic variation can influence epigen-
etic variation and the epimutation rate in several ways. Epigenetic variation can be
a significant source of natural phenotypic variation; therefore, it has the potential to
play a major role in adaptation to environmental change. Modern evolutionary the-
ory is primarily based on the inheritance of random genetic variation, so it has been
widely discussed whether evolutionary theory requires revision considering epi-
genetics (Jablonka and Raz 2009; Richards et al. 2010; Jablonka 2017).
Genomic instability and epigenetic changes induced by environmental pollutants
have been related to different effects on populations, such as the reduction of popu-
lation size due to loss of genetic variability and reproductive capacity. Pollution is
C. A. Matzenbacher (*)
Programa de Pós-Graduação em Genética e Biologia Molecular, Universidade Federal do Rio
Grande do Sul - UFRGS, Porto Alegre, Brazil
J. da Silva (*)
Universidade LaSalle – UNILASALLE and Universidade Luterana do Brasil - ULBRA,
Canoas, RS, Brazil
e-mail: juliana.silva@unilasalle.edu.br

https://doi.org/10.1007/978-3-030-61679-3_12
250 C. A. Matzenbacher and J. da Silva
any change in the environment that can be a natural or agrarian ecosystem, an urban
system, or even a micro scale. It can cause changes in the proportions or character-
istics of one of the elements that make up the environment itself, such as increased
carbon dioxide concentration. It may be the result of the introduction of natural
substances, although foreign to certain ecosystems, or artificial substances such as
the deposition of pesticides in the soil (Maximillian et al. 2019). To assess complex
chemical-biological interactions and predict the potential for chemical substances
that cause harmful effects and impact ecological communities and populations,
chemical and ecotoxicological methods are used to assess responses at both the
organism and cellular levels (Schaeffer 1991). This kind of research integrates data
from the field and laboratory biomarkers to understand and predict adverse effects;
this way of study looks at the whole system. Adverse outcome pathways (AOPs) are
conceptual frameworks that bring together what is known about chemical agents –
with limited safety data – and their adverse effects on human health and ecosystems
(Groh et al. 2015).
Contaminants transferred through the food chain play an important role in their
action and persistence in the ecosystem and can slowly accumulate in the tissues of
individuals over time. Many toxic substances such as metals and organic com-
pounds can be transferred from individuals’ tissues to their predators and reach a
higher concentration at higher trophic levels. Pollutants can also act on DNA, lead-
ing to teratogenic effects, mutations in germ cells, premature aging or induce neo-
plasia in somatic cells. In addition, DNA damage (mutations) can affect population
structure, in which genetic imbalance (change in genetic variability and allelic fre-
quency) has a direct effect on biodiversity decline, increasing vulnerability to envi-
ronmental stress and, consequently, possibly leading species to reduced survival and
extinction (Hemminki et al. 1979; Groh et al. 2015).
Toxicity is the ability of a particular substance or even the complex mixture of
elements present in the environment to affect a living organism. The effect will
depend on chemical to chemical and possible interactions, with different effects on
different types of cells and tissues. Environmental toxicology represents a complex
triangular interaction between anthropogenic chemicals, the environment, and
biota. A biomarker approach also involves the identification of molecules capable of
responding to anthropogenic (xenobiotic) chemicals by positive or negative regula-
tion, in order to use them to assess all environmental quality. In recent decades,
interest in using biomarkers or bioindicators as monitoring tools to assess environ-
mental pollution has increased considerably. This is because biomarkers are suitable
not only for providing information on the health status of exposed organisms but
also on the quality and/or quantity of the exposure situation. Thus, the biomarkers
can be used as toxicity measurements or as fingerprints for exposure to chemicals.
Depending on the character of the selected biomarkers, the evaluator will receive
more data on the adverse effects on organisms or about the exposure situation
(Sturla et al. 2014).
12 Effects of Environmental Pollution on the Conservation of Ctenomys 251
12.2 Biomarkers and Bioindicators
Different approaches are used to assess the effects and risks of exposure to chemi-
cals, physical, and biological agents. Biomarker is a term for analyzing the interac-
tion between a biological system and an environmental agent. In order to study the
effects of exposure to environmental genotoxins in wildlife species, many biomark-
ers have been applied which have been derived from human cancer risk assessment
studies, including markers of chemical DNA modifications such as DNA adducts,
markers of cytogenetic effects such as chromosomal aberrations and micronucleate
cells. Studies of markers of genotoxic effects in somatic or germ cells with wild
animal populations can help in the analysis of exposure to environmental mutagens.
It evaluates whether the agent is affecting biodiversity and the chance of survival,
inducing changes or selecting critical environments for the survival of current envi-
ronmental levels of pollution contributing to the assessment of ecological risks
(Kleinjans and Van Schooten 2002).
Many studies suggest that exposure to pollutants is associated with genotoxicity
biomarkers such as chromosomal aberrations, sister chromatic exchange (SCE),
micronuclei (MN; See Fig. 12.1), and DNA damage observed by the comet assay
(CA; See Fig. 12.2) (León-Mejía et al. 2011; Rohr et al. 2013; da Silva 2016;
Espitia-Pérez et al. 2018). MN formation is widely used in molecular epidemiology
as a biomarker of chromosomal damage, genome instability, and eventually cancer
(Fenech 2002). The occurrence of MN represents an integrated response to pheno-
types of chromosomal instability and altered cell viability caused by genetic defects
and/or exogenous exposures to genotoxic agents. The MN test detects aneugens
(numerical chromosomes aberrations) and clastogens (inducing breakages of chro-
mosomes) in the cytoplasm of interphase cells that have undergone cell division
during or after exposure to different agents (Fenech 2002, 2007; da Silva 2016).
Fig. 12.1 Images of

the Ctenomys torquatus
bone marrow cells
representing micronuclei
(MN). (Images from the
author’s archive)
Fig. 12.2 Images of comets assay of Ctenomys torquatus in blood cells representing damage
classes. (a) Class 0, undamaged; (b) class 1; (c) class 2; (d) class 3; (e) class 4, maximum damage.
(Images from the author’s archive)
The CA is a procedure for evaluating DNA lesions (i.e., strand breaks, DNA
adducts, excision repair sites, cross-links, and alkali-labile sites) and involves appli-
cation of an electrical current to cells, which results in the transport of DNA frag-
ments out of the nucleus. The image of DNA migration obtained resembles a comet
with a head and a tail, hence the term comet assay (Singh and Stephens 1998; Silva
et al. 2000). Since the DNA damage induced by toxic agents is often tissue and cell-
specific, CA is especially useful because it can detect DNA lesions in individual
cells obtained under a variety of experimental conditions. The technique can also be
used to evaluate DNA repair (Tice et al. 2000). The CA has already been used to
detect DNA damage in several native animals, especially small mammal species,
living near or in polluted areas (Petras et al. 1995; Salagovic et al. 1996).
Another biomarker that is being used recently is the telomere length (TL).
Telomeres are found at both ends of each chromosome, which protect the genome
from nucleolytic degradation, unnecessary recombination, repair, and interchromo-
somal fusion. Telomeres are nucleoprotein structures that protect the ends of
eukaryotic chromosomes with a noncoding repeatable DNA sequence (TTAGGG).

It has a single-strand 3’G adjacent nucleotide overhang linked by the shelterin hexa-
meric protein complex (Fig. 12.3), which makes them sensitive to oxidative stress
and single-stranded DNA breaks. Repeating units and shelter complexes harbor dif-
ferences between species, but their homology is established in almost every respect.
In normal somatic cells, telomeres decrease with each round of cell division due to
limitations of the replication mechanism to replicate the ends of linear DNA mole-
cules (De Lange 2005; Zakian 2012). When telomere length reaches a critical limit,
the cell undergoes senescence and/or apoptosis. Telomere length may therefore
serve as a biological clock to determine the lifespan of a cell and an organism, since
telomere length decreases with age. Once telomeric DNA is less capable of repair,
some agents associated with specific habits may expedite telomere shortening by
inducing damage to DNA (Kahl and da Silva 2016).
It is known that DNA methylation has a function in telomere length variability
(Blasco 2007; Yehezkel et al. 2008). DNA methylation patterns are formed by a
family of DNA methyltransferases enzymes that transfer a methyl group from
s-adenosyl-1-methionine (SAM) to the 5-position of cytosine forming 5-methyl-2-
deoxycytidine (5-mdC), primarily found in CpG dinucleotides (Liou et al. 2017).
Epigenetic mechanisms, such as DNA, RNA, histone modifications, and microR-
NAs have been shown to be potential links between the genetic and environmental
exposure, which can be determinant to health life or reduction in life span.
Environmental toxicants can alter epigenetic regulatory processes, and mediate spe-
cific mechanisms of toxicity and responses. Particularly, epigenetic modifications
can alter genome expression and function under exogenous influence (Kahl et al.
2019). Growing evidence suggests that at least 15 environmental chemicals may
lead directly to diseases via epigenetic mechanism-regulated gene expression
Fig. 12.3 Telomere with complex shelterin and telomerase. When TL reaches a critical limit, the
cell undergoes senescence or apoptosis. (Modify from Jacobs (2013) and Alenalee (2008))
changes, such as hydrocarbons and inorganic elements (Santoyo et al. 2011; Liou
et al. 2017). Other studies have brought evidence of the connection of global DNA
methylation and mechanisms such as oxidative stress (Pavanello et al. 2010).
As important as knowing some biomarkers used to know and evaluate the effects
of contaminants in free-living animals is knowing which are the best bioindicators
for each assessment. Bioindicator is the organism that provides information about
the environmental conditions of its habitat through its presence or absence or
through its behavior (Van Gestel and Van Brummelen 1996). It is also defined as a
species or group of species that readily reflect the abiotic or biotic state of an envi-
ronment, representing the impact of environmental changes on a habitat, commu-
nity or ecosystem. Either it is indicative of the diversity of a taxon subset or of all
diversity within an area (Gerhardt 2000). According to Altenburger et al. (2003),
bioindicators detect a biochemical aspect of toxic action (e.g., damage to the mem-
brane, inhibition of enzymes, and damage to DNA), providing rapid and direct indi-
cations of the toxic impact on the environment. Bioindicators can reveal a lot about
the mechanism of toxic action, which allows the extrapolation of related contami-
nants. However, bioindicators tend to be specific for toxic substances, as not all
compounds inhibit the same biological processes. Thus, it is important to choose
bioindicators relevant to the mechanisms of action to be known or evaluated.
The main anthropogenic activities studied using rodents refer to exhaustion of
motor vehicles (Degrassi et al. 1999; Heuser et al. 2007), industrial emissions
(Ieradi et al. 1998; Hazratian et al. 2017), polluted mining dump area (Andráš et al.
2006), and coal mining area (León et al. 2007). In general, contaminating com-
pounds are complex mixtures and contain heavy metals and hydrocarbons. However,
studies using subterranean rodents as bioindicators are rare. Subterranean rodents
are interesting as bioindicators of soil pollution, mainly because they maintain the
same territory for long periods and consume large amounts of vegetation. They play
an important role in the dynamics of the ecosystem, due to their ability to modify
the availability and dynamics of soil nutrients and resources for other species,
because of their excavation system (Nevo 1979; Busch et al. 2000; Reichman and
Seabloom 2002; Kerley et al. 2004; Dacar et al. 2010). Thus, studies with subter-
ranean rodents could provide better information on contamination of the environ-
ment, the food chain, and, principally, on bioaccumulation, as well as data on
potential exposures to wildlife and humans and on the effects of exposure to organic
and metallic pollutants, mainly due to their ecology.
12.3 he Role of Environmental Pollution

T
in Ctenomys Adaptation
The genus Ctenomys, popularly known as tuco-tucos, is composed of about 65 spe-

cies of subterranean rodents (Teta and D’Elia 2020, Chap. 2 this volume) that live
in burrows, but come to the surface to vocalize, clear the burrows, seek food, and
have morphological characteristics adapted to this lifestyle, such as back paw
bristles, reduced pinna, incisor teeth outside the mouth, as they use these teeth to aid
excavation, not letting sand in their mouth (Parada et al. 2011). They share some
common characteristics, including their solitary and territorial habits, small patchily
distributed populations, and small effective population sizes, which are associated
with low rates of adult dispersal and lead to a pattern of low genetic variation within
populations and high genetic divergence among populations. Furthermore, the spe-
cies commonly show high levels of karyotypic variation (Reig et al. 1990; Nowak
1999; Lacey et al. 2000).
The genus has a large geographic distribution extending from the extreme south
of the Neotropical region to the south of Peru, spreading all over the Patagonic
region with a great latitudinal variation, and recorded from the sea level up to
4000 m high in the Andean region (Reig et al. 1990). In Brazil, eight species of tuco-
tucos are described. Three of them, Ctenomys rondoni Miranda Ribeiro, 1914,
C. bicolor Miranda Ribeiro, 1914, and C. nattereri Wagner, 1848 are still poorly
investigated. All the other five species of tuco-tucos, Ctenomys torquatus
Lichtenstein, 1830, C. minutus Nehring, 1887, C. flamarioni Travi, 1981, C. lami
Freitas, 2001, and C. ibicuensis, de Freitas et al., 2012, occur in southern Brazil, in
the States of Rio Grande do Sul (RS) and Santa Catarina (SC).
C. torquatus is endemic to southern South America (Freitas and Lessa 1984).
Despite the wide geographic range, there is a more common chromosomal form
(2n = 44), and karyotype polymorphisms in the southern regions, restricted to the
limits of the Atlantic Ocean (2n = 46), and Western (2n = 40 and 42) of its distribu-
tion (Freitas and Lessa 1984; Fernandes et al. 2009; Gonçalves et al. 2009). Its
geographic distribution in RS almost coincides with the distribution of coal reserves
(Freitas 1995). This coincidence took our research group to these regions to analyze
possible genetic damage in the tuco-tucos that lived there. Because damage to DNA
is not immediately recognized in organisms and has broad-ranging effects, this
rodent could be an important system to monitor changes in environmental
genotoxicity.
Since the late 1990s, our research group has been studying C. torquatus (Da
Silva et al. 2000a, b; Silva et al. 2000a, b; Matzenbacher et al. 2019) and C. minutus
(Heuser et al. 2002) as an environmental quality bioindicators in Rio Grande do Sul.
Silva et al. (2000a, b) conducted a 2-year study to detect the effects of coal, com-
paring the results with MN assay and CA to C. torquatus (2n = 44). At the end of
2 years, 240 rodents had been analyzed (capture-mark-recapture method). The loca-
tions studied covered three locations in RS: Candiota, a region about 2 km from the
Presidente Médici coal power plant, Butiá, a region approximately 5 km from a strip
coal mine, and Pelotas, a region without a coal mine and power plant. Biological
hazards associated with Candiota coal field were investigated in a pilot study. The
results showed that coal and derivatives induced DNA and chromosomal lesions in
C. torquatus cells that were demonstrated by CA and MN test. The CA was more
sensitive and showed a direct relationship between age and damage, and an inverse
relationship between temperature and damage index. In addition to the authors dem-
onstrated higher concentrations of heavy metals for soil samples from coal regions
(Zn, Ni, Pb, Cd, V, and Cu), as well as hydrocarbons, and a relation of these
concentrations and DNA damage. Other studies using free living rodents, yellow-
necked mouse (Apodemus flavicollis), and bank vole (Clethrionomys glareolus),
which were exposed to a coal mining area (Czech Republic) also demonstrated
higher levels of DNA damage (Degrassi et al. 1999). Besides, León et al. (2007),
using CA assay in wild rodents Rattus rattus and Mus musculus, exposed in a coal
mining area (Cordoba, Colombia) show that mice and rats originating from the coal
mining area exhibited a significantly higher extent of DNA damage as assessed by
length of DNA migration, damage index, and percentage of damaged cells com-
pared to animals from a control area.
Another study was conducted at the same sites of Silva et al. (2000a, b), with the
same species of tuco-tucos, to evaluate the effect of exposure to coal and its deriva-
tives and to examine the relationship of coal exposure with variations in TL, global
DNA methylation, and genotoxicity (Matzenbacher et al. 2019). The study showed
a significant reduction in the TL of the exposed tuco-tucos compared to the unex-
posed ones. Moreover, it demonstrated no association to factors such as sex and age
with coal exposition. In this study, no relationship was found between global DNA
methylation and exposure to coal, as well as no correlation between TL and DNA
methylation, probably due to our small sample size. But a relation between more
damaged cells in adults may be related with the reduction in the adults’ number
from Candiota. The reduction in TL is normal and expected, but this reduction is
greater in exposed animals, which means that something is accelerating this loss in
this region of the chromosome leading to a premature senescence. Our results dem-
onstrated that C. torquatus suffer DNA damage/instability, as observed in the CA
(DNA damage), and telomere shortening, likely as a consequence of the oxidative
damage that results from their exposure to a complex mixture, including inorganic
and organic elements. To prove the possibility of this mechanism, metal analyzes
were carried out and Zn, Ni, Pb, Cd, V, and Cu were detected in soil. Martiniaková
et al. (2010) studies determine the concentrations of heavy metal in the liver, kidney,
and bone of yellow-necked mice (A. flavicollis) and bank voles (C. glareolus)
trapped in a region with a chemical plant, coal power station, and coal mines in
Nováky, Slovakia. Highest concentrations of Cd and Zn were found in the bone of
both yellow-necked mice and bank voles. Cu and Fe accumulated mainly in the
kidney and liver. There are many studies that report the relation between some coal
exposure and the high concentration of chemical elemental and oxidative stress
indexes such as the highest contents of S, Cl, Fe, Zn, and Br in frog Hypsiboas faber
(Zocche et al. 2014); the reduced survival to metamorphosis in exposed larvae in
grass shrimp (Palaemonetes pugio) (Kuzmick et al. 2007); a reduction in DNA
repair capacity in Mytilus edulis with increasing duration of exposure to genotoxic
agents (Steinert et al. 1998).
The other specie studied by our group was C. minutus which has a wide distribu-
tion, occurring from the south of “Farol de Santa Marta” (SC) to “São José do
Norte” (RS). Its distribution is related to the formation of the coastal plain in south-
ern Brazil and presents patterns of karyotype diversity that correspond to regions
where there were paleochannels limiting the dispersion between parapatric loca-
tions (Lopes et al. 2013). They have seven parental karyotypes (2n = 50a, 46a, 48a,
42, 46b, 48b, and 50b) and current hybridization zones with intermediate karyo-
types (Freygang et al. 2004; Marinho and De Freitas 2006; Lopes et al. 2013). These
species have been used in genetic and population studies because they exhibit high
karyotypic variability and deserves special attention in studies related to
conservation.
Heuser et al. (2002) evaluated a possible genotoxic effect of vehicle emissions in
C. minutus (2n = 46) on both sides of a highway on the coastal region (Amaral and
Weber), and Maribo, a control area. Peripheral blood of C. minutus was used to
perform MN test and CA, and the soil from their burrows to analyze the hydrocar-
bon concentration and the presence of some metals related to vehicle emission. In
addition, concentration of NO2 in the air also was measured. The study showed that
the DNA damage rate was higher near the highway, as well as the average NO2
concentration. Adult females showed greater DNA damage. The metals found in the
soil of the highway with higher concentrations were Cr, Ni, Cu, and Zn, and the
hydrocarbons were also shown higher in the two studied points in the highway in
relation to the control region. This study provided chemical and biological data
from areas exposed to automobile exhaust, indicating the association among envi-
ronmental agents with levels of damaged cells observed in the wild rodent C. minu-
tus. Similarly, Degrassi et al. (1999) found in the three parameters of measurement
of the MN test (total number, average, and frequency) increase in the exposed area
of Muro Torto, Rome (Italy) compared to the control area of the zoo under study
with house mice (Mus musculus domesticus); however, this difference was not sta-
tistically significant. Vehicular emission also demonstrated affect the vegetation
growth as shown in the study of Wagh et al. (2006) that evaluate pollution impact on
the vegetation along the road in Jalgaon City, Maharashtra (India), and observed
that vegetation at roadside with heavy traffic had less leaf area, total chlorophyll,
and total proteins in leaves. Hazratian et al. (2017) to assess the potential use of the
Norway rat, Rattus norvegicus, as a bioindicator for lead and cadmium accumula-
tion in 10 urban zones in Tehran, Iran. The anthropogenic activities and vehicular
emissions contribute to the entry of toxic metals to humans and other animal’s food
chains. The accumulation of heavy metals in some free-living rodents has been
extensively studied (Šumbera et al. 2003; Guerrero-Castilla et al. 2014). Other stud-
ies also showed the relation between inorganic elements present in vehicles exhaus-
tion and DNA damage in different species (Meireles et al. 2009; Brito et al. 2013).
Schleich et al. (2010) evaluated the concentrations of four heavy metals (Pb, Zn,
Fe, and Cu) in muscle and liver from the subterranean rodent Ctenomys talarum
from natural dunes, cultivated area, and military area of Buenos Aires Province,
Argentina. The study revealed a higher concentration of metals in the livers than in
the C. talarum muscles in the military (Fe and Cu) and cultivation areas (Pb and
Cu). In soil samples, the highest concentrations of metals found were Fe in both
military and cultivated areas. In the vegetation samples, low levels of metals were
found, with Cu being most abundant in the military area, Pb in the cultivation area,
and Zn in both cultivation and dunes areas.
In addition to the biomarkers that assess the effects of contamination on free-
living animals, other markers of equal importance for the conservation and
adaptation of these animals are interesting to be considered for a more comprehen-

sive assessment of these effects. Knowledge of the genetic structures of these popu-
lations is an example. Lopes and de Freitas (2012) studied the effects of habitat
reduction and fragmentation in four C. lami populations, due to human occupation,
progressive urbanization, and expansion of agriculture and livestock. Ctenomys
have a limited geographical distribution, as they are small populations distributed
irregularly and low levels of adult dispersal (Reig et al. 1990; Nowak 1999; Lacey
et al. 2000). In this study, it was demonstrated that the populations had no genetic
structure associated with the distinct karyotype groups. However, molecular mito-
chondrial and nuclear markers demonstrated the existence of two populations
instead of the four populations at the beginning of the study. These two populations
are not completely isolated but are probably reinforced by a geographical barrier.
The vulnerability of C. lami was greater than the authors previously assumed, then
the author suggested the designation of evolutionarily significant units (ESU) to one
population, based on their genetic differentiation for both molecular markers
(nuclear and mitochondrial), and a Management Unit (MU) to another one, which
considers statistically significant divergence in allele frequencies (nuclear or mito-
chondrial), no matter the phylogenetic differentiation of the alleles. In addition, they
suggested the inclusion of the conservation status of this species as vulnerable
(Lopes and de Freitas 2012).
Another interesting study involving conservation and habitat loss was carried out
with Magellan tuco-tuco. Magellan tuco-tuco (Ctenomys magellanicus) is the
southernmost Patagonian-Fueguian rodent, with a small distribution, which was
categorized as vulnerable due to a strong population decline caused by overexploi-
tation and loss and degradation of habitats produced by grazing sheep. In a study by
Lazo-Cancino et al. (2020), the authors estimated the appropriate habitat distribu-
tion for C. magellanicus and predicted the appropriate habitat distribution and
potential range changes under future climate change. According to the study, seven
climatic variables, associated with the water regime and temperature variation
between seasons in Patagonia, were the most important for the distribution of spe-
cies. Under most future climate change scenarios, suitable habitat for C. magellani-
cus would decline mainly in its current continental distribution, with drastic loss
and fragmentation of suitable habitats. This information is important for predicting
a bottleneck, which results in a decrease in genetic diversity, which is extremely
important for the viability of a population.
Animals such as tuco-tucos, which have reduced population size and still suffer
from the impact on their environments, such as habitat fragmentation, are more
susceptible to suffering DNA damage from exposure to pollutants and have a more
catastrophic effect on your population structure. Figure 12.4 shows us a summary of
the way that some chemical agents, in the form of a complex mixture, and frag-
mented habitats could be affecting small populations of mammals such as the tuco-
tucos. These habitats are also a source of reduced genetic diversity, due to the
degradation of the environment, and the emergence of new mutations that are lead-
ing them to an evolutionary adaptation to these effects (Matson et al. 2006; Pedrosa
et al. 2017). Species have developed adaptations that promote enough aptitude to
Fig. 12.4 Pressures that subterranean mammals suffer from anthropogenic actions
sustain different populations for long periods of time and environmental changes.
The adaptations reflect responses to the selection imposed by toxins that character-
ized the environment (Brady et al. 2017).
12.4 Future
Little is really known about the physiology of subterranean mammals, and much
less about the effects to which they are subjected in relation to the contamination
that their habitats are exposed to every day. Although underground environments
are considered more stable and simpler than those above ground, their characteris-
tics are important ecologically and evolutionarily at the level of development of the
species. The characteristics of underground niches have led to similar evolutionary
pressures that have resulted in morphological, physiological, and behavioral adapta-
tions converging on underground life, wherever they occur in the world. Subterranean
rodents (e.g., Geomys, Thomomys, Ctenomys) play an important role in the dynam-
ics of the ecosystem. They are considered ecosystem engineers because of their
ability to modify the availability of resources directly or indirectly for other species.
Its large excavation systems affect the texture, water holding capacity, soil nutrient
dynamics, and vegetation composition and abundance (Nevo 1979; Jones et al.
1994; Busch et al. 2000; Reichman and Seabloom 2002; Kerley et al. 2004; Dacar
et al. 2010).
Studies with conservation genetics or landscape genetics, conducted with popu-
lations of tuco-tucos, concluded that contemporary habitat fragmentation increases
population differentiation. Genetic analysis of the landscape suggests that habitat
quality and longitude were the most strongly associated environmental factors
(Lopes and de Freitas 2012; Mora et al. 2017). It may be time by now, given that our
knowledge of genetic toxicology has improved and that we also technically are bet-
ter able to investigate DNA damage making use of modern molecular biological
techniques, to start thinking on a new test strategy. Some examples are in silico
methods, transcriptome approach, as well as next generation screening and sequenc-
ing tests (Perkins et al. 2003; Barzon et al. 2011). Transcriptomic profiles obtained
from samples of wild animals considered to be environmental bioindicators may
highlight candidate genes associated with pollution tolerance. Searching selection
signatures using single nucleotide polymorphisms (SNP) genotyping offers a com-
plementary route to explore adaptive evolution and has the gain of being able to
provide information on selective pressures that affect all kinds of tissues (Hamilton
et al. 2016). In silico toxicology can complement the predominant in vitro and
in vivo toxicity tests, predicting toxicity, and prioritizing chemicals or drugs in
order to minimize harmful effects (Parthasarathi and Dhawan 2018). New DNA
sequencing techniques, known as “next generation sequencing” (NGS), offer high
speed and throughput that can produce a huge volume of DNA sequences with
many applications in research and diagnosis. The benefit is the determination of
sequence data from single DNA fragments from a library that are secreted into
chips, preventing the need for cloning into vectors before the acquisition of the
sequence. At this time, NGS technologies are applied for complete genomic
sequencing, research of genomic, metagenomic, epigenetic diversity, the discovery
of noncoding RNAs, and protein-binding sites and gene expression profile by RNA
sequencing (Barzon et al. 2011).
12.5 Conclusions
In conservation genetics, the analyses performed with Ctenomys described in this

chapter represent a new approach to assess the anthropogenic effect on natural
mammal populations. These studies are important due to the emphasis on the viabil-
ity of genotoxic assays and epigenetic tools in conservation studies. We are cur-
rently watching a growing trend of cooperation between ecotoxicologists and
conservationists, as evidenced by the rising number of studies with this approach,
which can improve research in the areas of conservation and evolution. Genotoxicity
and alteration in the methylation patterns in Ctenomys can result in environmentally
induced phenotypic plasticity, which may be transgenerationally inherited. More
studies, including modern molecular biological techniques, will always be needed
in the field of ecogenotoxicity and epigenetics to continue to shape our understand-
ing of the mechanisms of controlling and creating phenotypic variation and its
implications for evolution.
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Index
A Bolivian region, 61
Abrocomidae, 73 Boliviensis, 61
Adaptation, 141, 148, 152, 158, 160 Bone distribution, 160
Adaptive radiation, Ctenomys sp., 131, 133 Bones, 157, 158, 160
Adrenocorticotropic (ACTH) hormone, 225 Burrow system, 149, 151, 152
Adverse outcome pathways (AOPs), 250 Burrowing behavior, 160
Agouti, 171, 183
Alipio Miranda Ribeiro, 60
Allopatric process C
extrinsic factors, 43 Cafeteria tests, 215
Allopatric speciation model Caviomorpha, 152, 156, 157
C. australis and C. flamarioni, 45, 46 Central nervous system, 160
C. lami and C. minutus, 46, 47 Chemical DNA modifications, 251
differentiation events, 45 Chromosomal arms, 45
Allopatry, 69, 73, 79 Chromosomal differentiation, 50, 51
Amazonian forest, 218 Chromosomal numbers, 46
Amazonian group, 60 Chromosomal rearrangements, 34, 44
Ancestor, 183 Chromosomal speciation
Ancestral character, 183 Ctenomys sp., 45
Anthropogenic chemicals, 250 diploid number, 53
Apomorphy, 7 heterozygotes, 53
Apomorphy-based clade, 6 multiformity, 53
Apoptosis, 253 rearrangements, 43, 53
Axial tomographic images, 158 Chromosomal variation, 61
Cladogenesis, 89
Classical metapopulation model, 98
B Coastal dune systems, 95
Balanced energy, 227 Cohesive lineages, 7
Bartlets test, 173 Collaborative efforts, 36
Basal metabolism, 228, 230 Comet assay (CA), 251, 252, 255–257
Baupläne, 7 Complex craniomandibular system, 154
Bayesian approaches, 96 Conservation genetics
Bergmann’s rule, 77, 79 anthropogenic pressure, 102
Bioindicators, 250, 254, 255, 257, 260 distinct ecoregions, 102
Biomarkers, 250–252, 254, 257 endangered species management, 102
Biomonitoring, 250 environmental stochasticity, 103

https://doi.org/10.1007/978-3-030-61679-3
266 Index
Conservation genetics (cont.) analysis of significant variance, 179

habitat fragmentation, 102 burrowing systems, 167, 201
heterogeneity, 102 cattle grazing force, 217
narrow distributional range, 103 chromosomal evolution, 113–114
natural habitats exploitation, 103 C. ibicuensis, 255
objective, 102 clades, discriminant analysis, 125, 126
phylogeographic studies, 103 color patterns, 184
populations, 103 common characteristics, 255
Contaminants, 250 conservation genetics, 260
Coprophagy, 214 contact zones, 198
“Corrientes” group, 61, 89, 96, 101 convergence pattern, 169
“Corrientes species complex”, 101 convergent evolution, 181, 182, 184
Cranial morphology cripsis, 184
Ctenomys sp. C. rondoni, 255
ANOVA, 120 cryptic coloration, 177, 178, 180, 182
dorsal view, 134 C. talarum, 257
evolution, 129 dense taxonomic sampling, 33
geographical structure, 129 density, 173, 175, 176, 178
lateral view, 135 diet, 213, 215, 216, 218
LDA, 124 dietary composition, 217
mandible shape, 133 distributions, 193, 195, 197
MANOVA, 122 ecological function, 169
pairwise MANOVA, 123, 124 ecology, 196
phenogram, 131 effects, 200
ventral view, 135 evolutionary biologists, 168
Crypsis, 184 feeding behavior, 214
Cryptic coloration, 177, 178, 180, 182 feeding habits, 215, 218
Ctenomids, 178 food availability, 196
Ctenomyidae, 71, 113 fossils, 33
alternative definitions, 7 genotype, 171
apomorphy, 7 geographical distribution, 113, 115, 169,
apomorphy-based clade, 6, 7 255, 258
cohesive lineages, 7 geometric morphometrics, 116–118
conservative morphologies, 4 habitats, 169, 170, 194
evolutionary stages, 4 hair and skin color, 170–174
fossils, 3, 4 hybrid individuals, 198
history, 4 hybrids, 184
morphological differentiation, 7 inter- and intraspecific variation, 168
morphological diversification, 4 limitation, 195
paleontological ages, 8 living species, 22
phylogenetic analyses, 4 macroecological level, 184
recognition, 3 Mc1r, 170
rootless molars, 4 methodologies, 214
Sallamys, 8 microhabitats, 180
South American hystricomorph microhistological analysis, 215
rodents, 3, 12 molecular phylogenetic analysis, 115
stem, 6 morphological characteristics, 198
systematic and paleobiological, 3 morphological diversification, 116,
Ctenomyids, 213 133, 134
Ctenomys sp., 69, 73, 79 morphological skull variations, 114
adaptation, environmental morphological traits, 114
pollution, 254–257 pelage variation, 171–174
adaptive phenotypes, 185 phenotype, 171
adaptive variation, 170 phylogenetic relatedness, 169
Index 267
pigment distribution, 173, 175, 176, 178 evolutionary alternatives, 51

populations, 217 evolutionary process, 47
quantitative studies, 184 geographic distribution, 47, 50, 51
reproductive isolation, 133 habitats, 52
reproductive season, 199 haplogroups, 49
in size, 114 haplotypes, 49
skulls and mandibles, 116 hybridization zones, 50
skull size variation, 121 karyotypes, 47
social pattern, 199 mtDNA sequences, 49
social structure and ecological pericentric inversion, 47
features, 199 relationships, 50
soil coloration, 170–174 secondary analysis, 49
soil granulometry, 200 Southern Brazil, 51, 52
soil moisture, 196 sympatric speciation, 47
spatial scales, 200 tunneling, 51
species groups, 35, 114 Ctenomys rionegrensis, 92
specimens, 36, 214 Ctenomys sociabilis, 93
subgenera, 33 Ctenomys talarum, 92, 148
sympatric zone, 197 Ctenomys taxonomic status, 60
thermoregulation, 184
as tuco-tucos, 113, 184, 193, 196, 254
vegetation and nutrient content, 200 D
vegetation composition and Dasyprocta, 73
dynamics, 201 Density, 173, 175, 176, 178
See also Genus Ctenomys Digging
Ctenomys bicolor, 60, 61 in arid and semiarid ecosystems, 141
Ctenomys cytochrome b gene, 60 biomechanical approach, 148, 149
Ctenomys haigi, 93 bones, 157, 158, 160
Ctenomys lami, 46, 47 burrow excavation, 141
chromosomal polymorphism, 56 burrow system, 149, 151, 152
chromosomal rearrangements, 54 Ctenomys sp., 142
chromosomal variation, 53, 54, 56 foreclaws, 141, 142
diploid numbers, 54, 55 forefeet, 142
genetic structure, 54 mechanical advantage and forces, 148
haplotypes, 54, 55 morphological specializations, 161
heterozygous carriers, 55 musculoskeletal systems, 160
inhabits, 53 numerous terrestrial mammals, 141
meiosis, 54 physiological adaptations, 161
Mendocinus group, 56 predation pressure, 161
mitochondrial and nuclear molecular proximal pads, 142
markers, 56 scratch- and chisel-tooth, 142
morphometric approach, 56 scratch-digging, 152–154, 160
pericentric inversions, 53 skull and pectoral appendicular
populations and karyomorphs, 56 regions, 143–147
Robertsonian centric fusion, 54 structural bone resistance, 159
Torquatus group, 56 superficial masseter muscles, 158
Ctenomys minutus, 46, 47 tooth-digging, 154–157
Araranguá river, 49, 50 tuco-tucos, 150
Bayesian clustering and specimen, 52 Digging tools, 157
Camaquã and Jacuí rivers, 50 DNA-based methods, 215
channels, 48 DNA damage, 250–252, 256–259
chromosome evolution, 47, 48 DNA metabarcoding, 215
diploid number, 51 DNA methylation, 253, 254, 256
ecology and morphology, 52 DNA sequences, 30
268 Index
E mandibular fragment affine, 8

Ecological niche modeling (ENM), 45, 59 masseteric morphology, 8–9
Ecological physiology, 238 morphofunctional diversification, 12
Ecological processes, 213 morphologically cohesive, 8
Energy balance, 238 pampean region, 12
Energy intake challenges, 226 polytypic, 44
ENM-based approach, 59 speciation (see Speciation models)
Environmental pollution species and populations, 10
biomarkers and bioindicators, 251–254 species varieties, 61
in Ctenomys adaptation taxonomic and ecological
chemical agents, 258 diversification, 11
and conservation, 257 Geometric morphometrics
DNA damage, 258 Ctenomys sp., 116–118
morphological characteristics, 254 Geomys bursarius, 167
vehicular emission, 257 Glacial-interglacial cycles, 88
Environmental toxicants, 253 Glucocorticoids (GCs), 224
Environmental toxicology, 250 Grid per cell (GCs), 52
Epigenetic mechanisms, 253
Epigenetic modifications, 253
Epigenetics, 249 H
Epigenetic variation, 249 Habitat discontinuities, 44
Evolutionary perspective, 229 Habitat fragmentation, 102, 258, 259
Evolutionary radiation Haplogroup “Litoral”, 49
Ctenomys sp., 116, 131, 133 Haplogroup “Mostardas”, 49
Haplogroup “Sul”, 49
Haplogroup “Tavares”, 49
F Haplotype networks, 91
Feeding, 148, 215 Heat production, 228
Female familiarization, 234 Histocompatibility Complex (MHC), 234
Finite element analysis (FEA), 158 Hypothalamic-Pituitary-Adrenal (HPA)
Flooded systems, 96 axis, 224
Forelimb and mandibular muscles, 157
Forest environment, 60
Fossils, 4 I
Frater group, 11 Iberá region in Argentina, 61
Functional morphology, 142, 152 Immune responsiveness, 235
Ingestive and digestive processes, 226
In silico toxicology, 260
G Interspecific hybrid zone
GBG-band pattern, 45 chromosomal variability, 58
Generalized Procrustes analysis (GPA), 117 divergent populations, 58
Genotoxicity biomarkers, 251 ecological requirements, 59
Genus Ctenomys ENM, 59
apomorphies, 8 geomorphological evolution, 58
apomorphy clade, 9 habitat characteristics, 59
application, 11 homeology, 59
characteristics, 44 karyotypes, 59
crown clade, 9–11 mtDNA, 59
fossil record, 8, 9 Robertsonian mechanisms, 58
geographic distribution, 7, 10 Southern Brazil, 59
history, 8, 12 sympatry and allopatry, 59
hybrid zones, 56, 57 Interspecific interactions
interspecific hybrid zones, 58–59 demographic traits, 236
intraspecific hybrid zones, 57–58 experimental studies, 235
Index 269
immune response, 236 L

immune responsiveness, 237 Landscape genetics
MHC allelic and genotypic, 236 anthropic activities, 101
physiological processes, 236 anthropically fragmented landscape, 101
predators, 235 C. chasiquensis, 101
subterranean environment, 235 conservation and viability, 84
Intraspecific hybrid zones differentiation, 100
chromosomal polymorphisms, 58 elevations, 100
chromosomal rearrangements, 57, 58 environmental evolution, 101
chromosomal variability, 57 flood-prone habitats, 101
diploid numbers, 57 gene flow patterns, 100
introgressions, 58 geographical and environmental
microsatellite loci, 57 features, 99
2n and AN combination, 58 geospatial and genetic data, 100
Intraspecific interactions methodological tools, 83
behavioral perspective, 230 microsatellite loci, 100
courtship, 234 objectives, 84
environmental cues, 233 sampling locations, 100
female mate choice, 234 tuco-tucos, 99–101
internal development, 234 variables, 100
MHC alleles, 234 Last Glacial Maximum, 45
ovulation, 233 Late Quaternary, 88, 104
photoperiod, 233 Late Quaternary environmental
radiotelemetry studies, 233 changes, 94
reproduction, 233 Limited dispersal, 85
sociality continuum, 230 Linear discriminant analysis (LDA), 119,
solitary behavior, 230 124, 127
steroid hormones, 230 Linear regression, 178, 181
Isla Grande de Tierra del Fuego, 94 Locomotion, 148
Isolation by distance (IBD) pattern
allelic variants, 87
C. australis, 91 M
C. rionegrensis distribution, 86 Macroscopic property, 156
C. rionegrensis pattern, 88 Mechanical advantage (MA), 149
Ctenomys, 87 Melanocortin 1 receptor (MC1R), 169
demographic expansions, 87 α-Melanocyte stimulating hormone
dispersal and gene flow, 87 (α-MSH), 170
dispersion rates, 86 Mendelian diallelic character, 57
DNA variation, 86 Mendocinus species, 56, 61, 89, 182
gene flow and genetic drift, 86 Mesoscale, 156
genetic variation, 85 Microhistological technique, 216
microsatellite loci, 87 Micronuclei (MN), 251, 255
mtDNA variation, 86 MicroRNAs, 253
mutational rate, 87 Midwest region of Brazil, 61
nuclear molecular markers, 86 Minimum convex polygon (MCP), 52
phylogeographic studies, 86 Mitochondrial DNA (mtDNA), 86, 93
populations, 87 Mitochondrial genetic variation, 94
tuco-tucos, 86 MN test, 251, 255, 257
Modern evolutionary theory, 249
Modern molecular biological
K techniques, 259–260
Karyomorphs, 61 Molecular markers, 44, 258
Karyotypes, 46 Morphological differentiation, 3
270 Index
Morphological diversification demographic expansion, 94

Ctenomys sp., 133, 134 demographic stability, 96
Morphotype, 60 flooded systems, 96
mtDNA Cyt-b gene, 44 genetic variability, 93
Multivariate analyses, 23 genus Ctenomys, 95
Multivariate analysis of variance (MANOVA) glacial-interglacial cycles, 88
skull and mandible shape, Ctenomys, habitat availability, 95
119, 122 habitat specialization, 94
haplotypes, 90
historical gene, 96
N Holocene-Pleistocene boundary, 90
New DNA sequencing techniques, 260 mendocinus species, 89
Next generation sequencing (NGS), 260 Pleistocene-Holocene boundary, 97
Non-basal metabolism, 229 population expansions, 93
Non-key apomorphies, 8 range expansions, 88
relative isolation, 90
sister species, 90
O steppe environments, 94
Octodontidae, 73 Torquatus group, 88
Octodontoids, 6 tuco-tucos, 88, 89
Olfactory cues, 222 Phylogeographic structure, 83
Oligocene, 8 Phylogeographic studies, 83, 86
One-dimensional spatial systems, 87 Phylogeography, 83
Pigment distribution, 173, 175, 176, 178
Pigmentation, 171
P Pleistocene, 183
Pace of life syndrome (POLS), 238 Pleistocene stratigraphic sequence, 11
Paleochannels, 51 Plio-Pleistocene, 3
Pampa region, 61 Pollutants, 250
Parsimonious trees, 5 Pollution, 249
Patos Lagoon, 46 Polygyny, 234
Penial morphology, 35 Polytypic genus Ctenomys, 44
Phaeomelanin, 171 Population genetics structure
Pheomelanin, 170, 173 C. lami, 99
Photoperiod, 238 connectivity, 97
Phylogenetic analysis, 30, 31 demographic change, 98
Phylogenetic generalized least squared diversity, 97
regression (PGLS), 155 fragmented habitats, 97
Phylogeny, 61 gene flow, 97
Phylogeographic patterns, 45 genetic drift, 98
availability and connectivity, 88 landscapes, 97
C. australis, 92 metapopulation model, 98
C. chasiquensis, 90 microsatellite loci, 97, 98
cladogenesis, 89 peripheral areas, 98
C. lami, 95 revolutionized, 97
climatic conditions, 89 social factors, 98
coastal dune, 95 spatial distribution, 98
coastal sand dunes, 90 spatial scales, 98
C. minutus, 95, 96 Postacrosomic process, 35
Corrientes group, 89, 96 Principal components analysis (PCA), 119
C. pearsoni, 94, 95 Protadelphomys possess, 6
C. rionegrensis, 92
C. sociabilis, 93
C. talarum, 92 Q
demographic changes, 92 Qualitative and quantitative approach, 34
Index 271
R chromosomal, 53–56 (see also

Rapoport’s rule, 74 Chromosomal speciation)
Reproductive behavior, 233 classes, 43
Robertsonian rearrangements, 46 definition, 43
Robertsonian translocations, 43, 54 hybrid zones, 44
sympatric (see Sympatric speciation)
Species complex, 61
S Species conservation
Safety factor (SF), 159 geographical distribution, 202
Senescence, 253, 256 IUCN Red List, 203
Sensory biology knowledge, 203
chemical cues, 223 mammals, 202
olfactory cues, 222 subterranean rodents, 203
sensorial cues, 223 subterranean species, 202
subterranean ecotope, 223 tuco-tucos, 202
subterranean environment, 223 Species groups, 31
vocalizations, 223 Stress response
Sensory organs, 160 biological parameters, 225
Sensory signals, 221 cortisol levels, 225, 226
Sensory systems, 154 GCs, 224
Single nucleotide polymorphisms (SNP) homeostasis, 224
genotyping, 260 Subterminal band, 183
Skull geometry, 160 Subterranean and fossorial rodent species, 193
Skull morphological evolution, Ctenomys Subterranean mammals, 259
geographical structure Subterranean rodents, 254, 257, 259
cranial shape, 129 See also Ctenomys sp.
skull shape, 119, 120 Superstructures, 152
molecular phylogeny, 130–132 Surface-dwelling rodents, 84
sample and data collections, 116 Systematics
shape and size analysis, 119 Bolivian form, 21
size vs. shape, 132 candidate species, 24–26
skull shape chromosomic evidence, 19
morphological variation, 129, 131 cranial or dental characters, 18
phenogram, 129 distinctiveness, 28
species-level comparisons, 127 knowledge, 17
variation literature, 26
in cranium and mandible monophyly, 24
shape, 122–124 Neotropical rodents, 19
in cranium and mandible size, nomenclatorial, 23
120, 121 synonym, 28
Social and reproductive physiology, 231–232 taxonomic history, 18
Soil hardness, 229 taxonomic literature, 25, 27
Soil moisture, 196 taxonomic names, 28
Solitary species, 230 taxonomic status, 29
Spalax ehrenbergi, 167 taxonomy, 18
Spalax galili species, 52 tuco-tuco species, 29
Spatial autocorrelation pattern, 85 unavailable names, 27
Spatial cognition, 224 Sympatric speciation
Spatial memory formation, 224 C. lami (see Ctenomys lami)
Spatial orientation, 224 C. minutus, 45 (see also Ctenomys
Spatial performance, 224 minutus)
Spatial utilization, 195 intrinsic differentiation, 43
Speciation models process, 44
allopatric, 45–47 reproductive isolation, 43
272 Index
T macroecological analyses, 69
Taquara do Mundo Novo, 46 macroecological investigations, 70
Telomere length (TL), 252, 253 patterns, 69
Telomeres, 252, 253 phylogenetic and paleontological data, 79
Terminal and subterminal bands, 173 phylopatric behavior, 84
Territoriality, 222, 237 population genetic approaches, 84
Thermogenic capacity, 228 population genetic structure, 101
Thermoneutrality, 228 Southern South America, 84
Thermoregulatory, 227 species distribution, 70, 72, 86
Thomomys bottae, 167 syntopy, 69
Torquatus group, 46, 56, 88 territorial, 84
Toxicology evolution, 249 vegetation cover, 101
Tuco-tuco biology and ecology, 203
Tuco-tucos, 149, 151, 152, 193, 195
behavioral and eco-physiological U
characteristics, 85 Underground environments, 221, 259
body size distribution, 77, 78 Underground rodents
contiguous allopatry, 69 genus Ctenomys, 84
diversity, 69 radiation, 84
genetic differences, 85 subterranean rodents, 84
geographic ranges, 69 surface-dwelling, 84
geographic range size distribution, 71, 73,
74, 76, 79
habitat, 103 V
habitat specialization, 85 Vehicular emission, 257
hostile environments, 84 Ventral region, 174

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Thales Renato Ochotorena de Freitas

Gislene Lopes Gonçalves

ISBN 978-3-030-61678-6 ISBN 978-3-030-61679-3 (eBook)

© Springer Nature Switzerland AG 2021

Such begin Darwin’s comments on tuco-tucos, based on his encounters with

of the genome that are impacted by chromosomal rearrangements. Similarly, as our

Berkeley, USA Eileen Lacey

This book examines the biology of tuco-tucos (Ctenomys) from an evolutionary

than a triggering speciation in tuco-tucos, chromosomal rearrangements speed up

Part I Evolution of Ctenomys

Part II Geographic Patterns

Part III Organismal Biology

7 Biomechanics and Strategies of Digging������������������������������������������������ 141

Part IV Environmental Relationships

Alicia Álvarez Instituto de Ecoregiones Andinas, CONICET, Jujuy, Argentina

Pedro Cordeiro-Estrela Departamento de Sistemática e Ecologia, Centro de

Bruno Busnello Kubiak Programa de Pós-Graduação em Genética e Biologia

Aldo I. Vassallo Grupo Morfología Funcional y Comportamiento, Instituto de

Gislene Lopes Gonçalves has been a research collaborator in the Department of

Renan Maestri has been an assistant professor in the Department of Ecology,

Laboratory of Ecomorphology and Macroevolution at the Department of Ecology,

Diego H. Verzi, Nahuel A. De Santi, A. Itatí Olivares, Cecilia C. Morgan,

Ctenomyidae is a clade of South American hystricomorph rodents with a peculiar

D. H. Verzi (*) · N. A. De Santi · A. I. Olivares · C. C. Morgan

© Springer Nature Switzerland AG 2021 3

1.2  tem Ctenomyids and the Understanding

inclusion of †Protadelphomys within Echimyidae and have recovered †Sallamys as

members share few “non-key” apomorphies with their corresponding crown-group

1.3 The Genus Ctenomys

morphology, in the comparative context of the modern ctenomyids, support the

1.4 Final Considerations

Verzi DH (2002) Patrones de evolución morfológica en Ctenomyinae (Rodentia, Octodontidae).

Guillermo D’Elía, Pablo Teta, and Enrique P. Lessa

© Springer Nature Switzerland AG 2021 17

2.2.1 Currently Considered Distinct Species

The first species of Ctenomys to be described was C. brasiliensis Blainville, 1826.

Table 2.1 (continued)

After almost two centuries of taxonomic studies centered in Ctenomys, here we

“mariafarelli” would have priority over C. erikacuellarae. Bidau (2015) also

genealogical analysis of mitochondrial DNA sequences, including sequences of

2.2.2 Candidate Species

As it happens when any group that is taxonomically assessed, distinct candidate

Candidate species of Ctenomys have been identified based on distinct sources of

suggestion was reinforced by the results of Caraballo et al. (2012). In addition,

2.2.3 Unavailable Names

A common issue in the literature of Ctenomys is the extensive usage of nonavailable

addition to have never been formalized, to be best of our knowledge no evidence

2.2.5 Final Remarks of the Taxonomy Section

2.3 Phylogenetic Relationships

2.3.1 Early Phylogenetic Analyses

Three strictly phylogenetic analyses of species of Ctenomys were published before

2.3.2 DNA Sequence-Based Studies

2.3.3 Species Relationships and Species Groups

2.3.4 Subgenera of Ctenomys

2.3.6 Trait Evolution

2.3.7 Final Remarks on Phylogenetic Relationships

Much remains to be learned about the phylogenetic relationships of the species of

from the Holocene of central Argentina. J S Am Earth Sci 100. https://doi.org/10.1016/j.

Thales Renato Ochotorena de Freitas

Berkeley, USA Eileen Lacey

Part II Geographic Patterns

Part III Organismal Biology

7 Biomechanics and Strategies of Digging�� 141

Part IV Environmental Relationships

1.2 tem Ctenomyids and the Understanding

1.3 The Genus Ctenomys

1.4 Final Considerations

2.2.1 Currently Considered Distinct Species

2.2.2 Candidate Species

2.2.3 Unavailable Names

2.2.5 Final Remarks of the Taxonomy Section

2.3 Phylogenetic Relationships

2.3.1 Early Phylogenetic Analyses

2.3.2 DNA Sequence-Based Studies

2.3.3 Species Relationships and Species Groups

2.3.4 Subgenera of Ctenomys

2.3.6 Trait Evolution

2.3.7 Final Remarks on Phylogenetic Relationships

3.2 Allopatric Speciation Model

3.2.1 C. flamarioni and C. australis

3.2.2 C. minutus and C. lami

3.3 Sympatric Speciation Model

3.3.1 C. minutus

3.4 Chromosomal Speciation Model

3.4.1 C. lami

3.5 Hybrid Zones

3.6 Intraspecific Hybrid Zones

3.7 Interspecific Hybrid Zones

3.8 Other Processes

3.9 Final Comments

4.2 Patterns of Species Distribution

4.3 Patterns of Geographic Range Size Distribution

4.4 Patterns of Body Size Distribution

4.5 Concluding Remarks

5.1 n Overview of Phylogeography

5.2 hy Study Underground Rodents as Models

5.3 Patterns of Isolation by Distance

5.4 Phylogeographic Patterns

5.5 Spatial Scales of Population Genetic Structure

5.6 andscape Genetics: Which Factors Structure

5.7 What About Conservation Genetics?

6.2 Sample and Data Collections

6.3 Geometric Morphometric Analysis

6.4 Shape and Size Analysis

6.5 Geographical Structure of Skull Shape

6.6 Variation in Cranium and Mandible Size

6.7 Variation in Cranium and Mandible Shape

6.8 Geographical Structure of Cranial Shape