The bivalve superfamily Galeommatoidea is characterized by its symbiotic associations with other marine invertebrates. However, for many galeommatoideans, the host species remains unknown. Platomysia (Galeommatoidea) is a monotypic genus including a single species P. rugata, which is distinguished from other galeommatoideans in having distinct and evenly spaced commarginal ribs on its shell surface. This species was described based on a single right valve shell collected in Nanao Bay, Japan Sea, by Habe in 1951 and has been known only from Japanese waters. However, the biology of living animals has never been reported. We found that this species lives in the burrows of the sipunculan worm Sipunculus nudus in mud flats in the Seto Inland Sea, Japan. We investigated its host association and described its shell morphology and anatomy. In addition, we performed a phylogenetic analysis using two nuclear (18S and 28S ribosomal RNA) genes to determine its phylogenetic position in Galeommatoidea. The result suggests that this species belongs to the clade of commensal bivalves together with Pseudopythina, Byssobornia, and Pergrinamor. Platomysia rugata and other two groups of sipunculan-associated galeommatoideans were not monophyletic, suggesting that association with sipunculans occurred at least three times in the galeommatoid evolution.
The bivalve superfamily Galeommatoidea (Heterodonta) exhibits extremely high diversity in intertidal zones (Bouchet et al., 2002; Paulay, 2003; Lützen and Nielsen, 2005). As a distinct ecological characteristic, many members of this superfamily have symbiotic associations with other invertebrates in sediment bottom (Boss, 1965; Morton and Scott, 1989; Li et al., 2012; Goto et al., 2012). Most live in the burrows or on the body surface of host animals, whereas some live inside the host body (Boss, 1965; Kato, 1998; Morton and Scott, 1989; Goto et al., 2012). The known host taxa of galeommatoideans are highly diverse, belonging to Porifera, Cnidaria, Annelida, Mollusca, Brachiopoda, Bryozoa, Arthropoda, and Echinodermata (Boss, 1965; Morton and Scott, 1989; Goto et al., 2012). However, hosts remain unknown in many galeommatoideans.
Sipunculans are marine worms, which were previously assigned to their own phylum, but are now recognized as members of Annelida (Struck et al., 2007). Most sipunculans burrow in bottom sediments, whereas some groups live in the empty shell or narrow spaces in hard substrata (Stephen and Edmonds, 1972). They are often utilized as hosts by commensal galeommatoideans (Boss, 1965; Morton and Scott, 1989). To date, at least 14 species in 11 genera (Barrimysia,Epilepton, Fronsella, Jousseaumlella, Litigiella, Mioerycina,Montacuta, Mysella, Nipponomysella, Pseudopythina, andSalpocola) of galeommatoideans have been reported to have commensal associations with sipunculans (Bourne, 1906; Habe, 1958; Boss, 1965; Gage, 1979; Morton and Scott, 1989; Lützen and Kosuge, 2006; Jespersen et al., 2007; Lützen et al., 2008; Goto et al., 2012). Most live attached to burrow wall or body surface of sipunculans, whereas some species attach to gastropod shells occupied by sipuculans. Recent molecular phylogenetic analysis suggested that sipunculan-associated galeommatoideans are separated into two different lineages (Goto et al., 2012).
Platomysia is a monotypic genus including a single species P. rugata. The genus is distinguished from other galeommatoideans in having distinct and evenly spaced commarginal ribs on its shell surface (Habe, 1951). This species was described based on a single right valve collected in Nanao Bay, Toyama, Japan and is known only from Japanese waters (Habe, 1951; Fukuda, 2012). However, the biology of living animals has never been reported. The information on shell and anatomical characteristics and phylogenetic position of P. rugata remains poorly known (Huber, 2015).
In the present study, we found that P. rugata lives in the burrows of the sipunculan worm Sipunculus nudus (Annelida: Sipunculidae) in mud flats in the Seto Inland Sea, Japan. We described its host association, shell morphology, and anatomy. In addition, we performed a molecular phylogenetic analysis using the two nuclear (18S and 28S ribosomal RNA) genes to understand the phylogenetic position of this species in the superfamily Galeommatoidea.
MATERIALS AND METHODS
Sample collection and observations
We have investigated the diversity of commensal galeommatoideans in mud flats in Hakatajima, Ehime Prefecture and Takehara, Hiroshima Prefecture, Japan since 2009 by digging up various invertebrates' burrows (Goto et al., 2011, 2012, 2014). Sipunculus nudus (Sipunculidae) is one of the major burrowing invertebrates in these study sites. This sipunculan lives in temporal burrows, which are horizontal, not branched, and weakly lined with mucus, in bottom sediments. We collected P. rugata from the burrows of S. nudus in the middle or lower intertidal zone of mud flats at spring low tide during 2011 and 2012. The bivalves were preserved in 70–100% ethanol and brought back to the laboratory. The detailed observations of shell morphology and anatomy were performed under a dissecting microscope. We used a specimen collected from Takehara for molecular analysis (Table 1).
DNA extraction, PCR, and sequencing
Total genomic DNA was isolated from the bivalves following a previously described method (Goto et al., 2012). A small piece of soft tissue was homogenized in 800-µl lysis buffer and incubated at 55°C overnight, after which 80 µl of saturated potassium chloride was added to the lysate. This solution was incubated for 5 min on ice and then centrifuged for 10 min. The supernatant (700 µl) was transferred to a new tube, cleaned once with a phenol/chloroform solution, and precipitated with an equal volume of 2- propanol. The DNA pellet was rinsed with 70% ethanol, vacuum-dried, and dissolved in 100-µl TE buffer.
GenBank accession numbers of the specimens used in this study. The sequences obtained in this study are shown in bold with asterisk.
We sequenced fragments of the 18S and 28S genes. Polymerase chain reactions (PCRs) were used to amplify ∼1700 bp of 18S and ∼1000 bp of 28S. Amplifications were performed in 20-µl mixtures consisting of 0.4 µl of forward and reverse primers (20 µM each; Table 2), 2.0 µl of ExTaq buffer, 1.6 µl of dNTPs (2.5 µM each), 0.1 µl of ExTaq polymerase (TaKaRa, Otsu, Japan), and 15.1 µl of distilled water. Thermal cycling was performed with an initial denaturation for 3 min at 94°C, followed by 30 cycles of 30 s at 94°C, 30 s at a gene-specific annealing temperature (50–55°C), and 2 min at 72°C, with a final 3 min extension at 72°C. The sequencing reaction was performed using PCR primers and internal primers (Table 2) and a BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA); the reaction products were electrophoresed on an ABI 3130 sequencer (Applied Biosystems). The obtained sequences were deposited in the DDBJ/ EMBL/GenBank databases with accession numbers LC126832-LC126833 (Table 1).
In addition to the sequences of P. rugata obtained in this study, we also collected sequence data of other galeommatoideans and outgroups from GenBank (Table 1). Sequences of the 18S and 28S genes were aligned using the Muscle program (Edgar, 2004) with default settings in the software Seaview (Galtier et al., 1996; Gouy et al., 2010). We employed Gblocks v0.91b (Castresana, 2000; Talavera and Castresana, 2007) to eliminate the ambiguously aligned regions in 18S and 28S alignments. Phylogenetic trees were constructed using the Bayesian and maximum likelihood (ML) methods. Bayesian analyses were performed using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003) with substitution models chosen by Kakusan 4 (Tanabe, 2011). In the combined data set, substitution parameters were estimated separately for each gene partition (18S: SYM Gamma, 28S: GTR Gamma). Two independent runs of Metropolis-coupled Markov chain Monte Carlo were carried out simultaneously, sampling trees every 100 generations and calculating the average standard deviation of split frequencies (ASDSFs) every 1000 generations. Using the ‘stoprule’ option, analyses were continued until ASDSF dropped below 0.01, at which point the two chains were considered to have achieved convergence. As ASDSF was calculated based on the last 75% of the samples, we discarded the initial 25% of the sampled trees as burn-in. We confirmed that analyses reached stationarity well before the burn-in period by plotting the ln-likelihood of the sampled trees against generation time. Maximum likelihood analyses were performed using RAxML (Stamatakis, 2006) as implemented in raxmlGUI 1.31 (Silvestro and Michalak, 2012). The robustness of the ML tree was evaluated by 1000 bootstrap replications. Datasets were partitioned by gene and the GTRGAMMA model was implemented.
Primers used in this study.
We surveyed approximately 200 and 45 burrows of S. nudus in Hakatajima and Takehara, respectively, resulting that eight and three specimens of P. rugata were collected in each study site. All the bivalves were found as a solitary in the burrows of S. nudus (Fig. 1D-F). The occurrence rate of P. rugata in S. nudus burrows was ∼4 and ∼6.7% in Hakatajima and Takehara, respectively. The bivalves were attached to the burrow-wall surface mainly near the host body (Fig. 1E, F), whereas those directly attaching to the hosts were not found. We observed the bivalve behavior in an aquaria with its host for a while. The bivalve hided under the host body, but did not attach to the host. The other sipunculan Siphonosoma cumanense commonly inhabits mud flats in these study sites (Goto et al., 2011). We surveyed at least 20 burrows of S. cumanense in each study site. However, P. rugata was not collected from the S. cumanense burrows. We also surveyed the burrows of echiurans (Ikedosoma gogoshimense and Arhynchite hayaoi), holothurians (Protankyra bidentata), and echinoids (Echinocardium cordatum), but did not find any P. rugata.
Shell morphology and anatomy
Shell: The shell is very thin and fragile, its shape elongate-ovate and equivalve and nearly equilateral with beak in the middle (Figs. 1, 2). The anterior is slightly more expanded than posterior. Shell is covered by whitish, thin periostracum (Figs. 1, 2). Sculpture is prominent, consisting of distinct and evenly spaced commarginal ribs and close-set concentric striae at shell ventral margin. Radial ribs are absent. The pallial line is entire. Hinge of each valve consists of a single stout cardinal tooth in front of the umbo and well-developed oblique internal ligament posterior to the umbo (Fig. 2F, G).
Anatomy: A single large inner demibranch is present and covers the frequently branched ovary (Fig. 2B, C). Small labial palps are attached to the anterior end of the demibranch. Anterior and posterior adductor muscles are ovate, anterior is more elongate and larger than posterior (Fig. 2B). Foot is moderate-sized, tongue-shaped, with a small, prominent heel and a large rounded toe (Fig. 1A, B). The mantle edges narrowly extend beyond the margin of the shell and bear numerous regularly arranged very short papillae (Fig. 1A, B). No prominent tentacles were observed.
To determine the phylogenetic position of P. rugata in Galeommatoidea, we performed molecular phylogenetic analyses using 41 galeommatoideans and eight outgroup species (including five heterodont and three non-heterodont bivalve species) (Table 1). In the resulting tree (Fig. 3), P. rugata was monophyletic with Pseudopythina, Peregrinamor, and Byssobornia (Bayesian posterior probability = 1.00, Bootstrap support value = 100). Sipunculan-associated species was not recovered as monophyletic (Fig. 3). Platomysia rugata is relatively closely related with another sipunculan-associated species Pseudopythina aff. nodosa (Fig. 3), although they were not monophyletic (Fig. 3).
We found that P. rugata is a commensal, living in the burrow of the sipunculan S. nudus. This bivalve probably feeds on plankton or small organic particles in the water currents caused by host sipunculans, using the host burrows as a shelter from predators. Platomysia rugata was always attached to the host burrow wall and has never been found from the host body surface in this study. Such a host-use pattern is different from those of other galeommatoideans associated with S. nudus; Pseudopythina nodosa and P. aff. nodosa are attached to the host body surface (Morton and Scott, 1989; Goto et al., 2012), Salpocola philippinensis and Fronsella ohshimai are attached to the posterior end of the host (Habe, 1951, 1958; Lützen et al., 2008), and Litigiella pacifica is attached to both host body surface and burrow wall (Lützen and Kosuge, 2006). Such niche partitioning in S. nudus-associated galeommatoideans may be the result of adaptations that reduce the interspecific competition for the host. On the other hand, recent molecular phylogeny showed that S. nudus includes not a few cryptic species (Kawauchi and Giribet, 2013). Thus, it is also possible that difference of host-use mode among S. nudus-associated galeommatoideans is related with specialization to different host species. Except for P. rugata, four galeommatoideans are associated with S. nudus in Japan: L. pacifica, P. aff. nododa, S. philippinensis, and F. ohshimai (Habe, 1958; Lützen and Kosuge, 2006; Lützen et al., 2008; Goto et al., 2012). Platomysia rugata has been known from temperate coasts of Japan (Habe, 1951; Fukuda, 2012), whereas L. pacifica, P. aff. nodosa, and S. philippinensis are distributed in subtropical to tropical coasts of Japan (mainly the Ryukyu Islands) (Lützen and Kosuge, 2006; Lützen et al., 2008; Goto et al., 2012; Goto pers. obs.). Thus, the distribution of P. rugata does not overlap those of the latter three species. The holotype of F. ohshimai was collected with S. nudus in Amakusa, Nagasaki Prefecture, Japan (Habe, 1958). However, no living animals have been recorded since the first description.
Habe (1977) suggested that, although P. rugata resembles thracids, it belongs to Galeommatoidea because it has no pallial sinus. Contrary to this, Huber (2015) doubted its assignment in Galeommatoidea and perceived it as a juvenile of another Japanese heterodont species, e.g., Lutrophora, or even Panomya or Panopea. However, our phylogenetic analyses suggest that P. rugata definitely belongs to Galeommatoidea (Fig. 3). Our analyses also suggest that sipunculan-associated galeommatoideans are not monophyletic (Fig. 3). Platomysia rugata is relatively closely related with other sipunculan-associated species P. aff. nodosa (Fig. 3). However, whether evolutionary origin of their symbiotic associations with sipunculans is same or not remains unclear. On the other hand, P. rugata belongs to the clade of Pseudopythina, Peregrinamor, and Byssobornia (Fig. 3). All of the members of this clade are commensally associated with burrowing invertebrates (Morton and Scott, 1989; Goto and Kato, 2012). Some are attached to the host body surface (Kato and Itani, 1995), whereas the others are attached to the host burrow-wall surface (Morton, 1972; Morton and Scott, 1989; Goto and Kato, 2012). In this clade, the host taxa are highly diverse, including mantis shrimps (P. subsinuata), crabs (P. macrophthalmensis), echiurans (P. ochetostomae and B. yamakawai), sipunculans (P. rugata), and holothurians (P. aff. ariake) (Morton and Scott, 1989; Goto and Kato, 2012; Goto et al., 2012; this study). This suggests that host switching across different animal groups have occurred frequently in this clade.
Habe (1951) described P. rugata based only on a single right valve shell. In this study, we described the hinge structure of both of right and left valves, each of which has one anterior cardinal and one oblique internal ligament posterior to the umbo (Fig. 2F, G). This hinge structure is basically similar to those of Pseudopythina described in Morton and Scott (1989). In addition, this species has a single demibranch like Pseudopythina (Jespersen et al., 2009). Thus, close relatedness of this species with Pseudopythina is supported by both the shell morphology and anatomical characteristics.
Platomysia rugata is a new example of commensal galeommatoidean from the Seto Inland Sea. Until now, various commensal galeommatoideans have been reported from around this area, including Arthritica japonica, Divariscintilla toyohiwakensis, Koreamya setouchiensis, Nipponomysella subtruncata, Basterotia gouldi, Curvemysella paula, Peregrinamor oshimai, Pseudopythina subsinuata, and Pseudopythina aff. ariakensis (Kato and Itani, 1995; Lützen and Takahashi, 2003; Goto et al., 2007, 2011, 2012, 2014; Yamashita et al., 2011). Each of these galeommatoideans uses a different burrowing invertebrate as a host. Thus, the diversity of symbiotic galeommatoideans in the Seto Inland Sea is probably a function of a rich burrowing invertebrate fauna in mud flats.
We wish to express our sincere gratitude to S. Kojima (University of Tokyo), Y. Kano (University of Tokyo), and D. Ó Foighil (University of Michigan) for hosting the first author as a research fellow for this study and permission to use the facilities in the laboratory. We also thank T. Lee (University of Michigan) for helping the first author to set up the laboratory facilities to observe specimens. This study was partially supported by grants to RG from the Japan Society for the Promotion of Science (JSPS) Research Fellowship for Young Scientists and the JSPS Postdoctoral Fellowships for Research Abroad.