phylogenomic analysis of echinoderm class...

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rspb.royalsocietypublishing.org 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] Electronic supplementary material is available at http://dx.doi.org/10.1098/rspb.2014.0479 or via http://rspb.royalsocietypublishing.org. Phylogenomic analysis of echinoderm class relationships supports Asterozoa Maximilian J. Telford 1 , Christopher J. Lowe 2 , Christopher B. Cameron 3 , Olga Ortega-Martinez 4 , Jochanan Aronowicz 5 , Paola Oliveri 1 and Richard R. Copley 6,7 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 De ´partment de Sciences Biologiques, Universite ´ de Montre ´al, Pavillion Marie-Victorin, C.P. 6128, Succ. Centre-ville, Montre ´al, Que ´bec, Canada H3C 3J7 4 Department of Biological and Environmental Sciences, Sven Love ´n Centre for Marine Sciences, University of Gothenburg, Kristineberg 566, Fiskeba ¨ckskil 451 78, Sweden 5 Department of Organismal Biology and Anatomy, University of Chicago, Chicago, IL 60650, USA 6 CNRS, Laboratoire de Biologie du De ´veloppement de Villefranche-sur-mer (LBDV), Observatoire Oce ´anographique, 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 Cryptosyr- ingida. 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) sub- optimal, 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 phylo- genetic 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 Echinoderms are composed of five extant classes, sea urchins (echinoids), star- fish (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. on May 7, 2018 http://rspb.royalsocietypublishing.org/ Downloaded from

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ResearchCite 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]

Electronic supplementary material is available

at http://dx.doi.org/10.1098/rspb.2014.0479 or

via http://rspb.royalsocietypublishing.org.

& 2014 The Author(s) Published by the Royal Society. All rights reserved.

Phylogenomic analysis of echinodermclass relationships supports Asterozoa

Maximilian J. Telford1, Christopher J. Lowe2, Christopher B. Cameron3,Olga Ortega-Martinez4, Jochanan Aronowicz5, Paola Oliveri1

and Richard R. Copley6,7

1Department of Genetics, Evolution and Environment, University College London, Darwin Building, Gower Street,London WC1E 6BT, UK2Hopkins Marine Station, Department of Biology, Stanford University, 120 Oceanview Boulevard, Pacific Grove,CA 93950, USA3Department de Sciences Biologiques, Universite de Montreal, Pavillion Marie-Victorin, C.P. 6128,Succ. Centre-ville, Montreal, Quebec, Canada H3C 3J74Department of Biological and Environmental Sciences, Sven Loven Centre for Marine Sciences,University of Gothenburg, Kristineberg 566, Fiskebackskil 451 78, Sweden5Department of Organismal Biology and Anatomy, University of Chicago, Chicago, IL 60650, USA6CNRS, Laboratoire de Biologie du Developpement de Villefranche-sur-mer (LBDV), ObservatoireOceanographique, Villefranche-sur-mer 06230, France7Sorbonne 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 Cryptosyr-

ingida. 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) sub-

optimal, 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 phylo-

genetic 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. IntroductionEchinoderms are composed of five extant classes, sea urchins (echinoids), star-

fish (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

Echinoidea

Holothuroidea

Ophiuroidea

Asteroidea

Crinoidea

Asterozoan Cryptosyringid

Figure 1. The two hypotheses of echinoderm evolution discussed in thiswork. The Asterozoan hypothesis unites ophiuroids and asteroids on thebasis of a five-rayed body plan. The cryptosyringid hypothesis places ophiur-oids as sister group to the holothurians and echinoids on the basis of, forexample, closed ambulacral grooves (see Introduction).

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(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 echino-

derm 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 hypo-

thesis, 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 pos-

sess 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 con-

trast 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 develop-

ment of the larval skeleton in the sea urchin Strongylocentrotuspurpuratus 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

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 top-

ology [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 con-

cluded 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 (cri-

noids, 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 support-

ing 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 aster-

oids, two holothurians, four echinoids and a crinoid as well

as data from three hemichordates and a cephalochordate as

outgroup taxa.

2. Material and methodsOur analysis combined sequences from novel data sources and

pre-existing protein and expressed sequence transcript databases,

as outlined below.

(a) Sources of dataAmphiura 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 construc-

ted 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 flow-

through aquaria at Friday Harbor Laboratories. Brooded juveniles

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at a range of stages of adult body plan formation were dissec-

ted 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 repro-

ductive 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 obtai-

ned from the GenBank Sequence Read Archive (accession

no. SRX043529).

All other echinoderm nucleotide sequences were taken from

the NCBI est_others database.

(b) Preprocessing of nucleotide sequencesHigh coverage and short read lengths mean that sequences pro-

duced 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. tribuloidesreads were assembled using the Newbler assembler, to give 53 028

and 21 969 contigs, respectively [21].

(c) Orthologue identification and alignmentconstruction

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 expres-

sed sequences from the NCBI est_others database, with the

addition of the Florometra, Amphipholis, Amphiura, Solaster and

Eucidaris processed datasets described above and Ptychoderaflava 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

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 echi-

noderms were retained (i.e. no echinoderm sequences were

allowed to branch within the hemichordates or between hemichor-

dates 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].

(d) Additional filters on final alignmentOnly 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 pres-

ence 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.

(e) Phylogenetic analysesPHYLOBAYES 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 replace-

ment 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 con-

vergence 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 phyloge-

nies on 50 bootstrap replicates (i.e. sampling with replacement to

create an alignment the same size as the original) and 100 jack-

knife 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 con-

sensus trees calculated using bpcomp, with 200 cycles discarded

as burn-in.

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( f ) Cross-validationWe performed statistical comparisons of the CAT þ GTR þ G, the

WAG þ G and GTRþ 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.

(g) Phylogenetic signal dissection: site rate rankingRecapitulating 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 echino-

derm 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 hetero-

geneously 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.

(h) Phylogenetic signal dissection: gene rate rankingUsing 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. 3.2We partitioned the dataset into individual genes and, by unlink-

ing 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 top-

ology and branch length but to be scaled independently and

also use individually best fitting empirical model as before.

3. Results(a) Data assemblyWe 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 align-

ment and were removed from subsequent analysis, namely

Patiria miniata (asteroid), Heliocidaris erythrogramma (echinoid),

Pseudocentrotus depressus (echinoid) and Strongylocentrotus

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).

(b) Echinoderm phylogenyAnalysis 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 þholothurians) are recovered as sister clades. Within the echi-

noids, 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 site-

heterogeneous 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 þGTR 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 top-

ology. As with the bootstrap samples, the next most common

result in the jack-knife set (25%) contained the unlikely group-

ing of holothurians and ophiuroids. Cryptosyringids sensustricto ((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 sup-

port 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,

Paracentrotus lividus

Strongylocentrotus purpuratus

Hemicentrotus pulcherrimus

Eucidaris tribuloides

Holothuria glaberrima

Apostichopus japonicus

Solaster stimpsoni

Patiria pectinifera

Amphipholis squamata

Amphiura filiformis

Florometra serratissima

Saccoglossus kowalevskii

Ptychodera flava

Balanoglossus clavigerus

0.1

Branchiostoma floridae

Figure 2. Full tree from complete dataset (see text for description).

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the recent work by Pisani et al. addressing the possibility of sys-

tematic error due to unequal rates of evolution and saturation

using an approach they call phylogenetic signal dissection

supports, instead, the cryptosyringid clade (EchinozoaþOphiuroidea). This result was supported in the Pisani et al.analysis only when using the portion of the alignment remain-

ing 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 align-

ment (constant and most variable sites removed), we still find

strong support for the Asterozoa–Echinozoa topology (elec-

tronic 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 align-

ment) 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 iden-

tical 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 stron-

gest support for Asterozoa (pp¼ 1, figure 3a). The second

and third quartiles still support this clade but with diminishing

support (second quartile pp¼ 0.83, figure 3b; third quartile

pp¼ 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 (pp¼ 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

Paracentrotus lividus(a) (b)

(c) (d )

Strongylocentrotus purpuratus

Strongylocentrotus purpuratus

Hemicentrotus pulcherrimus

Eucidaris tribuloides

Holothuria glaberrima

Apostichopus japonicus

Solaster stimpsoni

Hemicentrotus pulcherrimus

Eucidaris tribuloides

Holothuria glaberrima

Apostichopus japonicus

Solaster stimpsoni

Patiria pectinifera

Amphipholis squamata

Amphiura filiformis

Patiria pectinifera

Amphipholis squamata

Amphiura filiformis

Florometra serratissima

Florometra serratissima

Saccoglossus kowalevskii

Saccoglossus kowalevskii

Ptychodera flava

Ptychodera flava

Balanoglossus clavigerus

Branchiostoma floridae

0.1

0.99

0.71

Paracentrotus lividus

Strongylocentrotus purpuratus

Hemicentrotus pulcherrimus

Eucidaris tribuloides0.82

0.83

0.99

0.1

Holothuria glaberrima

Apostichopus japonicus

Solaster stimpsoni

Patiria pectinifera

Amphipholis squamata

Amphiura filiformis

Florometra serratissima

Saccoglossus kowalevskii

Ptychodera flava

Balanoglossus clavigerus

Branchiostoma floridae

Paracentrotus lividus

Strongylocentrotus purpuratus

Hemicentrotus pulcherrimus

Eucidaris tribuloides

Holothuria glaberrima

Apostichopus japonicus

Solaster stimpsoni

Patiria pectinifera

Amphipholis squamata

Amphiura filiformis

Florometra serratissima

Saccoglossus kowalevskii

Ptychodera flava

Balanoglossus clavigerus

Branchiostoma floridae

Balanoglossus clavigerus

Branchiostoma floridae

Paracentrotus lividus

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 evolvingquartile.

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and allowed genes to adopt firstly gene-specific empiri-

cal 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 phylogenyAll of our analyses unequivocally support a clade of Ophiuroi-

dea 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 rela-

tive 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 ortho-

logues 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 pro-

blematic if ophiuroids and echinoids shared a complex

molecular character state [38], such as a Hox cluster rearrange-

ment 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 sys-

tematic 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 skeletonThe most recent molecular study to address echinoderm

phylogeny [14] stated that their result (i.e. supporting crypto-

syringids) ‘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 with-

out making assumptions about the relative ease of gain or loss

events (electronic supplementary material, figure S3). Under

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the scenario of a single origin for the larval skeletons (the evi-

dence for which we review briefly below), the cryptosyringid

hypothesis is indeed more parsimonious than the asterozoan,

requiring a single loss of the skeleton in holothurians (and con-

comitant reversal to a auricularia/bipinnaria type of larva),

with the asterozoan hypothesis requiring an additional loss/

reversal in asteroids (electronic supplementary material,

figures S3a and S3c). If a dual origin of the larval skeleton is

favoured, the two phylogenetic scenarios are indistinguishable

on parsimony grounds (so far as it relates to the skeleton)

(electronic supplementary material, figures S3b and S3d).

Although the larval skeleton seems intuitively to be a

complex trait, in the sense that it might be regarded as unli-

kely to have evolved twice, all echinoderm classes share an

adult skeleton, and the evolution of the larval skeleton may

be the result of a relatively simple redeployment of this,

making the possibility of convergence more likely. Gao &

Davidson [39], comparing larval and adult skeletogenesis,

have identified the genes specifically required for embryonic

activation of the skeletogenesis network in the echinoid

S. purpuratus, showing that this is only a small component

of the overall skeletogenic GRN. It is worth noting that the

key gene in the embryonic skeleotogenic GRN, Pmar1,

involved in the establishment of the double negative gate

that de-represses skeletogenesis [5] currently appears to be

an echinoid autapomorphy. We could not find evidence of

Pmar1 orthologue presence in any taxa in our echinoderm

EST database (or non-echinoderm databases) except for

S. purpuratus. Although Vaughn et al. [40] mention an unpub-

lished ophiuroid Pmar1 orthologue, their inability to find

Pmar1 transcripts expressed in any stage of brittlestar devel-

opment supported their conclusion that ‘embryonic skeleton

formation in sea urchins and brittle stars represents conver-

gent evolution by independent co-optation of a shared

pathway used in adult skeleton formation’ [40, p. 14].

The suggestion that the echinoid larval skeleton may be a

derived state is further supported by McCauley et al.’s [41]

analysis of holothurian embryogenesis. Based on an analysis

of the embryonic expression patterns of mesodermal regulatory

genes in Parastichopus parvimensis, they suggest ‘that the ances-

tor of the sea cucumber þ sea urchin clade may have had a

mesoderm with a regulatory state similar to that of extant sea

cucumbers and may have developed only a rudimentary

larval skeleton’ [41, p. 5], and argue that, as such, holothurians

are unlikely to have lost a complex larval skeleton. If we accept

the sister group relationship of echinoderms and holothurians,

this in turn suggests that the larval skeleton was not a

plesiomorphy of these two taxa and thus unlikely to be a plesio-

morphy of any possible clade including the ophiuroids. Other

authors have argued on morphological grounds that the two

kinds of plutei are convergent forms [42,43], but as the defining

feature of the pluteus is its skeleton, most desirable would be a

comparison of the fine details of the developmental genetics

responsible for initiating production of the larval skeletons,

which we would expect to more exactly overlap in homologous

plutei. We emphasize that such evidence would be useful for

ruling out the scenario of a single origin for the pluteus

within cryptosyringids (electronic supplementary material,

figure S3c), not for distinguishing between the dual origin scen-

arios—however, our molecular evidence clearly favours the

Asterozoa (i.e. electronic supplementary material, figure S3b).

(c) Adult morphologyThe cryptosyringids owe their name to the fact that their

radial water vascular system and radial nerve become cov-

ered 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 phyloge-

netic 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 echi-

noids. Under the Asterozoa hypothesis, we would predict

differing molecular mechanisms, suggesting parallel evol-

ution 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 þholothurian 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 Exobiol-ogy 1-EXO11-0135. C.B.C. acknowledges funding from the NSERCdiscovery grants programme. M.J.T. is supported by a Royal SocietyWolfson Research Merit Award.

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