Downloaded from rspb.royalsocietypublishing.

org on September 30, 2014

Phylogenomic analyses of deep gastropod

relationships reject Orthogastropoda
Felipe Zapata1, Nerida G. Wilson3,*, Mark Howison2, Sónia C. S. Andrade4,†,
Katharina M. Jörger5,6, Michael Schrödl5,6, Freya E. Goetz1,‡, Gonzalo Giribet4
rspb.royalsocietypublishing.org and Casey W. Dunn1
1
Department of Ecology and Evolutionary Biology, and 2Center for Computation and Visualization,
Brown University, Providence, RI 02906, USA
3
Western Australian Museum, Welshpool, WA 6106, Australia
4
Research Museum of Comparative Zoology, Department of Organismic and Evolutionary Biology, Harvard University,
Cambridge, MA 02138, USA
5
SNSB-Bavarian State Collection of Zoology, Munich 81247, Germany
Cite this article: Zapata F, Wilson NG, 6
Department Biology II, BioZentrum, Ludwig-Maximilians-Universität, Planegg-Martinsried 82152, Germany
Howison M, Andrade SCS, Jörger KM, Schrödl
MH, 0000-0002-0764-4090
M, Goetz FE, Giribet G, Dunn CW. 2014
Phylogenomic analyses of deep gastropod Gastropods are a highly diverse clade of molluscs that includes many familiar
relationships reject Orthogastropoda. animals, such as limpets, snails, slugs and sea slugs. It is one of the most abun-
Proc. R. Soc. B 281: 20141739. dant groups of animals in the sea and the only molluscan lineage that has
http://dx.doi.org/10.1098/rspb.2014.1739 successfully colonized land. Yet the relationships among and within its constitu-
ent clades have remained in flux for over a century of morphological, anatomical
and molecular study. Here, we re-evaluate gastropod phylogenetic relationships
by collecting new transcriptome data for 40 species and analysing them in com-
Received: 12 July 2014
bination with publicly available genomes and transcriptomes. Our datasets
Accepted: 21 August 2014 include all five main gastropod clades: Patellogastropoda, Vetigastropoda,
Neritimorpha, Caenogastropoda and Heterobranchia. We use two different
methods to assign orthology, subsample each of these matrices into three
increasingly dense subsets, and analyse all six of these supermatrices with
Subject Areas:
two different models of molecular evolution. All 12 analyses yield the same
evolution, taxonomy and systematics unrooted network connecting the five major gastropod lineages. This reduces
deep gastropod phylogeny to three alternative rooting hypotheses. These results
Keywords: reject the prevalent hypothesis of gastropod phylogeny, Orthogastropoda. Our
Gastropoda, Mollusca, Heterobranchia, dated tree is congruent with a possible end-Permian recovery of some gastropod
phylogenomics clades, namely Caenogastropoda and some Heterobranchia subclades.

Author for correspondence: 1. Introduction

Felipe Zapata Gastropoda, the clade of molluscs that includes snails, slugs and their relatives,
e-mail: felipe_zapata@brown.edu is hyperdiverse with respect to species number, morphology, habitat and many
other attributes. They radiated in marine, freshwater and terrestrial systems,
and display extensive body plan disparity [1]. A total of 32 000–40 000
*
marine species of gastropods have been described, but this is thought to rep-
Present address: Centre for Evolutionary resent only between 23 and 32% of the total estimated number of marine
Biology, University of Western Australia, species of gastropods [2]. In addition, there are a large number of limno-terres-
Crawley, WA 6009, Australia. trial snails and slugs [3], many of which are threatened to a degree unparalleled
†
Present address: Departamento de Zootecnia, among other invertebrate groups [4]. The overall magnitude of the gastropod
diversity is extremely hard to estimate; in a survey of a New Caledonian
ESALQ-USP, Piracicaba-SP 13418 – 900, Brazil.
‡ coral reef lagoon, gastropods represented almost 80% of the 2738 species of
Present address: Smithsonian National
molluscs found (excluding cephalopods) [5], with many undescribed species.
Museum of Natural History, Department of Gastropods are characterized by having a single shell and an operculum, at
Invertebrate Zoology, Washington, DC, USA. least in the larval stage, and by undergoing torsion during development. They
range in size from less than 1 mm to almost 1 m, and their shell has been modified
Electronic supplementary material is available enormously in many groups, including the common coiled and torted (usually
dextrally) snail-like, the highly convergent limpet-like, or the rare tubular or even
at http://dx.doi.org/10.1098/rspb.2014.1739 or
bivalved shells [6]. Many lineages have reduced the shell or it has been entirely lost.
via http://rspb.royalsocietypublishing.org.
Gastropod relationships have been at the centre of molluscan research and have
been in flux for decades (figure 1). Many authors have employed cladistic methods

& 2014 The Author(s) Published by the Royal Society. All rights reserved.
 Downloaded from rspb.royalsocietypublishing.org on September 30, 2014

(a) (d) Patellogastropoda 2

Patellogastropoda
Vetigastropoda
Neritimorpha

rspb.royalsocietypublishing.org
OMA1-B/ML
Neritimorpha OMA2-B/ML
Vetigastropoda
Orthogastropoda Caenogastropoda OMA3-B/ML
Caenogastropoda
Apogastropoda Heterobranchia
Apogastropoda Heterobranchia
(b) Patellogastropoda (e) Patellogastropoda
Vetigastropoda Archaeogastropoda Vetigastropoda ABA1-B
Neritimorpha Neritimorpha ABA2-B
Orthogastropoda ABA3-B/ML
Caenogastropoda Caenogastropoda
Apogastropoda Heterobranchia Apogastropoda Heterobranchia

Proc. R. Soc. B 281: 20141739

(c) Neritimorpha (f) Vetigastropoda
Patellogastropoda Patellogastropoda
Vetigastropoda Neritimorpha ABA1-ML
ABA2-ML
Caenogastropoda Caenogastropoda
Apogastropoda Heterobranchia Apogastropoda Heterobranchia
Figure 1. (a,b) Hypotheses for the internal relationships of Gastropoda. Not all listed studies find monophyly of all taxa, as Vetigastropoda is often paraphyletic or
diphyletic in earlier studies. Apogastropoda (i.e. Caenogastropoda þ Heterobranchia) is monophyletic in nearly all published studies. Hypotheses on the left do not
have support from the analyses presented here. Hypotheses on the right are consistent with the analyses presented here and differ only in their rooting. Matrix
construction (ABA, OMA), subsampling strategy (1,2,3) and inference method (Bayes, ML) supporting each of these hypotheses is indicated.

to analyse morphological data [6–10]. This work supports the clades in this group by creating a comprehensive taxonomic
monophyly of gastropods and the division of the group into dataset from 40 novel transcriptomes and 16 publicly available
five main clades—Patellogastropoda, Vetigastropoda, Neriti- genomes or transcriptomes. Using information from multiple
morpha, Caenogastropoda and Heterobranchia—in addition nuclear protein-coding genes provides large amounts of data
to the less understood Cocculinida and the so-called ‘hot-vent that can provide key phylogenetic insights [26] as well as
taxa’ (Peltospiridae and Cyathermiidae). The first numerical cla- facilitating several aspects of phylogenetic inference.
distic analysis included 117 morphological characters coded for
40 taxa, dividing gastropods into Eogastropoda (Patellogastro-
poda and Neolepetopsoidea; but several authors now find
Neolepetopsoidea nested within Patellogastropoda [11]) and
2. Material and methods
Orthogastropoda (all remaining gastropods; figure 1a [10]). (a) Taxon sampling, RNA isolation and sequencing
Other well-supported clades recovered in these analyses We collected new transcriptome data for 40 species, including 34
included Patellogastropoda, Vetigastropoda, Neritimorpha, gastropods and six other molluscs. All new datasets are paired-
Caenogastropoda and Heterobranchia, the last two forming end Illumina reads, except for single-end Illumina datasets for
the clade Apogastropoda (figure 1a–f). However, the Eogastro- Hinea brasiliana, Philine angasi and Strubellia wawrai. Samples
poda/Orthogastropoda division has not been supported in were prepared for sequencing with TruSeq RNA Sample Prep
Kit (Illumina) or a previously described custom protocol [27].
other analyses combining morphology with molecules
We deposited all these new sequence data, along with associated
(figure 1b [6]) or in molecular analyses (e.g. [12–18]), which
specimen collection information, voucher accession numbers,
tend to find support for Thiele’s [19] clade Archaeogastropoda RNA extraction methods and library preparation details, in
(with or without Neritimorpha) (see figure 1 for a summary of NCBI Sequence Read Archive (BioProject PRJNA253054). Vou-
hypotheses). chers for most specimens were deposited at the Museum of
Heterobranchia comprises the most diverse and ecologi- Comparative Zoology, Harvard University (Cambridge, MA,
cally widespread gastropod clades, including the informal USA) and Scripps Institution of Oceanography (La Jolla,
groups allogastropods, opisthobranchs and pulmonates [20]. CA, USA). The publicly available data for Siphonaria pectinata
With conservative estimates suggesting more than 40 000 are here shown in the figures as Siphonaria naufragum, according
species, heterobranchs are abundant in habitats ranging from to a recent revision [28].
the benthic realm to pelagic, intertidal to deep sea, tropical
to polar, and freshwater to terrestrial [3,21]. These transitions (b) Data analyses
are not evenly spread across lineages, and the concomitant These data were analysed in combination with publicly available
morphological specializations have made defining homologies data for 16 additional species to generate 56-taxon matrices. All
Illumina reads (new and publicly available) were assembled
difficult in many cases [22]. Although a consensus of relation-
with AGALMA (v. 0.3.4 – 0.3.5) [29], 454 datasets were assembled
ships among heterobranch groups is emerging [23,24], and
externally with NEWBLER (v. 2.3 and 2.5p1) and gene predictions
Panpulmonata [25] has been recently supported [26], the mono-
from Lottia gigantea [30] and Pinctada fucata [31] were imported
phyly and relationships of other higher taxa (e.g. Nudipleura, directly into AGALMA. Source code for most analysis steps as
Tectipleura) have not been evaluated with next-generation well as sequence alignments, tree sets, summary trees and vou-
data. In this study, we address the evolution of Gastropoda cher information are available in a git repository at https://
and evaluate the relationships among and within major bitbucket.org/caseywdunn/gastropoda.
 Downloaded from rspb.royalsocietypublishing.org on September 30, 2014

(a) 3
supermatrix 1
0 genes 300 862

rspb.royalsocietypublishing.org
supermatrix 2
species

supermatrix 3
57

(b)

Proc. R. Soc. B 281: 20141739

0 genes 364 1245
species

57
Figure 2. The six matrices that were considered here. Supermatrices were assembled with two methods: (a) ABA and (b) OMA. Three matrices were constructed for
each of these methods. Supermatrix 1 is the full set of genes and species. From Supermatrix 1, Supermatrix 2 is constructed as a subset of the best-sampled species.
From Supermatrix 2, Supermatrix 3 is constructed as a subset of the best sampled genes. See Material and methods for additional details. Black indicates sampled
genes for each taxon. Genes and species are sorted by sampling, with the best-sampled in the upper left.

Two methods were used to generate the supermatrices scores, time-series plots of the cumulative split frequencies, maxi-
within AGALMA. In method 1, after assembly, translation and mum bipartition discrepancies across chains less than 0.1, and an
removal of mtDNA loci, the sequences from all taxa were com- estimated effective sample size of tree likelihoods of at least 100.
pared to each other using an All-By-All BLASTP search, and a Post-burn-in sampled trees were combined and summarized
phylogenetic approach to identify orthologous sequences [32]. with a majority rule consensus tree.
We refer to this method as ABA. In method 2, the sequences Tree dating was conducted with MCMCTREE v. 4.7 [37] using
from all taxa were compared using OMA v. 0.99t [33] to directly the approximate likelihood calculation algorithm [38], and the
assign sequences to groups of orthologues using an entirely WAGþ G model of evolution. A birth–death speciation process
phenetic approach [34]. We refer to this method as OMA. was specified as tree prior with default parameters (death and
For each method (ABA and OMA), we constructed three pro- growth rate parameters equal 1, and sampling parameter equals
gressively smaller and denser amino acid supermatrices, creating 0). Rate heterogeneity among lineages was modelled using an
a total of six matrices (figure 2). Supermatrix 1 was constructed uncorrelated lognormal relaxed molecular clock [39] with a diffuse
by concatenating all orthologue sequences until the cumulative gamma G(1,1) prior for the substitution rate and the rate-drift par-
gene occupancy was 50% (49 752 sites/862 loci for ABA and 190 ameter. We used fossil calibrations to set prior densities on the
752 sites/1245 loci for OMA; 425 loci in common between ABA ages of five nodes (see figure 4) using minimum soft bounds
and OMA) and then removing Pyropelta sp. and Paralepetopsis with a left tail probability of 2.5% [40]. Because MCMCTREE
sp., which were poorly sampled. Supermatrix 2 was constructed always needs a calibration point on the root [37], we used
by removing taxa with less than 20% gene occupancy from Super- 550 Ma (ca Terreneuvian; [41]) to set a prior density on the root
matrix 1. The removed taxa include Haliotis kamtschatkana, age using a maximum soft bound with 2.5% tail probability. We
Perotrochus lucaya, Littorina littorea, Siphonaria naufragum, Chaeto- ran MCMCTREE twice each time for 1.2  107 generations,
derma sp. and Pomacea diffusa for both OMA and ABA matrices, sampling every 1.0  103 and discarding 20% of the samples as
as well as Amphiplica gordensis for the ABA Supermatrix burn-in. Convergence was determined with time-series plots of
2. This taxon was removed from the ABA Supermatrix 2 boot- the likelihood scores and assessing for correlation of divergence
strap replicates and the ABA Supermatrix 2 posterior probability times between runs.
tree sets prior to summary so that they could be consistently
displayed (electronic supplementary material, figures S1 and S2). (c) Hypothesis testing for Orthogastropoda
Supermatrix 3 was constructed by trimming genes from We statistically compared the Orthogastropoda hypothesis to our
Supermatrix 2 until the cumulative gene occupancy reached 70% maximum-likelihood tree using the SOWH test [42]. To carry out
(15 735 sites/300 loci for ABA and 45 084 sites/364 loci for this analysis, we used SOWHAT [43] specifying a constraint tree
OMA; 110 loci in common between ABA and OMA). and the WAGþ G model on supermatrix 1 (OMA). We used
We inferred phylogenetic relationships using both maximum- the automatic stopping criterion implemented in SOWHAT to
likelihood (ML) and Bayesian approaches, for a total of 12 determine an appropriate sample size for the null distribution.
phylogenetic analyses on the six supermatrices. For ML, we used
EXAML v. 1.0.11 [35] with a WAGþ G model of amino acid evol-
ution. Bootstrap values were estimated with 200 replicates.
Bayesian analyses were conducted with PHYLOBAYES MPI v. 1.4e 3. Results and discussion
[36] using the CAT-Poisson model of amino acid evolution. Two
independent MCMC chains were run on each matrix, adjusting (a) Deep relationships among major gastropod clades
the number of cycles until convergence was achieved. Conver- Our datasets strongly support the monophyly of gastropods.
gence was determined with time-series plots of the likelihood This result is not surprising in itself but has only recently
 Downloaded from rspb.royalsocietypublishing.org on September 30, 2014

(a) Polyplacophora 4
Chiton olivaceus
0.5/1/1/1
Aplacophora Chaetoderma sp.

rspb.royalsocietypublishing.org
0/0.93/0.93/0.93
Greenland neomeniomorph
Monoplacophora Neomenia megatrapezata
0.04/0.55/0.67/0.91 Cephalopoda Laevipilina hyalina
Octopus vulgaris
Scaphopoda Gadila tolmiei
0.5/1/1/0.99
Ennucula tenuis
0.5/1/1/1
Bivalvia 0.5/1/1/1 Solemya velum
Pinctada fucata
1/1/0.95/0.91
Neotrigonia margaritacea
0.5/0.71/0.99/0.29
Astarte sulcata
1/0.61/1/0.44
1/1/1/0.99
Myochama anomioides

Proc. R. Soc. B 281: 20141739

Patellogastropoda Mercenaria campechiensis
Lottia gigantea
1/1/0.11/0.58
0.49/0.99/1/0.94 Perotrochus lucaya
Granata imbricata
Vetigastropoda Lepetodrilus fucensis
Amphiplica gordensis
Haliotis kamtschatkana
0/0.43/0.03/0.47
Phasianella ventricosa
1/1/1/0.95
Prothalotia lehmanni
Gastropoda Monodonta labio
Neritimorpha
Nerita peloronta
Titiscania limacina
Caenogastropoda Pomacea diffusa
Hinea brasiliana
0.99/0.98/1/0.98 Urosalpinx cinerea
1/0.58/0.97/0.09
Sorbeoconcha Ilyanassa obsoleta
gene sampling Hypsogastropoda Janthina janthina
1/0.99/1/0.91
150 genes Rubyspira osteovora
1/1/0.47/0.83
Euspira heros
624 genes 1/1/1/0.89
Littorina littorea
1109 genes Architectonica perspectiva
Nudipleura Pleurobranchaea
public data californica
Fiona pinnata
1/1/1/0.99
new data Bathydoris clavigera
Heterobranchia
Doris kerguelenensis
Hydatina physis
Rissoella caribaea
OMA-Bayes/OMA-ML/ABA-Bayes/ABA-ML Tylodina fungina
= 1/1/1/1 Euopisthobranchia
Philine angasi
alternative rooting locations 0/1/0/0.92
Haminoea antillarum
Clione antarctica
Aplysia californica
Tectipleura
Oxynoe viridis
(b) Turbonilla sp.
0/0.27/0/0
Microhedyle glandulifera
Vetigastropoda Heterobranchia Panpulmonata 1/0.94/1/0.95
Strubellia wawrai
f 1/0.46/1/0.3
Ophicardelus sulcatus
d e Onchidella floridana
0/0.2/0/0.04
Phallomedusa solida
0/0.24/0/0.16
1/1/0.97/0.58 1/1/1/1
0/0.44/0/0.35
Biomphalaria glabrata
Patellogastropoda Caenogastropoda Siphonaria naufragum

Neritimorpha 0.07
Figure 3. Summary tree for analyses of Supermatrices 1. (a) Rooted phylogram of the maximum-likelihood OMA analysis, including outgroup taxa. Branch support
values are shown on descendent nodes. The areas of the lollipops, which are centred on the branch tips, are proportional to the number of genes sampled in OMA
Supermatrix 1. (b) Unrooted cladogram of the ingroup taxa. Branch support values are shown, and alternative rooting locations are indicated with orange arrows.
These support values were calculated by removing the outgroup taxa from the tree sets used to generate (a) and regenerating consensus trees. The letter on the
rooting arrow corresponds to the hypotheses shown in figure 1.

been supported by molecular analyses of large datasets therefore can be reduced to a 5-taxon problem (figures 1
[32,44,45] (see also [14,15,17,18]), or in the total evidence and 3b). Our 12 analyses (two inferences methods on two
analysis of Aktipis et al. [6]. Our analyses also support the types of supermatrices each subsampled in three different
monophyly of all major gastropod clades represented by ways) all recover the same unrooted ingroup relationships
multiple taxa: Vetigastropoda, Neritimorpha, Caenogastro- for these five clades (figure 3b; electronic supplementary
poda and Heterobranchia (figure 3a). Patellogastropoda is material, figures S1b and S2b). These ingroup relationships
represented by a single species, so its monophyly could not are strongly supported by all methods except the ABA
be evaluated. The deep internal relationships of gastropods ML analyses, which have lower support than the other
 Downloaded from rspb.royalsocietypublishing.org on September 30, 2014

methods for a bipartition Vetigastropoda þ Patellogastropoda Because we ran 12 phylogenetic analyses, we can explore 5
(58, 75 and 56% for Supermatrices 1, 2 and 3) and recover the differences in support between these three alternative

rspb.royalsocietypublishing.org
Vetigastropoda þ Neritimorpha in a minority of replicates. hypotheses for gastropod rooting across inference method
The lower support in these analyses may be due to the poor (Bayes and ML), matrix construction method (OMA and
sampling of Patellogastropoda. These ingroup relationships ABA) and matrix subsampling (Supermatrices 1, 2 and 3;
allow us to reject the hypotheses for gastropod relationships figure 2). Matrix subsampling had little effect on deep relation-
indicated in figure 1a,b. ships. Analyses of Supermatrix 1 (figure 3) and Supermatrix 2
Although the ingroup relationships found broad consist- (electronic supplementary material, figure S1) were consistent
ent support, the rooting of gastropods is still not well with all three rooting positions (figure 1d–f ). Analyses of
resolved. Our results are congruent with three possible root- Supermatrix 3 (electronic supplementary material, figure S2)
ings (orange arrows in figure 3b; electronic supplementary found support for only two of these rootings (figure 1d,f ).
material, figures S1b and S2b). This is akin to other recalci- Unlike the other analyses, it did not recover Apogastropoda

Proc. R. Soc. B 281: 20141739

trant animal phylogeny questions, including the root of as the sister group to all other gastropods (figure 1e). This par-
Metazoa [46,47] and the root of arthropods [48]. Though ticular hypothesis (figure 1e) is interesting because it includes
the hypothesis indicated in figure 1c is compatible with the Archaeogastropoda, which was proposed nearly a century ago
ingroup relationships supported here, we never recover this by Thiele [19]. Bayesian analyses recovered Neritimorpha as
rooting and it can be excluded. This reduces the possible the sister group to Apogastropoda (figure 1d,f) in all analyses,
alternatives for deep gastropod relationships to the three but ML analyses found very low support for this relation-
hypotheses (figure 1d–f ). Two of these remaining hypotheses ship. Analyses of OMA matrices provided strong support
have been proposed before [6,14]; the other (figure 1f ) is for a clade comprising Patellogastropoda and Vetigastropoda
recovered for the first time here. (figure 1d,e), but analyses of ABA matrices did not.
The rejection of several widely held hypotheses for deep These results suggest clear strategies for distinguishing
gastropod phylogeny (figure 1a–c) has major implica- between the remaining hypotheses for deep gastropod
tions for the understanding of gastropod evolution. All our relationships. Since these hypotheses differ only in their root-
analyses reject the Orthogastropoda hypothesis (a clade com- ing, improved outgroup sampling will be critical. To
prised Vetigastropoda, Neritimorpha, Caenogastropoda and maximize gene sampling and matrix density, we limited our
Heterobranchia) and the placement of Patellogastropoda sampling of non-gastropod molluscs to those for which Illu-
as the sister group to other gastropods (figure 1a,b). Even mina data [32] or genomes are available. Future analyses of
in the minority of ABA ML replicates that recover an gastropod relationships will need to include more outgroups
ingroup partition Vetigastropoda þ Neritimorpha, the root- to resolve the remaining open questions. Previous phyloge-
ing is inconsistent with Orthogastropoda. The broadly nomic analyses of molluscs that also included extensive 454
accepted Orthogastropoda hypothesis has been proposed in and Sanger data [32,44] had much broader non-gastropod
multiple configurations [6,9,10,49,50]. The placement of Patel- sampling but minimal gastropod sampling. The rooting of gas-
logastropoda as the sister group to Orthogastropoda has been tropods was not fully supported in these analyses either, but
driven by considerable anatomical research. One potential the strongly supported ingroup relationships are compatible
character supporting this placement is the ciliary ultrastruc- with the three hypotheses supported here. In addition,
ture of the cephalic tentacles, which also occurs in Bivalvia improved sampling of Patellogastropoda (here represented
and Solenogastres but is lacking from other gastropods [51]. by a single species with a complete genome) and Neritimor-
In this scenario, this character is plesiomorphic for Mollusca, pha, and the addition of the unsampled Neomphalina and
retained in Patellogastropoda and was lost a single time in Cocculiniformia, will be critical.
Orthogastropoda. However, because enforcing the mono-
phyly of Orthogastropoda is significantly worse (SOWH
test: n ¼ 152, D-likelihood ¼ 374.0137, p ¼ 0) than our most (b) Relationships within major gastropod clades
likely tree (figure 3), our results indicate that this character Although our sampling is focused on resolving deep relation-
may be convergent between Patellogastropoda and outgroup ships between the major gastropod clades, our results do find
taxa, or was lost more than once within Gastropoda. We also strong support for some previously unresolved relationships
reject another recent hypothesis for gastropod rooting, the within Vetigastropoda, Caenogastropoda and Heterobranchia.
sister group relationship of Neritimorpha to other Gastropoda A key question within Vetigastropoda is the placement
(figure 1c) [15,17,18]. of Pleurotomarioidea (Perotrochus in our analyses), which
Our reduction of deep gastropod phylogeny to three alterna- appears outside Vetigastropoda in some previous studies
tive hypotheses (figure 1d–f) clarifies multiple open questions. [14,15]. Here we find strong support for the placement of
All three of these hypotheses include the monophyly of Apogas- Pleurotomarioidea as the sister group to all other vetigastro-
tropoda (i.e. Heterobranchia þ Caenogastropoda), reinforcing pods (figure 3a), resolving this issue. We also resolve the
this widely accepted aspect of gastropod relationships. position of Seguenzioidea (Granata) as the next clade to diverge
Other relationships supported here have been found earlier from other vetigastropods (figure 3a; electronic supplementary
(figure 1d,e; see review by Aktipis et al. [6]; for different material, figures S1 and S2). Our analyses also recover a
views, see e.g. the gastropod classification by Bouchet et al. well-supported clade of deep-sea taxa (Pseudococculinidae
[52] and the mitogenomic study by Grande et al. [53]; see also (Amphiplica) and Lepetodriloidea (Lepetodrilus); figure 3a). We
[6,14]). However, it is now acknowledged that mitogenomic also find strong support (figure 3a; electronic supplementary
data are not appropriate for resolving deep gastropod relation- material, figures S1 and S2) for a clade comprising Phasianelli-
ships [54]. To the best of our knowledge, no molecular analysis dae (Phasianella) and Trochoidea (Prothalotia and Monodonta).
supported the placement of Vetigastropoda as sister group to The position of Haliotis is not resolved (figure 3a), perhaps
all other gastropods. due to relatively poor gene sampling.
 Downloaded from rspb.royalsocietypublishing.org on September 30, 2014

Pro. Cam. Ord. Si. De. Car. Per. Tri. Jur. Cre. Cen. 6
A Chiton olivaceus
Chaetoderma sp.

rspb.royalsocietypublishing.org
Greenland neomeniomorph
Neomenia megatrapezata
B Laevipilina hyalina
Octopus vulgaris
Gadila tolmiei
Ennucula tenuis
Solemya velum
C Pinctada fucata
Neotrigonia margaritacea
Astarte sulcata
Myochama anomioides
Mercenaria campechiensis
D Lottia gigantea
Perotrochus lucaya
Granata imbricata
Lepetodrilus fucensis

Proc. R. Soc. B 281: 20141739

Amphiplica gordensis
Haliotis kamtschatkana
Phasianella ventricosa
Prothalotia lehmanni
Monodonta labio
Nerita peloronta
Titiscania limacina
Pomacea diffusa
Hinea brasiliana
Urosalpinx cinerea
Ilyanassa obsoleta
Janthina janthina
E Rubyspira osteovora
Euspira heros
Littorina littorea
Architectonica perspectiva
Pleurobranchaea californica
Fiona pinnata
Bathydoris clavigera
Doris kerguelenensis
Hydatina physis
Rissoella caribaea
Tylodina fungina
Philine angasi
Haminoea antillarum
Clione antarctica
Aplysia californica
Oxynoe viridis
Turbonilla sp.
Microhedyle glandulifera
Strubellia wawrai
Ophicardelus sulcatus
Onchidella floridana
Phallomedusa solida
Biomphalaria glabrata
Siphonaria naufragum
600 500 400 300 200 100 0
time before present (Ma)

Figure 4. Chronogram with estimates of divergence times for internal nodes. Bars correspond to 95% credibility intervals. Fossil constraints were set on nodes A – E:
node A, 231 Ma (Leptochiton davolii [66]); node B, 505 Ma (Plectronoceras cambria [67]); node C, 475 Ma (Glyptarca serrata, Arenigian [68]); node D, 530 Ma
(Fordilla troyensis from the Tommotian of Siberia [69 – 71]); node E, 418 Ma (Sublitoidea [72]). We used 550 Ma (ca Terreneuvian; see [41]) to set a prior density
on the root age using a maximum soft bound with 2.5% tail probability. Geological periods abbreviated on top: Pro., Proterozoic; Cam., Cambrian; Ord., Ordovician;
Si., Silurian; Dev., Devonian; Car., Carboniferous; Per., Permian; Tri., Triassic; Jur., Jurassic; Cre., Cretaceous; Cen., Cenozoic.

Caenogastropoda is a megadiverse clade comprising about remaining nudibranch lineages [26]. Our results recovered a
60% of living gastropod species [55], so our limited sampling monophyletic Cephalaspidea (Philine þ Haminoea), sister
can address only a small fraction of open questions about internal group to Anaspidea (Aplysia) þ Pteropoda (Clione). Umbra-
relationships of this group. The relationships we can test are lar- culoidea (Tylodina) was sister group to Cephalaspidea þ
gely in agreement with prior morphological [10,55–57] and Anaspidea þ Pteropoda; all four taxa together represent the
molecular [58–60] analyses. We find a sister group relationship well-supported Euopisthobranchia (figure 3a; electronic sup-
of Ampullarioidea (represented by Pomacea) to Sorbeoconcha, plementary material, figures S1 and S2). We find support for
which comprise the remaining sampled caenogastropods Panpulmonata (figure 3a; electronic supplementary material,
(figure 3a). Within Sorbeoconcha, Cerithioidea (Hinea) is the figures S1 and S2), but their internal relationships are mostly
sister group to Hypsogastropoda, the latter dividing into a sipho- unresolved and clearly require future attention. Like previous
nate (in our case the two Neogastropoda: Urosalpinx and Sanger sequencing-based studies, our analyses consistently
Ilyanassa) and an asiphonate group (Janthina, Littorina, Euspira recover a Panpulmonata þ Euopisthobranchia clade or Tecti-
plus Rubyspira), similar to the findings of Ponder et al. [55]. pleura [25,62]. The relationship of Tectipleura to other
The basic structure of internal Heterobranchia relation- heterobranchs has been of particular interest. We recover
ships has only recently gained some agreement [23,24]. Our two conflicting hypotheses for these relationships, neither
strong support for the placement of Architectonica as sister of which has been previously proposed. Our likelihood ana-
group to the other sampled heterobranchs is consistent with lyses place the unnamed clade Rissoelloidea (Rissoella) þ
most other analyses [24]. Nudibranchia (Fiona þ Bathydoris þ Acteonoidea (Hydatina) as the sister group to Tectipleura
Doris) and Nudipleura (Pleurobranchaea þ Nudibranchia) (figure 3a; electronic supplementary material, figures S1
were monophyletic [61,62], despite some suggestion that and S2). Our Bayesian analyses, however, place Nudipleura
Pleurobranchoidea may not be the sister group to the and this Rissoelloidea þ Acteonoidea clade together with
 Downloaded from rspb.royalsocietypublishing.org on September 30, 2014

strong support, and place this clade as sister to Tectipleura (figure 4). However, denser sampling will be required to 7
(electronic supplementary material, figure S3). Previous ana- derive accurate diversification curves to test these hypotheses.

rspb.royalsocietypublishing.org
lyses have instead favoured Euthyneura, a clade comprising
Data accessibility. Raw sequence data: NCBI Sequence Read Archive
Tectipleura and Nudipleura (but excluding Rissoelloidea þ
BioProject PRJNA253054, accession nos. SRR1505101–SRR1505105,
Acteonoidea). We do not recover Euthyneura in any of our SRR1505107–SRR1505141. Analysis scripts, phylogenetic alignmets,
analyses. Tectipleura is united by a monaulic reproductive tree sets, summary trees and voucher information: https://bitbucket.
system [23], but even Euthyneura is not entirely defined by org/caseywdunn/gastropoda. The most recent commit at the time of
euthyneury, as there are internal reversals [63] and the con- submission is available at https://bitbucket.org/caseywdunn/gastro-
poda/src/b93fce3bf8e90cc0124327f5f7d3d0353ee4d295. Phylogenetic
dition is known in other groups (Rhodopemorpha [64]).
data also available at http://dx.doi.org/10.5061/dryad.5bc98.
Rissoelloidea þ Acteonoidea and Euthyneura share giant neur-
Acknowledgements. Thanks to Alicia R. Pérez-Porro and Ana Riesgo for
ons in macroscopic animals [63], and if necessary, a simple
laboratory assistance, Vanessa González for contributing unpublished
redefinition of the taxon Euthyneura to include Rissoelloidea þ libraries for Neotrigonia, Astarte, Myochama and Mercenaria Stephen

Proc. R. Soc. B 281: 20141739

Acteonoidea would maintain stability. Smith for preliminary data analyses, Samuel Church for help running
SOWHAT and Robert Vrijenhoek at Monterey Bay Aquarium Research
Institute (MBARI) for providing the Rubyspira and Amphiplica samples.
(c) Chronogram N.G.W., G.G. and C.W.D. conceived of and designed the study.
Our dated phylogeny (figure 4) shows a Cambrian origin of N.G.W., G.G., K.J., M.S. and F.E.G. collected samples. S.C.S.A., F.E.G.,
stem gastropods with crown diversification into its five main K.J. and N.G.W. prepared samples for sequencing. F.Z. designed and
ran analyses. M.H. implemented software and assisted with data man-
lineages during the Ordovician to the Devonian, as well
agement. G.G., F.Z., N.G.W. and C.W.D. wrote the manuscript. All
shown in the gastropod fossil record [72]. From the well- authors discussed/contributed to the final manuscript version.
sampled groups, crown Vetigastropoda diversified first, Funding statement. This research was supported by the US National Science
around the Devonian–Carboniferous, followed by Neriti- Foundation through the Systematics Program (awards 0844596, 0844881
morpha and Heterobranchia at similar periods. Crown and 0844652) and the Alan T. Waterman Award. Field work was
Caenogastropoda seem to have diversified later, around the supported by the US National Science Foundation through the Assem-
Permian–Triassic, perhaps initiating its explosive diversifica- bling the Tree of Life program BivAToL grant (award 0732903), and by
the German Research Foundation (SCHR667/9–1 and 13–1). Sequen-
tion after the end-Permian mass extinction ca 254 Ma, cing at the Brown Genomics Core facility was supported in part by
responsible for the extinction of 95–99% of marine species and NIH P30RR031153 and NSF EPSCoR EPS-1004057, and sequencing at
to change the ecosystems and their faunal composition forever. the Harvard Center for Systems Biology was supported with internal
Such drastic post-extinction diversifications have been recently funds from the Museum of Comparative Zoology, and by the Volkswa-
gen Foundation to K.M.J. Data transfer was supported by NSF RII-C2
shown for other modern clades of marine organisms (e.g.
EPS-1005789. Analyses were conducted with computational resources
Crinoidea [73] and Protobranchia [74]). This could also explain and services at the Center for Computation and Visualization at
other explosive radiations in gastropods, especially within the Brown University, supported in part by the NSF EPSCoR EPS-1004057
euthyneuran Heterobranchia clades such as Nudipleura and the State of Rhode Island.

References
1. Bieler R. 1992 Gastropod phylogeny and the Streptoneura (Mollusca). J. Moll. Stud. 54, 13. Colgan DJ, Ponder WF, Eggler PE. 2000 Gastropod
systematics. Annu. Rev. Ecol. Syst. 23, 311– 338. 367 –441. (doi:10.1093/mollus/54.4.367) evolutionary rates and phylogenetic relationships
(doi:10.1146/annurev.es.23.110192.001523) 8. Lindberg DR, Ponder WF. 1996 An evolutionary assessed using partial 28S rDNA and histone H3
2. Appeltans W et al. 2012 The magnitude of global tree for the Mollusca: branches or roots? In Origin sequences. Zool. Scr. 29, 29 –63. (doi:10.1046/j.
marine species diversity. Curr. Biol. 22, 2189– 2202. and evolutionary radiation of the Mollusca (ed. 1463-6409.2000.00021.x)
(doi:10.1016/j.cub.2012.09.036) J Taylor), pp. 67 –75. Oxford, UK: Oxford University 14. Aktipis SW, Giribet G. 2010 A phylogeny of
3. Barker GM. 2001 Gastropods on land: phylogeny, Press. Vetigastropoda and other ‘archaeogastropods’:
diversity, and adaptive morphology. In The biology 9. Salvini-Plawen LV, Steiner G. 1996 Synapomorphies re-organizing old gastropod clades. Invertebr. Biol.
of terrestrial molluscs (ed. GM Barker), pp. 1–146. and plesiomorphies in higher classification of 129, 220–240. (doi:10.1111/j.1744-7410.2010.
Wallingford, UK: CABI Publishing. Mollusca. In Origin and evolutionary radiation of the 00198.x)
4. Collen B, Böhm M, Kemp R, Baillie JEM (eds). 2012 Mollusca (ed. JD Taylor), pp. 29 –51. Oxford, UK: 15. Aktipis SW, Giribet G. 2012 Testing relationships
Spineless: status and trends of the world’s invertebrates. Oxford University Press. among the vetigastropod taxa: a molecular
London, UK: Zoological Society of London. 10. Ponder WF, Lindberg DR. 1997 Towards a approach. J. Moll. Stud. 78, 12– 27. (doi:10.1093/
5. Bouchet P, Lozouet P, Maestrati P, Heros V. 2002 phylogeny of gastropod molluscs: an analysis using mollus/eyr023)
Assessing the magnitude of species richness in tropical morphological characters. Zool. J. Linn. Soc. 119, 16. Giribet G, Okusu A, Lindgren AR, Huff SW, Schrödl
marine environments: exceptionally high numbers of 83 –265. (doi:10.1111/j.1096-3642.1997.tb00137.x) M, Nishiguchi MK. 2006 Evidence for a clade
molluscs at a New Caledonia site. Biol. J. Linn. Soc. 75, 11. Nakano T, Sasaki T. 2011 Recent advances in composed of molluscs with serially repeated
421–436. (doi:10.1046/j.1095-8312.2002.00052.x) molecular phylogeny, systematics and evolution of structures: monoplacophorans are related to chitons.
6. Aktipis SW, Giribet G, Lindberg DR, Ponder WF. patellogastropod limpets. J. Moll. Stud. 77, Proc. Natl Acad. Sci. USA 103, 7723–7728. (doi:10.
2008 Gastropoda: an overview and analysis. In 203 –217. (doi:10.1093/mollus/eyr016) 1073/pnas.0602578103)
Phylogeny and evolution of the mollusca (eds 12. Colgan DJ, Ponder WF, Beacham E, Macaranas JM. 17. Wilson NG, Rouse GW, Giribet G. 2010 Assessing the
WF Ponder, DR Lindberg), pp. 201 –237. Berkeley, 2003 Gastropod phylogeny based on six segments molluscan hypothesis Serialia
CA: University of California Press. from four genes representing coding or non-coding (Monoplacophora þ Polyplacophora) using
7. Haszprunar G. 1988 On the origin and evolution of and mitochondrial or nuclear DNA. Moll. Res. 23, novel molecular data. Mol. Phylogenet. Evol. 54,
major gastropod groups, with special reference to 123 –148. (doi:10.1071/MR03002) 187–193. (doi:10.1016/j.ympev.2009.07.028)
 Downloaded from rspb.royalsocietypublishing.org on September 30, 2014

18. Stöger I, Sigwart JD, Kano Y, Knebelsberger T, 32. Smith S, Wilson NG, Goetz F, Feehery C, Andrade Phylogenet. Evol. 67, 223– 233. (doi:10.1016/j. 8
Marshall BA, Schwabe E, Schrödl M. 2013 The SCS, Rouse GW, Giribet G, Dunn CW. 2011 Resolving ympev.2013.01.010)

rspb.royalsocietypublishing.org
continuing debate on deep molluscan phylogeny: the evolutionary relationships of molluscs with 47. Dunn CW et al. 2008 Broad phylogenomic
evidence for Serialia (Mollusca, Monoplacophora phylogenomic tools. Nature 480, 364–367. (doi:10. sampling improves resolution of the animal tree
plus Polyplacophora). Biomed Res. Int. 2013, 1038/nature10526) of life. Nature 452, 745 –749. (doi:10.1038/
407072. (doi:10.1155/2013/407072) 33. Altenhoff AM, Gil M, Gonnet GH, Dessimoz C. 2013 nature06614)
19. Thiele J. 1925 Gastropoda. In Handbuch der Inferring hierarchical orthologous groups from 48. Giribet G, Richter S, Edgecombe GD, Wheeler WC.
Zoologie, 5 (1,2) (ed. W Kukenthal), pp. 38 –155. orthologous gene pairs. PLoS ONE 8, e53786. 2005 The position of crustaceans within the
Berlin, Germany: Walter de Gruyer. (doi:10.1371/journal.pone.0053786) Arthropoda—evidence from nine molecular loci
20. Haszprunar G. 1985 The Heterobranchia—a new 34. Fernández R, Laumer CE, Vahtera V, Libro S, and morphology. In Crustacean Issues 16: Crustacea
concept of the phylogeny and evolution of the Kaluziak S, Sharma PP, Pérez-Porro AR, Edgecombe and Arthropod Relationships. Festschrift for Frederick
higher Gastropoda. Z. Zool. Syst. Evol.-forsch. 23, GD, Giribet G. 2014 Evaluating topological conflict in R. Schram (eds S Koenemann, RA Jenner),

Proc. R. Soc. B 281: 20141739

15 –37. (doi:10.1111/j.1439-0469.1985.tb00567.x) centipede phylogeny using transcriptomic data sets. pp. 307–352. Boca Raton, FL: Taylor & Francis.
21. Wägele H, Klussmann-Kolb A, Vonnemann V, Mol. Biol. Evol. 31, 1500–1513. (doi:10.1093/ 49. McArthur AG, Harasewych MG. 2003 Molecular
Medina M. 2008 Heterobranchia I: The molbev/msu108) systematics of the major lineages of the Gastropoda.
Opisthobranchia. In Phylogeny and evolution of the 35. Aberer AJ, Stamatakis A. 2013 ExaML: Exascale In Molecular systematics and phylogeography
mollusca (eds WF Ponder, DR Lindberg), pp. 385– maximum likelihood: program and documentation. of mollusks (eds C Lydeard, DR Lindberg),
408. Berkeley, CA: University of California Press. See http://sco.h-its.org/exelixis/web/software/ pp. 140–160. Washington, DC: Smithsonian Books.
22. Schrödl M, Neusser TP. 2010 Towards a phylogeny examl/index.html. 50. Ponder WF, Lindberg DR. 1996 Gastropod
and evolution of Acochlidia (Mollusca: Gastropoda: 36. Lartillot N, Rodrigue N, Stubbs D, Richer J. 2013 phylogeny—challenges for the 90s. In Origin and
Opisthobranchia). Zool. J. Linn. Soc. 158, 124–154. PhyloBayes MPI: phylogenetic reconstruction with evolutionary radiation of the Mollusca (ed. J Taylor),
(doi:10.1111/j.1096-3642.2009.00544.x) infinite mixtures of profiles in a parallel pp. 135–154. Oxford, UK: Oxford University Press.
23. Schrödl M, Jörger KM, Klussmann-Kolb A, Wilson environment. Syst. Biol. 62, 611–615. (doi:10.1093/ 51. Künz E, Haszprunar G. 2001 Comparative
NG. 2011 Bye bye ‘Opisthobranchia’! A review on Sysbio/Syt022) ultrastructure of gastropod cephalic tentacles:
the contribution of mesopsammic sea slugs to 37. Yang ZH. 2007 PAML 4: phylogenetic analysis by Patellogastropoda, Neritaemorphi and
euthyneuran systematics. Thalassas 27, 101–112. maximum likelihood. Mol. Biol. Evol. 24, 1586– Vetigastropoda. Zool. Anz. 240, 137 –165. (doi:10.
24. Wägele H, Klussmann-Kolb A, Verbeek E, Schrödl M. 1591. (doi:10.1093/Molbev/Msm088) 1078/0044-5231-00017)
2014 Flashback and foreshadowing—a review of 38. dos Reis M, Yang ZH. 2011 Approximate likelihood 52. Bouchet P, Rocroi JP, Frýda J, Hausdorf B, Ponder
the taxon Opisthobranchia. Org. Divers. Evol. 14, calculation on a phylogeny for Bayesian estimation WF, Valdés A, Warén A. 2005 Classification and
133–149. (doi:10.1007/s13127-013-0151-5) of divergence times. Mol. Biol. Evol. 28, 2161– nomenclator of gastropod families. Malacologia 47,
25. Jörger KM, Stöger I, Kano Y, Fukuda H, 2172. (doi:10.1093/molbev/msr045) 1–397.
Knebelsberger T, Schrödl M. 2010 On the origin of 39. Drummond AJ, Ho SY, Phillips MJ, Rambaut A. 53. Grande C, Templado J, Zardoya R. 2008 Evolution of
Acochlidia and other enigmatic euthyneuran 2006 Relaxed phylogenetics and dating with confidence. gastropod mitochondrial genome arrangements.
gastropods, with implications for the systematics of PLoS Biol. 4, e88. (doi:10.1371/journal.pbio.0040088) BMC Evol. Biol. 8, 61. (doi:10.1186/1471-2148-8-61)
Heterobranchia. BMC Evol Biol. 10, 323. (doi:10. 40. Yang Z, Rannala B. 2006 Bayesian estimation of 54. Stöger I, Schrödl M. 2013 Mitogenomics does not
1186/1471-2148-10-323) species divergence times under a molecular clock resolve deep molluscan relationships (yet?). Mol.
26. Kocot KM, Halanych KM, Krug PJ. 2013 using multiple fossil calibrations with soft bounds. Phylogenet. Evol. 69, 376– 392. (doi:10.1016/j.
Phylogenomics supports Panpulmonata: Mol. Biol. Evol. 23, 212–226. (doi:10.1093/ ympev.2012.11.017)
Opisthobranch paraphyly and key evolutionary steps molbev/msj024) 55. Ponder WF, Colgan DJ, Healy JM, Nützel A, Simone
in a major radiation of gastropod molluscs. Mol. 41. Edgecombe GD, Giribet G, Dunn CW, Hejnol A, LRL, Strong EE. 2008 Caenogastropoda. In Phylogeny
Phylogenet. Evol. 69, 764–771. (doi:10.1016/j. Kristensen RM, Neves RC, Rouse GW, Worsaae K, and evolution of the mollusca (eds WF Ponder,
ympev.2013.07.001) Sørensen MV. 2011 Higher-level metazoan DR Lindberg), pp. 331–383. Berkeley, CA:
27. Riesgo A, Andrade SC, Sharma PP, Novo M, Pérez- relationships: recent progress and remaining University of California Press.
Porro AR, Vahtera V, González VL, Kawauchi GY, questions. Org. Divers. Evol. 11, 151 –172. 56. Strong E. 2003 Refining molluscan characters:
Giribet G. 2012 Comparative description of ten (doi:10.1007/s13127-011-0044-4) morphology, character coding and a phylogeny of
transcriptomes of newly sequenced invertebrates 42. Swofford DL, Olsen GJ, Waddell PJ, Hillis DM. 1996 the Caenogastropoda. Zool. J. Linn. Soc. 137,
and efficiency estimation of genomic sampling in Phylogenetic inference. In Molecular systematics 447–554. (doi:10.1046/j.1096-3642.2003.00058.x)
non-model taxa. Front. Zool. 9, 33. (doi:10.1186/ (eds DM Hillis, C Moritz, BK Mable), pp. 407–514. 57. Simone LRL. 2011 Phylogeny of the
1742-9994-9-33) Sunderland, MA: Sinauer Associates. Caenogastropoda (Mollusca), based on comparative
28. Giribet G, Kawauchi GY. Submitted. How many 43. Church SH, Ryan JF, Dunn CW. 2014 Automation morphology. Arq. Zool. 42, 161– 323.
species of Siphonaria pectinata are there? J. Moll. and evaluation of the SOWH test for phylogenetic 58. Harasewych MG, Adamkewicz SL, Plassmeyer M,
Stud. topologies with SOWHAT. bioRxiv 1–17. (doi:10. Gillevet PM. 1998 Phylogenetic relationships of the
29. Dunn CW, Howison M, Zapata F. 2013 Agalma: an 1101/005264) lower Caenogastropoda (Mollusca, Gastropoda,
automated phylogenomics workflow. BMC 44. Kocot KM et al. 2011 Phylogenomics reveals deep Architaenioglossa, Campaniloidea, Cerithioidea) as
Bioinform. 14, 330. (doi:10.1186/1471-2105- molluscan relationships. Nature 477, 452–456. determined by partial 18S rDNA sequences. Zool.
14-330) (doi:10.1038/nature10382) Scr. 27, 361 –372. (doi:10.1111/j.1463-6409.1998.
30. Simakov O et al. 2013 Insights into bilaterian 45. Meyer A, Witek A, Lieb B. 2011 Selecting ribosomal tb00467.x)
evolution from three spiralian genomes. Nature protein genes for invertebrate phylogenetic 59. Colgan DJ, Ponder WF, Beacham E, Macaranas J.
493, 526–531. (doi:10.1038/nature11696) inferences: how many genes to resolve the 2007 Molecular phylogenetics of Caenogastropoda
31. Takeuchi T et al. 2012 Draft genome of the pearl Mollusca? Methods Ecol. Evol. 2, 34– 42. (doi:10. (Gastropoda: Mollusca). Mol. Phylogenet. Evol. 42,
oyster Pinctada fucata: a platform for understanding 1111/j.2041-210X.2010.00052.x) 717–737. (doi:10.1016/j.ympev.2006.10.009)
bivalve biology. DNA Res. 19, 117– 130. (doi:10. 46. Nosenko T et al. 2013 Deep metazoan phylogeny: 60. Zou S, Li Q, Kong L. 2011 Additional gene data and
1093/dnares/dss005) when different genes tell different stories. Mol. increased sampling give new insights into the
 Downloaded from rspb.royalsocietypublishing.org on September 30, 2014

phylogenetic relationships of Neogastropoda, within Australia. J. Moll. Stud. 77, 375–387. (doi:10.1093/ Science 180, 866–868. (doi:10.1126/science.180. 9
the caenogastropod phylogenetic framework. Mol. mollus/eyr028) 4088.866)

rspb.royalsocietypublishing.org
Phylogenet. Evol. 61, 425–435. (doi:10.1016/j. 65. Laghi GF. 2005 Upper Triassic chitons from the 71. Nützel A, Erwin DH, Mapes RH. 2000 Identity and
ympev.2011.07.014) Italian Dolomites. Societa Veneziana di Scienze phylogeny of the Late Paleozoic Subulitoidea
61. Wägele H, Willan RC. 2000 Phylogeny of the Naturali Lavori 30, 79 –84. (Gastropoda). J. Paleontol. 74, 575–598. (doi:10.
Nudibranchia. Zool. J. Linn. Soc. 130, 83 –181. 66. Nishiguchi MK, Mapes RH. 2008 Cephalopoda. In 1666/0022-3360(2000)074,0575:IAPOTL.2.0.CO;2)
(doi:10.1111/j.1096-3642.2000.tb02196.x) Phylogeny and evolution of the mollusca (eds 72. Frýda J, Nützel A, Wagner PJ. 2008 Paleozoic
62. Dinapoli A, Klussmann-Kolb A. 2010 The long way to WF Ponder, DR Lindberg), pp. 163–199. Berkeley, Gastropoda. In Phylogeny and evolution of the
diversity—phylogeny and evolution of the CA: University of California Press. mollusca (eds WF Ponder, DR Lindberg), pp. 239–
Heterobranchia (Mollusca: Gastropoda). Mol. Phylogenet. 67. Cope JCW. 1997 The early phylogeny of the class 270. Berkeley, CA: University of California Press.
Evol. 55, 60–76. (doi:10.1016/j.ympev.2009.09.019) Bivalvia. Palaeontology 40, 713 –746. 73. Rouse GW et al. 2013 Fixed, free, and fixed: the
63. Brenzinger B, Haszprunar G, Schrödl M. 2013 At the 68. Parkhaev PY. 2008 The Early Cambrian radiation of fickle phylogeny of extant Crinoidea

Proc. R. Soc. B 281: 20141739

limits of a successful body plan-3D microanatomy, Mollusca. In Phylogeny and evolution of the (Echinodermata) and their Permian –Triassic origin.
histology and evolution of Helminthope (Mollusca: mollusca (eds WF Ponder, DR Lindberg), pp. 33 – Mol. Phylogenet. Evol. 66, 161– 181. (doi:10.1016/j.
Heterobranchia: Rhodopemorpha), the most worm- 69. Berkeley, CA: University of California Press. ympev.2012.09.018)
like gastropod. Front. Zool. 10, 37. (doi:10.1186/ 69. Pojeta Jr J, Runnegar B. 1974 Fordilla troyensis and 74. Sharma PP, Zardus JD, Boyle EE, González VL, Jennings
1742-9994-10-37) the early history of pelecypod mollusks. Am. Sci. 62, RM, McIntyre E, Wheeler WC, Etter RJ, Giribet G. 2013
64. Brenzinger B, Wilson NG, Schrödl M. 2011 3D 706 –711. Into the deep: a phylogenetic approach to the bivalve
microanatomy of a gastropod ‘worm’, Rhodope 70. Pojeta Jr J, Runnegar B, Kriz J. 1973 Fordilla subclass Protobranchia. Mol. Phylogenet. Evol. 69,
rousei n. sp. (Heterobranchia) from southern troyensis Barrande: the oldest known pelecypod. 188–204. (doi:10.1016/j.ympev.2013.05.018)