Musculature in sipunculan worms: ontogeny and ancestral states

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Musculature in sipunculan worms: ontogeny and ancestral states

Anja Schulzeand Mary E. Rice

Smithsonian Marine Station, 701 Seaway Drive, Fort Pierce, FL 34949, USA

Author for correspondence (email: [email protected]).

Present address: Department of Marine Biology, Texas A & M University at Galveston, 5007 Avenue U, Galveston, TX 77551, USA.

SUMMARY Molecular phylogenetics suggests that the Sipuncula fall into the Annelida, although they are morphologically very distinct and lack segmentation. To understand the evolutionary transformations from the annelid to the sipunculan body plan, it is important to reconstruct the ancestral states within the respective clades at all life history stages. Here we reconstruct the ancestral states for the head/

introvert retractor muscles and the body wall musculature in the Sipuncula using Bayesian statistics. In addition, we describe the ontogenetic transformations of the two muscle systems in four sipunculan species with different developmental modes, using F-actin staining with fluorescent-labeled phalloidin in conjunction with confocal laser scanning microscopy. All four species, which have smooth body wall musculature and less than the full set of four

introvert retractor muscles as adults, go through developmental stages with four retractor muscles that are eventually reduced to a lower number in the adult. The circular and sometimes the longitudinal body wall musculature are split into bands that later transform into a smooth sheath.

Our ancestral state reconstructions suggest with nearly 100%

probability that the ancestral sipunculan had four introvert retractor muscles, longitudinal body wall musculature in bands and circular body wall musculature arranged as a smooth sheath. Species with crawling larvae have more strongly developed body wall musculature than those with swimming larvae. To interpret our findings in the context of annelid evolution, a more solid phylogenetic framework is needed for the entire group and more data on ontogenetic transformations of annelid musculature are desirable.

INTRODUCTION

Sipuncula have long been regarded as a distinct protostome phylum (Hyman 1959; Stephen and Edmonds 1972; Cutler 1994). Most analyses have placed them in the lophotrochozoan clade (Zrzavy´ et al. 1998; Giribet et al. 2000; Peterson and Ernisse 2001; Passamaneck and Halanych 2006), but until re- cently relationships within the lophotrochozoans were largely unresolved. Today there is a growing consensus that sipunculans fall into the annelids (Boore and Staton 2002; Bleidorn et al. 2005; Struck et al. 2007; Dunn et al. 2008), sparking a renewed interest in their morphology and development.

As adults, their body is divided into a trunk and a re- tractable introvert with a crown of tentacles at its anterior end, showing little resemblance to any known annelid group.

More similarities to annelids are apparent in the larval stages, such as the trochophore larva with prototrochal and metatrochal ciliary bands, the retention of the egg envelope to form the larval cuticle and the paired ventral nerve cord in the pelagosphera larva of several species (Rice 1985). On the other hand, the absence of morphological segmentation in any life history stage is striking (Wanninger et al. 2005).

In general, to understand the morphological transformations from one body plan to another, it is important to determine the ancestral states, that is character states at the

roots of the respective clades. Whenever possible this should encompass all life history stages, because some stages may retain ancestral states more readily than others. This is ex- empliﬁed in the Echiura, in which only the larvae retain a segmented nervous system, indicative of their annelid origins (Hessling and Westheide 2002). Ontogenetic transformations can also be informative characters themselves.

In the Sipuncula, the body wall musculature and the introvert retractor muscles are important taxonomic characters.

The longitudinal and circular body wall muscles are either arranged in continuous sheaths or are broken up into numerous bands. The number of introvert retractor muscles in the adults varies from one to four. They originate in the body wall of the trunk and insert in the head region.

Here we reconstruct the ancestral states of the two muscle systems using Bayesian statistics. For this purpose, we rean- alyze a simplified dataset of sipunculan sequence and morphological data previously generated by the first author and collaborators (Schulze et al. 2007). In addition, we analyze the ontogenetic transformations of the musculature using phalloidin-staining of F-actin and confocal laser scanning microscopy. As these transformations may depend on the developmental mode of the species, we studied them in four sipunculan species displaying different developmental patterns.

DOI: 10.1111/j.1525-142X.2008.00306.x

&2009 The Author(s)

Journal compilation&2009 Wiley Periodicals, Inc.

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Rice (1967, 1975a, b, 1976) describes four distinct developmental patterns in the Sipuncula (Fig. 1): (1) direct development; (2) one pelagic larval stage: lecithotrophic trochophore;

(3) two pelagic larval stages: lecithotrophic trochophore and lecithotrophic pelagosphera; (4) two pelagic larval stages: lecithotrophic trochophore and planktotrophic pelagosphera.

The majority of sipunculan species of which development has been studied, develop according to mode IV. The lecithotrophic trochophore stage is generally short lived, but the pelagosphera may remain pelagic for 4–8 months (Scheltema and Hall 1975). Pelagosphera larvae swim by means of a strongly developed metatrochal ciliary band. When disturbed, their head region, including the metatroch, completely retracts into the trunk by a contraction of the head retractor muscles.

The head retractor muscles eventually transform into the introvert retractor muscles of the juvenile.

The four species we chose for the present study all have less than the full set of four introvert retractor muscles and continuous layers of circular and longitudinal body wall musculature. Phascolion cryptum(Fig. 2A) displays developmental mode I (Rice 1975a). In the adult stage, a single introvert retractor muscle is present with two separate roots at the I

II

III

IV

Fig. 1. The four developmental modes in the Sipuncula. I. Direct development. II. Indirect development with a single pelagic stage, the lecithotrophic trochophore. III. Indirect development with a lecithotrophic trochophore and a lecithotrophic pelagosphera. IV.

Indirect development with a lecithotrophic trochophore and a planktotrophic pelagosphera. (Modiﬁed from Rice 1975a, b.)

A

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dr C

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ee

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G cm

lm

np a

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dr vr Fig. 2. Developmental stages ofPhascolion cryptum; (A) Introvert

of adult emerging from gastropod shell, macrophotography; (B) Drawing of interior anatomy of adult; note single introvert retractor muscle with two roots (arrows); (C) scanning electron micrograph of juvenile in the process of elongation; 36 h post fertilization (p.f.); egg envelope still clearly visible in anterior part; (D–G) juvenile stages, confocal laser scanning projections after ﬂuorescent staining for F-actin with phalloidin Alexa 488. (D) Juvenile, 36 h p.f., dorsal slice. (E) Same specimen as (D), ventral slice. (F) Crawling stage, dorsal view, 3 days p.f.; note distinct bands of longitudinal and circular body wall musculature. (G) Same specimen, interior slice; note four head retractor muscles. a, anus; cm, circular body wall musculature; dr, dorsal head retractor muscles;

e, esophagus; ee, egg envelope; ir, introvert retractor muscle; j, juvenile; lm, longitudinal body wall musculature; nc, nerve cord;

np, nephridium; vr, ventral head retractor muscles. Scale bars:

20mm.

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posterior end of the trunk (Fig. 2B). Themiste lageniformis (Fig. 3A) develops according to mode III (Pilger 1987). Its pelagosphera larva is capable of swimming short distances but spends most of its time crawling on the bottom. The adult has two introvert retractor muscles originating in the posterior third of the trunk (Fig. 3B).Phascolion psammophilum (Fig.

4A) also displays developmental mode III (Rice 1993a), but its pelagosphera larva is purely pelagic. As an adult, the species has a single large dorsal retractor muscle and a weaker ventral muscle with two separate roots (Fig. 4B). The fourth species,Nephasoma pellucidum(Fig. 5A) develops according to mode IV (M. E. Rice, personal observation). It has two introvert retractor muscles as an adult (Fig. 5B).

MATERIAL AND METHODS

Ancestral state reconstruction

Ancestral state reconstruction was performed by reanalyzing a simpliﬁed version of the dataset ﬁrst presented by Schulze et al.

(2007), with a constrained topology, using Bayesian statistics. The analysis included the same four gene regions (18S ribosomal RNA, 28S ribosomal RNA, Histone H3, and cytochromecoxidase subunit I) (Table 1) and 58 morphological characters and utilized the same mixed models for the diﬀerent data partitions. One diﬀerence to the original analysis was that due to computing constraints only 1,000,000 generations of Monte Carlo Markov chains were performed instead of 1,500,000. Of the 1,000,000 generations the initial 500,000 were discarded as burn-in. Another change from the pre- vious analysis was that for most species only single representatives were included in the analysis, except for two species that clearly appeared to be polyphyletic in the initial analysis. The two species were Aspidosiphon parvulus and Phascolosoma granulatum (see comments on their status in Schulze et al. 2007).

Ancestral states were reconstructed for the root node of all Sipuncula. To accomplish ancestral state reconstruction, the topology was constrained to render the Sipuncula monophyletic. The high support for sipunculan monophyly in all recent analyses (Maxmen et al. 2003; Staton 2003; Schulze et al. 2005, 2007) jus- tiﬁed constraining the topology. Ancestral states were only reconstructed for the morphological characters.

Collecting adults

Adults of the four species were collected in March and April 2003.

P. cryptuminhabits abandoned gastropod shells, which were sieved from intertidal sand in the vicinity of the Harbor Branch Ocean- ographic Institution, Fort Pierce, FL. Specimens ofT. lageniformis were retrieved from the crevices of oyster shells around Jack Island, Indian River Lagoon, near the Fort Pierce Inlet.P. psammophilum, an interstitial species, was collected 6 miles oﬀshore from Fort Pierce, using sediment dredges on the R/V SUNBURST and

Fig. 3. Developmental stages ofThemiste lageniformis. (A) Adult, macrophotography; (B) Drawing of interior anatomy; note two introvert retractor muscles; (C, D, F, H, I) Confocal laser scanning projections after ﬂuorescent staining with phalloidin Alexa 488, (E, G) Scanning electron micrographs. (C) Early pelagosphera, 37 h post fertilization (p.f.), dorsal slice. (D) Same specimen as (C), ventral slice. (E) Pelagosphera larva, 5 days p.f., lateral view. (F) Pelagosphera larva, 5 days p.f.; note strongly developed circular body wall musculature, lateral view. (G) Juvenile, 15 days p.f., head retracted, anterior is up; (H) Juvenile, 20 days p.f., head retracted.

Two dorsal head retractors are strongly developed. At least one ventral retractor is still present but less strongly developed than dorsal retractors. (I) Juvenile, 20 days p.f. No sign of the ventral head retractors is detectable in this specimen. Arrows indicate dorsal retractors; circular body wall musculature less clearly organized in bands. a, anus; cm, circular body wall musculature; dr, dorsal head retractor muscles; e, esophagus; ey, eye; hr, head retractor muscles; ir, introvert retractor muscles; m, mouth; mt, metatroch; np, nephridium; pt, prototroch; t, tentacles; to, terminal organ; tr, terminal organ retractor muscle; vr, ventral head retractor muscles. Scale bars: 50mm.

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subsequent sieving of the sediment. Specimens ofN. pellucidum were collected from rubble (including mollusc shells, sand dollar tests, and rocks), 4 miles oﬀshore from Fort Pierce, FL, using an echinoderm dredge on the R/V SUNBURST.

Spawning and rearing larvae

Multiple adults of each species were kept in glass dishes in approximately 200 ml seawater at room temperature. None of the species shows sexual dimorphism.T. lageniformisreproduces pri- marily parthenogenetically (Pilger 1987), whereas the other species display bisexual reproduction. Spawning occurred in the labora-

tory, generally after changing the water. Whenever eggs were observed in the culture dishes, they were pipetted into a clean dish and observed for development. Larval cultures were kept for up to six weeks. Water was changed at least every 2 days. The larvae were periodically fed with unicellular algae or diatoms (Isochrysis, Dunaliella, orNanochloropsis).

Sample preparation for confocal laser scanning microscopy

Live specimens were relaxed by ﬂoating menthol dissolved in 95%

ethanol on the surface of a shallow dish with seawater at 41C for 20 min. They were ﬁxed for up to 24 h in a 4% solution of pa- raformaldehyde in 0.1MSo¨rensen’s phosphate buﬀer (PBS) (Clark et al. 1981) at 41C. Samples were then washed in PBS and per- meabilized for 1 h in a solution of 0.2% Triton X-100 in PBS (PBT). Fluorescent staining was accomplished in 1.5 ml microcen- trifuge tubes wrapped in aluminum foil using a 1:20 solution of Phalloidin Alexa 488 in PBT. Staining times were either 1 h at room temperature or 12–24 h at 41C. Following staining, specimens were washed in PBS and transferred to 30% isopropanol. They were then pipetted onto poly-L-lysine-coated microscope slides to which they attached. The slides with the attached specimens were taken through a dehydration series of isopropanol (30%, 50%, 70%, 95%, and 100%). Specimens were cleared and mounted nonpermanently with Murray’s clear (2:1 benyl benzoate:benzyl acohol) and viewed with a Radiance 2100 confocal laser scanning microscope. Z-series of optical slices were produced for each specimen with slices every 0.1–0.5mm, depending on the specimen. Im- age projections were generated in Volocity version 3.6.1 for MacIntosh (improvision) or Confocal Assistant version 4.02 (Brelje 1994–1996).

Sample preparation for scanning electron microscopy Specimens were relaxed as described above. They were fixed in 2.5% glutaraldehyde in Millonig’s phosphate buffer (Millonig 1964) for at least 12 h at 41C. Fixation was followed by three washes in a 1:1 mixture of Millonig’s phosphate buffer and 0.6M

sodium chloride and postﬁxation in 1% osmium tetroxide (1:1:2 mix of 4% OsO4:Millonig’s buﬀer:0.75M NaCl). Samples were then dehydrated in an ethanol series up to 100% and critical point

Fig. 4. Developmental stages of Phascolion psammophilum, (A) Scanning electron micrograph of adult introvert and tentacles; (B) Drawing of interior anatomy of adult; note single large dorsal introvert retractor and smaller ventral introvert retractor muscle;

(C, D, E) Confocal laser scanning projections after ﬂuorescent staining with phalloidin Alexa 488. (C) Late trochophore, dorsal view, 24 h post fertilization (p.f.). Note four head retractor muscles (arrows). (D) Pelagosphera larva, 2 days, dorsal view. Four head retractor muscles of similar size are obvious in anterior body region (arrows). Circular body wall musculature in bands. (E) Pelagosph- era larva 5 days (contracted). Dorsal pair of head retractor muscles much more strongly developed than ventral (only one ventral retractor observed at this stage). a, anus; cm, circular body wall musculature; dr, dorsal head retractor muscles; h, hooks; i, intestine; np, nephridium; t, tentacles; vr, ventral head retractor muscles.

Scale bars: (A) 200mm, (B-F) 50mm.

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dried. Specimens were mounted on SEM stubs using double-sticky tape and viewed in a JEOL 6400 Visions scanning electron microscope (Peabody, MA, USA). All images were stored digitally.

RESULTS

Ancestral states could be reconstructed with nearly 100%

probability for the musculature-related characters. The average probability over 1000 sampled trees (500,000 generations of Monte Carlo Markov chains in a Bayesian analysis with every 500th tree sampled) for the longitudinal body wall musculature to be arranged in distinct bands at the root node of the sipunculan tree is 97.5%. With an average probability of 98.1% the circular body wall musculature was smooth.

With 98.1% probability, the ancestral sipunculan had two pairs of introvert retractor muscles.

The phylogenetic analysis with the constrained topology resulted in a very similar tree (Fig. 6) to the one presented in Schulze et al. (2007). The same ﬁve clades were recovered, except thatPhascolosoma capitatumfalls into clade VI instead of V as in the original analysis.

P. cryptum

The ontogenetic transformations of the musculature of P. cryptum are illustrated in Fig. 2.P. cryptum omits both the trochophore and the pelagosphera stage. A crawling, highly contractile juvenile emerges from the egg envelope (Fig.

2C) approximately 36 h post fertilization (p.f.). Although partially obscured by yolk, a dorsal and a ventral pair of introvert retractor muscles can be distinguished (Fig. 2, D and E). At 48 h p.f. the juvenile crawls on the bottom of the dish by contractions of its body wall musculature. At this stage, both the circular and the longitudinal body wall musculature are strongly developed and organized in bands (Fig. 2F). Four separate retractor muscles are apparent at this stage (Fig. 2G).

T. lageniformis

Figure 3 shows the development of T. lageniformis, which lacks the trochophore stage. Although the early pelagosphera at 37 h p.f. has four head retractors (Fig. 3, C and D), this is no longer the case at 5 days p.f. The pelagosphera at 5 days p.f. (Fig. 3, E and F) shows weakly deleloped prototroch and metatroch but very strongly developed body wall musculature, especially the circular body wall musculature which shows an organization in broad bands (Fig. 3F). Dorsal and ventral head retractor muscles cannot clearly be distinguished at this stage. The retractor musculature consists of loosely arranged individual ﬁbers that reach from the head region to about 3/4 toward the posterior end where they insert into the body wall. At about 15 days the larva loses its ability to swim and starts to undergo metamorphosis. Most notably, the head A

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Fig. 5. Developmental stages ofNephasoma pellucidum; (A, D, G) scanning electron micrographs; (B) Drawing of interior anatomy of adult; note two introvert retractor muscles; (C, E, F, H) confocal laser scanning projections after ﬂuorescent staining for F-actin with phalloidin Alexa 488. (A) Anterior introvert of adult, showing arrangement of the tentacles. (C) Late trochophore stage, 40 h post fertilization (p.f.) (see supporting information for rotational view which clearly shows four separate head retractor muscles). (D) Early pelagosphera, 40 h p.f., ventral view. (E) Early pelagosphera, 40 h p.f., ventral slice of larva. (F) Same specimen as E, dorsal slice of larva. (G) Pelagosphera, 5 days p.f., ventral view. (H) Pelagosph- era, 5 days p.f., ventrolateral view. a, anus; bo, buccal organ; cm, circular body wall musculature; dr, dorsal head retractor muscles;

hr, head retractor muscles; i, intestine; ir, introvert retractor muscles; m, mouth; mt, metatroch; nc, nerve cord; no, nuchal organ;

np, nephridium; t, tentacles; to, terminal organ; tr, terminal organ retractor muscles; vr, ventral head retractor muscles; scale bars:

20mm.

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Table 1. GenBank accession numbers for all sipunculan and outgroup sequences included in the phylogenetic analysis and reconstruction of ancestral states

Species MCZ catalogue # 18S rRNA 28S rRNA Histone H3 COI

Sipunculidae

Phascolopsis gouldii DNA100199 AF123306 AF519272 AF519297 DQ300134

Siphonosoma cumanense DNA100991 DQ300002 DQ300047 DQ300089 DQ300157

Siphonosoma vastum DNA100625 DQ300003 AY445137 AY326297 DQ300158

Sipunculus(S.)norvegicus DNA101069 DQ300004 DQ300090 DQ300159

Sipunculus(S.)nudus DNA100246 AF519240 AF519270 AF519295 DQ300161

Sipunculus(S.)phalloides DNA101337 DQ300009 DQ300094 DQ300165

Sipunculus(S.)polymyotus DNA101121 DQ300010 DQ300095 DQ300166

Xenosiphon branchiatus DNA101086 DQ300016 DQ300050 DQ300101 DQ300172

Golﬁngiidae

Golﬁngia elongata DNA100465 DQ299969 DQ300031 DQ300065 DQ300121

Golﬁngia margaritacea DNA100738 DQ299973 DQ300032 DQ300069 DQ300126

Golﬁngia vulgaris DNA100207 AF519244 AF519273 DQ300127

Nephasoma diaphanes DNA101072 DQ299975 DQ300071 DQ300128

Nephasoma ﬂagriferum DNA100440 DQ299976 DQ300033 DQ300072 DQ300129

Nephasoma pellucidum DNA101009 DQ299978 DQ300131

Thysanocardia catherinae DNA101068 DQ300015 DQ300099

Thysanocardia nigra DNA100606 AF519247 AF519274 DQ300100

Themistidae

Themiste(T.)dyscrita DNA101095 DQ300011 DQ300167

Themiste(T.)hennahi DNA100627 DQ300012 DQ300096 DQ300168

Themiste(L.)lageniformis DNA100229 AF519249 AF519276 AF519302 DQ300169

Themiste(L.)minor DNA101083 DQ300013 DQ300097 DQ300170

Themiste(T.)pyroides DNA101084 DQ300014 DQ300098 DQ300171

Phascolionidae

Onchnesoma steenstrupii DNA101080 DQ299979 DQ300034 DQ300074

Phascolion(L.)cryptum DNA101007 DQ299980 DQ300035 DQ300075 DQ300132

Phascolion(I.)gerardi DNA101002 DQ299981 DQ300076

Phascolion(P.)psammophilum DNA101006 DQ299982 DQ300036 DQ300133

Phascolion(P.)strombus DNA100101 AF519248 AF519275 AF519301

Phascolosomatidae

Antillesoma antillarum DNA101008 DQ299951 DQ300051 DQ300102

Apionsoma(A.)misakianum DNA100737 DQ299952 DQ300017 DQ300052 DQ300103

Apionsoma(A.)murinae DNA100446 DQ299953 DQ300018

Apionsoma(E.)pectinatum DNA100624 AY326293 AY445142 AY326300 DQ300104

Phascolosoma(P.)agassizii DNA101096 DQ299985 DQ300037 DQ300078 DQ300135

Phascolosoma(P.)albolineatum DNA100396 AF519251 AF519278 DQ300136

Phascolosoma(F.)capitatum DNA101070 DQ299986 DQ300079 DQ300137

Phascolosoma(P.)granulatum DNA100201 AF519252 AF519279 AF519304 DQ300138

Phascolosoma(P.)granulatum X79874

Phascolosoma(P.)nigrescens DNA100822 DQ299988 DQ300039 DQ300081 DQ300141

Phascolosoma(P.)noduliferum DNA100208 AF519253 AF519280 AF519305 DQ300144

Phascolosoma(P.)perlucens DNA100228 AF519254 AF519281 AF519306 DQ300145

Phascolosoma(P.)scolops DNA100813 DQ299998 DQ300044 DQ300085 DQ300152

Phascolosoma(P.)stephensoni DNA100469 AF519256 AF519283 AF519310 DQ300153

Phascolosoma(P.)turnerae DNA100230 DQ300000 DQ300046 DQ300087 DQ300154

Aspidosiphonidae

Aspidosiphon(A.)albus DNA101017 DQ299954 DQ300053 DQ300105

Aspidosiphon(A.)elegans DNA101016 DQ299957 DQ300020 DQ300056 DQ300106

Aspidosiphon(P.)ﬁscheri DNA100620 AY326294 AY326301 DQ300107

Aspidosiphon(A.)gosnoldi DNA101014 DQ299959 DQ300022 DQ300057 DQ300109

Aspidosiphon(A.)gracilis schnehageni DNA101087 DQ299960 DQ300023 DQ300058 DQ300110

Aspidosiphon(P.)laevis DNA100467 AF519261 DQ300024 DQ300059 DQ300111

Aspidosiphon(A.)misakiensis DNA100205 AF119090 AF519288 AF519312

Aspidosiphon(A.)muelleri DNA100206 DQ299962 DQ300025 DQ300060 DQ300113

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is mostly retracted and the metatroch is lost (Fig. 3G). At 20 days p.f., two strongly developed dorsal head retractors are clearly visible (Fig. 3, H and I). In one case, a less strongly developed ventral retractor muscle was present (Fig. 3H). The density of ﬁbers in the body wall musculature has increased and an arrangement into bands is much less obvious than in earlier stages.

P. psammophilum

The development of this species, with a trochophore and a lecithotrophic pelagosphera is shown in Fig. 4. In the late trochophore, approximately 24 h p.f., a dorsal and a ventral pair of retractor muscles are visible (Fig. 4C). Some circular body wall musculature is discernible in the central part of the larva but is still rudimentary and not clearly in bands. At 2 days p.f. (Fig. 4D) the circular body wall musculature is most strongly developed in the constriction between head and trunk and in the posterior area of the trunk. Some longitudinal muscle fibers are present in the body wall. The four retractor muscles are still clearly distinguishable and of approximately equal size. At five days p.f. (Fig. 4E) the number of individual muscle fibers has increased greatly in the dorsal pair of retractor muscles. We have not observed how the number of dorsal retractor muscles is reduced to one. The circular body wall musculature is still discernible in rings.

N. pellucidum

N. pellucidumshows the most common developmental mode in sipunculans, with a lecithotrophic trochophore and a

planktotrophic pelagosphera (Fig. 5). The introvert retractor muscles and the terminal organ retractor muscles start form- ing in the late trochophore, ca. 40 h p.f. In the early pelagosphera, a ventral and a dorsal pair of retractor muscles can be distinguished (Fig. 5C, also see animation in supporting information). The body wall musculature is indistinct.

At 40 h p.f. (Fig. 5, D–F), the two pairs of retractor muscles are still discernible, although individual ﬁbers from the dorsal and ventral muscle on each side have almost approached each other. Similar to P. psammophilum, the circular body wall musculature forms rings and is most strongly developed at the constriction between head and trunk region (Fig. 5, E and F).

In the later stage pelagosphera ofN. pellucidum(Fig. 5, G and H) the terminal organ and the associated musculature are very strongly developed (Fig. 5H).

DISCUSSION

Traditionally, parsimony has been the only method available to reconstruct ancestral states but the results can depend on the preference settings in the reconstruction algorithm and leave no room for uncertainty. In case of ambiguities, the

‘‘accelerated transformation’’ algorithm (ACCTRAN in PAUP; Swoﬀord 2003) places character state changes close to the root of the tree whereas ‘‘delayed transformation’’

algorithm (DELTRAN in PAUP) places them closer to the leaves. The probabilities calculated with Bayesian statistics, as applied in this study, allow a better judgement of the degree of Table 1. (Contd.)

Species MCZ catalogue # 18S rRNA 28S rRNA Histone H3 COI

Aspidosiphon(P.)parvulus DNA100202 AF119075 DQ300026 DQ300061

Aspidosiphon(P.)parvulus DNA100982 DQ299964 DQ300027 DQ300063 DQ300115

Aspidosiphon(P.)steenstrupii DNA100232 AF519262 AF519291 AF519315 DQ300116

Cloeosiphon aspergillus DNA100825 DQ299968 DQ300030 DQ300120

Lithacrosiphon cristatus DNA100623 AY326295 AY445142 AY326302

Nemertea

Amphiporussp. AF119077 AF519265 AF519293 AJ436899

Argonemertes australiensis AF519235 AF519264 AF519293 AY428840

Mollusca

Lepidopleurus cajetanus AF120502 AF120565 AY070142 AF120626

Rhabdus rectius AF120523 AF120580 AY070144 AY260826

Yoldia limatula AF120528 AF120585 AY070149 AF120642

Annelida

Lumbrineris latreilli AF519238 AF519267 AF185253 AY364855

Lamellibrachiaspp. AF168742 AF185235 U74055

Owenia fusiformis AF448160 AY428824 AY428832 AY428839

Urechis caupo AF119076 AF519268 X58895 U74077

Lumbricus terrestris AJ272183 F AF185262 NC_001673

Entoprocta

Loxosomella murmanica AY218100 AY218129 AY218150 AY218083

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conﬁdence in the reconstructions. Here the ancestral states of the musculature at the sipunculan root node could be reconstructed with nearly 100% posterior probability.

Direct evidence from fossils would be the most reliable source of information about ancestral states. However,

the only unambiguously identiﬁed fossils of sipunculans are uninformative with regard to musculature, although parts of the internal anatomy are well preserved (Huang et al. 2004). No fossils of sipunculan larvae have ever been discovered.

0.1

Loxosomella Owenia

Lepidopleurus

Yoldia 78

Rhabdus

Argonemertes Amphiporus 81

Lumbricus Urechis

Lumbrineris Lamellibrachia

66 Sipunculus norvegicus 101069

Sipunculus nudus 100246 87 Xenosiphon branchiatus 101086

Sipunculus phalloides 101337 Sipunculus polymyotus 101121 93

Siphonosoma cumanense 100991 Siphonosoma vastum 100625

Phascolosoma turnerae 100230

Phascolion gerardi 101002 Nephasoma pellucidum 101009

Phascolion psammophilum 101006

Phascolion cryptum101007 Onchnesoma steenstrupii 101080 Phascolion strombus 100101 66

Nephasoma diaphanes 101072 Nephasoma flagriferum 100440

Golfingia elongata 100465 Golfingia vulgaris 100207

Phascolopsis gouldii 100199

79 Apionsoma murinae 100446

Golfingia margaritacea 100738

Thysanocardia catherinae 101068 Thysanocardia nigra 100606

Themiste lageniformis 100229 Themiste minor 101083 Themiste hennahi 100627

Themiste dyscrita 101095 Themiste pyroides 101084 70

89 65 52 72 59 72 72

Phascolosoma capitatum 101070

Phascolosoma granulatum 100201 Phascolosoma agassizii 101096

Phascolosoma nigrescens 100822 Phascolosoma granulatum x79874 Phascolosoma stephensoni 100469 Phascolosoma noduliferum 100208

Phascolosoma scolops 100813 Phascolosoma albolineatum 100396 Phascolosoma perlucens 100228 63

99 65

Apionsoma misakianum 100737 Apionsoma pectinatum 100624

Cloeosiphon aspergillus 100825 Antillesoma antillarum 101008

Lithacrosiphon cristatus 100623 Aspidosiphon muelleri 100206

Aspidosiphon albus 101017 Aspidosiphon misakiensis 100205

Aspidosiphon steenstrupii 100232 63 98

Aspidosiphon parvulus 100202

Aspidosiphon laevis 100467 Aspidosiphon parvulus 100982 Aspidosiphon gosnoldi 101014 Aspidosiphon gracilis 101087 99Aspidosiphon elegans 101016

Aspidosiphon fischeri 100620 98

68 79 72 66

95

* *

*

* *

*

* *

*

* *

*

* Clade II: IRM - 4, LMB - y; CMB - y

Clade III:

IRM - 1-4 LMB - n*

CMB - n Clade I:

IRM - 4 LMB - y CMB - y

Clade IV:

IRM - 4 LMB - y*

CMB - n

Clade V:

IRM - 2-4 LMB - y/n CMB - n

*except P. capitatum

*except P. gouldii

Fig. 6. 50% Majority rule consensus tree resulting from the Bayesian analysis of four gene regions and morphology. Shaded boxes outline the ﬁve major clades. Branch support is given as Bayesian posterior probability. Asterisks: 100% posterior probability. Black box indicates the root node of the Sipuncula for which the ancestral states were reconstructed. IRM, number of introvert retractor muscles; CMB, cirucular muscle bands (yes/no); LMB, longitudinal muscle bands (yes/no).

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The re-analysis of the simpliﬁed dataset from Schulze et al.

(2007) resulted in a very similar tree as the analysis with the full dataset. The placement of P. capitatum (clade V in Schulze et al. 2007, clade IV in the present analysis) is the only diﬀerence with regard to the composition of the major clades, but it has weak support in both of the analyses (56% and 65% posterior probability, respectively). The clades that branch oﬀ early in the Sipuncula (clades I and II) are com- posed of species with four introvert retractor muscles and circular and longitudinal muscle bands as adults. All of the more derived clades have a continuous sheath of circular body wall musculature. Within Clade III there is variation in the number of introvert retractors, but the body wall musculature is generally organized in smooth layers, with the exception of Phascolopsis gouldii. In this species, the longitudinal body wall musclulature is split into anastomosing bands, that is bands that may fuse to neighboring bands at intervals throughout the length of the trunk. Members of clade IV consistently have four introvert retractor muscles. All exceptP. capitatum have longitudinal muscle bands. Members of clade V vary in both the number of retractor muscles as well as in the arrangement of the longitudinal body wall musculature.

Within the ﬁve clades there are some minor discrepancies between the analysis of the reduced and the full dataset (for details consult Schulze et al. 2007). The support for the monophyly of clade III is lower in this study than in the original analysis (72% pp vs. 100% pp) whereas that of clade V is higher (79% pp vs. 56% pp).

Head/introvert retractor muscles

Our analysis indicates with high probability (98.1%) that the presence four separate introvert retractor muscles is the ancestral state for adult Sipuncula. This condition is present in all representatives of clades I and II, as well as in the earliest branches in clades III and V, represented by Phascolosoma turnerae and two Apionsoma species, respectively. It is also present in most representatives of clade IV, except in Phascolosoma captitatumfor which the position is still uncer- tain (see above). Our ﬁndings agree with Cutler and Gibbs (1985) and Cutler (1994) who proposed a hypothetical ancestral sipunculan with four introvert retractor muscles based on morphological analyses.

We were not able to reconstruct the ancestral states for the larvae in the same way as for the adults because larval anatomy has only been studied for a limited number of sipunculan species. However, in almost all known cases, two pairs of head retractor muscles are present in the pelagosphera larvae (A˚kesson 1958a, b; Hall and Scheltema 1975) (Table 2). The exceptions are ‘‘type J’’ with a single pair and ‘‘type S’’ with three pairs (Hall and Scheltema 1975). With regard to ‘‘type J,’’ Hall and Scheltema (1975) mention that additional muscles are sometimes present. For ‘‘type S,’’ the second pair of

dorsal retractors might actually be a pair of protractor muscles. Scheltema and Rice (1990) describe a very similar, pos- sibly the same, larval type, named ‘‘leura’’ type ‘‘i’’ and only mention one pair of dorsal and ventral retractors each.

P. cryptum belongs to the subgenus Lesenka which is characterized by a single retractor muscle with two origins in the posterior body wall. In the adult ofP. cryptumboth origins are to the left of the ventral nerve cord (Fig. 2B). Four retractors are present in the juvenile until at least 3 days p.f.

(Fig. 2, D, E, and G). We have not observed when fusion or loss of muscles occurs.

In T. lageniformis, it appears that the ventral pair of retractors is reduced (Fig. 3, H and I) and the dorsal pair transforms into the adult introvert retractors.

P. psammophilum belongs to the subgenus Phascolion, characterized by a single strong dorsal retractor muscle and a more weakly developed ventral retractor with two separate origins in the body wall (Fig. 4B). We have shown that in the larvae of this species the head retractor muscles are of equal size until the early pelagosphera stage (Fig. 4E). In later pelagosphera larvae, the dorsal retractors are already more strongly developed than the ventral retractors (Fig. 4E). We have not been able to observe the process by which the number of dorsal retractors is reduced to one.

A˚kesson (1958a, b) describes the larva of Phascolion (Phascolion) strombus, which has four introvert retractor muscles and a pair of protractor muscles. He reports that the reduction in the number of retractor muscles does not occur until the larvae are about 1 month old.

In the 5-day-old pelagosphera of N. pellucidum all four retractor muscles are of similar size (Fig. 5H). We have not observed which muscles are reduced to yield the adult condition with a single pair of retractor muscles.

Body wall musculature

Our reconstructions suggest that at the root of the Sipuncula the circular body wall musculature was a continuous sheath, whereas the longitudinal musculature was split into bands. In contrast, Cutler and Gibbs (1985) and Cutler (1994) con- cluded that the ancestral sipunculan had both longitudinal and circular body wall musculature organized as continuous sheaths. This was based on their understanding of sipunculan phylogeny, which diﬀered in several respects from our more recent analyses. In particular, Cutler (1994) assumed that the Sipunculidae (with longitudinal and circular muscles in bands) were a derived clade and thatApionsoma(with circular muscles always and longitudinal muscles mostly in continuous sheaths) was morphologically closest to the sipunculan root.

Consequently, he assigned continuous body wall musculature to his ‘‘revised hypothetical ancestral sipunculan.’’

The longitudinal body wall musculature generally develops later than the circular body wall musculature. None of the

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early pelagic stages we examined had any longitudinal body wall musculature but Table 2 shows that many later-stage pelagic pelagosphera larvae have longitudinal muscle bands, even if they have smooth longitudinal body wall musculature as adults, such asApionsoma misakianum. We only observed longitudinal musculature in the crawling larval and juvenile stages ofT. lageniformisandP. cryptum. An arrangement in bands was obvious in the 3-day-old juvenile ofP. cryptum.

The circular musculature forms in bands in all examined species. Wanninger et al. (2005) observed the same in P.

strombus. They report that the number of circular muscle bands does not increase during initial growth of this species, an indication that they are not segmental structures. Later larval stages (Table 2) and adult sipunculans rarely have circular muscle bands. The condition is only present in adults of clades I and II. Even though these are early branches in the phylogeny, our ancestral state reconstruction indicates that the condition of the circular body wall musculature is not ancestral. This example shows that an extant group, which

has its evolutionary origins close to the root of the tree, does not necessarily look like its ancestor; it evolved over the same time period as other members of the clade and may have accumulated just as much morphological change.

CONCLUSIONS

Even though Haeckel’s biogenetic law (‘‘ontogeny recapitu- lates phylogeny’’) has been discredited, parallels between ontogeny and phylogeny are not uncommon. In the case of the Sipuncula, the four head/introvert retractor muscles reﬂect the ancestral state of the group and are retained in nearly all of the pelagosphera larvae, even though adults may have a reduced number.

The situation is more complex with regard to the body wall musculature. The ancestral state for the longitudinal musculature, an arrangement in separate bands, is not obvious in the Table 2. Summary of published data on musculature in sipunculan larvae and respective adults

Larval type Species

Longitudinal body wall musculature

Circular body wall musculature

Head/introvert retractors

Reference

Larva Adult Larva Adult Larva Adult

Phascolion strombus

Continuous Continuous Bands Continuous 2 pairs (11 pair of protractors)

Single dorsal and single ventral retractor

1,2

Type A (‘‘Baccardia citronella’’)

Aspidosiphonsp. Continuous ? Continuous Continuous 2 pairs 1 pair 3

Type B (‘‘Smooth’’)

Xenosiphon branchiatus

Bands Bands Continuous Bands 2 pairs

(11 pair of protractors)

2 pairs (11 pair of protractor muscles)

3,4

Type C (‘‘Baccardia oliva’’)

Apionsoma misakianum

16 bands Continuous Continuous Continuous 2 pairs 2 pairs 3

Type E Siphonosoma cumanense

18 bands ? Continuous ? 2 pairs 2 pairs 3,5

Type F ? 24 bands ? Continuous ? ? ? 3

Type J ? 22 bands ? Continuous ? 1 pair

(1metatrochal retractors)

? 3

Type L ? Poorly developed ? Poorly

developed

? 2 pairs

(11 pair of protractors)

? 3

Type O ? 28 bands ? Continuous ? 2 pairs ? 3

Type P ? Bands ? Continuous ? 2 pairs ? 3

Type S Sipunculus polymyotus

50 bands Bands Poorly

developed

Bands 3 pairs (2 dorsal11 ventral pair)

2 pairs 3

1Wanninger et al. (2005);²A˚kesson (1958);³Hall and Scheltema (1975);⁴Ja¨gersten (1963);⁵Rice (1988).

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early larval stages, but is common in most of the later pelagic stages (Table 2) and in the crawling juvenile ofP. cryptum.

The circular body wall musculature forms early and always in bands which later transform into a continuous sheath. Our analyses indicate that the continuous sheath is the ancestral condition for adult Sipuncula, but we have no information about the ancestral condition in the larvae. It is possible that the transformation from separate bands in the larva to a continuous sheath in the adult is ancestral.

The condition of the body wall musculature is also in part dependent on the developmental mode. Pelagic larval forms generally have less strongly developed body wall musculature than crawling larval stages. In our case, the pelagic pelagosphera stages of P. psammophilum andN. pellucidum have rel- atively poorly developed body wall musculature. We ﬁnd the most prominently developed body wall musculature in the benthic larva and juvenile ofP. cryptum.T. lageniformis, with a larva that both swims and crawls is intermediate. These diﬀerences are easily explained from a functional morphology perspective: pelagic stages mostly move by ciliary action of their metatroch, whereas benthic stages move by contractions of their body.

To interpret our ﬁndings in the context of annelid phylogeny, the position of the Sipuncula within the Annelida needs to be determined with more certainty. There is some indication for a sister group relationship between Sipuncula and the Terebelliformia but it is only weakly supported (Struck et al. 2007). An increasing amount of information is available on the diversity of annelid musculature, but a solid phylogenetic framework necessary to determine plesiomor- phic versus apomorphic states is still lacking. The longitudinal body wall muscles are usually more developed that than the circular muscles, which are even absent in a number of taxa (Tzetlin and Filippova 2005; Purschke and Mu¨ller 2006). The longitudinal muscles in annelids are most commonly arranged in four to six distinct bands. The formation of a continuous sheath, as in the Clitellata, is regarded as derived (Purschke and Mu¨ller 2006). The longitudinal muscle bands in sipunculans are more numerous than in annelids, but more detailed ontogenetic studies of the muscular system in diﬀerent annelid groups might reveal more similarities to the Sipuncula than obvious in the adults.

Acknowledgments

This work was partially supported by a MarCraig grant at Harvard University to G. Giribet and E. B. Cutler, which included a postdoctoral fellowship to A. S. It was continued under a Smithsonian Marine Station fellowship to A. S. (contribution number XXX).

We are grateful to the staﬀ and postdoctoral fellows at the Smith- sonian Marine Station for their technical and ﬁeld support, notably J. Piraino, H. F. Reichardt and S. Santagata. Carolyn Gast is ac- knowledged for Fig. 2B, 3B, and 5B and Charissa Baker for Fig. 4B.

Figures 2B, 3B, 4B, and 5B were reproduced with permission from Rice (1993a, Fig. 2G) and Rice (1993b, Figs. 11–13).

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