Coloniality has evolved once in Stolidobranch Ascidians (original) (raw)

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Friday Harbor Laboratories, University of Washington

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From the symposium “Complex Life-Histories of Marine Invertebrates” presented at the annual meeting of the Society for Integrative and Comparative Biology, January 4–8, 2005, San Diego, California.

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Liyun Zeng, Molly W. Jacobs, Billie J. Swalla, Coloniality has evolved once in Stolidobranch Ascidians, Integrative and Comparative Biology, Volume 46, Issue 3, June 2006, Pages 255–268, https://doi.org/10.1093/icb/icj035
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Abstract

Ascidians exhibit a rich array of body plans and life history strategies. Colonial species typically consist of zooids embedded in a common test and brood large, fully developed larvae, while solitary species live singly and usually free-spawn eggs that develop into small, undifferentiated larvae. Ascidians in the order Stolidobranchia include both colonial and solitary species, as well as several species with intermediate morphologies. These include social species, which are colonial but do not live completely embedded in a common test, and a few solitary species that brood embryos and larvae until they are competent to metamorphose. We examined how many times coloniality has evolved within the Stolidobranchia, with phylogenetic analyses using full-length 18S rDNA and partial cytochrome oxidase B sequences for taxa in the families Molgulidae, Styelidae, and Pyuridae. Tunicata orders Phlebobranchia and Stolidobranchia are sister groups, and the family Molgulidae is a monophyletic group and should be raised to the subordinal level, as shown previously by analyses from this lab with partial 18S sequences. In contrast to previous studies, styelids and pyurids are separated into monophyletic groups by ML and Bayesian analyses. We show a single clade within the family Styelidae that contains two colonial (compound) botryllid species, a Symplegma (colonial compound), a colonial (social) species Metandrocarpa taylori, as well as four solitary species, thus confirming that the botryllids are a subfamily of the Styelidae. These results suggest that the ancestor of the Stolidobranchia was solitary and that coloniality has evolved only once within this clade of ascidians. Further phylogenetic analyses of aplousobranch and phlebobranch ascidians will be necessary to understand the number of times that coloniality has evolved within the class Ascidiacea.

Introduction

Deuterostomes are a monophyletic group of animals (Cameron and others 2000) and can be further subdivided into two clades (Fig. 1I and II). One clade includes the phyla Echinodermata, Hemichordata, and Xenoturbellida, while the other consists of the chordates (Cameron and others 2000; Peterson and Eernisse 2001; Bourlat and others 2003; Smith and others 2004; Blair and Hedges 2005; Zeng and Swalla 2005). Phylum Chordata was originally divided into three subphyla: the Tunicata, Cephalochordata, and Vertebrata, which share five morphological characters (Rychel and others 2006). However, it has been suggested that the Tunicata should be raised to the phylum level (Kozloff 1990; Cameron and others 2000; Zeng and Swalla 2005).

Fig. 1

Major phylogenetic relationships of the deuterostomes are shown, modified from Zeng and Swalla (2005) and Smith and colleagues (2004). Deuterostomes include two major clades: (I) Phylum Echinodermata (classes Crinoidea, Asteroidea, Ophiuroidea, Holothuroidea, Echinoidea), phylum Hemichordata (family Harrimaniidae, order Pterobranchia, family Ptychoderidae), and phylum Xenoturbellida. Hemichordata and Echinodermata have ciliated, feeding larvae. (II) Phylum Chordata, including subphyla Tunicata, Cephalochordata, and Vertebrata. Tunicata include class Appendicularia, class Thaliacea, and class Ascidiacea (suborders Phlebobranchia, Aplousobranchia, and Stolidobranchia). Family Molgulidae is traditionally within suborder Stolidobranchia but should be a separate suborder. Suborder Aplousobranchia (Class Ascidiacea) and class Appendicularia have highly divergent rRNA sequences and have been difficult to place phylogenetically. Our analyses suggest two different placements of the Appendicularia, shown by dotted lines.

Many phylogenetic and developmental studies suggest that Cephalochordata and Vertebrata are sister groups, more closely related to each other than either is to the Tunicata (Turbeville and others 1994; Wada and Satoh 1994; Cameron and others 2000; Winchell and others 2002). However, recent genome analyses suggest that tunicates may be more closely related to vertebrates than cephalochordates (Blair and Hedges 2005; Philippe and others 2005; Delsuc and others 2006). Also, tunicates possess neural crest cells (Jeffery and others 2004) and placodes (Manni and others 2004; Bassham and Postlethwait 2005; Mazet and others 2005) that are lacking in cephalochordates. Tunicates typically have long branch-lengths, which confound phylogenetic analyses and create artifacts (Blair and Hedges 2005; Zeng and Swalla 2005; Delsuc and others, 2006). In summary, the placement of the tunicates within deuterostomes has been problematic (Winchell and others 2002; Blair and Hedges 2005; Zeng and Swalla 2005; Delsuc and others 2006), even though studies have shown that tunicates are monophyletic (Swalla and others 2000; Stach and Turbeville 2002; Winchell and others 2002).

Ascidians, or sea squirts, are members of the class Ascidiacea, within Tunicata, that exhibit diverse life history strategies (Satoh 1994; Burighel and Cloney 1997; Davidson and others 2004). Ascidian tadpoles have key chordate characteristics such as a notochord and a dorsal hollow nerve cord (Swalla 2004a, 2004b; Fig. 2A), but these traits are lost after metamorphosis. Adult ascidians may be solitary and sexual or colonial and alternating between sexual and asexual reproduction by budding (Fig. 2) (Berrill 1935, 1936; Nakauchi 1982; Burighel and Cloney 1997). Colonial ascidians (Fig. 2B and C) tend to be ovoviviparous, producing large eggs and releasing adultated larvae that stay in the water column for only a short period of time before settling and initiating metamorphosis into the adult form (Fig. 2A) (Berrill 1935, 1936; Jeffery and Swalla 1992; Burighel and Cloney 1997; Davidson and others 2004). Solitary ascidians (Fig. 2D–G) either release large numbers of relatively small eggs into the water column, where fertilization and subsequent development into tadpole larvae takes place, or brood large, highly differentiated larvae (Berrill 1935).

Fig. 2

Photographs of selected species from class Ascidiacea used in the study: (A–F) are in the suborder Stolidobranchia; G is in the suborder Phlebobranchia. (A) The tadpole of Botrylloides violaceus, family Styelidae, colonial (compound). (B) An adult B. violaceus, Styelidae, colonial (compound). Adults of (C) Metandrocarpa taylori, Styelidae, colonial (social). (D) Cnemidocarpa finmarkiensis, Styelidae, solitary. (E) Styela gibbsii, Styelidae, solitary. (F) Halocynthia igaboja, Pyuridae, solitary. (G) Corella inflata, Corellidae, solitary. Our study shows that (A), (B), (C), and (D) belong to a Styelidae clade which contains both colonial and solitary species. (E) belongs to another clade of Styelidae, which are all solitary species. (F) H. igaboja is in the clade Pyuridae, a family related to Styelidae. (G) C. inflata is in a different order, Phlebobranchia.

Ascidians were originally divided into colonial and solitary species by taxonomists, but in the early part of the 20th century classification based on branchial sac and gonad morphology became universally accepted (Van Name 1945; Berrill 1950; Nishikawa 1990; Kott 1998; Monniot F and Monniot C 2001; Monniot and others 2001; Lambert 2005). Recently, phylogenies based on DNA sequences have helped to clarify some evolutionary relationships among the tunicates, although most phylogenies are entirely consistent with the taxonomic relationships (Wada and others 1992; Hadfield and others 1995; Wada 1998; Cameron and others 2000; Swalla and others 2000; Stach and Turbeville 2002; Winchell and others 2002; Turon and López-Legentil 2004). Wada and colleagues (1992) examined the evolution of coloniality and concluded that coloniality has evolved several times within class Ascidiacea, but did not identify particular clades within orders.

The ascidian order Stolidobranchia contains three widely acknowledged families: Molgulidae, Pyuridae, and Styelidae. Recent studies have shown that solitary molgulids are a monophyletic group, but styelids and solitary pyurids have remained unresolved by previous analyses (Wada and others 1992; Swalla and others 2000; Stach and Turbeville 2002). Colonial species are currently distributed among several taxonomically separate groups within the family Styelidae (Berrill 1950; Kott 1985). Many Tunicata taxonomists presently include the colonial (compound) botryllids as a subfamily within styelids (Kott 1998; Monniot F and Monniot C 2001; Monniot and others 2001; Saito and others 2001), while Nishikawa (1990, 1995) considers Botryllidae a separate family, partially because of their coloniality.

The diversity of life histories and morphologies among the ascidians make them an excellent group in which to examine life history evolution. For example, some species within the families Molgulidae and Styelidae bypass the tadpole stage altogether and exhibit direct (anural) development (Jeffery and Swalla 1992; Hadfield and others 1995; Huber and others 2000). When the developmental mode is mapped onto a phylogeny, then it is clear that direct (anural) development has evolved more than once within the Molgulidae, suggesting the possibility that anural development may be mediated by a conserved switch that can be activated with relative ease in evolutionary time (Hadfield and others 1995; Swalla and Jeffery 1996; Huber and others 2000).

Kott (1989) has suggested an additional family within the Stolidobranchia of deep-sea tunicates, the Hexacrobylidae, while the Monniots have created a separate class, Sorberacea, for them (Monniot and others 1975; Monniot C and Monniot F 1990). Unfortunately, we have not been able to obtain any Sorberaceans for phylogenetic studies, but the described species are solitary and morphologically similar to the Molgulidae (Monniot and others 1975; Kott 1989; Monniot C and Monniot F 1990).

The family Styelidae is particularly interesting because it contains both colonial and solitary species as well as a number of species with intermediate morphologies (Figure 2, Table 1, Van Name 1945). For example, Metandrocarpa taylori and other social colonial species reproduce clonally, brood large larvae, and have reduced adult body size similar to other colonial species, but zooids are connected only by stolons rather than being completely embedded together in a common test (Fig. 2C; Van Name 1945). Styelidae also contain Dendrodoa grossularia, which does not reproduce clonally but does brood very large larvae and grows in large dense clusters that superficially resemble social colonies (Van Name 1945); however, the individuals in these clusters are not necessarily close relatives (Bishop and Ryland 1993). The styelid species Polycarpa pomaria has also been observed to brood larvae under laboratory conditions, although the larvae are small and it is unclear whether this normally occurs in nature (Berrill 1950; Svane and Young 1989). It is intriguing that the evolution of brooding and large offspring size may be related to the evolution of coloniality, but to date there has been no phylogenetic evidence for this hypothesis.

Examination of relationships within botryllids, which are all colonial, are interesting in light of understanding allorecognition (Cohen and others 1998) and population dynamics (Stoner and others 1997; Stoner and others 2002) as well as higher taxonomic level phylogenies. Our study uses complete 18S rDNA sequences and the partial mitochondrial cytochrome oxidase B (cob) gene for twenty-eight species in thirteen genera to construct a phylogeny of the Stolidobranchia (Table 1). We use this phylogeny to test the hypotheses that coloniality has evolved multiple times in the Stolidobranchia. We show that two different botryllid species and one Symplegma group closely together in phylogenetic analyses, suggesting that these colonial compound species are monophyletic. We show that coloniality is likely to have evolved only once within the Stolidobranchia because the colonial social species M. taylori falls as a sister group to the colonial compound clade.

Table 1

18S rDNA sequences and references for all Tunicata (Urochordata)

Table 1

18S rDNA sequences and references for all Tunicata (Urochordata)

Materials and methods

Biological materials, DNA isolation, and DNA sequencing

M. taylori was collected on Tatoosh Island, WA or by dredging near San Juan Island and maintained in a tank of recirculating or running seawater. Boltenia villosa, Botrylloides violaceus, and Corella inflata were collected off the docks at Roche Harbor, WA, on San Juan Island. Styela gibbsii and Cnemidocarpa finmarkiensis were collected from the docks at Friday Harbor Laboratories in Friday Harbor, WA. Botryllus schlosseri was collected from the docks of Shilshole Marina in Seattle, WA. Dendrodoa grossularia was collected from rocks at Roscoff, France. Botryllus planus, Symplegma viride, and Polycarpa papillata were collected by BJS in Puerto Rico, while teaching an Evolution and Development course at University of Puerto Rico. For colonial ascidians, individual zooids were dissected by hand, taking care to discard parasites and food items in the colony. Solitary ascidians were dissected free of their tunics with similar care, and either gonad or mantle (in non-gravid individuals) was dissected out, macerated, and used for extraction. Genomic DNA was isolated according to Hadfield and colleagues (1995) and amplified and purified according to Swalla and colleagues (2000). Primers used to amplify mitochondrial cob gene sequences from genomic DNA were CobF 5′-TGR GGN CAR ATG WSN TTY TG-3′ and CobR 5′-GGR AAN ARR AAR TAY CAY TC-3′ (Turon and others 2003). GenBank accession numbers for the mitochondrial cytochrome oxidase B sequences are DQ345907–DQ34592. Ciona intestinalis (GenBank accession no. NC004447; Gissi and others 2004), Ciona savignyi (GenBank accession no. NC004570; Yokobori and others 2003), and Doliolum nationalis (NC006627; Yokobori and others 2005) sequences were taken from the entire mitochondrial sequence. Sequencing was performed at the Biochemistry Sequencing Facility and the Biology Comparative Genomics Facility at the University of Washington.

Sequences, alignments, and phylogenetic analyses

The 18S rDNA sequences used for this study were mostly sequenced in our lab, but we also included a few additional species from GenBank (Table 1). Alignment of the ascidian 18S rDNAs was performed using Clustal W (Thompson and others 1994). There will be few gaps in the 18S rDNA alignment for tunicates if aplousobranch sequences are not included. Mitochondrial cytochrome oxidase B sequences were translated with an ascidian mitochondrial code in MacVector, and then aligned with Clustal W. Protein alignments were used to accurately align the nucleotide sequences. One Appendicularian, Oikopleura dioica, was included in the analysis. Sites containing gaps were excluded from phylogenetic analyses to reduce systematic errors. Alignments were analyzed with PAUP*4.0b2 (Swofford 1999) to produce bootstrap maximum parsimony (MP) trees and neighbor-joining (NJ) trees (Saitou and Nei 1987). NJ trees were built using a Kimura two-parameter model in PAUP (Kimura 1980). We used α = 0.50 for the γ distribution model. A Minimum Evolution (ME) tree was produced by heuristic searches in PAUP* under the same models of nucleotide substitution described above for NJ tree. Bootstrap maximum parsimony was calculated with PAUP*. We used the program MODELTEST 3.06 to find the best model and parameters to build the Maximum Likelihood (ML) trees. Confidence in NJ, ME, and MP trees were determined by analyzing 1000 bootstrap replicates and ML was determined by analyzing 100 bootstrap replicates (Felsenstein 1985). We built the Bayesian trees using MrBayes with 1 × 106 generation repeats with the nucleotide model 4by4, Nst = 6, rates =invgamma, Ngammacat = 4, and Burnin = 155 (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003; Ronquist and others 2005). Tree reliability was also determined by comparing trees based on the same data, but produced with different tree-making algorithms (NJ, ME, MP, ML, and Bayesian).

Results

We constructed expanded molecular phylogenies of the Styelidae and Pyuridae in order to examine the evolution and speciation of colonial and solitary species within the ascidian order Stolidobranchia. We included two colonial (compound) botryllids, (Botrylloides, Botryllus), one colonial (compound) Symplegma, the colonial (social) M. taylori, and several new solitary species in our analyses (Fig. 2; Table 1). A total of forty-one tunicate species were analyzed in the present study (Table 1). Twenty-eight taxa are in order Stolidobranchia, including eleven species from suborder or family Molgulidae, twelve species from family Styelidae (including three botryllids) and five species from family Pyuridae; nine taxa are in the order Phlebobranchia, and four are tunicates from outside of class Ascidiacea, including three species from class Thaliacea and one from class Appendicularia (Fenaux 1993; Table 1). The complete alignment of 18S rDNA of forty-seven chordate species with thirty-seven ascidians and six outgroup taxa (five vertebrate species and one cephalochordate species) contained ∼1810 sites including gaps. This alignment is available on-line at Author Webpage. Full-length 18S rDNA sequences were subjected to phylogenetic analysis using MP, NJ, and Bayesian algorithms. The MP, NJ, ME, ML, and Bayesian trees were very similar and are available on-line (Supplementary Data). Fifteen equally parsimonious MP trees were recovered by using heuristic searches and 1000 bootstrap replicates with PAUP. The NJ analysis, calculated with Kimura two-parameter evolutionary distances and 1000 bootstrap pseudoreplicates was completely congruent with the tree resulting from a ME analysis with 1000 bootstrap pseudoreplicates. Figure 3 shows the combined tree resulting from the MP analysis with bootstrap values from MP, NJ, and ME with branches drawn to scale.

Fig. 3

Maximum parsimony (MP), neighbor-joining (NJ), and minimum evolution (ME) combined trees generated from the 18S rRNA dataset. Majority rule consensus with 1000 bootstrap replicates and 1000 random-addition full heuristic search of a MP tree recovered by using PAUP. NJ tree generated by PAUP using complete 18S rDNA sequences of forty-one tunicate species, branches drawn to scale. Five species of Vertebrata and one species of Cephalochordata were used to root the tree. Bootstrap values are shown as percentages of 1000 replicates at each node only when ≥50%. The 1000 replicates bootstrap ME tree has the same topology as the NJ tree. The bootstrap values of MP, NJ, and ME are put at each node of the tree, separated by slashes. Ascidian species' life histories are marked as compound, social, and solitary with icons.

In this MP, NJ, and ME combined tree, Stolidobranchia contains two distinct clades, the suborder Molgulidae and the families Styelidae +Pyuridae. Colonial compound (B. violaceus, B. planus, S. viride) and colonial social (M. taylori) species group together with the solitary brooding species D. grossularia, the solitary occasional brooder P. pomaria, solitary P. papillata, and the solitary free spawner C. finmarkiensis. The other clade in family Styelidae is made up entirely of solitary free-spawning species. The node separating the two clades within the Styelidae is supported by 99, 100 and 100% bootstrap values, indicating high support for this node. The clustering of compound and social species within one clade of the Styelidae suggests a single evolutionary origin of coloniality within the Stolidobranchia. In MP, NJ, and ME trees, Appendicularia always falls as a sister group to the rest of the tunicates, although with low bootstrap support (52%/62%/58%). The MP tree shown (Fig. 3) is consistent with the NJ and ME trees with only minor differences (supplementary materials). In MP analyses, family Pyuridae is paraphyletic, but the Styelidae are a monophyletic group with high bootstrap support (supplementary materials). In contrast, trees constructed with NJ and ME algorithms recover family Pyuridae as a monophyletic group, with 68 and 80% bootstrap support (supplementary materials, see also Zeng and Swalla 2005).

Figure 4 shows a combined tree of ML and Bayesian analysis. The ML tree had 100 bootstrap replicates (Fig. 4 and supplementary material) with a reduced dataset (complete alignment, but only twenty-eight taxa). The Bayesian tree had a full dataset 1 × 106 generation repeats (Fig. 4 and supplementary material) and was mostly congruent within the stolidobranchs. The interesting placement of taxa in the ML and Bayesian trees is the position of class Appendicularia, as shown in the combined tree in Fig. 4. In NJ, ME, and MP analyses (Fig. 3), the Appendicularia fall as a sister group to the rest of the Tunicata, as suggested by previous analyses (Swalla and others 2000; Wada 1998; Stach and Turbeville 2002). In contrast, the ML and Bayesian trees place the Appendicularia as a sister group to the suborder Stolidobranchia, rather than a sister group to the rest of the tunicates, with bootstrap support of 85% and a posterior probability value of 67% (Fig. 4 and supplementary material). However intriguing, these results should be interpreted with caution because of the long branches of the Appendicularia taxa (Swalla and others 2000).

Fig. 4

Bayesian and Maximum Likelihood (ML) combined tree generated form the 18S rDNA dataset. Bayesian Tree was built by MrBayes 3.1 with 1 000 000 generation repeats using 4by4 nucleotide model and Nst = 6, rates = invgamma, Ngammacat = 4, and burnin = 155. ML tree was recovered by using TrN+I+G model in PAUP. The parameters used to build the ML tree are tested by the program MODELTEST 3.06. Nodes with <50% bootstrap support are shown collapsed. The ML bootstrap value is placed on the right side of the posterior probability value at the supported node. The Bayesian/ML combined tree has almost the same structure as NJ, ME, and MP trees. Styelidae species with colonial and social life histories are in a single clade with a few solitary species, which is supported by high posterior probability and ML bootstrap value. Unlike the MP/NJ/ME combined tree (Fig. 3), Oikopleura grouped as a sister group with stolidobranchs although the posterior probability and bootstrap values are low. The Bayesian/ML combined tree strongly supports a single origin of coloniality in stolidobranch ascidians. Ascidian species' life histories are marked as compound, social, and solitary with icons.

The 18S rDNA was not divergent enough to resolve relationships well within the styelid clade that contains the colonial species (Figs 3 and 4). We first attempted to clarify relationships within this clade by sequencing the hypervariable D-loop of 28S rDNA, but there was much less variability within Styelidae than previous studies had shown within Molgulidae (Hadfield and others 1995; Huber and others 2000). We found 591 total characters within the 28S rDNA fragment, 145 characters were constant, 326 variable characters were parsimony-uninformative, and only 120 were parsimony informative, so the trees are not shown. Mitochondrial genomes evolve much faster than the nuclear genome, so we sequenced part of the mitochondrial protein-coding gene, cytochrome oxidase B (cob). The sequenced fragment of cob is only ∼420 bp long, but it has 287 parsimony informative sites, approximately three times as much variation as the 28S rDNA gene fragment.

Figure 5 shows the phylogenetic trees of stolidobranch families Styelidae (twelve species), Pyuridae (four species), phlebobranch family Cionidae (two species), and a single member of class Thaliacea using NJ and MP algorithms. We also built DNA and protein trees using Bayesian algorithms, but the results had almost the same topology as NJ and MP trees shown in Figure 5. NJ tree shows the result of 1000 bootstrap replicates and the MP tree generated by 1000 bootstrap repeat with 1000 random replication in PAUP* (Fig. 5). In these trees, the styelid and botryllid clades have the same topology, and the trees differ only by whether the pyurids come out monophyletic (MP) or paraphyletic (NJ) (Fig. 5). M. taylori (colonial, social), B. violaceus (colonial, compound), B. planus (colonial, compound), B. schlosseri (colonial, compound), S. viride (colonial, compound), P. papillata (solitary), and C. finmarkiensis (solitary) group together, while the solitary P. pomaria and D. grossularia form a second clade. In summary, whether the pyurids are monophyletic or paraphyletic is unresolved by these analyses, but the mitochondrial phylogenetic trees show a single clade within the Stolidobranchs that contains all the colonial species, both compound and social. These data strongly support the single origin of coloniality in Stolidobranch ascidians.

Fig. 5

NJ and MP combined trees showing phylogenetic relationships within the suborder Stolidobranchia, using 420 bp of a mitochondrial cob gene and rooted with two species of Cionidae and one species of Doliolidae (Class Thaliacea). The Kimura two-parameter distance estimation method was used with 1000 bootstrap replicates. MP tree was generated with 1000 bootstrap repeats and 1000 random heuristic searches. NJ and MP's bootstrap values are located at each node of tree. Styelidae species with compound, social, and solitary lifestyles are indicated with icons.

Discussion

18S rRNA genes have become popular tools for phylogenetic inferences because they are relatively easy to isolate, ubiquitous, and not prone to lateral gene transfer (Olsen 1988). Questions have been raised about the suitability of 18S data for reconstructing deep phylogenetic relationships because 18S rRNA exhibits variation in evolutionary rates, both between taxa and between sites on the molecule itself (Abouheif and others 1997). Within the Tunicata, however, a conserved 1 kb portion of the 18S rRNA resolves relationships between ascidian families much better than mitochondrial genes (Hadfield and others 1995; Wada 1998; Swalla and others 2000; Stach and Turbeville 2002). This is likely due to large genetic distances within the Tunicata that have not been previously appreciated (Swalla and others 2000; Zeng and Swalla 2005). Relative rate tests have shown that the suborder Molgulida and class Appendicularia (Fenaux 1993) are evolving significantly faster than other tunicate clades (Huber and others 2000), but both of these clades are morphologically and molecularly distinct monophyletic groups. Since neither of these groups was used as an outgroup, this variation in rates is unlikely to substantively affect our results.

This study used the entire 18S rDNA molecule and partial mitochondrial cytochrome oxidase B sequences to examine phylogenetic relationships within one tunicate order, the Stolidobranchia. Including the more variable 5′ and 3′ regions of the 18S rDNA molecule increased the resolution of the analyses significantly relative to previous analyses using only a conserved 1 kb portion (Hadfield and others 1995; Swalla and others 2000; Stach and Turbeville 2002), but the Tunicata 18S sequences are easy to align, as there are few gaps, insertions, or deletions (see TreeBase Author Webpage) (study accession number S1440; matrix accession numbers M2591 and M2592). Our phylogenies show stolidobranchs to be monophyletic, although we would very much like to include the benthic deep-sea Hexacrobylidae (Kott 1989; Monniot C and Monniot F 1990) if specimens were available to us. All member of this class described so far are solitary, so the inclusion of this group of tunicates would not significantly affect the conclusions of this article.

In all analyses presented here, colonial (compound and social) species group together with the solitary brooder D. grossularia, the possibly brooding P. pomaria, and the solitary non-brooders P. papillata and C. finmarkiensis. Therefore, coloniality evolved only once within the Stolidobranchia and the stolidobranch common ancestor was likely to be solitary. M. taylori, the morphologically ambiguous social species, may be in the process of becoming less or more integrated as a colony. M. taylori buds from oozooids, (Watanabe and Newberry 1976), similar to colonial ascidians (Nakauchi 1982), so it is likely that the process of asexual reproduction is conserved between these species. Similarly, large larval size and brooding in D. grossularia and possibly brooding in P. pomaria may represent transitional states en route to the loss or gain of coloniality. Further phylogenetic efforts should focus on gaining better resolution of the “colonial” clade within the Stolidobranchia.

The solitary Molgulidae are a single monophyletic group that should be raised from the familial to the subordinal level. We propose renaming it Molgulida. The other recognized families of the Stolidobranchia are Styelidae (which includes botryllids for most taxonomists) and Pyuridae. There is some conflict in the data concerning the monophyly of the Pyuridae, but each analysis recovered Pyuridae + Styelidae as a monophyletic group with the botyllids coming out as a monophyletic group within the Styelidae, thus confirming its status as a subfamily (Kott 1998; Monniot F and Monniot C 2001; Monniot and others 2001; Saito and others 2001). The clade containing Styela gibbsii, Styela montereyensis, Styela plicata, and Pelonaia corrugata was recovered in all analyses, suggesting that this is a closely related monophyletic group of solitary species. It is an interesting grouping because P. corrugata has tailless larvae while the rest of the species in the clade have tailed larvae (Hadfield and others 1995).

Coloniality and the ability to reproduce asexually may be strongly selected for in certain environments (Nakauchi 1982; Satoh 1994). However, only a few phyla within the invertebrates contain colonial species (Davidson and others 2004). Shifting between colonial and solitary lifestyles involves alteration of a whole suite of life history characteristics, and it is possible that developmental or morphological constraints make that transition difficult to accomplish (Davidson and others 2004). Groups such as Styelidae, which contain a range of species from solitary to compound colonial, are particularly exciting from the point of view of an evolutionary developmental biologist.

Aplousobranchia, an order within the ascidians, are all colonial but have been particularly problematic to place phylogenetically (Stach and Turbeville 2002; Winchell and others 2002; Turon and López-Legentil 2004; Zeng and Swalla 2005). These species have long branches, and the zooids are small, leaving them prone to contamination artifacts (Stach and Turbeville 2002) as discussed in Yokobori and others (2006). There are three major hypotheses of tunicate evolution (Fig. 6). The first hypothesis (Fig. 6A) suggests that Appendicularia and Aplousobranchia are grouped together as a sister group of the rest of the tunicates, which is supported by Stach and Turbeville (2002). This relationship may be suspect because of the long-branch attraction of Appendicularia and Aplousobranchia 18S rDNA sequences. The second view (Fig. 6B) is supported by mitochondrial COI gene analysis (Turon and López-Legentil 2004) and suggests that Aplousobranchia is a sister group to Stolidobranchia, and then Phlebobranchia and Thaliacea are sister groups to them. Unfortunately, this analysis did not include any Appendicularia. A final hypothesis (Fig. 6C) shows Aplousobranchia grouped with Thaliacea and then grouped with Phlebobranchia, and collectively are a sister group to Stolidobranchia and Appendicularia. This hypothesis is partially supported by our analysis in the 18S rDNA Maximum Likelihood tree and Bayesian tree (Fig. 4), but the bootstrap and posterior probability values are not high enough (85 and 67%). These relationships are also supported by the recent trees published with Aplousobranchia 18S (Yokobori and others 2006). The position of Aplousobranchia and Appendicularia is still somewhat unresolved within the tunicates and needs further analysis. The placement of the colonial Aplousobranchia within the rest of the tunicates is critical to understand whether the tunicate ancestor was solitary or colonial (Zeng and Swalla 2005). When the entire Tunicate phylogeny is better resolved, detailed comparative analysis of development may begin to elucidate the critical steps in life history evolution that can lead to a switch between solitary and colonial lifestyles.

Fig. 6

There are three hypotheses concerning the position of Aplousobranchia within the Tunicata. (A) Aplousobranchia is grouped with Appendicularia, which together fall as a sister group to the rest of the tunicates. (B) Aplousobranchia is the sister group to the Stolidobranchia. (C) Appendicularia is the sister group to Stolidobranchia and Aplousobranchia are a sister group to Thaliacea. Hypothesis B is supported by recently published mitochondrial data (Turon and Lopez-Legentil 2004). (C) is partially supported by this article and Yokobori's recently Aplousobranch sequence data (Yokobori and others 2006). (Stolido = Stolidobranchia, Phlebo = Phlebobranchia, Thalia = Thaliacea, Aplouso = Aplousobranchia, and Appendi = Appendicularia).

This article is dedicated to the spirit of Dr Larry McEdward, the “Larval Marvel.” Larry was a great colleague and a dear friend. A warm thanks to Dr Eduardo Rosa-Molinar and the Evo-Devo class of 2001 at the University of Puerto Rico for helping collect some of the species used in these analyses (BIOL 6999: Special Topics in Modern Biology; Author Webpage). We would like to thank Dawn Vaughn, who contributed to this work during a rotation in the Swalla Lab in the fall of 2003, and J. Muse Davis, who helped with some of the initial sequencing. Bob Paine and Chris Harley are thanked for collecting samples of M. taylori on Tatoosh Island. Cory Bishop and Bryan Crawford are thanked for the beautiful underwater photos of adult ascidians shown in Figure 2B–E. We would like to thank Professor Ken Warheit, Professor Scott Edwards, and Chris Hess for their help in performing initial analyses in Molecular Evolution classes at the University of Washington. Dr Dennis Willows, former Director of Friday Harbor Laboratories, is thanked for his encouragement throughout the project. The FHL staff members, especially Scott Schwinge, Kathleen MacDanold, and Blanche Bybee, are thanked for lab space, housing, and supplies, respectively. Sequencing was performed in the Comparative Genomics Center in the Biology Department at the University of Washington, funded in part by Major Research Instrumentation Grant no. 2002236 from the Murdock Foundation. This work was supported by an International Graduate Fellowship to L.Z., an NSF graduate research fellowship to M.W.J., and by a Seaver Institute and UW Department of Biology grant to B.J.S. Conflict of interest: none declared.

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Author notes

From the symposium “Complex Life-Histories of Marine Invertebrates” presented at the annual meeting of the Society for Integrative and Comparative Biology, January 4–8, 2005, San Diego, California.

© The Author 2006. Published by Oxford University Press on behalf of The Society for Integrative and Comparative Biology. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org.

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