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Phylogenomic analysis of echinoderm
class relationships supports Asterozoa
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Research
Cite this article: Telford MJ, Lowe CJ,
Cameron CB, Ortega-Martinez O, Aronowicz J,
Oliveri P, Copley RR. 2014 Phylogenomic
analysis of echinoderm class relationships
supports Asterozoa. Proc. R. Soc. B 281:
20140479.
http://dx.doi.org/10.1098/rspb.2014.0479
Received: 27 February 2014
Accepted: 17 April 2014
Subject Areas:
bioinformatics, evolution, taxonomy
and systematics
Keywords:
echinoderm, phylogeny, phylogenomic,
Asterozoa, Cryptosyringida
Authors for correspondence:
Maximilian J. Telford
e-mail: [email protected]
Richard R. Copley
e-mail: [email protected]
Maximilian J. Telford1, Christopher J. Lowe2, Christopher B. Cameron3,
Olga Ortega-Martinez4, Jochanan Aronowicz5, Paola Oliveri1
and Richard R. Copley6,7
1
Department of Genetics, Evolution and Environment, University College London, Darwin Building, Gower Street,
London WC1E 6BT, UK
2
Hopkins Marine Station, Department of Biology, Stanford University, 120 Oceanview Boulevard, Pacific Grove,
CA 93950, USA
3
Départment de Sciences Biologiques, Université de Montréal, Pavillion Marie-Victorin, C.P. 6128,
Succ. Centre-ville, Montréal, Québec, Canada H3C 3J7
4
Department of Biological and Environmental Sciences, Sven Lovén Centre for Marine Sciences,
University of Gothenburg, Kristineberg 566, Fiskebäckskil 451 78, Sweden
5
Department of Organismal Biology and Anatomy, University of Chicago, Chicago, IL 60650, USA
6
CNRS, Laboratoire de Biologie du Développement de Villefranche-sur-mer (LBDV), Observatoire
Océanographique, Villefranche-sur-mer 06230, France
7
Sorbonne Universites, UPMC Univ Paris 06, Laboratoire de Biologie du Developpement de Villefranche-sur-mer,
Observatoire Oceanographique, Villefranche-sur-mer 06230, France
While some aspects of the phylogeny of the five living echinoderm classes are
clear, the position of the ophiuroids (brittlestars) relative to asteroids (starfish),
echinoids (sea urchins) and holothurians (sea cucumbers) is controversial.
Ophiuroids have a pluteus-type larva in common with echinoids giving
some support to an ophiuroid/echinoid/holothurian clade named Cryptosyringida. Most molecular phylogenetic studies, however, support an ophiuroid/
asteroid clade (Asterozoa) implying either convergent evolution of the pluteus
or reversals to an auricularia-type larva in asteroids and holothurians. A recent
study of 10 genes from four of the five echinoderm classes used ‘phylogenetic
signal dissection’ to separate alignment positions into subsets of (i) suboptimal, heterogeneously evolving sites (invariant plus rapidly changing)
and (ii) the remaining optimal, homogeneously evolving sites. Along with
most previous molecular phylogenetic studies, their set of heterogeneous
sites, expected to be more prone to systematic error, support Asterozoa. The
homogeneous sites, in contrast, support an ophiuroid/echinoid grouping,
consistent with the cryptosyringid clade, leading them to posit homology of
the ophiopluteus and echinopluteus. Our new dataset comprises 219 genes
from all echinoderm classes; analyses using probabilistic Bayesian phylogenetic methods strongly support Asterozoa. The most reliable, slowly
evolving quartile of genes also gives highest support for Asterozoa; this support
diminishes in second and third quartiles and the fastest changing quartile places
the ophiuroids close to the root. Using phylogenetic signal dissection, we find
heterogenous sites support an unlikely grouping of Ophiuroidea þ Holothuria
while homogeneous sites again strongly support Asterozoa. Our large and
taxonomically complete dataset finds no support for the cryptosyringid
hypothesis; in showing strong support for the Asterozoa, our preferred
topology leaves the question of homology of pluteus larvae open.
1. Introduction
Electronic supplementary material is available
at http://dx.doi.org/10.1098/rspb.2014.0479 or
via http://rspb.royalsocietypublishing.org.
Echinoderms are composed of five extant classes, sea urchins (echinoids), starfish (asteroids), sea cucumbers (holothurians), brittlestars (ophiuroids) and sea
lilies (crinoids). Although the modern classes appear in a relatively short time
interval early in the fossil record (525–480 Ma) [1], extant crown group species
in each class have more recent origins: present-day crinoids and echinoids
radiated in large part after the Permian/Triassic extinction event for instance
& 2014 The Author(s) Published by the Royal Society. All rights reserved.
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Asterozoan
Cryptosyringid
Echinoidea
Ophiuroidea
Crinoidea
Figure 1. The two hypotheses of echinoderm evolution discussed in this
work. The Asterozoan hypothesis unites ophiuroids and asteroids on the
basis of a five-rayed body plan. The cryptosyringid hypothesis places ophiuroids as sister group to the holothurians and echinoids on the basis of, for
example, closed ambulacral grooves (see Introduction).
(approx. 250 Ma) [2,3], with asteroids also likely to have
undergone an evolutionary bottleneck at this time [4]. The
resulting long stem lineages leading to the living forms
mean that the monophyly of each class is not in doubt. The
rapid and ancient appearance of the classes, however,
means that the resolution of the relationships between them
is challenging.
Dealing first with the uncontroversial aspects of echinoderm class-level phylogeny, consideration of morphology and
previous molecular analyses strongly support a monophyletic
group of echinoids, asteroids, holothurians and ophiuroids
(Eleutherozoa). Within this eleutherozoan clade, most authors
also accept a close relationship between the globular echinoids
and holothurians (Echinozoa). The major point of remaining
dispute revolves around the position of the ophiuroids, with
two plausible solutions. According to the Asterozoa hypothesis, ophiuroids are the sister group of the asteroids,
whereas the Cryptosyringida hypothesis links the ophiuroids
instead to the Echinozoa (figure 1).
The relationship of the echinoids and ophiuroids is of
particular evolutionary interest, as members of both classes possess a pluteus-type larva (electronic supplementary material,
figure S1). These are characterized by a mesodermally derived
calcite skeleton, which supports the elongated, ciliated arms
that are used for feeding and swimming. Pluteus larvae contrast with non-skeleton-forming, generic dipleurula type that
characterizes the early development of crinoids, asteroids and
holothurians. The dipleurula has been proposed to represent
the larval form ancestral to all ambulacrarians, as it is also
shared with hemichordates, the sister group to echinoderms.
The gene regulatory network (GRN) involved in the development of the larval skeleton in the sea urchin Strongylocentrotus
purpuratus is one of the best understood developmental
networks in biology [5], but has yet to be characterized
in ophiuroids.
Among morphologists, Hyman was ‘of the opinion that the
closer relationship of ophiuroids to echinoids rather than to
2. Material and methods
Our analysis combined sequences from novel data sources and
pre-existing protein and expressed sequence transcript databases,
as outlined below.
(a) Sources of data
Amphiura filiformis were collected by Peterson mud grabs at a
depth of approximately 40 m close to the Sven Loven Centre
for Marine Sciences, Kristineberg, on the Gulmar fjord, Sweden
(588150 N, 118250 E). A normalized cDNA library was constructed from mRNA of arm tissues from A. filiformis (regenerated
and intact tissues) and sequenced using Roche 454 GS-FLX
Titanium technology.
Adult Amphipholis squamata were collected in Argylle Creek
on San Juan Island, Washington, and maintained in flowthrough aquaria at Friday Harbor Laboratories. Brooded juveniles
Proc. R. Soc. B 281: 20140479
Asteroidea
2
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Holothuroidea
asteroids, as usually supposed, is not to be doubted’ on the
basis of the shared pluteus larva, ambulacral canals enclosed
by epineural folds and biochemical criteria [6, pp. 699–700].
Littlewood et al. [7] also found support for this scenario with
their most parsimonious tree based on combined larval and
adult morphology, although they note that the alternative
asterozoan tree was only two steps longer.
The first molecular analyses using 18S rRNA genes found
monophyletic echinoderms, but a questionable internal topology [8]; subsequent reanalysis of these data using more
modern phylogenetic methods supports cryptosyringids [9].
Using 28S and 18S rRNA genes, Mallatt & Winchell [10]
found strong support for Asterozoa. An analysis of 13
protein-coding sequences encoded in mitochondrial genomes
[11] identified three lineages within the echinoderms—
crinoids, ophiuroids and a clade consisting of (asteroids,
(echinoids, holothurians)). Inspection of this tree shows that
the ophiuroids are particularly rapidly evolving, raising the
likelihood that their position as sister to other eleutherozoan
classes is the result of a long-branch attraction (LBA) artefact
[12]. Janies and co-workers analysed combined molecular
(seven loci) and morphological datasets from a large set of
echinoderms. They produced trees supporting asterozoans
and cryptosyringids (as well as other scenarios) and concluded that the overall phylogeny was sensitive to the
choice of analytical methods [13].
Most recently, Pisani and co-workers presented an analysis
of seven nuclear protein-coding genes and three nuclear
ribosomal RNAs from four of the five echinoderm classes (crinoids, ophiuroids, asteroids and echinoids). Analysis of their
full dataset supported the Asterozoa hypothesis (ophiuroids,
asteroids) [14]. By careful removal of subsets of data expected
to be problematic, they produced, in contrast, a tree supporting the cryptosyringid (ophiuroid, echinoid) clade. Their
analysis did not include any holothurian sequences. There is
the possibility that this omission might have resulted in an
unnecessarily long echinoid branch rendering the dataset
more susceptible to branch positioning artefacts.
We have re-examined the question of echinoderm class
relationships with a large dataset (219 nuclear protein-coding
genes) including representatives from all five extant classes of
echinoderms including data from two ophiuroids, two asteroids, two holothurians, four echinoids and a crinoid as well
as data from three hemichordates and a cephalochordate as
outgroup taxa.
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High coverage and short read lengths mean that sequences produced using so-called next generation (i.e. Illumina, 454)
technologies benefit from being preassembled using dedicated
software.
We assembled a dataset of 46 312 223 2 101 bp paired-end
sequences from the crinoid F. serratissima using the Inchworm
program from the Trinity package [19]. This resulted in a set of
1 681 253 sequences.
Amphiura filiformis reads were assembled in MIRA v. 3.2.0rc3
[20], giving 35 742 sequences. Amphipholis squamata and E. tribuloides
reads were assembled using the Newbler assembler, to give 53 028
and 21 969 contigs, respectively [21].
(d) Additional filters on final alignment
Only proteins for which at least four out of five echinoderm
classes were represented were included in the concatenated
alignment (echinoids were invariably present owing to the presence of a S. purpuratus protein in the seed orthologue trios). We
finally processed the alignment by visual inspection to remove
unreliably aligned sections. The final concatenated alignment
contained 219 genes.
(c) Orthologue identification and alignment
construction
(e) Phylogenetic analyses
We used a set of 12 625 Saccoglossus kowalevskii (Skow) sequences
retrieved from the NCBI protein sequence database to search the
predicted proteomes of the chordate Branchiostoma floridae (Bflo),
retrieved from the JGI and the sea urchin S. purpuratus
(Spur) retrieved from the NCBI. We identified trios of sequences
that formed consistent reciprocal best matches in each other’s
proteomes and were therefore likely orthologues. Best matches
were identified using blastp [22].
For all Saccoglossus sequences that were members of an
orthologous trio (Skow, Bflo and Spur), we used tblastn to
search a database of all echinoderm and hemichordate expressed sequences from the NCBI est_others database, with the
addition of the Florometra, Amphipholis, Amphiura, Solaster and
Eucidaris processed datasets described above and Ptychodera
flava sequences taken directly from the NCBI trace archive
without preprocessing.
Significant hits to each Saccoglossus protein were extracted from
the echinoderm nucleotide sequence databases and placed into
species-specific groups. The nucleotide sequences in each species
group were then assembled in isolation using CAP3 [23].
Assembled sequences thus produced were searched against
PHYLOBAYES analyses were performed with the computationally
intensive site-heterogeneous CAT þ GTR þ G mixture model
taking approximately one month per analysis. This model
accounts for across-site heterogeneities in the amino acid replacement process and is implemented in an MCMC framework by
the program PHYLOBAYES v. 3.3d [29]. Two independent runs
were performed with a total length of 6000 cycles, saved every
10 cycles. To construct the tree, the first 2000 points were discarded
as burn-in, and the topology and posterior consensus support was
computed on the 4000 remaining trees. We also checked for convergence of the two independent runs following burn-in of 2000
using bpcomp, which in each case showed a ‘maxdiff’ (maximum
discrepancy) below 0.1.
In order to assess the reliability of trees, we calculated phylogenies on 50 bootstrap replicates (i.e. sampling with replacement to
create an alignment the same size as the original) and 100 jackknife replicates (i.e. sampling without replacement, alignments
25% original length), in both cases using PHYLOBAYES with the
CAT þ GTR þ G model. Chains were run for 1000 cycles, and consensus trees calculated using bpcomp, with 200 cycles discarded
as burn-in.
3
Proc. R. Soc. B 281: 20140479
(b) Preprocessing of nucleotide sequences
metazoan protein sequences from the NCBI NR protein sequence
database (blastp) and translated against the best match using the
estwise program from the genewise package [24].
Each of the resulting assembled protein sequences was
then searched against the combined proteomes of Saccoglossus,
Branchiostoma and Strongylocentrotus using blastp, and only
those for which the best hits were consistent with the members
of the initial Skow, Bflo and Spur orthologous trio were retained.
Sets of proteins for each Skow, Bflo and Spur trio were
then aligned with all predicted orthologous echinoderm and
hemichordate proteins using PROBCONS [25]. A phylogeny was
constructed for each protein alignment using PHYML [26], using
the WAG model [27], estimating the proportion of invariable
sites, estimating the gamma distribution parameter allowing four
gamma rate categories (WAG þ IG4) and optimizing the tree
topology and branch lengths. Each phylogeny was then rooted
with the Branchiostoma sequence (the chordate outgroup). Only
those alignments that then yielded a monophyletic cluster of echinoderms were retained (i.e. no echinoderm sequences were
allowed to branch within the hemichordates or between hemichordates and the root). This test will have the effect of excluding gene
sets that include paralogous echinoderm sequences. Paralogous
sequences would cluster outside a set of orthologous genes in a
correct phylogeny. Rooting with the Branchiostoma orthologue
will cause any paralogous echinoderm sequences to branch
between this root and hemichordate (the presence of which is
inevitable given the composition of our seed orthologue trios).
Branching order of echinoderm classes within the echinoderm
monophyletic group was not constrained during this process.
Tree re-rooting and tests for monophyletic groups were performed
using custom perl and python scripts. Final alignments were also
screened for RNA contamination against all RNA sequences from
the Rfam database [28], using tblastn [22].
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at a range of stages of adult body plan formation were dissected out of the bursal sacs of the adult animals and flash frozen in
liquid nitrogen. Following total RNA extraction, cDNA library
construction was carried out by Express Genomics, Maryland,
and sequencing of excised inserts was carried out using Roche
454 GS-FLX technology.
Solaster stimpsoni adults were collected by SCUBA at Eagle
Cove San Juan Island, Washington, in April during their reproductive season. Adults were spawned by injection of 1-methyl
adenine as described [15]. The positively buoyant embryos
were cultured in running seawater tables as described [16] with
Sanger EST sequencing as described in [17,18].
Adult Florometra serratissima were collected by SCUBA at
Bamfield Marine Station Sciences Centre, British Columbia,
Canada. Mature oocytes were dissected from the adult female
pinnules using forceps and fertilized with sperm obtained by
rupturing testes. Embryos were cultured in glass bowls. Gastrula,
early doliolaria and late doliolaria developmental stages were
frozen in liquid nitrogen and total RNA prepared using the
Ambion RNA aqueous mini kit. RNA samples were pooled
and prepared for sequencing using the Illumina TruSeq stranded
mRNA sample prep with oligo-dT selection then sequenced
using HiSeq 101 bp paired-end reads.
Eucidaris tribuloides transcriptome sequences were obtained from the GenBank Sequence Read Archive (accession
no. SRX043529).
All other echinoderm nucleotide sequences were taken from
the NCBI est_others database.
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(f ) Cross-validation
(g) Phylogenetic signal dissection: site rate ranking
(h) Phylogenetic signal dissection: gene rate ranking
Using an alignment for each gene, a maximum-likelihood tree
was calculated using PHYML [26] and the total length of that
tree (in estimated substitutions per position across all branches)
was divided by the number of taxa on the tree to give an estimate
of the rate of evolution for each gene. Genes were concatenated in
order of their evolutionary rates and the resulting dataset
divided into four approximately equal-sized quartiles containing
genes ranging from the slowest to the fastest. Each of these four
datasets was analysed separately and the results compared.
(i) Partition by gene using MRBAYES v. 3.2
We partitioned the dataset into individual genes and, by unlinking the partitions, we allowed each gene to find its own best
fitting empirical model from among the 10 possible in MRBAYES
( poisson, jones, dayhoff, mtrev, mtmam, wag, rtrev, cprev, vt
or blosum). We ran two runs of four chains each for a total of
500 000 generations well beyond convergence of the two runs.
We additionally unlinked the rate multiplier associated with
each gene partition allowing the genes to share the same topology and branch length but to be scaled independently and
also use individually best fitting empirical model as before.
3. Results
(a) Data assembly
We gathered a set of 3872 Saccoglossus, Branchiostoma and
Strongylocentrotus orthologous trios and used these to search
our echinoderm dataset, producing 3537 alignments including
additional echinoderm data. Of these, 2630 passed tests for
echinoderm monophyly—that is, the echinoderm sequences
formed a subtree that included all echinoderm sequences and
none of the outgroup species.
Several species that were present in the NCBI est_others
sequence database, and thus captured by our automated
procedures, had particularly low representation in the final alignment and were removed from subsequent analysis, namely
Patiria miniata (asteroid), Heliocidaris erythrogramma (echinoid),
Pseudocentrotus depressus (echinoid) and Strongylocentrotus
(b) Echinoderm phylogeny
Analysis using PHYLOBAYES [29] on the full dataset of 45 818
reliably aligned amino acids from 15 taxa produced the tree
shown in figure 2. As widely recognized, the crinoids diverge
first and form a sistergroup to the four other classes—the
Eleutherozoa. Within the Eleutherozoa, the Asterozoa clade
(asteroids þ ophiuroids) and the Echinozoa (echinoids þ
holothurians) are recovered as sister clades. Within the echinoids, the cidaroid E. tribuloides is the deepest branching and
within the hemichordates, the ptychoderids clustered together,
as expected. All clades have a posterior probability of 1. This
topology is supported by analyses using both the widely
employed site-homogeneous WAG þ G and GTR þ G models
(gamma with five rate categories) and the more complex siteheterogeneous CAT þ GTR model (with the default Dirichlet
process for estimating rates across sites [32]). We verified by
cross-validation that the CAT þ GTR þ G model had a better
fit to our dataset than the GTR þ G (DlnL 228.36 + 23.5159)
and that the GTR þ G had a better fit than WAG þ G (DlnL
290.48 + 17.613).
We performed 50 bootstrap replicates using the CAT þ
GTR þ G model: 90% of replicates supported Asterozoa. Of
the remainder, 8% placed ophiuroids as sister to holothurians,
with only 2% supporting Cryptosyringida. We also performed
100 jack-knife replicates using just 25% of the full dataset: the
results with such a reduced dataset were naturally less
robust; nevertheless 57% of trees support the asterozoan topology. As with the bootstrap samples, the next most common
result in the jack-knife set (25%) contained the unlikely grouping of holothurians and ophiuroids. Cryptosyringids sensu
stricto ((H,E),O) were observed in only 8% of trees, with a
further 5% containing monophyletic cryptosyringids although
with ophiuroids and holothurians as sister groups ((H,O),E). In
summary, both bootstrap and jack-knife replication provide
support for asterozoans. The cryptosyringids were the third
best supported topology with both approaches, and less well
supported than a grouping of holothurians and ophiuroids,
which, to the best of our knowledge, has no serious support from other sources and can likely be discounted as a
long-branch artefact.
The Asterozoa–Echinozoa topology has been supported by
the majority of molecular analyses of this problem. By contrast,
4
Proc. R. Soc. B 281: 20140479
Recapitulating the approach of Pisani et al. [14], the number of
changes at each site was calculated over a maximum-parsimony
tree constructed with unresolved relationships between echinoderm classes. The number of changes for each character over the
tree was normalized to correct for positions with missing data,
by dividing by the number of changes by the number of taxa
with a character at that position. The positions in the alignment
were then divided into one sub-alignment containing heterogeneously evolving sites (comprised all invariant positions plus
the most frequently substituting quarter of the variable sites)
and a second sub-alignment containing the more homogeneously
evolving positions comprised the remaining 75% of the more
slowly evolving variable sites. These two datasets were analysed
separately and the results compared.
intermedius (echinoid). We also excluded the hemichordate
Rhabdopleura compacta, again owing to low coverage. Although
incidental to the question of echinoderm phylogeny, this meant
we were unable to address the issue of enteropneust monophyly
(but see [31] for a convincing conclusion based on miRNAs).
In total, 219 genes were found to have representatives
from at least four echinoderm classes. Fifteen taxa were
included in the final alignment of 80 666 columns. No hits
to the Rfam database were found in the sequences included
in these alignments. After filtering for confidently aligned
regions, 45 818 positions remained. The full dataset is
described in the electronic supplementary material, table S1.
Of the individual gene trees for genes represented in the
final alignment, 31 contained monophyletic Cryptosyringida
and 49 contained monophyletic Asterozoa. Requiring both
crinoid and holothurian sequences to be present (which
a priori we would expect to produce more reliable results)
enriched Asterozoa (10 trees) relative to Cryptosyringida
(three trees).
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We performed statistical comparisons of the CAT þ GTR þ G, the
WAG þ G and GTR þ G models using cross-validation tests as
described in [30]. Ten replicates were run: 9/10 of the positions
randomly drawn from the alignment were used as the learning
set and the remaining 1/10 as the test set. For each model,
MCMC were run for 1100 cycles, 100 being discarded as burn-in.
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Paracentrotus lividus
5
Strongylocentrotus purpuratus
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Hemicentrotus pulcherrimus
Eucidaris tribuloides
Holothuria glaberrima
Apostichopus japonicus
Patiria pectinifera
Amphipholis squamata
Amphiura filiformis
Florometra serratissima
Saccoglossus kowalevskii
Ptychodera flava
Balanoglossus clavigerus
Branchiostoma floridae
0.1
Figure 2. Full tree from complete dataset (see text for description).
the recent work by Pisani et al. addressing the possibility of systematic error due to unequal rates of evolution and saturation
using an approach they call phylogenetic signal dissection
supports, instead, the cryptosyringid clade (Echinozoa þ
Ophiuroidea). This result was supported in the Pisani et al.
analysis only when using the portion of the alignment remaining after the constant and the most variable sites had been
removed. We set out to replicate this approach with our larger
and more taxonomically complete dataset. We used two
approaches to test the effects of removing faster evolving and
less reliable data: first, the site-based method as described,
which we term site rate ranking (SRR); second, we classified
each gene in our alignment into one of four quartiles according
to its rate of evolution and analysed each of these quartiles in
turn—we call this gene rate ranking (GRR). The GRR method
has the advantage that it does not depend on pre-specifying
any topology, whereas the SRR approach has the advantage
that it treats each site independently.
Using either of these approaches with our dataset, we find no
support for the cryptosyringid clade. Electronic supplementary
material, figure S2, shows the results using the site-based SRR
method. With the supposedly optimal portion of the alignment (constant and most variable sites removed), we still find
strong support for the Asterozoa–Echinozoa topology (electronic supplementary material, figure S2a). The support is
slightly lower than when using the whole alignment (support
for Asterozoa pp ¼ 0.94), presumably because the alignment is
shorter (16 678 sites compared to 46 913 for the complete alignment) and hence less informative. Using the suboptimal sites
(constant and variable), we recover an improbable topology
with Holothuroidea and Ophiuroidea as sister taxa (both are
long branches) and the relative positions of echinoids, asteroids
and the holothurian/ophiuroid group are unresolved (electronic
supplementary material, figure S2b).
Earlier support for cryptosyringids came from a dataset
that did not include holothurians [14] and so we repeated
the SRR method with this class excluded. We recovered identical relative positions for each of the remaining classes and so
this difference in taxonomic representation does not explain
the difference between our results and those of Pisani et al.
[14] (results not shown).
Figure 3 shows the results from the gene-based GRR
approach. The slowest quartile (most reliable) shows the strongest support for Asterozoa (pp ¼ 1, figure 3a). The second
and third quartiles still support this clade but with diminishing
support (second quartile pp ¼ 0.83, figure 3b; third quartile
pp ¼ 0.71, figure 3c) The fourth quartile (fastest evolving
genes and likely least reliable) places the ophiuroids as sister
group to the Echinozoa þ Asteroidea, presumably due to
LBA between the relatively long-branched ophiuroids and the
outgroups (figure 3d). All other aspects of the tree (including
the Echinozoa clade) do not change as the datasets get faster
and all other clades receive the maximum support (pp ¼ 1) in
analyses of all four quartets. The difference in rate is very
evident when comparing these trees (shortest (figure 3a) to
longest (figure 3d)), which are all drawn to the same scale.
We conducted two experiments using analyses performed
with MRBAYES in which we partitioned the dataset by genes
Proc. R. Soc. B 281: 20140479
Solaster stimpsoni
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(a)
(b)
Paracentrotus lividus
6
Paracentrotus lividus
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Strongylocentrotus purpuratus
Strongylocentrotus purpuratus
Hemicentrotus pulcherrimus
Hemicentrotus pulcherrimus
Eucidaris tribuloides
0.82
Holothuria glaberrima
Eucidaris tribuloides
Holothuria glaberrima
Apostichopus japonicus
Apostichopus japonicus
Solaster stimpsoni
Solaster stimpsoni
Patiria pectinifera
Patiria pectinifera
0.83
Amphipholis squamata
Amphipholis squamata
Amphiura filiformis
Amphiura filiformis
Florometra serratissima
Saccoglossus kowalevskii
Ptychodera flava
Ptychodera flava
Balanoglossus clavigerus
Balanoglossus clavigerus
Paracentrotus lividus
Branchiostoma floridae
Paracentrotus lividus
Branchiostoma floridae
Strongylocentrotus purpuratus
0.1
Strongylocentrotus purpuratus
0.1
Hemicentrotus pulcherrimus
(c)
0.99
Eucidaris tribuloides
Hemicentrotus pulcherrimus
Eucidaris tribuloides
(d)
Holothuria glaberrima
Apostichopus japonicus
Holothuria glaberrima
0.99
Apostichopus japonicus
Solaster stimpsoni
Solaster stimpsoni
Patiria pectinifera
Patiria pectinifera
0.71
Amphipholis squamata
Amphipholis squamata
Amphiura filiformis
Amphiura filiformis
Florometra serratissima
Florometra serratissima
Saccoglossus kowalevskii
Saccoglossus kowalevskii
Ptychodera flava
Ptychodera flava
Balanoglossus clavigerus
Branchiostoma floridae
Balanoglossus clavigerus
Branchiostoma floridae
Figure 3. Results from our data stratified by overall length of gene trees (gene rate ranking), from (a), the slowest evolving quartile, to (d ) the fastest evolving
quartile.
and allowed genes to adopt firstly gene-specific empirical models of amino acid substitution and secondly both
gene-specific empirical models and a gene-specific rate. In
both experiments, the topology was the same as in figure 2
with posterior probability of 1.0 for all nodes.
4. Discussion
(a) Echinoderm phylogeny
All of our analyses unequivocally support a clade of Ophiuroidea plus Asteroidea: Asterozoa. Despite using two methods to
stratify our data by evolutionary rate, we found no support for
the alternative Cryptosyringida hypothesis of a sister group
relationship of Ophiuroidea and Echinoidea þ Holothuroidea.
Our dataset is considerably larger than others that have been
brought to bear on this question, includes at least two members
of each of the relevant classes and deep sampling of the closest
outgroup (the crinoid). A priori, we might expect such a dataset
to produce a more reliable result, and we have not been able to
identify confounding factors. Our trees do reveal very short
branches leading to the asterozoan and echinozoan clades,
however. These divergences appear to have taken place in a
compressed time interval, a fact perhaps underscored by the
absence of any miRNAs that may have helped resolve the relative positioning of these taxa [14]. Further molecular testing of
our topology might come from even deeper taxon sampling or
new types of data. The order of the echinoid Hox genes, for
instance, at least as observed in S. purpuratus, is derived relative
to that seen in hemichordates, and apparently coupled with
this, the HOX4 orthologue has been lost [33,34]. HOX4 orthologues have, however, been identified in crinoids [35] and
asteroids [36]. Their status in ophiuroids is uncertain (the
Stegophiura sladeni sequence referred to as SS-11 in the previous
study [37] is not convincing), but our phylogeny would be problematic if ophiuroids and echinoids shared a complex
molecular character state [38], such as a Hox cluster rearrangement and gene loss, to the exclusion of asteroids. We are not
aware of any other complex molecular character states that
might be informative for this phylogenetic question: the systematic exploration of their existence, presence and absence
must await complete genome sequences of representatives of
other echinoderm classes.
(b) Asterozoa and the origins of the larval skeleton
The most recent molecular study to address echinoderm
phylogeny [14] stated that their result (i.e. supporting cryptosyringids) ‘confirms that that the morphologically similar
pluteus larval stages of echinoids and ophiuroids are indeed
homologous rather than convergent’. It is not clear, however,
that the larval skeleton can be used as a phylogenetic character
to distinguish between the alternative phylogenetic trees without making assumptions about the relative ease of gain or loss
events (electronic supplementary material, figure S3). Under
Proc. R. Soc. B 281: 20140479
Florometra serratissima
Saccoglossus kowalevskii
Downloaded from http://rspb.royalsocietypublishing.org/ on June 15, 2017
(c) Adult morphology
The cryptosyringids owe their name to the fact that their
radial water vascular system and radial nerve become covered during development, a trait that was viewed as a
synapomorphy of the group [44]. In later work, however,
Smith and co-workers [7] pointed out that the developmental
mechanism by which this occurs differs within the echinoids,
suggesting that its presence in some instances may be due to
convergence, and as such, it would not be a reliable phylogenetic marker. To the best of our knowledge, there has been no
work on the molecular mechanisms leading to covered radial
nerves and water vascular system in ophiuroids and echinoids. Under the Asterozoa hypothesis, we would predict
differing molecular mechanisms, suggesting parallel evolution of the trait in echinoids and ophiuroids, unless the
trait were secondarily absent from asteroids, in which case
the fossil record may be the best source of evidence.
If our inference of monophyletic Asterozoa is correct, it
means that the hypothesis of a star-like common ancestor of
Eleutherozoa can neither be rejected nor supported. This in
turn has implications for the interpretation of the fossil
record: in particular, based on our analysis, there is no
reason to rule out a non-stellate ancestor for the echinoid þ
holothurian stem group.
Acknowledgements. We thank Sam Dupont (University of Gothenburg,
Kristineberg) and David Dylus (UCL) for helpful comments.
Funding statement. C.J.L. acknowledges funding from: NASA Exobiology 1-EXO11-0135. C.B.C. acknowledges funding from the NSERC
discovery grants programme. M.J.T. is supported by a Royal Society
Wolfson Research Merit Award.
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