Zoosyst. Evol.

95 (2) 2019, 543–556  |  DOI 10.3897/zse.95.38727

The evolutionary terrestrialization of planarian flatworms

(Platyhelminthes, Tricladida, Geoplanidae):
a review and research programme
Ronald Sluys1
1 Naturalis Biodiversity Center, P.O. Box 9517, 2300 RA Leiden, The Netherlands

http://zoobank.org/49967409-C3F7-4005-9F7B-7EAECBB93AA8

Corresponding author: Ronald Sluys (Ronald.Sluys@naturalis.nl)

Academic editor: A. Schmidt-Rhaesa  ♦  Received 1 August 2019  ♦  Accepted 20 September 2019  ♦  Published 29 October 2019

Abstract
The terrestrialization of animal life from aquatic ancestors is a key transition during the history of life. Planarian flatworms form an
ideal group of model organisms to study this colonization of the land because they have freshwater, marine, and terrestrial represen-
tatives. The widespread occurrence of terrestrial flatworms is a testament to their remarkable success occupying a new niche on land.
This lineage of terrestrial worms provides a unique glimpse of an evolutionary pathway by which a group of early divergent aquatic,
invertebrate metazoans has moved onto land. Land flatworms are among the first groups of animals to have evolved terrestrial adap-
tations and to have extensively radiated. Study of this terrestrialization process and the anatomical key innovations facilitating their
colonization of the land will contribute greatly to our understanding of this important step in Metazoan history. The context and scien-
tific background are reviewed regarding the evolutionary terrestrialization of land flatworms. Furthermore, a framework of a research
programme is sketched, which has as its main objective to test hypotheses on the evolution of land planarians, specifically whether
particular anatomical and physiological key innovations have contributed to their evolutionary successful terrestrial colonization
and radiation. In this context special attention is paid to the respiration in aquatic and terrestrial planarians. The research programme
depends on a comprehensive phylogenetic analysis of all major taxa of the land flatworms on the basis of both molecular and anatom-
ical data. The data sets should be analyzed phylogenetically with a suite of phylogenetic inference methods. Building on such robust
reconstructions, it will be possible to study associations between key innovations and the evolutionary terrestrialization process.

Key Words
adaptations, evolution, key innovations, land flatworms, model organisms, respiration, terrestrialization

Introduction
Charles Darwin was fascinated by planarian flatworms, have discovered terrestrial flatworms. It was only in
and he was particularly struck by the fact that there is a 1846 that it came to his attention that already in 1774
group of planarians that actually live on land. As he wrote the Danish naturalist O. F. Müller had described the land
in a letter from 23 July 1832 to his mentor Henslow: flatworm Microplana terrestris (Müller, 1774) (Porter
“Amongst the lower animals, nothing has so much and Graham 2016).
interested me as finding 2 species of elegantly coloured Darwin (1983: 25) appropriately and succinctly
true Planariae inhabiting the dry forest.” And in a letter described land flatworms as follows: “In general form
from 15 August 1832: “I have today to my astonishment they resemble little slugs, but are very much narrower
found 2 Planariae living under dry stones….”. Darwin in proportion, and several of the species are beautifully
thought for a long time that he was the first person to coloured with longitudinal stripes.” He took some of

Copyright Ronald Sluys. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
544 Sluys, R.: The evolutionary terrestrialization of planarian flatworms

the land flatworms that he had collected in Tasmania on Key innovations afford enhanced performance, promot-
board of the Beagle and managed to keep them alive for ing ecological opportunity. These innovations enable
two months. He experimented on the animals by cutting subsequent species diversification and radiation. For ex-
some animals into half, and he discovered that after 25 ample, marine gastropod snails in several cases evolved
days each piece had regenerated to an almost complete terrestriality independently from amphibious ancestors
animal (Darwin 1983; Sluys 2016). Darwin was so much by side-stepping a major constraint on land snail evo-
intrigued by his flatworm findings that in a letter of 22 lution, viz., the need to produce mucus for locomotion
May 1833, written when he was in Tierra del Fuego, he (Rosenberg 1996). Interestingly, mucus plays an impor-
asked his sister Catherine to send him Dalyell’s (1814) tant role in land flatworms: forming a slime trail for lo-
paper on several species of British freshwater planarians comotion, slime threads by which they can cross spaces
(Thomson 2009). In this paper Dalyell (1814) reported or lower themselves from heights, and a protective coat
many observations on the biology of these worms, against drying. Apparently, the worms have followed a
including a detailed study of their fission and regeneration different adaptive pathway than the molluscs and were
(Ball and Reyoldson 1981). Since the days of Darwin and able to colonize the land without conserving mucus.
Dalyell our knowledge on the regenerative capacity of The land flatworms or planarians (Fig. 1) likely
land planarians, and that of triclad flatworms in general, represent one of the first groups of animals that during
has greatly increased (cf. Reddien 2018; Rink 2018), as evolution have colonized the land and have extensively
well as our knowledge on the morphology, taxonomy, radiated (see below: Impact and innovative aspects).
phylogeny, and distribution of land flatworms (cf. Sluys Study of the evolutionary terrestrialization of the land
and Riutort 2018 and references therein). The fact that flatworms and the key anatomical innovations facilitating
delicate organisms such as free-living flatworms, most of this process will contribute greatly to our understanding
which occur in freshwater or marine environments, are able of the early steps onto land of the Metazoa. Planarian
to survive on land has received little attention. Thus far, no flatworms (Platyhelminthes Claus, 1887, Tricladida
detailed studies have been undertaken that attempted to Lang, 1884) form an ideal group of model organisms to
analyze how and when during their evolutionary history study this process because they have freshwater, marine,
planarians conquered the terrestrial environment, which of and terrestrial representatives. Preadaptations for life on
their anatomical and physiological features enabled their land are the conditions that (1) flatworms have internal
colonization of the land, and which adaptations currently fertilization, (2) the cocoons enveloping the eggs have a
facilitate their occupation of the terrestrial niche. relatively hard shell, (3) the young hatch directly as young
Therefore, in the following I do not so much present worms and not as larvae (Little 1983), and that (4) the
the results of such studies, but provide a review of this worms produce mucus. Traditionally, three major groups
subject and sketch the context, scientific background, and of triclads were recognized: Paludicola Hallez, 1892
framework of a research programme in which land flat- (freshwater planarians), Maricola Hallez, 1892 (marine
worms form the model group through which we may not triclads), Terricola Hallez, 1892 (land planarians). A fourth
only learn about their own terrestrialization but may be clade, the Cavernicola Sluys, 1990, was proposed by Sluys
enlightened also on the early evolutionary terrestrializa- (1990). More recently, molecular phylogenetic studies
tion of animal life in general. In this context, special at- have shown that one of the three paludicolan families is
tention is paid to the respiration in aquatic and terrestrial more closely related to the land planarians than to the other
planarians. In addition, the results obtained during this freshwater planarians (Baguñà and Riutort 2004; Álvarez-
putative research programme will also provide data for Presas et al. 2008; see also Sluys and Riutort 2018).
some collateral topics, such as biodiversity assessment
and historical biogeography.

Early evolutionary terrestrialization

of animal life, as exemplified by land
flatworms
The terrestrialization of animal life from marine or
freshwater ancestors is a key event in the history of life
on earth, particularly because in the course of evolution
“…transitions among physically different habitats… are
rare” (Vermeij and Dudley 2000: 546; see also Vermeij
2010). Moving from an aquatic to a terrestrial niche is
challenging due to the dramatically different demands
each environment places on the physiology and structure Figure 1. Photograph of the South American land planarian
of an organism, thus requiring numerous innovations. Polycladus gayi (from Grau and Carbayo 2010).

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Zoosyst. Evol. 95 (2) 2019, 543–556 545

Model organism: land planarians Key innovations, adaptive radiation, and

Terrestrial planarians (Platyhelminthes, Tricladida, Geo-
terrestrialization
planidae Stimpson, 1857) are a relatively species-rich
group (approx. 910 nominal species) with a worldwide, Understanding of the evolutionary dynamics of the following
mainly pan-tropical, distribution (Fig. 2). The animals live presumed key innovations, for example, may contribute
typically in tropical jungles and wooded areas. Terrestri- to our insight in the evolution of land flatworms: (1)
al planarians colonized the land hundreds of millions of colonization of the land (on which occasions did the transition
years ago, but they still lack any special mechanism for from water to land occur and were there reversals?); (2)
water conservation (Kawaguti 1932). Although they re- contribution of the various kinds of anatomically complex
quire a humid environment, they cannot endure long sub- creeping soles and (3) of mesenchymal body musculature
mersion in water, in contrast to their marine and freshwa- (absent in freshwater and marine forms) to the effective
ter relatives (Froehlich 1955). The animals cannot endure terrestrialization; (4) contribution of cephalic specializations
heat and direct sunlight, otherwise they desiccate quickly for the capture of prey to the adaptive radiation process; (5)
(Kawaguti 1932). Because of these limitations they tend the relation between the ecology and anatomy of the various
to remain hidden during the day in humid, but not wet, taxa and their various types of pharynges (frequently totally
refuges, only emerging at night when the relative humid- different from freshwater and marine forms) for capturing
ity of the air is high. Land planarians are fully terrestrial and digesting prey; (6) the extent to which the various
cryptozoic organisms because they are not tied to aquatic kinds of multi-cellular eyes of land flatworms (completely
systems for reproduction. The worms are part of the soil different from marine and freshwater forms) facilitated
ecosystem, living within the habitat of their prey (Ogren terrestrialization and adaptive radiation; (7) adaptation of
1955; Ball and Sluys 1990; Ogren and Sheldon 1991). particular sense organs, such as olfactory chemoreceptors,
Terrestrial planarians are successful top-predators of other to the humid air of the terrestrial environment, in contrast
invertebrates such as snails, slugs, earthworms, isopods, to taste chemoreceptors that evolved in aquatic habitats; (8)
insect larvae, and springtails; they themselves are rarely the way in which the worms are able to cope with a major
predated upon by other organisms, although some of their evolutionary constraint: the need to produce mucus for their
predators may be beetles, snails, and other land planarians locomotion, mucus for the most part being water; (9) the
(Boll and Leal-Zanchet 2018 and references therein). Land correlation between various body shapes (cylindrical, flat,
planarians search for, attack, and capture prey much larg- etc.) on the one hand and water conservation and various
er than themselves, employing various techniques such as terrestrial habitats (ranging from humid to rather dry) on
physical force, adhesive mucus, pharyngeal action, and the other hand, a cylindrical body considered to be more
pouring very effective digestive secretion over the surface economical in terms of water conservation (Clark and
of the live prey, or into it, by the protrusible pharynx. Cowey 1958); (10) the manner in which the protonephridia
The widespread occurrence of these terrestrial flat- adapted from an osmoregulatory system in aquatic ancestors
worms (Fig. 2) is a testament to their remarkable success to a resorptive system (saving water and/or eliminating
occupying a new niche on land for hundreds of millions metabolic wastes) in land flatworms; (11) the change from
of years. This lineage of flatworms thus provides a unique aquatic to terrestrial respiration.
glimpse of an evolutionary pathway by which a group
of early divergent aquatic, invertebrate metazoans has
moved onto land. Impact and innovative aspects
Phylogenetic studies, including those on flatworms in
Objectives general and land flatworms in particular, are generally
based on one-sided approaches, incorporating either
The main objective of the research programme described morphological/anatomical data, or molecular data.
here is to test hypotheses on the evolution of land flat- Molecular studies might plot some morphological data
worms, specifically whether particular anatomical and on the resulting phylogenies to legitimize the molecular
physiological key innovations have contributed to their trees (‘pseudo-morphology’; cf. Mooi and Gill 2010;
successful terrestrial colonization and subsequent radia- Assis and Rieppel 2011; see also Williams and Ebach
tion (see below: Hypotheses testing). This first requires a 2010). But for the land flatworms there are no studies
comprehensive phylogenetic analysis of all major taxa of that take an integrative approach with both kinds of
the land flatworms (e.g., for the current 55 genera; cf. Sluys data. This contrasts with the fact that many interesting
et al. 2009). The project needs to construct this phylogeny scientific questions in this group of animals concern
on the basis of both molecular and anatomical data. This the evolution of their structures, key innovations, and
reconstruction forms the necessary basis and framework adaptations (see above: Key innovations, adaptive
for subsequent comparative studies of key innovations, radiation, and terrestrialization).
adaptive radiation, and historical biogeography of land Land flatworms likely are among the first groups of
flatworms, and on the early terrestrialization of animal life. animals that during evolution have colonized the land

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546 Sluys, R.: The evolutionary terrestrialization of planarian flatworms

Figure 2. Map of species richness in land planarians on an equal area grid map; maximum in red, minimum in dark blue (from
Sluys 1998). Over the past 20 years new discoveries have added to the number of native and introduced species. Nevertheless, the
current pattern of biodiversity remains very similar to the one shown in this map, although now, for example, about 20 new species
are recognized in Europe, 40 in southeastern and southern Brazil and northeastern Argentina, nine in the Australian territories, and
six in New Zealand (Sluys 2016).

and have subsequently radiated extensively. According stricted to the Ecdysozoa Aguinaldo et al., 1997 and the
to fossil information arthropods would be the first ani- Deuterostomia Grobben, 1908 (cf. Labandeira 2005).
mals to have colonized the land, with atmospheric oxy- Such issues may only be adequately addressed with the
gen levels as the major driver of successful colonization help of the phylogenetic trees generated during the re-
(Ward et al. 2006). However, phylogenetic trees suggest search programme outlined here. These phylogenetic
flatworms as one of the early colonizers (cf. Hedges and reconstructions will also form the long-awaited robust
Kumar 2009). This is generally neglected and, therefore, backbone for conservation biology studies in which land
the study proposed here will contribute to a more bal- planarians function as indicator taxa (see Sluys 1999).
anced representation of the evolutionary history of ter- Furthermore, triclad flatworms are a key group in his-
restrial animal life. torical biogeography because they do not possess larval
The fossil record of flatworms is sparse and hard- dispersive stages. Therefore, they are excellent models
ly provides adequate calibration points for a molecular for vicariance scenarios as explanations for current bio-
clock (cf. Pierce 1960; Alessandrello et al. 1988; Ruiz geographic patterns. Thus, the research programme will
and Lindberg 1989; Poinar 2003); calibration points may facilitate tests of the hypothesis that plate tectonics has
have to be based on paleogeographical information, am- been a major factor in their historical biogeography (cf.
ber fossils, coprolites, or estimates of mutation rates (cf. Sluys 1994, 1995).
Blaxter 2009 and references therein). Thus, the proposed For the morphological data I envision this programme
project will also form a first step towards our understand- to develop a formal knowledge representation (ontology)
ing of the absolute timing of the early terrestrialization of of planarian phenotypes and character states that leverages
animal life. It will facilitate the test of the hypothesis that progress in this field (for a review, see Mabee et al. 2007)
terrestrialization is linked to atmospheric oxygen levels, such that one of the outcomes will be a ‘Rosetta stone’ of
as was proposed for arthropods (Ward et al. 2006). The concepts in planarian morphology that can be used not just
project may thus contribute to the first few insights into to disambiguate characters and their states in the putative
the terrestrialization of the Lophotrochozoa Halanych et study but also to inform subsequent research questions in
al., 1995 because our current understanding is mainly re- connecting genomic data and planarian phenotypes.

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Zoosyst. Evol. 95 (2) 2019, 543–556 547

Phylogeny suggested that the second approach, i.e., combined anal-

ysis, is applied.
Knowledge on the phylogenetic relationships within the From an empirical perspective, it has been shown
Tricladida is based on phylogenetic analyses of molecular that morphology can have a profound effect on the com-
and morphological datasets (for a review, see Sluys and bined analysis, irrespective of the fact that the number
Riutort 2018). Attention has been paid mostly to: (1) of molecular characters generally exceeds the number of
the relationships between the higher taxa within the morphological features (Jenner 2004; Assis 2009). Com-
Tricladida (cf. Sluys 1989a; Carranza et al. 1998; Álvarez- bined analyses may be different from the analyses of the
Presas et al. 2008); (2) the affinities within the suborder separate data matrices and consensus trees may hide a
of the Maricola (Sluys 1989b); (3) the higher taxon phylogenetic signal that is generated by a total evidence
relationships within the freshwater planarians in general analysis. Positive contributions of morphology to quanti-
and within the family Dugesiidae Ball, 1974 in particular tative clade support measures in combined analyses have
(Ball 1974; De Vries and Sluys 1991; Sluys 2001; Lázaro been observed for a large number of taxa (Jenner 2004).
et al. 2009). Sluys and Kawakatsu (2006) explored the
phylogenetic relationships within the freshwater families
Dendrocoelidae Hallez, 1892 and Kenkiidae Hyman, Timetree calibration
1937. More recent studies have resulted in an expanded
character state matrix, including several new species and As the fossil record of flatworms is sparse and does not
gene sequences, and in a robust phylogenetic tree for the provide adequate calibration points for a molecular clock,
higher taxa (Álvarez-Presas et al. 2008). calibration of the phylogenetic timetrees has to be based
For decades the evolutionary relationships within the on other kinds of data, such as, for example, paleogeo-
group of land flatworms have been neglected. Partial graphical information (see above: Impact and innovative
taxonomic revisions of the group have been published aspects). This means, for example, that we will be look-
but these were rarely based on a phylogenetic analysis ing for closely related taxa a and b that are endemic to
(cf. Ogren and Sluys 1998; Kawakatsu et al. 2005). It was the areas A and B, respectively. In addition, we will be
only with the advent of molecular techniques that studies looking for those areas A and B inhabited by endemic
started to address the phylogenetic structure within the taxa for which paleogeographic data indicate the time
group (cf. Álvarez-Presas et al. 2008; Carbayo et al. 2013). since the two areas have fragmented from a single ances-
On the basis of the results of these molecular studies, and tral area. These two pieces of information, together with
in combination with anatomical data, a revised higher the molecular clock hypothesis, will enable one to date
classification of planarian flatworms was proposed (Sluys all cladogenetic and biogeographic events in the entire
et al. 2009), in which the subordinal taxonomic rank of lineage of which a and b only form a part.
the terrestrial planarians was downscaled to the level of Within the triclad flatworms there is a good number
family, viz., the Geoplanidae. However, these molecular of such disjunctions, due to vicariance events, within
studies suffer from the fact that they only incorporate species or between closely related species, that may be
relatively few exemplar species and then only for a small tested as possible calibration points. For example, land
selection of land planarian taxa. planarians of the genus Othelosoma Gray, 1869 are
restricted to Africa and India and have attained their
current distribution when India and Africa started to
Methodology separate at about 150 Mya. Table 1 specifies the taxa, and
their presumed vicariance events and divergence times
Separate and combined analyses that may profitably be used for calibration.
One may perhaps be inclined to consider paleogeo-
It is proposed here to use both molecular and morpho- graphical calibrations to be less ideal than fossil calibra-
logical data sets. To characterize their phylogenetic tions. In point of fact, the opposite may be the case. Fos-
signal the character state matrices should be analyzed sil-calibrated molecular clocks at best provide minimum
separately, using parsimony (morphology) and Bayesian dates (Wilke et al. 2009). Therefore, it has been argued
methods (morphological as well as molecular). In addi- that clocks are best calibrated with reference to the distri-
tion, molecular and morphological data sets should be bution of molecular clades and associated tectonics (e.g.,
combined into one joint analysis. With respect to mor- Azuma et al. 2008; Heads 2014).
phological and molecular characters two approaches Molecular dating is a rapidly developing field and there-
may be followed: (1) morphology is merely optimized fore there is currently no single best method; each approach
in post-tree analysis of the molecular results, or (2) mor- has its advantages and disadvantages. It is also important
phological and molecular characters are combined into to note that in the phylogenetic tree of the Platyhelminthes,
one data matrix. The first approach is favoured in many the triclads constitute one of the crown groups (cf. Baguñà
recent studies and considered to be the only contribution and Riutort 2004). This implies that the nodes below the
of morphology to phylogenetic analysis by Scotland et Tricladida represent older taxa that therefore might be used
al. (2003); but see Jenner (2004) for a rebuttal. It is here as potential outgroups in the phylogenetic analysis.

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548 Sluys, R.: The evolutionary terrestrialization of planarian flatworms

Datasets and phylogenetic analysis and Dr F. Carbayo (2003, University of Sao Paulo). Both
researchers made detailed character codings and scorings
The molecular data matrix for this project may be for taxa of land planarians. Dr Winsor mostly focussed on
derived from published data from GenBank, and new Australian taxa, while Dr Carbayo concentrated on South
molecular sequences generated from fresh material of American species, although neither used these scorings
new taxa examined during the project. Previous studies for a subsequent phylogenetic analysis. During the
have shown that the following genes provide the best project character codings and scorings may be extracted
resolution at the hierarchical levels of the phylogenetic from these two doctoral theses and, subsequently, be
tree of the triclads that form the focus of the putative combined, refined and also supplemented by scorings for
research programme: nuclear 18S rDNA and 28S rDNA taxa not examined by Winsor or Carbayo. Definition and
for resolving the deeper, more ancient branches in the scoring of morphological character states should comply
tree, and mitochondrial COI to contribute signal on more with the most recent insights (cf. Sereno 2007).
recent splits (e.g. between and within genera) (Álvarez- The morphological, molecular, and combined data
Presas et al. 2008). Subsequently, another nuclear matrix may be analyzed using maximum parsimony
marker, the elongation factor 1-alpha (EF), was added to (e.g., TNT; Goloboff et al. 2003), maximum likelihood
the list of informative markers for an intermediate level (e.g., RaxML; Stamatakis 2014; IQtree; Nguyen et al.
between family and species, contributing to resolution of 2015), and Bayesian Inference (MrBAYES, Ronquist et
the relationships between members of the Geoplaninae al. 2012, and BEAST, Drummond et al. 2012). Inform-
subfamily (Carbayo et al. 2013). ative genes should first be analyzed separately, but also
It has been suggested that many of the problems combined in the form of concatenated datasets. Substi-
associated with the amplification and sequencing of tution model parameters may either be selected using
planarian molecular markers may be solved by applying nested likelihood ratio tests or explored using reversible
next generation sequencing (NGS) methodologies (Sluys jump Markov chain Monte Carlo methods (jModeltest,
and Riutort 2018). This new era has started already, and Darriba et al. 2012; Partitionfinder, Lanfear et al. 2017).
in recent years genomes and transcriptomes of freshwater Saturation tests should be performed using DAMBE
planarians have already been sequenced (Egger et al. (Xia 2017).
2015; An et al. 2018; Grohme et al. 2018; Rozanski et Molecular data may be analyzed also under a dynamic
al. 2018), contributing new anonymous nuclear markers approach to homology. In the dynamic approach deline-
that can be used as phylogenetic tools. There are even ations are dependent upon the topology of the phyloge-
bioinformatic pipelines that can help in the discovery of netic trees on which they are optimized. In this context
these markers using NGS data as input (Frías-López et hypotheses of homology are part of phylogenetic hypoth-
al. 2016). This has already been applied in freshwater eses and are subject to the same optimality criteria as the
planarians, and will soon be used also for terrestrial trees, viz., minimisation of evolutionary transformation
flatworms. Moreover, there is now available also events. The computer program POY (ver. 4.0 beta 2635;
information on other mitochondrial markers, in addition Wheeler et al. 2006, Varón et al. 2010) allows for phy-
to COI, as whole mitogenomes were sequenced and logenetic tree searches under this dynamic homology or
annotated (Sakai and Sakaizumi 2012; Solà et al. 2015; direct optimization approach.
Gastineau et al. 2019; Yang et al. 2019), contributing to Aquatic planarians should form one of the outgroups
the combination of nuclear and mitochondrial markers, for the phylogenetic analysis. Particularly the freshwater
thus obtaining better resolved phylogenies. family Dugesiidae has been shown to share a close rela-
The project requires a large morphological data matrix tionship with the land flatworms (see Sluys et al. 2009).
of all major taxa of land planarians (e.g., for the current But other freshwater groups, as well as marine triclads,
55 genera; cf. Sluys et al. 2009). For this, the programme should be included also as outgroups. Comparison with
may be taking as leads the doctoral dissertations of both these freshwater and marine forms will reveal the adapta-
Dr L. Winsor (2003, James Cook University, Townsville) tions that made possible the transition to land.

Table 1. Taxa, and their presumed vicariance events and divergence times that may be used for calibration of the timetrees.
Taxon Vicariant distribution Divergence time (Mya)
Othelosoma species Africa (31 sp.)/India (7 sp.) ≤150
Bipalium species Madagascar (23 sp.)/India & SE Asia (160 sp.) 90-50
Girardia species N. America (4 sp.)/ S. America (39 sp.) 3,5
genus Girardia/genus Dugesia N. & S. America (42 sp.)/Africa (21 sp.) 130–100
Dugesia species E. Med. (12 sp.)/W. Mediterranean (10 sp.) 38-3
Procerodes littoralis E. Atlantic/W. Atlantic 150
Foviella affinis E. Atlantic/W. Atlantic 150
Uteriporus vulgaris E. Atlantic/W. Atlantic 150
genus Amblyplana/genus Geoplana Africa (9 sp.)/S. America (64 sp.) 100
Romankenkius species S. South America (1 sp.)/Australia (12 sp.) 120

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Hypotheses testing for the root of the phylogeny with alternately polarized
state changes reconstructed nearer the tips. Likewise,
Creeping sole expectations can also be formulated for body shape:
if cylindrical body shapes indeed conserve water one
would expect transitions to this morphology to coincide
The research proposed here seeks to explore the evolu- with, or follow terrestrialization when reconstructed on
tion of terrestrialization of land flatworms in time and the phylogeny. In a similar way, all of the hypotheses
in correlation with presumed key morphological adap- mentioned above (see above: Key innovations, adaptive
tations. As regards the time axis, this will be important radiation and terrestrialization), as well as others that may
both for direct reconstructions of when terrestrializa- arise during the study, can be tested.
tion happened, as well as in the subsequent analysis of
whether hypothesized key innovations co-vary in their
location on the phylogeny with elevated diversification Respiration in planarians
rates under a model of adaptive radiation (e.g., using the
method of Ree 2005). It is here proposed that divergence Introduction
dates estimates may be reconstructed from molecular A particular crucial feature that may be among the most
data calibrated using previously published node ages difficult to examine and to plot its character states on the
(Hedges and Kumar 2009) in a relaxed molecular clock phylogenetic trees concerns the adaptation to terrestrial
approach as implemented in BEAST (Drummond et al. respiration. Planarian flatworms possess neither circula-
2012). As for covariance between traits (see above: Key tory nor respiratory systems for transporting oxygen or
innovations, adaptive radiation and terrestrialization), digested food substances to the internal tissues. In these
the general model will be that the radiation of flatworms, animals, oxygen is absorbed across the entire body wall
with presumably repeated terrestrializations, constitutes a and for this diffusion process water is required to dissolve
natural experiment such that morphological changes hy- oxygen and carbon dioxide in order to cross cell mem-
pothesized to be associated with terrestrialization co-vary branes. Clearly, this poses no problem for freshwater and
with and co-occur in ancestral state reconstructions on a marine planarians as they live in an aquatic habitat, but
phylogenetic tree. Sophisticated comparative methods to when ancestral planarians colonized the land, leaving this
correct for autocorrelation (because the different lineages aquatic milieu must have formed a major hurdle, as the
are related to each other) have been developed, including physical properties of water and air are so different. It is
Bayesian statistical methods that allow for phylogenetic true that many land planarians live in habitats with a high
uncertainty (e.g., Pagel and Meade 2006). Understanding humidity, but still these conditions greatly differ from a
of the evolutionary dynamics and consequences of such fully aquatic environment, while there are also terrestrial
innovations will contribute to the unraveling of their ge- planarians that occur in mesophile and xerophile habitats
netic basis and their role in speciation events and, conse- (Froehlich 1955; Winsor et al. 1998).
quently, the adaptive radiation of this group of animals.
To give an example, the study will provide insight into Thickness of flatworms
the evolution of creeping soles, the latter defined as: “A The analysis of this subject is complicated by the fact that
flat or ridged modified strip of epithelium on the ventral not much is known about the respiratory physiology of
surface of geoplanid triclad flatworms characterized by free-living flatworms in general and planarians in par-
the presence of cilia …. which provides propulsive forces ticular. Furthermore, most of these studies concern aquat-
by ciliary or muscular action, or by a combination of ic species. A striking example is McNeill Alexander’s
both” (Sluys et al. 2009: 1773). Traditionally, a narrow (1979) conclusion that the maximum possible thickness
creeping sole is considered as the primitive character of a free-living flatworm is 1.0 mm or at most 1.5 mm.
state, while medium to broad soles and loss of a creeping His conclusion is based on an argument that involves the
sole are seen as derived states. However, narrow soles calculation of the diffusion rate of oxygen. In philosoph-
may also secondarily result from partial reduction, while ical context McNeill Alexander’s type of functional ex-
absence of a creeping sole altogether may be the ancestral planation has been presented as an example of viability
state, as aquatic forms lack one. However, the evolution explanations, which are distinct from causal or historical
of creeping soles has never been analyzed within a explanations (Wouters 1995).
phylogenetic context; the research programme developed McNeill Alexander’s (1979) calculation is based on a
here will be the first to do so. If the traditional view number of factors, viz., (a) partial pressure of oxygen in
holds true, one would expect to infer the narrow creeping water, (b) rate of gas diffusion, (c) the fact that oxygen
sole as the character state at the root of the tree, with mostly will diffuse through the dorsal body surface, as
transitions to medium or broad soles, or loss of a creeping the ventral surface is in contact with the substrate, (d)
sole, nearer the tips. Under the opposite scenario, the the diffusion constant for oxygen diffusing through con-
absence of a creeping sole being the ancestral state, one nective tissue, (e) density of flatworm tissue. When the
would consequently expect this state to be reconstructed known or estimated values for these variables are used

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550 Sluys, R.: The evolutionary terrestrialization of planarian flatworms

in a derivation using Fick’s law of diffusion, “This cal- the time the flatworms hide under stones or fallen logs,
culation indicates that the maximum possible thickness etc. and that during this resting period they are highly
for a flatworm … is about 0.5 mm if oxygen diffuses contracted, thus presumably approaching the condition
in only from the dorsal surface, or 1.0 mm if it diffuses of preserved specimens.
equally from the ventral surface…. A similar calculation Another example of a thick flatworm-like animal is
for a cylindrical turbellarian indicates that the maximum the basal bilaterian Xenoturbella bocki Westblad, 1950,
possible diameter would be 1.5 mm …. This is proba- which may reach a length of 2–3 cm and a thickness of
bly why large flatworms are flat.” (McNeill Alexander 5 mm (Franzén and Afzelius 1987).
1979: 185). Another objection that may be raised against
As flatworms rely entirely on oxygen diffusion through McNeill Alexander’s (1979) calculation concerns his
the surface, it is indeed advantageous to have a large sur- assumption that diffusion in flatworms is more or less
face area:volume ratio, i.e., to be flat and not cylindrical. equal to that in frog muscle and connective tissue,
In general, this holds true for aquatic species, while with- i.e., 2 × 10−5mm2atm−1s−1. However, it is doubtful that
in species their thickness hardly or not at all increases the mesenchyme and gut tissue—forming the major
with an increase in plan area of the body (Calow 1987). component of the planarian body—have the same
Another interpretation of the broad bodies of large land diffusion constant as frog muscles and connective tissue.
flatworms is that this facilitates capture and subdual of Evidently, the diffusion constant of planarian tissue will
prey (cf. Cseh et al. 2017), i.e., forms an evolutionary ad- not be equal to that in water (6 × 10−5mm2atm−1s−1) but
aptation to new kinds of terrestrial prey not encountered perhaps 4 × 10−5mm2atm−1s−1 would be a value that is
by their aquatic ancestors. more realistic for flatworms. This would then imply a
For an organism that relies on direct diffusion of higher rate of diffusion and, consequently, would allow
oxygen Prosser (1973) provided a formula for the the worms to be thicker.
calculation of the thickness of the animal, based on For the rate (m) of oxygen consumption per unit vol-
the oxygen concentration of the medium, a diffusion ume of tissue, McNeill Alexander (1979: 185) used 0.1
coefficient K, and the rate of oxygen consumption. When cm3 oxygen g−1h−1, “or a little more”. However, accord-
the calculation is done for both water and air (Table 2) it ing to Hyman (1951: 207 and references therein) this
reveals that air-breathers can be six times thicker because may be higher, viz., 0.2–0.31 cm3 oxygen g−1h−1, that
there is more oxygen in air than in water (it should be is “0.2–0.3 cc per gram per hour in adult worms.” Ac-
noted that there is uncertainty about the units in which cording to Moore (2006) it would be about 0.1–0.2 ml
thickness is expressed). oxygen g−1h−1 at 15 °C.
Nevertheless, the conclusion of McNeill Alexander A variable that is not taken into account by McNeill
(1979), as well as the calculation on which it is based, is Alexander (1979) is the relationship between size and
open to a number of criticisms. First of all, it is certainly metabolic rate in flatworms. Although data are scant,
not the case that large flatworms are always very flat or generally respiratory rate decreases as size increases
thin. For example, “giant” freshwater species from Lake (Vernberg 1968).
Baikal, such as Bdellocephala bathyalis Timoshkin & Although McNeill Alexander’s view on the maxi-
Porfirjeva, 1989, are certainly thicker than 0.5–1.0 mm mum thickness of flatworm-like animals has been ex-
(B. bathyalis measuring 3.3 mm in thickness in pre- plicitly or implicitly endorsed (e.g., Ruppert et al. 2004;
served condition; Sluys et al. 1998). And also the large Moore 2006), he considered it merely “a very rough cal-
land planarians are usually thicker, such as Polycladus culation in a textbook” and he conceded that “There are
gayi Blanchard, 1845 (4 mm thick), Pseudogeoplana many doubtful assumptions in the calculation” (McNeill
lumbricoides (Schirch, 1929) (3.5 mm), P. nigrofusca Alexander in litt., 22 August 1996).
(Darwin, 1844) (3 mm), Geoplana rufiventris Schultze
& Müller, 1857 (2.3 mm) (Von Graff 1899). Although Respiratory pigments
such measurements on the thickness are usually made on Apart from the doubtful assumptions in McNeill
preserved specimens, and that thus fully stretched live Alexander’s calculation, another explanation for
specimens will be thinner, this does not necessarily com- the empirical fact that flatworms frequently are
promise the picture. It should be realized that most of thicker than 0.5–1.0  mm may lie in the presence of

Table 2. Data and results of thickness calculation according to Prosser’s (1973) formula: thicknesss = √(8CO(K/VO2)); oxygen con-
centration in medium (Co) and K value (for muscle) from Prosser (1973); oxygen consumption (Vo2) converted from value of 0.2
ml/g/hr (= 0.0033 ml/g/min; see this paper).
Medium Oxygen medium (ml/ml) K (muscle; cm2/min/atm) Oxygen consumption (ml/g/min) Thickness (units ?)
Water 0.00651 0.000014 0.0033 0.014864234
Air 0.2095 0.000014 0.0033 0.084322613

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respiratory pigments, transporting oxygen across Rates of oxygen consumption may be determined and
tissues. Hemoglobin is the most common respiratory expressed in different ways and are generally influenced
pigment among invertebrates in general (Brusca and by the temperature of the habitat. The type of response to
Brusca 2003). With respect to flatworms it has been changes in temperature varies per species, as some spe-
reported mostly from parasitic helminths (Weber and cies are eurythermal and others much more stenothermal
Vinogradov 2001) as well as for a number of ecto- in their ecological requirements (Vernberg 1968 and ref-
and endosymbiotic turbellarians, such as Paravortex erences therein). Therefore, respiration rate frequently is
Wahl, 1906, Triloborhynchus Bashiruddin & Karling, expressed as microliters-O2-per-(milli-)gram-wet weight-
1970, Cleistogamia Faust, 1924, Serita Cannon, 1982, per hour (µlO2/(m)g/WW/h) at a particular temperature.
Paranotothrix Cannon, 1982 (Jennings and Cannon Another way to express oxygen consumption is by calcu-
1987; Jennings 1988, 1997). Hemoglobin is known lating the coefficient b, or the regression slope of a particu-
also from the free-living flatworms, viz., a species of lar power function for the relation between body size and
Phaenocora Ehrenberg, 1835 (Vernberg 1968; Weber respiration. Unfortunately, the value of b varies much as it
and Vinogradov 2001). It has been hypothesized that greatly depends on the size of the animals, their physiolog-
presence of hemoglobin in the entosymbiotic graffilid ical condition, and many external factors (Heitkamp 1979).
rhabdocoel Paravortex scrobiculariae (Graff, 1882) For five species of freshwater planarians (Dugesia
represents an adaptation to the oxygen-poor conditions gonocephala (Dugès, 1830), Crenobia alpina (Dana, 1766),
to which this species is subjected, in contrast to the Polycelis nigra (Müller, 1774), P. felina (Dalyell, 1814),
well-aerated habitats of other species, such as P. cardii Schmidtea polychroa (Schmidt, 1861)) the following values
(Hallez, 1908) and Graffilla buccinicola Jameson, 1897, were found for oxygen consumption (µlO2/g/WW/h),
which lack hemoglobin (Jennings 1981). Unfortunately, measured at a temperature of about 15 °C: 170, 240, 135,
oxygen carrying components such as hemoglobin have 199, 116, respectively (Vernberg 1968). Unfortunately, these
not been studied in planarians. Winsor (1998) mentioned values for triclad flatworms cannot be compared directly
the possible respiratory role of uroporphyrins (other than with those determined for various microturbellarians as
hemoglobin) present in the rhabdoids of Platydemus these were calculated per milligram wet weight (µlO2/
manokwari de Beauchamp, 1962. Potential presence of mg/WW/h), resulting in the following values, measured
hemoglobin may be deduced from the presence of non- at a temperature of 15 °C: 1.242 (Dalyellia viridis (Shaw,
epidermal pigments, often situated in or around specific 1791)), 0.688 (Opistomum pallidum Schmidt, 1848), 0168
organs, such as the brain and the pharynx (Jennings (Mesostoma ehrenbergi (Focke, 1836)), ranging between
1981, 1988; Jennings and Cannon 1987). In planarians, 0.298 and 0.700 (eight different populations of Mesostoma
body colouration generally is due to the presence of lingua (Abildgaard, 1789)) (Heitkamp 1979).
granular pigments located in the mesenchyme directly The habitat temperature of the tropical terrestrial land
below the dorsal epidermis (Sluys and Riutort 2018), planarian Bipalium kewense Moseley, 1878 is usually
while in most species of the freshwater genus Girardia much higher than that of the aquatic triclads mentioned
Ball, 1974 the pharynx is also pigmented. Therefore, red above, albeit that this invasive species has established
pigment at other locations in the planarian body may itself outdoors in, for example, several North American
point to the presence of hemoglobin. However, I have states, the West Indies, Portugal, French Guiana,
rarely observed pigment to be present at such other and France (Sluys 2016; Justine et al. 2018). The
locations, at least in histological preparations, suggesting oxygen consumption of B. kewense specimens from
that hemoglobin is absent in triclad flatworms. outdoor localities in Arkansas, USA was determined at
temperatures varying between 27–33 °C, which yielded
Respiration in triclads and other free-living turbel- respiration rates (µlO2/g/WW/h) ranging between 113–
larians 290 (Daly and Matthews 1982). It is noteworthy that
Oxygen consumption in planarians and other free-living these values are in the same order of magnitude as those
turbellarians has been studied chiefly in freshwater spe- determined for freshwater planarians.
cies (cf. Vernberg 1968; Heitkamp 1979) and only once in The coefficient b based on oxygen consumption of
a terrestrial species (Daly and Matthews 1982). An impor- entire specimens of B. kewense ranged between 0.686–
tant result that emerged from these studies is the relation- 0.753, as measured at temperatures ranging between
ship between body size and respiration, in that generally 27–33 °C (Daly and Matthews 1982). These values are
smaller animals have a higher rate of oxygen consumption in the same order of magnitude as those determined for
than larger specimens, when determined on a weight-spe- the freshwater triclads Crenobia alpina (0.66), Dugesia
cific basis; however, some species showed the reverse gonocephala (0.82), and Polycelis felina (0.82) and the
correlation, while for others no relationship could be es- microturbellarians Mesostoma ehrenbergi (0.625), M.
tablished between body size and oxygen uptake (Vernberg lingua (0.850), and Opistomum pallidum (0.880) (Heit-
1968 and references therein; Heitkamp 1979). kamp 1979 and references therein).

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Terrestrial respiration that it may not provide a lubricant for locomotion in the
Terrestrial flatworms face two problems that involve land planarians but form a moisture-retaining sealant in
mutually conflicting adaptations, viz., desiccation a resting animal (Winsor 1998). However, one function
and respiration. A cylindrical body, with less surface does not exclude the other.
area:volume ratio, will minimize water loss but restricts Production of surface secretions may also have
diffusion of oxygen to the internal tissues. Probably this formed a preadaptation for respiration in the terrestrial
is the reason why smaller terrestrial species tend to be environment as it covers the body with a “watery”
round or oval in cross-section, e.g., species of the land layer that presumably improves the uptake of oxygen.
planarian genus Microplana Vejdovsky, 1890. Therefore, This may be related to the possible role, including
large species tend to be flattened to create a large surface respiration, of substances (porphyrins) in rhabdoids that
area:volume ratio in order to facilitate diffusion of oxygen are conspicuous in the dorsal/dorso-lateral epithelium
to the deep tissues. Nevertheless, the generally large and (and microrhabdites over the ventral surface) of land
particularly long species of the land planarian subfamily planarians (see above). To the best of my knowledge,
Bipaliinae Von Graff, 1896 also have a more or less this aspect of mucus secretion and respiration in land
cylindroid body in cross-section. flatworms has never received any attention.
The partial pressure of oxygen in well-aerated water Evidently, uptake of oxygen through the body wall is
in equilibrium with air is 0.21 atm (McNeill Alexander only the first step in the respiration process. Hereafter,
1979) and results in about 9 mgO2/l at 20 °C. Evidently, the oxygen needs to be transported to tissues deeper
the actual amount of dissolved oxygen available at a inside the planarian body. In the absence of respiratory
particular aquatic habitat depends on the temperature, pigments (see above: Respiratory pigments) this can be
depth, altitude, and the mixing properties of the water achieved only by means of diffusion. This implies that
(e.g., running water in shallow streams mixing better with also the internal tissues must have a sufficient amount of
air). The amount of oxygen in air is about 30 times that water in order to be able to dissolve oxygen and carbon
of water (Little 1990; Moore 2006), amounting to about dioxide. Maintaining a sufficient level of hydration may
210  ml O2, weighing 280 mg, in 1 L of air (Schmidt- be unproblematic for freshwater planarians but may
Nielsen 1975), while the diffusion rate of oxygen through require certain adaptations in marine and terrestrial forms.
air is much faster than through water (3 million times In particular, land planarians have no physiological or
faster through air; Prosser 1973). anatomical adaptations for water retention (Kawaguti
In the present context it suffices to realize that, thus, 1932). On the one hand land planarians have practically
availability of sufficient oxygen would not have formed no water-saving adaptations, while on the other hand
a stumbling-block during an evolutionary transition they avoid wet environments and thus are considered to
from water to a terrestrial environment. But, clearly, be stenohygric hygrocoeles (Froehlich 1955). It should
the organisms needed to evolve respiratory adaptations here be noted that the situation may not be as absolute
enabling them to extract oxygen from the air, as opposed as suggested by Kawaguti’s (1932) findings, in that
to their ancestors, which had evolved in an aquatic habitat. land flatworms may prevent desiccation by encasing
One such adaptation may be the production of themselves in mucus. Further, there may be also
mucus, which is secreted by both aquatic and terrestrial biochemical adaptations (secondary metabolism; see
planarians. Mucus plays several important roles in the Campbell 1965) in land planarians for the elimination
life of a planarian flatworm and is produced by various of carbon dioxide and nitrogenous waste that appear to
kinds of gland. Secretions from glands at the body margin facilitate water retention, such as the secretion of calcium
produce a slime trail that facilitates the gliding movement salts (Percival 1925), which is consistent with being a
of both aquatic and terrestrial triclads, effectuated by stenohygric hygrocoele. In contrast to land planarians,
the propulsive force of cilia on the ventral body surface freshwater species are subjected to osmotic influx of water
(Jones 1978). Many species of land planarians do not and therefore must continuously regulate its volume with
have such dedicated marginal glands, and various types the help of their protonephridial system.
of mucus are secreted from their creeping sole and/or One way to assess the hydration of the planarian
ventral surface. Sticky mucus discharged by cephalic body is to determine the osmolarity of the tissues,
glands partakes in the capture of prey and has neurotoxic which is expected to vary inversely with the degree
properties (cf. Thielicke and Sluys 2019 and references of hydration (Jones et al. 2004). The osmolarity of the
therein). The mucus of triclad flatworms also has freshwater planarians Schmidtea polychroa and Girardia
repellent properties as the worms are only rarely eaten dorotocephala (Woodworth, 1897) was 125–128 mOsm
by other animals since their surface secretions appear to kg−1 and 126  mOsm kg−1, respectively, while that of the
have repugnatorial function (Hyman 1951; Winsor 1998). marine triclad Procerodes littoralis (Ström, 1768) ranged
As mucus is produced also in aquatic triclads it could between 217–272 mOsm kg−1 (Jones et al. 2004 and
well be that these various functions already formed references therein). Tissue osmolarity for the terrestrial
evolutionary preadaptations of similar functions in land planarian Arthurdendyus triangulatus (Dendy, 1896)
planarians. Perhaps the secretion produced by the marginal ranged between 187.8–257.5 mOsm kg−1, depending upon
adhesive zone is an exception as it has been suggested the laboratory conditions (relatively dry or fully hydrated)

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Zoosyst. Evol. 95 (2) 2019, 543–556 553

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