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Home , Balanoglossus, Deuterostome, Hemichordate

EVOLUTION & DEVELOPMENT 1:3, 166Ð171 (1999)

Hemichordates and evolution: robust molecular phylogenetic support for a ؉ clade

Lindell D. Bromham* and Bernard M. Degnan Department of and Entomology, University of Queensland, Brisbane 4072, Australia *Author for correspondence (email: [email protected] or [email protected])

SUMMARY were traditionally allied to the the parametric bootstrap, to reanalyze DNA data from com- , but recent molecular analyses have suggested plete mitochondrial genomes and nuclear 18S rRNA. This that hemichordates are a sister group to the , a approach provides the first statistically significant support for relationship that has important consequences for the inter- the hemichordate ϩ echinoderm clade from molecular data. pretation of the evolution of deuterostome body plans. How- This grouping implies that the ancestral deuterostome had ever, the molecular phylogenetic analyses to date have not features that included an adult with a and a dorsal provided robust support for the hemichordate ϩ echinoderm nerve cord and an indirectly developing dipleurula-like . clade. We use a maximum likelihood framework, including

INTRODUCTION a phylogenetic analysis independent of the major develop- mental and morphological differences between phyla, could Molecular data have provided a new view of metazoan phy- clarify deuterostome relationships. logeny, challenging phylogenetic relationships inferred from Previous molecular phylogenies based on the 18S rRNA morphological and developmental data (reviewed in Raff 1998; gene (Holland et al. 1991; Wada and Satoh 1994) and mito- Zrzavy et al. 1998; Valentine et al. 1999). Molecular studies, chondrial DNA (Castresana et al. 1998) have suggested that particularly those based on the 18S rRNA nuclear gene, have hemichordates fall on the echinoderm lineage, forming the provided support for four major superphyletic clades within Ambulacralia (ϭ Hemichordata ϩ Echinodermata) clade. the Metazoa: the diploblastic prebilaterians (which includes Combined molecular and morphological data sets (Turbeville , ctenophores, cnidarians, and placozoans), the ecdyso- et al. 1994; Zrzavy et al. 1998) also support the hemichordate zoan (includes and allies, and nema- ϩ echinoderm clade. This relationship is gaining acceptance todes and allies), the lophotrochozoan protostomes (molluscs, and is informing interpretations of deuterostome evolution , and other spiralians, lophophorates, rhabodocoel flat- and development (e.g., Tagawa et al. 1998; Ogasawara et al. worms, and some ), and the 1999; Peterson et al. 1999a, 1999b). However, while many (chordates, hemichordates and echinoderms) (e.g., Turbev- studies have supported a hemichordate ϩ echinoderm clade, ille et al. 1994; Wada and Satoh 1994; Halanych et al. 1995; none has provided robust support for the grouping (low Aquinaldo et al. 1997; Cohen et al. 1998; Littlewood et al. bootstrap values suggest that data do not unequivocally sup- 1998; Ruiz-Trillo et al. 1999). However, the relationships of port the clade). Deep, -level phylogenies can pro- the phyla within these four superphyla are less certain. duce questionable results (Abouheif et al. 1998; Takezaki The deuterostomes are an informative case in point. and Gojobori 1999), so it is important to assess the level of While the monophyly of the chordates, hemichordates, and support for the hemichordate ϩ echinoderm clade and to ask echinoderms is supported by developmental, morphological, whether this grouping could have arisen by an artifact of and molecular data (Garstang 1928; Berrill 1955; Jefferies analysis. 1986; Holland et al. 1991; Turbeville et al. 1994; Wada and We have evaluated the strength of the molecular phyloge- Satoh 1994; Halanych et al. 1995; Gee 1997; Peterson et al. netic signal for a hemichordates ϩ echinoderm clade by re- 1997; Castresana et al. 1998; Zrzavy et al. 1998), the rela- analyzing the available DNA sequence using the statistical tionships among these phyla remain debatable. The widely framework of maximum likelihood (including the paramet- varying body plans displayed by members of these phyla ric bootstrap) to ask whether the hemichordate ϩ echino- makes assessment of phylogeny from morphological charac- derm grouping is a significantly better fit to the data than al- ters difficult and has led to the generation of multiple phylo- ternative topologies. Our analysis has several advantages genetic hypotheses (see Fig. 1). Molecular data, by offering over previous molecular phylogenetic studies of the position

© BLACKWELL SCIENCE, INC. 166

Bromham and Degnan Hemichordates and deuterostome evolution 167

Fig. 1. The “traditional” hypothesis places hemichordates (H) on the (C) lineage, supported by shared U-shaped pharyngeal slits, and possibly a dorsal nerve cord (DNC) and stomachord/-like structure (Basler and Ruppert 1990). However, recent analysis of the expression of the Brachyury gene in an enteropneust hemichordate does not lend support to the contention that the stomachord and notochord are homologous (Tagawa et al. 1998; Peterson et al. 1999a). The second hypothesis groups hemichordates and echinoderms (E) based on similar larval forms (Ruppert and Basler 1986; Peterson et al. 1997) and adult axial complex (Basler and Ruppert 1990). A third hypothesis groups echinoderms and chordates to the exclusion of the hemichordates, based on stylophoran fossils which Jefferies (1986) interprets to be chordates possessing the echinoderm-like features of a calcite skeleton and dexiothetism (asym- metrical reduction of structures on the right side). of hemichordates. We use two independent sequences, con- and the alternative hypothesis is compared to an expected distribu- servative alignment of which provides a total of over 5000 tion in likelihood differences given the null hypothesis (Goldman base pairs (bp) of both mitochondrial and nuclear DNA se- 1993a, 1993b; Huelsenbeck et al. 1996; Huelsenbeck and Rannala quence data. We use maximum likelihood because, in addi- 1997). For certain cases, this distribution of expected variance can tion to being more generally accurate and robust than other be assumed (Goldman 1993a, 1993b), but otherwise a null distribu- tion can be generated using Monte Carlo simulation of data phylogenetic inference methods (Huelsenbeck 1995; Huelsen- (Huelsenbeck et al. 1996; Huelsenbeck and Rannala 1997). This beck and Rannala 1997), maximum likelihood allows the use produces an expected distribution of difference in likelihood values of reasonably sophisticated models of DNA sequence evolu- between the alternative and null hypotheses if the null hypothesis is tion and provides a tractable statistical framework for assess- true (see Fig. 2). If the difference in likelihoods between the maxi- ing the relative support for alternative phylogenetic hypoth- mum likelihood solution and the null hypothesis is greater than 95% eses. Parametric bootstrap analysis allows us to assess the of the expected values, then the null hypothesis can be confidently probability that the molecular phylogenetic signal that rejected. This procedure is known as parametric bootstrapping (for a groups hemichordates with echinoderms could have arisen useful review see Huelsenbeck and Rannala 1997). by chance (Huelsenbeck et al. 1996). This hypothesis-testing approach allows us to provide the first strong molecular phy- Data logenetic support for this clade and to confidently reject the 18S rRNA has been much favored for “deep phylogeny,” and con- “traditional” hypothesis (hemichordates ϩ chordates) and sequently it is available for a large range of taxa, although the use of RNA genes in phylogenetic studies is complicated by the heter- the “calcichordate” theory (echinoderms ϩ chordates; Jef- ogenous substitution patterns that are a consequence of the three- feries 1986) as explanations of these molecular data. dimensional structure of the gene product (Dixon and Hillis 1993; Vawter and Brown 1993; Rzhetsky 1995; Tillier and Collins 1998). The recently sequenced whole mitochondrial genome of an en- MATERIALS AND METHODS teropneust hemichordate, carnosus, has now permit- ted assessment of deuterostome phylogeny using mitochondrial Evolutionary history can be inferred from DNA sequences by using DNA (Castresana et al. 1998). We used a concatenated alignment a substitution matrix to calculate for every site in the sequence the of 11 protein-coding genes from the mitochondrial genome. Both probability that any given produced the DNA se- the 18S rRNA and mitochondrial protein-coding sequences were quences observed for extant taxa (Felsenstein 1981). On the assump- aligned against sequences from a previous analysis of the origins of tion that all base changes are independent, the overall likelihood of metazoans (Bromham et al. 1998; http://evolve.zoo.ox.ac.uk/Align- a given phylogenetic tree is the product of probabilities across all ments/.html). Because these alignments included only sites. The tree that has the highest likelihood is deemed the best fit sites that are informative over much deeper divergences, they are to the data, and therefore the most probable scenario for the evolu- conservative for this analysis. tion of those sequences given that model of sequence evolution. The Analyzing large numbers of simulated data sets can be compu- likelihood ratio test provides a “universal framework” for compar- tationally demanding (in terms of processor time), so the parametric ing the fit of phylogenetic hypotheses to a given dataset; the ratio of bootstrap is most easily applied to small trees. To focus on the po- the likelihoods of the data maximized under both the null hypothesis sition of the hemichordates with respect to the echinoderm and

168 EVOLUTION & DEVELOPMENT Vol. 1, No. 3, Nov.–Dec. 1999

Fig. 2. We use the parametric bootstrap to ask, If the traditional hypothesis was true would we have got the hemichordate ϩ echinoderm grouping by chance? Assessing whether the maximum likelihood (i.e., “best-fit tree”; H1) is a significantly better fit to the data than an alternative topology requires an expected distribution of the difference in likelihood values between the alternative topologies if the null hypothesis is true. We generate a null distribution by Monte Carlo simulation of data under the null hypothesis (using SeqGen; Rambaut and Grassly 1997). If the difference in likelihoods between the maximum likelihood solution and the null hypothesis is greater than 95% of the expected values, then the null hypothesis can be confidently rejected (Huelsenbeck et al. 1996).

chordate lineages, we chose six mitochondrial genomes and nine of both 18S rRNA and mitochondrial genes for six was also 18S rRNA sequences to represent these three phyla, plus outgroups analyzed (one of the echinoderm sequences being a concatenation (Fig. 3). Given that the hemichordate ϩ echinoderm grouping has of two Asteroidea species, the 18S rRNA of Asterias amurensis and been supported by previous analyses using many more taxa (e.g., the mitochondrial DNA of Asterina pectinifer). Holland et al. 1991; Turbeville et al. 1994; Wada and Satoh 1994), including the analysis on which the present study is based which Phylogenetic analysis used 40 18S rRNA sequences from 11 phyla including 17 deu- For each of the alignments, the maximum likelihood tree was terostome sequences (Bromham et al. 1998), we are confident that found, with an HKY85ϩ⌫ substitution model, which allows for un- the result obtained in this study is not simply an artifact of reduced even base composition and for different rates of transitions and sample size. The two alignments (1538 bp of 18S rRNA and 3688 transversions (Hasegawa et al. 1985), with gamma-distributed rates bp of mitochondrial protein coding genes, first and second codon across the sequence (Yang 1994; Yang et al. 1994). We estimated positions only) were analyzed separately, but a combined alignment base composition, transition/transversion ratio (ti/tv), and gamma

Bromham and Degnan Hemichordates and deuterostome evolution 169

Fig. 3. For both the mitochondrial and 18S rRNA sequences, the maximum likelihood tree (H1) groups the hemichordates (Balanoglo- ssus carnosus, kowalevskii) with the echinoderms (Asterina pectinifer, Arbacia lixula, Asterias amurensis), to the exclusion of the chordate clade containing the (Branchiostoma lanceolatum) and the (Latimeria chalumnae, Sebas- tolobus altivelis). Outgroup sequences are an insect (Drosophila melanogaster) and a (Artemia franciscana). Each maximum likelihood tree (H1) is compared with the “next best” tree (H0) which does not contain the hemichordate ϩ echinoderm grouping. The log likelihood values (lnL) and estimated transition/transversion ratio (ti/tv) and gamma shape parameters (␣) are given for each tree. The scale is in substitutions per site. shape parameter (␣) from the sequence data. Note that because all the hemichordate ϩ echinoderm grouping (such that H0 ϭ not third codon positions were excluded from the mitochondrial align- (H1)). H0 was produced by constraining the maximum likelihood ment, a codon-based model was not appropriate for this data. The solution to exclude trees containing the hemichordate ϩ echino- small number of taxa in the mitochondrial alignment made an ex- derm grouping. For both sequences (and the combined alignment), haustive search of all trees possible, but for the larger 18S rRNA H0 conformed to the traditional hypothesis, placing the hemichor- tree a heuristic search was performed using TBR branch swapping dates on the chordate lineage (Fig. 3). We did not explicitly test Jef- from an initial neighbor joining tree (see Swofford 1999). The max- feries’ (1986) calicichordate hypothesis, but given that the tree rep- imum likelihood solution for both 18S rRNA and mitochondrial se- resenting the calcichordate hypothesis has an even lower likelihood, quences, and for the combined alignment, groups the hemichor- rejection of H0 strongly suggests that the calcichordate hypothesis dates with the echinoderms (Fig. 3). would also be rejected for this data. To assess whether the maximum likelihood tree is significantly The difference in likelihood between the maximum likelihood better than alternative topologies, we needed to assess whether the solution (H1) and the null hypothesis (H0) is described by the test maximum likelihood topology could have arisen by chance if an al- statistic ␦ ϭ 2(lnLH1ÐlnLH0). To test whether this observed differ- ternative phylogeny was true. We defined the maximum likelihood ence in likelihood between H1 and H0 is significant—that it could tree (which contained the hemichordate ϩ echinoderm grouping) as not simply be the result of phylogenetic “noise”—a null distribution H1 and defined the null hypothesis, H0, as any tree not containing of expected variation in ␦ was generated by simulating the evolu-

170 EVOLUTION & DEVELOPMENT Vol. 1, No. 3, Nov.–Dec. 1999 tion of 100 sets of sequences along the H0 tree, with the same se- Holland, 1995; Ogasawara et al. 1999). Therefore, the hemi- quence length and estimated gamma shape parameter, transition/ chordate ϩ echinoderm grouping suggests that the pharynx transversion ratio, and base compositions as the original data (Fig. must have been present in the deuterostome ancestor rather 2). Each simulated dataset was analyzed as for the real data, finding than independently derived in both the chordate and hemi- the highest likelihood score and the score of the tree constrained to chordate lineages. H0 (hemichordates not with the echinoderms), giving a value of ␦ This suggests that early steps in chordate evolution would for each simulated data set. This distribution of 100 ␦ values repre- sents the expected degree of variation in likelihood scores between have included the acquisition of a notochord-supported mus- H1 and H0 if H0 is true. cularized tail, , and the loss of the dipleurula larval phase and adoption of direct development. The bipha- sic history of ascidians and salps appears to be second- arily derived (Wada and Satoh 1994; Wada 1998). Because RESULTS AND DISCUSSION the deuterostome ancestor must have possessed a pharynx, the chordate postanal muscularized tail complex must have For both mitochondrial and 18S rRNA (and the combined developed independently of the pharynx (Hinman and Deg- alignment) the difference in likelihoods between H1 and H0 nan, 1999). The hemichordate ϩ echinoderm grouping also is greater than that for any of the simulated datasets, so we implies that the echinoderm adult body plan characters of can conclude that the likelihood value of H1 would not have pentameral symmetry, a water vascular system, an endoskel- arisen if H0 was true. We therefore reject the null hypothesis eton, and ring-shaped surrounding the and conclude that the hemichordate ϩ echinoderm grouping that radiates along the arms were not present in the an- is a significantly better fit to the data than any alternative to- cestral deuterostome and so these characters must be part of pology for these data. a dramatic autapomorphic transformation in the echinoderm Significant support for the hemichordate ϩ echinoderm lineage after it diverged from the hemichordate lineage. Fix- clade has important implications for understanding the last ing the phylogenetic position of the urochordates within the common ancestor from which echinoderms, hemichordates, deuterostomes may shed further light on the features of the and chordates evolved. Consideration of the possible out- ancestral deuterostome. groups to the deuterostome clade gives little indication of the features of the ancestral deuterostome, beyond the most basic We are indebted to Andrew Rambaut, for advice and assistance characters: possession of a eucoelomate bilaterian body plan throughout this project, and thank Ziheng Yang and Nick Goldman and probably indirect development (Peterson et al. 1997). for their helpful comments. Fixing the hemichordate lineage with respect to the root of the deuterostome clade allows inference of the presence of major body plan characterisitics in the ancestral deuterosome REFERENCES on the basis of shared derived characters. 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