Deciphering deuterostome phylogeny: molecular, morphological, and palaeontological perspectives

9.1 Introduction

Deuterostomes form one of the three major divi- sions of the Bilateria and are sister group to the Lophotrochozoa plus Ecdysozoa (Eernisse and Peterson, 2004; Philippe et al., 2005a; Telford et al.,
2005; Dunn et al., 2008). Traditionally the group was recognized on the basis of a shared embryonic development pattern: gastrulation occurs at the vegetal pole and the blastopore becomes the anus, while the mouth forms secondarily (Chea et al.,
2005). Analysis of molecular data has consistently found the deuterostome grouping, with generally high levels of support (Turbeville et al., 1994; Wada and Satoh, 1994; Halanych et al., 1995; Cameron et al.,
2000). Five major clades make up the Deuterostomia: craniates, cephalochordates, echinoderms, hemi- chordates, and tunicates (see Figure 9.1). Because vertebrates, including ourselves, belong to the cra- niates, there has long been a fascination about their invertebrate origins. Theories of chordate evolution have abounded for over 100 years, but it is only in the last 10 to 15 years that deuterostome relation- ships have come into sharp focus, driven largely by new data from molecular genetics and the fossil record, and new analyses of traditional morpho- logical and ontogenetic data. From this plethora of information, some complementary, others sup- porting contradictory conclusions, a more coherent picture of the phylogeny and early evolution of deu- terostomes is starting to emerge.
Here we review four key areas where there has been the most heated debate in the last 5 years: phylogenetic relationships of the major deu- terostome groups; the earliest fossil record and
divergence times of deuterostome groups; the evolution of body axes; and the characteristics of the ancestral deuterostome body plan.


9.2 Deuterostome phylogenetic relationships

Until 10 years ago there was little consensus about the relationships of the major deuterostome groups (see reviews by Gee, 1996, and Lambert, 2005). Depending upon whether emphasis was given to comparative adult morphology, embryology, or the fossil record, different sister-group relationships could be argued. Larval traits provided support for a grouping of echinoderms and hemichordates (Hara et al., 2006; Swalla, 2006), adult traits pro- vided support for a grouping of hemichordates and chordates (Cameron et al., 2000) while palaeonto- logical data were used to support an echinoderm– chordate pairing (Gee, 1996). Probably the most widely accepted view in the mid-1990s was that echinoderms were sister group to the rest and that chordates and hemichordates were sister taxa [i.e. (echinoderms {hemichordates [tunicates (cephalo- chordates + craniates)]})].
With the arrival of molecular data the problem of deuterostome relationships seemed to be solved. Early results were based on analyses of ribosomal gene sequences and pointed to echinoderms and hemichordates as sister groups (Turbeville et al.,
1994; Wada and Satoh, 1994) and to a monophyletic Chordata comprising tunicates, cephalochordates, and craniates (Turbeville et al., 1994). Within the chordates, tunicates were identified as sister group

80

D E U T ERO S TO M E P H Y L O G EN Y 81


Xenoturbellida


Crinoidea

Asteroidea

Ophiuroidea

Holothuroidea

I
Echinoidea

Pterobranchia

Harrimaniidae

Ptychoderidae
Cephalochordata

Phlebobranchia

Thaliacea

II Aplousobranchia

Appendicularia

Stolidobranchia

Molgulidae

Vertebrata

Figure 9.1 Current deuterostome phylogeny, according to available molecular and morphological data. Dotted lines show clades of uncertainty where conflicting data have been obtained; I and II mark clades where the evidence for a monophyletic group is very high. I, Ambulacraria is made up of hemichordates and echinoderms. Mitochondrial, ribosomal and genomic evidence are in agreement for this grouping. The Ambulacraria develop similarly through gastrulation and share larvae that feed by ciliated bands, strengthening their sister- group relationship. Genomic evidence suggests that Xenoturbellids may be a sister group to the Ambulacraria, but it is also possible that they are an outgroup to the rest of the deuterostomes. II, Chordates are a monophyletic group that share a specific body plan, but mitochondrial and genomic evidence are in conflict about the position of the tunicates. Mitochondrial and ribosomal evidence place cephalochordates as sister group to the vertebrates, whereas genomic evidence places tunicates as the sister group to the vertebrates. Figure modified from Zeng and Swalla (2005).


to Euchordates (= craniates plus cephalochordates) (Turbeville et al., 1994; Wada and Satoh, 1994; Adoutte et al., 2000; Cameron et al., 2000; Winchell et al., 2002; Bourlat et al., 2003). Reassuringly these relationships appeared to be robust to tree-building
methods (Furlong and Holland, 2002) and morpho- logical support for these groupings was forthcom- ing (Peterson and Eernisse, 2001). The pairing of echinoderms and hemichordates is further sup- ported by shared Hox gene motifs (Peterson et al.,

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2004), mitochondrial genes and gene arrangements
(Bromham and Degnan, 1999; Lavrov and Lang,
2005), and amino acid sequences of selected nuclear
genes, such as actins (Swalla, 2007).
However, as data accumulated and more care-
ful probing of the molecular phylogenetic signal
was carried out, some parts of the deuterostome
tree came into question. Although the clades
Ambulacraria and Chordata remained well sup-
ported (Figure 9.1), the position of tunicates was
found to be unstable and varied according to
which gene was used (Winchell et al., 2002). A new
group, Xenoturbellida, was also added to the deu-
terostomes (Bourlat et al., 2003, 2006). Furthermore,
in the last 2 years phylogenies have been increas-
ingly constructed from concatenated amino acid
sequences of large numbers of protein-coding
genes (Blair and Hedges, 2005; Philippe et al.,
2005a; Bourlat et al., 2006; Delsuc et al., 2006; Dunn
et al., 2008). Most of these phylogenies place ech-
inoderms and hemichordates as sister groups, and
recognize Chordata as a clade, but place tunicates,
not cephalochordates, as sister group to chordates
(a grouping termed Olfactores). At the same time,
an extensive cladistic reanalysis of morphological
data found strong support for Olfactores (Ruppert,
2005). Figure 9.1 summarizes the current emerging
view of deuterostome relationships.
While higher-level relationships have attracted
most attention, extensive progress has also been
made in constructing detailed phylogenetic
hypotheses for taxa within each of these major
groups. A summary of echinoderm relationships
was given by Smith et al., (2004) and, for the most
part, morphological and molecular data point to
the same branching order. Only the position of
ophiuroids with respect to other eleutherozoan
taxa remains problematic. The match between
molecules, morphology, and the fossil record is
particularly strong for echinoids (Smith et al.,
2006).
There is less certainty about the relationships
within hemichordates. Traditionally the group has
been divided into the worm-like enteropneusts
and the tube-dwelling pterobranchs, with enterop-
neusts considered as derived from a pterobranch-
like ancestor (e.g. Barrington, 1965). Morphological
analysis places pterobranchs and enteropneusts
as sister groups (Halanych et al., 1995; Cameron et al., 2000). However, molecular phylogenies based on ribosomal genes provide a mixed signal. The
18S rRNA gene data nest pterobranchs within a paraphyletic enteropneust grade while 28S rRNA gene sequence data place pterobranchs and enter- opneusts as sister groups, but this may be due to the lack of informative sites in 28S rRNA for Hemichordata (Cameron et al., 2000; Winchell et al.,
2002). As discussed below, whether pterobranchs are derived or basal with respect to other hemi- chordates is critical for reconstructing the ancestral body plan of the Ambulacraria.
Zeng et al., (2006) provide a detailed 18S rDNA phylogeny of tunicates that mostly agrees with earlier molecular and morphological ana- lyses (Swalla et al., 2000). Within the tunicates, morphological and molecular data are largely con- gruent (Swalla et al., 2000; Stach and Turbeville,
2002). Five separate clades of tunicates, corre- sponding to traditionally recognized groupings, are supported by molecular data: The ascid- ian clades Stolidibranchia, Phlebobranchia, and Aplousobranchiata, and the pelagic tunicates Appendicularia and Thaliacea (Figure 9.1). The Thaliacea are all colonial and are sister group to the Phlebobranchia plus Aplousobranchiata clade. In molecular phylogenies, the solitary, pelagic free- floating Appendicularia has a very long branch and falls either as outgroup to the rest of the tuni- cates (Swalla et al., 2000; Stach and Turbeville, 2002) or as a sister group to the Stolidobranchia (Zeng et al., 2006). If the tunicate ancestor resembles the Appendicularia plus Stolidobranchia clade, then it would be likely to be solitary. However, if the ancestor resembled the Phlebobranchia, Aplousobranchiata, and Thaliacea clade, then it would be more likely to be colonial (Zeng and Swalla, 2005). Whatever the tunicate ancestor, the tunicate adult body plan bears little resemblance to the rest of the chordates, suggesting that major changes in adult body plan happened early in tuni- cate evolution (Swalla 2006, 2007).
There are so few living cephalochordates that this group is represented by a single taxon in most molecular analyses, and the different extant gen- era are morphologically very similar (Zeng and Swalla, 2005). By contrast, extensive molecular

D E U T ERO S TO M E P H Y L O G EN Y 83



and morphological phylogenies exist for craniates (Rowe, 2004). The basal relationships of hagfishes, lampreys, and other primitive craniates such as conodonts, has been detailed thoroughly by Donoghue et al., (2000). The most recent molecular phylogeny based on protein-coding genes (Blair and Hedges, 2005) places hagfishes and lampreys as sister taxa (Cyclostomata) right at the base of the craniate tree, as in previous ribosomal-based analyses (Mallatt and Sullivan, 1998).


9.3 Insights from the fossil record

In the last 10 years a number of new and poten- tially important fossil deuterostomes have been recovered from Early and Middle Cambrian deposits. However, progress has been more difficult here than for molecular studies because of problems arising from incomplete preservation and ambiguity of interpretation. Fossils provide data on morphologies that once existed and, when included in phylogenetic analyses with extant taxa, can aid in recognition of ancestral charac- ter states and can alter relationships by displaying character combinations not seen in living forms. However, missing data can be a major problem for the interpretation of fossils, and even with the best soft-tissue preservation in the Cambrian we have only the outline of internal organs preserved without structural detail. Furthermore, fossil anatomy can only be interpreted through refer- ence to extant organisms. For taxa without obvi- ous modern counterparts (as for example in the case of vetulicolians discussed below), the choice of which modern analogue to select as a reference for interpretation can make a huge difference. The fact that the taxonomic placement of most Cambrian soft-bodied deuterostome taxa remains disputed attests to the difficulty of interpretation posed by these fossils.
An even more crippling problem for palaeon- tologists is that many of the high-ranking taxa are recognized on the basis of just a few, mostly embryological, biochemical, or genetic char- acters, or from molecular data alone, such as Xenambulacraria (Bourlat et al., 2006). Even for classes and phyla, fossils can only be placed with certainty once one or more derived morphological
synapomorphies have evolved, so that early stem- group members are commonly much more difficult to identify, simply because there are no emergent features! This is a general problem of ancestral taxa in the fossil record—we know they probably exist, but we simply do not know how to identify them. Here we review the fossil evidence for the oldest members of each phylum or subphylum and then examine the case for more primitive deuterostome stem-group members.


9.3.1 Hemichordates

Of the two hemichordate morphologies, the colo- nial, tube-dwelling pterobranchs have by far the best fossil record. The tubes of fossil ptero- branchs preserve well and show two excellent synapomorphies; a characteristic fusellar struc- ture and an internal stolon. It has long been recog- nized that the extinct Graptolithina are a primarily planktonic group of colonial hemichordates closely related to the extant pterobranchs (Maletz et al.,
2005; Rickards and Durman, 2006). Indeed, as bet- ter knowledge of the Cambrian faunas has accrued, it has become increasingly difficult to draw a clear division between pterobranchs and graptolites (Maletz et al., 2005). For example, the Ordovician Cephalodiscus-like genus Melanostrophus preserves the morphological ultrastructure of the tube in spectacular detail and displays a mixture of extant cephalodiscid and extinct graptolite features (Mierzejewski and Urbanek, 2004). It now seems likely that many of the primitive benthic dendroid graptolites are probably better classified as ptero- branchs (Maletz et al., 2005). Pterobranch tubes showing clear fusellar structure and an internal stolon are now recorded from the Middle Cambrian (Maletz et al., 2005; Rickards and Durman, 2006), the oldest coming from the early Middle Cambrian (Bengtson and Urbanek, 1986). Both rhabdopleu- rids and cephalodiscids are present by the end of the Middle Cambrian (Rickards and Durman,
2006) showing that crown-group divergence had
occurred by then.
Fossil enteropneust worms have proven to be
much more elusive to identify. The most character-
istic apomorphies likely to be seen in fossils are the
proboscis and collar, but to date no such Cambrian

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worm-like organisms have been formally described from the Cambrian, although Conway Morris (1979) lists enteropneusts as present in the Burgess Shale. Shu et al., (1996a) initially interpreted the fossil chordate Yunannozoon from the Lower Cambrian of China as a fossil enteropneust, but this was later corrected (Shu et al., 2003b). Wignall and Twitchett (1996) recorded possible burrows of enteropneusts from the Lower Triassic of Poland, and the oldest body fossil of an enteropneust is from the Lower Jurassic of Italy (Arduini et al., 1981).


9.3.2 Echinoderms

Echinoderms have a calcitic skeleton with a very












Pharyngeal gill slits


Posterior


Mouth
Anterior














Muscular bilateral stalk

distinctive structure termed ‘stereom’. The genes responsible for stereom deposition are unique to echinoderms (Bottjer et al., 2006). Thus although upstream regulator genes of skeletogenesis might be common to all deuterostomes (Ettensohn et al.,
2003), the specific skeletal construction of echino- derms is a reliable synapomorphy. Furthermore, all members of the crown group show radiate symmetry, so stereom-bearing fossils without radial symmetry are best considered as stem- group echinoderms. The morphology and rela- tionships of stem-group echinoderms have been reviewed by Smith (2005) and some information on their soft-tissue anatomy can be deduced con- fidently because of the close correspondence between stereom microstructure and investing tissue (Clausen and Smith, 2005). Stem-group echinoderms are diverse and include a variety of asymmetrical forms. These reveal that the most basal echinoderms had external gill openings, a bilaterally symmetrical muscular stalk (solutes and stylophorans; Figure 9.1), and, less certainly, a pair of tentacles and a pharyngeal basket with atrium (cinctans) (Smith, 2005). These fossils thus provide an important window onto the early his- tory of echinoderms, revealing key stages in the origins of the echinoderm body plan, one of the most divergent of any bilaterian. The oldest skeletal elements with stereom are mid Lower Cambrian, and both crown-group (eleutherozoan and pel- matozoan) and stem-group (solutes) members are present by the latest Lower Cambrian, showing
Figure 9.2 The stem group echinoderm Cothurnocystis
(Ordovician, Scotland), showing the presence of pharyngeal gill slits.

that diversification of crown-group echinoderms
had already occurred.
Vetulocystids, from the late Lower Cambrian of
China, were described as stem-group echinoderms
by Shu et al., (2004). Three taxa were erected but, of
these, two were badly preserved and not convin-
cingly different from the better-known Vetulocystis.
Furthermore, Vetulocystis shows close similarity to
vetulicolans in having a bipartite body plan with
a cuticular sheath, articulated posterior append-
age, and at least one lateral pouch (‘gill opening’—
possibly paired but no specimen shows part and
counterpart). These fossils do not share a single
echinoderm synapomorphy so it is not clear why
these were placed in the Ambulacraria.


9.3.3 Xenoturbellids

This small-bodied taxon has no known morpho- logical synapomorphies and lacks a skeleton; not surprisingly, there is no known fossil record.

9.3.4 Tunicata

There are a number of synapomorphies that should allow firm identification of tunicates from the fos- sil record: their tunicine cuticle, pharyngeal basket, inhalent and exhalent openings to their atrium, and endostyle. However, they are soft-bodied and their fossil record is very sparse. Shu et al., (2001a)

D E U T ERO S TO M E P H Y L O G EN Y 85



reported a fossil tunicate (Cheungkongella) from the Lower Cambrian of China, but this was based on a unique and incomplete specimen that later, with the collection of additional material, turned out to be a junior synonym of Phlogites, a taxon with paired branched tentacles whose affinities are uncertain (Chen et al., 2003; Xian-Guang et al., 2006). However, another Lower Cambrian fossil from China, Shanouclava, is a tunicate and shows typical ascidian characters, including a pharyngeal basket and possible endostyle (Chen et al., 2003). This is described as being a crown-group tunicate, related to aplousobranch ascidians. The fossil record thus clearly shows that, like echinoderms, the tunicate body plan was well established, and crown-group tunicate divergence had already occurred by the end of the Lower Cambrian.
It is also vaguely possible that the relatively diverse group of problematic Lower Cambrian fos- sils termed vetulicolians are appendicularian-like tunicates, but this is much less certain (Aldridge et al., 2007). The problem associated with vetulicol- ians is discussed in more detail below.


9.3.5 Cephalochordata

This small group of fish-like invertebrates is read- ily identified from adult morphology, being jawless and having a continuous dorsal fin, myotomes, post-anal tail, and barred gill slits. Pikaia, from the Middle Cambrian Burgess Shale, has simple gill slits but is widely accepted to be a member of this clade. The Lower Cambrian Cathaymyrus is also attributed to this clade (Shu et al., 1996b), though it is less well preserved and, consequently, the case here is less convincing.


9.3.6 Craniata

Hagfish, lampreys, and an important extinct group, the conodonts, branch close to the base of the craniate crown group (Donoghue et al., 2000). The character combinations used to place fossil members within this clade are a well-developed brain with eyes, notochord, myotomes, and gill slits. Fossil agnathan fishes are present in the Lower Cambrian (Shu et al.,
1999; Zhang and Xian-Guang, 2004) and show evi-
dence of a complex brain and well-developed eyes
(Shu et al., 2003b). Two taxa were originally described (Myllokunmingia and Haikouichthys), but subse- quently these have been shown to be synonymous (Xian-Guang et al., 2002). There seems little dispute about their taxonomic affinities.


9.3.7 Potential representatives of more basal deuterostomes

Fossil representatives of the common stem of two or more phyla are difficult to recognize with confidence because adult morphological syn- apomorphies are lacking. Over the last 10 years various fossils have been championed as deu- terostomes more basal than any of the five major groups, but few if any of these claims have stood up to detailed scrutiny. Vetulicolids are a clear case in point. First described as arthropods then later as basal deuterostomes, tunicates, or even possibly kinorhynchs, these animals have been the focus of much attention and debate, as recently reviewed (Aldridge et al., 2007). If vetulicolids were indeed basal deuterostomes they would provide critical information on the ancestral body plan. However, the recent cladistic analysis of Aldridge et al. (2007) has highlighted just how tenuous the anatomical interpretations of these fossil remains are. Earlier reports of a vetulicolian mesodermal skeleton (Shu et al., 2001a) are now considered unlikely. Indeed Vetulicola itself shows clear evidence of a jointed exoskeleton, and it is this that makes some (Caron,
2006) hesitant of rejecting arthropod affinities, des- pite their apparent lack of limbs. Most have a ser- ies of ‘lateral pouches’ which may or may not open externally and whose detailed structure is far from clear. These have been variously interpreted as gill slits, pouch-like arthropod gills, or midgut glands. Aldridge et al. (2007) accepted these as gills but could not determine whether they were internal or external openings. A third character, the presence of an endostyle, is unprovable, since the structure in question is no more than a dark line picked out by iron oxide—it exists, but its identification requires knowledge of taxonomic affinities and guesswork based on shape and position. Finally the nature of the thin tube running to the tip of the posterior is also critical. Shu et al., (2001a) interpret this as the gut, making the anus terminal, whereas Aldridge

86 AN I M AL EV O L UTI O N



et al., (2007) raise the more likely possibility that it is another structure (possibly the notochord). In the face of such uncertainty the taxonomic placement of this group remains very much in doubt.
As Lacalli (2002) and Aldridge et al., (2007) stress, vetulicolids share many similarities with tunicates, especially appendicularians, although there are still significant problems of interpretation. The jointed exoskeleton would have to be composed of tunicin and the lateral pouches would be internal gill slits, but both of these are potentially provable from the fossil record. Given that the interpretation of soft tissue imprints will always remain ambiguous, it seems likely that palaeontologists will continue to have to interpret fossils in the light of theories of deuterostome origins based on data from living organisms (contra Shu et al., 2001b).
A potential stem group ambulacrarian, Phlogites, was recently redescribed by Xian-Guang et al., (2006). These specimens have a pair of branched and erect tentacles, a large U-shaped gut and a muscular stalk. Xian-Guang et al., (2006) compared Phlogites with various groups, noting its close similarity to uncalcified echinoderms, but eventu- ally opting for it being a lophotrochozoan. It may, however represent a stem-group ambulacrarian, although the lack of obvious gill slits in Phlogites makes this somewhat less compelling.
The ancestral chordate was likely to have been fishlike with a post-anal tail, open pharyngeal slits, a notochord, and a single fin, with limited devel- opment of eyes and brain. The best contenders for representative stem-group chordates are the yunna- nozoons. Yunannozoons were originally described as hemichordates (Shu et al., 1996a) then later as stem deuterostomes (Shu et al., 2003). However, one yunnanozoon, Haikouella, has been restudied by Mallatt and Chen (2003) and Mallatt et al., (2003), and has been shown to be encephalized, with small eyes, a single fin, internal gill arches, and lateral and ventral myotomes. Haikouella was interpreted as a cephalochordate-like suspension feeder with an endostyle and tentacles forming a screen across the mouth. Detailed cladistic assessment (Mallatt and Chen, 2003) found that yunnanozoons lack any specific hagfish characters and fall closer to craniates than to cephalochordates. They are either stem-group craniates, stem-group Olfactores or
possibly late stem-group chordates. Again the disagreement over interpretation of structures (Mallatt and Chen, 2003; Shu et al., 2003) highlights just how tenuous identification of soft-tissue anat- omy in these forms can be. Basal deuterostomes are even less easy to diagnose, and no fossil has been convincingly identified.
In summary, members of all five major deutero- stome groups can be distinguished on adult syn- apomorphies by the end of the Lower Cambrian, and crown-group divergence had taken place in at least three (echinoderms, hemichordates, tunicates) (Figure 9.3). By contrast, a lack of adult morpho- logical criteria and the general difficulty of inter- preting fossils that preserve only outline traces of soft-tissue organs has meant that no common stem- group member of two or more of these groups has yet been convincingly demonstrated, except pos- sibly yunnanozoons, whose exact position within Chordata remains problematic.


9.4 When did deuterostome groups originate?

The palaeontological data discussed above, com- bined with our best molecular and morphological phylogenies, demonstrate that all five major clades of deuterostomes were already present in the Lower Cambrian, c. 520 million years ago (Ma) (Figure 9.3). Indeed, for some, crown-group divergence had already begun. There is no evidence from the rock record of an older fauna when just stem-group members existed, so unless one wishes to take a direct reading of the fossil record and argue for an almost instantaneous origin, the fossil record tells us only about the latest time by which these groups arose. Consequently, we must turn to molecular data to get estimates of their times of origin. This, however, is by no means without problems.
Most early attempts to use molecular similar- ity to estimate divergence times all pointed to a very deep divergence of deuterostomes (and other metazoans), pre-dating the first fossil evidence by several hundred million years (Wray et al., 1996; Feng et al., 1997; Gu and Li, 1998; Wang et al., 1999; Nei et al., 2001). These studies all calculate diver- gence times using a single molecular rate of change across the tree, and thus rely on all taxa having

D E U T ERO S TO M E P H Y L O G EN Y 87



similar rates of molecular evolution. However, both Ayala et al., (1998) and Peterson et al. (2004) noted that there was a significant decrease in the rate of molecular evolution in vertebrates that could mislead molecular clock estimates. To avoid this problem Ayala et al., (1998) generated a linearized molecular tree after carefully checking for rate het- erogeneity, and found that chordates and echino- derms split around 600 Ma. Peterson et al., (2004) used a different approach, avoiding all vertebrates, and calculated divergence dates using a series of fixed calibration points based on the arthropod and echinoderm fossil records. This gave a minimum estimated divergence between hemichordates and echinoderms at 526–567 Ma.
Recently, methods of analysis that accommodate rate variation using a ‘relaxed clock’ model have been developed. These methods allow for rate vari- ation across the tree to be modelled. A variety of methods are now available and can produce highly convincing results, both in simulation studies and with empirical data (Ho et al., 2005; Near et al., 2005; Smith et al., 2006; Yang and Rannala, 2006), so long as the prior probabilities used are realistic. Early application of a Bayesian relaxed clock method to the problem of metazoan divergence times by Aris-Brosou and Yang (2003) found phylum-level splits in the Deuterostomia at around 520–530 Ma, matching Peterson et al.’s (2004) results. However, this used an unrealistic model of molecular evolu- tion (Ho et al., 2005), and other analyses that apply more appropriate models (Douzery et al., 2004) have continued to find deeper divergence dates for the Bilateria. Using a much larger data set, Bayesian methodology, and multiple fossil calibration points, Blair and Hedges (2005) estimated the divergence of crown group craniates as 652 (605–742) Ma, the divergence of Olfactores at 794 (685–918) Ma and the divergence of Ambulacraria at 876 (725–1074) Ma. Based on the echinoderm divergence of ech- inoids from starfish, the more recent lower bound for these molecular estimates is in best accord with fossil evidence (Figure 9.3).
In summary, although there remains some debate about timing due to the methodological uncertain- ties associated with calculating divergence dates from molecular data, neither rapid diversification nor very deep diversification seem supported by
current studies. Molecular data provide evidence that the earliest record of deuterostome evolution is missing from the fossil record, though whether this gap is only a few tens of millions of years or maybe as much as 200 million years remains unre- solved.
If we cannot be precise about the timing of the event is it possible to determine from molecular data whether diversification occurred rapidly or slowly? Rokas et al., (2005) raised an old argument in favour of metazoan diversification being com- pressed in time. They noted that whereas fungal relationships are clearly resolved by molecular data, those of some metazoans are not. As both groups evolved at around the same time, the early history of metazoans may have been a radiation compressed in time, in agreement with a direct reading of the palaeontological record. Baurain et al., (2007), however, showed that their result was an artefact of inadequate taxon sampling and/or model of sequence evolution. By increasing the number of species, replacing fast-evolving species by slowly evolving ones, and using a better model of sequence evolution, Baurain et al., (2007) found that resolution amongst metazoan clades was markedly improved. Thus with the right models and good taxon sampling, molecular data support the idea of a relatively gradual unfolding of deuter- ostome diversity over time.


9.5 Conserved gene networks pattern deuterostome axes and germ layers

The Hox complex is a duplicated set of genes that is frequently found in a single cluster on the chromo- some, and is important for anterior to posterior patterning in bilaterian animals (Lemons and McGinnis, 2006). Within the deuterostomes, Hox clusters have been characterized from all of the major phylogenetic groups except xenoturbellids (Swalla, 2006). It is probable that the ancestral chordate had 14 genes linearly aligned on the chromosome, and that they were expressed col- linearly in an anterior to posterior manner, as both cephalochordates and vertebrates have 14 genes, with the posterior six genes showing independent duplication from the protostome posterior genes

88 AN I M AL EV O L UTI O N





6

9


8


1 2 3 4 5 7

3 Tunicates
B
7 Craniates
6 Cephalochordates
A
4 5
Echinoderms
C
2
Hemichordates


Cryogenian Ediacaran
Pre-Cambrian
C G B
Early M Late
Cambrian

680
600

Time (million years)
500

Figure 9.3 The fossil record of deuterostomes and molecular-clock estimates of divergence times. Thick black lines indicate known occurrence; thin lines indicate inferred range. Error bars show 95% confidence intervals on molecular estimates. Cambrian deuterostomes and possible deuterostomes as follows: 1, Phlogites (Lower Cambrian; possible stem-group ambulacrarian); 2, Rhabdopleura (Middle Cambrian pterobranch); 3, Shanouclava (Lower Cambrian ?aplousobranch ascidian tunicate); 4, Trochocystites (Middle Cambrian stem-
group echinoderm); 5, Stromatocystites (Lower Cambrian crown-group echinoderm); 6, Cathaymyrus (Lower Cambrian cephalochordate);
7, Haikouichthys (Lower Cambrian craniate); 8, Haikouella (Lower Cambrian chordate; possible stem-group craniate); 9, Vetulicolia (Lower Cambrian problematica). Molecular dating for nodes A–C as follows. A, craniate–echinoderm split based on a linearized gene tree (from Ayala et al., 1998); B, craniate–tunicate split based on the Bayesian relaxed clock model (from Douzery et al. 2004); C, echinoderm– hemichordate split based on a linearized gene tree (from Peterson et al., 2004). Grey bands are deposits with soft bodied preservation:
C, Chengjiang Formation; G, Guanshan Formation; B, Burgess Shale.


(Minguillón et al., 2005; Lemons and McGinnis,
2006; Swalla, 2006).
A duplication of the posterior genes into three
genes called Hox 11/13a, Hox 11/13b, and Hox
11/13c characterizes both echinoderm and hemi-
chordate Hox gene clusters (Peterson, 2004; Morris
and Byrne, 2005; Cameron et al., 2006; Lowe et al.,
2006). The separate posterior genes share amino
acid motifs, strongly suggesting that echinoderms
and hemichordates share a common ancestor that
differed from the chordate ancestor (Peterson,
2004). In addition, the sea urchin genome has been
sequenced (Sodergren et al., 2006) and the Hox clus-
ter has an inversion, with Hox 3 juxtaposed against
Hox 11/13c, and the subsequent loss of Hox 4

(Cameron et al., 2006). It will be very interesting to examine the arrangement of the Hox complex in other echinoderms to see if they have a similar inversion. If all echinoderms have this Hox inver- sion, then it will be interesting to examine enterop- neust hemichordates, because they have collinear Hox expression in the ectoderm (Aronowicz and Lowe, 2006). The fact that hemichordates, ceph- alochordates, and vertebrates all show collinear expression of the Hox genes in an anterior to pos- terior manner, beginning directly after the first gill slit, suggests that the deuterostome ancestor had an antero-posterior axis determined by the Hox complex immediately posterior to the first gill slit formed (Swalla, 2006).

D E U T ERO S TO M E P H Y L O G EN Y 89



Echinodermata
Hemichordata Cephalochordata Tunicata Vertebrata
D D a

Lateral view

Oral

Aboral Oral
Aboral A
P A P A P




Dorsal view
V V V








Ambulacraria Chordata

= Nodal
= Dorsal (chordin)
= BMP

Figure 9.4 Summary of body axis determination in Deuterostomata: A, anterior; P, posterior; a, animal; v, vegetal. Expression of nodal is in red for all phyla. Dorsal in hemichordates is shown by BMP expression in yellow. In Echinodermata the aboral (dorsal) axis is shown by a yellow strip, with nodal expression marked red on the right side of the larvae, opposite where the adult rudiment will form. Dorsal in chordates is marked in blue (lower pictures) while expression of chordin is shown during gastrulation in Cephalochordata, Tunicata, and
Vertebrata. The BMP–chordin axis is reversed in chordates from the Ambulacraria. Note that nodal expression is on the left side in chordates, and on the right side in echinoderms. Expression patterns taken from Duboc et al , (2005) (urchin nodal), Lowe et al. (2006) (hemichordate chordin), Yu et al., (2007) (cephalochordate BMP and chordin), Darras and Nishida (2001) (ascidian chordin), and Sasai and De Robertis (1997) (frog BMP and chordin). (See also Plate 7.)


In contrast, the chordate dorsoventral axis is inverted when compared to the hemichordate dorsoventral axis (Figure 9.4 and Plate 7; Ruppert,
2005; Lowe et al., 2006; Swalla, 2007). The hemi- chordate gill slits and bars are located dorsally, opposite to the ventral mouth, while in cephalo- chordates and vertebrates the gill slits and bars are located on the same side of the mouth (Swalla, 2007). Gill openings are also dorsal in stem-group echi- noderms (Smith, 2005). Recent results have shown that the dorsal side of a juvenile hemichordate is determined by a strip of bone morphogenetic protein (BMP) expressed dorsally, and a ventral expression stripe of BMP antagonists, including chordin, (Lowe et al., 2006), as has been reported for arthropods (Sasai and De Robertis, 1997). In contrast, in vertebrates, BMP is expressed ventrally, and chordin is expressed dorsally (Sasai and De Robertis, 1997; Yu et al., 2007), suggesting that the chordate dorsoventral axis is inverted compared with a hemichordate or arthropod dorsoventral axis (Lowe et al., 2006). Considering these results,

evolution of the chordates would entail moving the mouth from the side opposite the gill slits to the same side as the gill slits, while also evolving a notochord and dorsal central nervous system from an enteropneust-like nerve net.
Nodal is so far one of the few described deuteros- tome-specific genes, evolving from a duplication of a BMP-like ancestor (Duboc et al., 2004; Chea et al.,
2005). In deuterostomes, nodal signalling results in left–right asymmetry in bilateral embryos. In all chordates, nodal is expressed on the left side during development, specifying asymmetry (Chea et al.,
2005). However, in sea urchins, nodal is expressed on the right side (Duboc et al., 2004, 2005). If nodal is expressed on the right side during hemichordate development, then it would give further weight to the evidence that the Ambulacraria have a dorso- ventral axis similar to flies, and that chordates have an inverted dorsoventral axis (Figure 9.4).
Identifying the homologous body axes in ech- inoderms has long been problematic because of their lack of obvious anteroposterior or dorsoventral axes

90 AN I M AL EV O L UTI O N



as adults. Indeed it has been traditional for echino- derm workers to avoid using such terms when refer- ring to anatomical orientation, referring instead to oral–aboral and radial–interradial. This major innov- ation in body plan seems to have been triggered by a shift from posterior facultative to anterior obligate larval attachment (Smith, 2008). Anterior attachment necessitated the introduction of a phase of torsion in development to bring the mouth into a more appro- priate orientation for filter feeding, which in turn rotated the axis of the developing adult 90º out of alignment with Hox and other body patterning genes. As a result the developing echinoderm rudiment came to receive a complex mosaic of antero-poste- rior signalling, and extensive co-option of signalling pathways was able to take place, allowing innov- ation. The fossil record provides important insights into both pre-attachment and post-attachment stages in this evolutionary process (Smith, 2008) .



9.6 What was the body plan of the earliest deuterostome?

By combining evidence from the most primitive fos- sil members of each clade with our best molecular phylogenetic hypotheses and current ideas of hom- ology derived from both genetic network data and traditional comparative morphology we can start to build up a picture of the body plan of the earliest deuterostomes (e.g. Cameron et al., 2000; Ruppert,
2005; Zeng and Swalla, 2005). Here we review the evidence relating to the latest common ancestors of Ambulacraria, Chordata, and Deuterostomia.


9.6.1 Chordata

The discovery that tunicates and craniates may be sister groups to the exclusion of cephalochor- dates implies that characters common to cephalo- chordates and craniates are basal to all Chordata. Tunicata, so distinct in many ways (Zeng and Swalla,
2005), have undergone considerable morphological and genomic simplification, including loss of meta- meres, nephridia, and some Hox genes (Swalla,
2006, 2007). As cephalochordates and the cartil- aginous fishes both contain intact Hox clusters that are expressed collinearly along the antero-posterior
axis, the chordate ancestor presumably had a similar Hox cluster that determined the antero-posterior axis (Minguillón et al., 2005). If tunicates and vertebrates are sister taxa, then stri- ated heart muscle and recently discovered neural crest-like tissue were present in the latest common ancestor of tunicates and vertebrates. Based on shared features between cephalochordates and cra- niates, the latest common ancestor of all chordates might be expected to be a worm-like segmented organism with metamerism and myotomes, a dor- sal notochord, dorsal hollow nerves, pharyngeal mucous net filtration with endostyle, undivided fins, locomotory post-anal tail, a pre-oral hood, and a hepatic portal system (Ruppert, 2005; Swalla,
2007). As neither Ambulacraria nor cephalochor- dates have a well-developed brain, encephalization may have been limited and sensory facilities poorly developed in the very earliest chordates (Brown et al., 2008). However, the absence of some sensory systems in cephalochordates may be secondary if Haikouella, with its small eyes and weak enceph- alization, represents a late stem-group chordate. Reproduction was likely through direct develop- ment. Amongst fossils, yunnanozoons display the closest morphological similarity to this body plan.


9.6.2 Ambulacraria

Living echinoderms are so highly derived com- pared with other deuterostomes that meaningful comparisons of adult body plans have been more or less futile. Even determining the antero-poste- rior axis in adult echinoderms has proved to be very difficult until recently (Peterson et al., 2000; Morris and Byrne, 2005; Swalla, 2006). However, with the inclusion of pre-radiate fossil stem-group echinoderms, comparison is greatly simplified and clarified. There is a clear antero-posterior axis, and structures that have been lost from all crown-group echinoderms, such as the pharyngeal openings and muscular stalk, are evident (Smith, 2005). There is another problem, however, and that is deter- mining whether the enteropneust or pterobranch body plan is primitive for hemichordates. Because lophophorates were suggested as ancestral, the pterobranch model has traditionally been taken as primitive (Gee, 1996). Echinoderms and pterobranchs

D E U T ERO S TO M E P H Y L O G EN Y 91



share a muscular stalk used for attachment and locomotion, a hollow, branched tentacular system derived from the same mesocoel and used for the same purpose, so these features would instead favour a stalked, tentaculate hemichordate form rather than a worm-like latest common ancestor (Smith, 2005). More recently, however, the enterop- neust model has been suggested because the ante- ro-posterior and dorsoventral axes in enteropneust hemichordates are determined by similar genetic pathways to the chordates (Cameron et al., 2000; Zeng and Swalla, 2005).
Irrespective of whether an enteropneust or ptero- branch body plan is ancestral for hemichordates, the latest common ancestor of echinoderms and hemichordates is expected to have a diffuse nerve network, no mesodermal skeleton, tricoelomic development, a planktotrophic dipleurula larva and abrupt metamorphosis, a nephridial system, and pharyngeal gill slits.


9.6.3 Deuterostomia

There are three competing models for how the latest common ancestor to the deuterostome body was con- structed: an ambulacrarian model, a chordate model, or a xenoturbellid model. Given this uncertainty it is not yet clear whether the ancestral developmental mode would have been direct or indirect. Although both tunicates and ambulacrarians have larvae that are planktotrophic, they are very different in form and in the gene networks that are deployed dur- ing development (Swalla, 2006). Ambulacraria have dipleurula-type larvae that feed using ciliary bands, and there has been longstanding recognition that echinoderms and hemichordates larvae are hom- ologous (Hara et al., 2006). The tunicate larva is very different and clearly non-homologous, as well as non-feeding (Swalla, 2006). Ascidian tadpoles share a common plan with vertebrates including a noto- chord centred in the tail flanked dorsally by the ven- tral nerve chord, laterally by muscles, and ventrally by endoderm (Swalla, 2007). Both planktotrophic larval types presumably evolved independently in the late Precambrian at a time when the ocean sur- face waters were becoming rich in phytoplankton for the first time and benthic predation was dramat- ically increasing (Butterfield, 2007).
Ettensohn et al. (2003) have shown that there is a very similar homeodomain protein, Zlx1, in echi- noderms and chordates that controls downstream genes required for biomineralization. This sug- gests that the ancestral deuterostome possessed a mesenchymal cell lineage that was able to engage in biomineralization and that a Zlx1-like protein was involved in the specification of these cells. However, the genes used for biomineralization in echinoderms and vertebrates are a completely different (Bottjer et al., 2006), so the detailed biom- ineralization process evolved independently in echinoderms and vertebrates.
Given Xenoturbella’s phylogenetic position as basal to Ambulacraria (Bourlat et al., 2003, 2006) it is possible that the latest common ancestor to deu- terostomes was a small, delicate, ciliated marine worm with a simple body plan lacking a through gut, organized gonads, excretory structures, and coelomic cavities. Although this would accord with the long hidden history of deuterostomes suggested by molecular clocks (e.g. Davidson et al., 1995) and may indeed pertain to the very earliest stem-group deuterostomes, it is definitely not parsimonious to have shared derived features, such as pharyn- geal feeding and gill slits, a post-anal muscular tail, or stalk evolving independently in Chordates and Ambulacraria, and such features were surely present in the latest members of their stem group. There are therefore two realistic contenders:
The latest common ancestor might have been a benthic filter-feeding worm with gill slits similar to extant enteropneust worms (Cameron et al., 2000). The common features are a simple nerve plexus with- out regionalization, a pharynx with gill slits used in filter feeding, well-developed circular and longitu- dinal muscles, and direct development. However, this would imply that tentacles were independently evolved in echinoderms and pterobranchs.
Alternatively a tentaculate pterobranch-like organism was long a popular model for the primi- tive deuterostome based on the idea that hemi- chordates were primitive and lophophorates were ancestral (Gee, 1996). With modern molecular phy- logenies this has become less popular. However, it cannot be entirely dismissed if the latest common ancestor to Ambulacraria was pterobranch-like rather than enteropneust-like.

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9.7 Summary

Deuterostomes are a monophyletic group of ani- mals that include the vertebrates, invertebrate chor- dates, ambulacrarians, and xenoturbellids. Fossil representatives from most major deuterostome groups are found in the Lower Cambrian, sug- gesting that evolutionary divergence occurred in the late Precambrian, in agreement with molecular clocks. Molecular phylogenies, larval morphology, and the adult heart/kidney complex all support echinoderms and hemichordates as a sister group- ing (Ambulacraria). Xenoturbellids represent a relatively newly identified deuterostome phylum that lacks a fossil record, but molecular evidence suggests that these animals are a sister group to
the Ambulacraria. Within the chordates, lancelets share large stretches of chromosomal synteny with the vertebrates, have an intact Hox complex, and are sister group to the vertebrates according to riboso- mal and mitochondrial gene evidence. In contrast, tunicates have a highly derived adult body plan and are sister group to the vertebrates by phylogen- etic trees constructed from concatenated genomic sequences. Lancelets and hemichordates share gill slits and an acellular cartilage, suggesting that the ancestral deuterostome also shared these features. Gene network data suggest that the deuterostome ancestor had an anteroposterior axis specified by Hox and Wnt genes, a dorsoventral axis specified by a BMP/chordin gradient, and a left–right asym- metry determined by expression of nodal.