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Contributions to Zoology, 80 (3) – 2011

Molecular phylogeny and shell microstructure of Fungiacava eilatensis Goreau et al. 1968, boring into mushroom corals (Scleractinia: Fungiidae), in relation to other mussels (Bivalvia: Mytilidae)

Masato Owada1,3, Bert W. Hoeksema2

1.  Department of Biological Sciences, Kanagawa University, 2946 Tsuchiya, Hiratsuka, Kanagawa, Japan

2.  Department of Marine Zoology, Netherlands Centre for Biodiversity Naturalis, P.O. Box 9517, 2300 RA Leiden, the Netherlands

3. pt125365@kanagawa-u.ac.jp

Keywords: 18s rRNA, Fungiacava, Leiosolenus, mushroom corals, rock-boring bivalves, shell microstructure

Abstract


Research on the evolution of the symbiosis between the boring mussel Fungiacava eilatensis (Bivalvia: Mytilidae) and its mushroom coral hosts (Scleractinia: Fungiidae), which requires phylogenetic reconstructions of both the Mytilidae and the Fungiidae, contributes to the understanding of the complexity of coral reef ecosystems. Previously, Fungiacava was regarded as a genus that had descended from Leiosolenus or as belonging to the subfamily Crenellinae, but no phylogenetic support has been obtained for this hypothesis. In the present study, the 18s rRNA sequences of ten mytilid species and the shell microstructures of 12 mytilids were investigated. The phylogenetic position of F. eilatensis is discussed in relation to its associations with its host species. The results of the molecular phylogenetic analysis indicate that F. eilatensis forms a sister group with Leiosolenus simplex, a live-coral-boring species within the Leiosolenus clade. Scanning electron microscope observations indicate that the shell of F. eilatensis is constructed of 3 layers: the outer shell layer with a homogeneous structure, the middle shell layer as a sheet nacreous structure and the inner shell layer with an irregular simple prism structure. This shell microstructure of F. eilatensis is similar to that of Leiosolenus malaccanus and L. simplex. These findings show that F. eilatensis has descended from a coral-boring Leiosolenus species and that it would be adequate for Fungiacava to be treated as Leiosolenus. However, because of its extraordinary shell shape it has been kept as a separate genus.

Introduction


Bivalves of the genera Fungiacava and Leiosolenus are endosymbiotic mytilids that bore into living corals (Goreau et al., 1970; Mokady et al., 1998). Shells of Fungiacava are typically flat and heart-shaped (Hoeksema and Kleemann, 2002), whereas those of Lithophaga are more or less cylindrical and torpedo-shaped (Kleemann and Hoeksema, 2002). As endosymbionts they show very little intraspecific variation in shell morphology in comparison to attached species of, for instance, Mytilus (Groenenberg et al., 2011).

Fungiacava eilatensis Goreau, Goreau and Neumann, 1968, bores into 14 species of living corals that belonged to four genera and six subgenera of Fungiidae (Hoeksema and Achituv, 1993; Hoeksema and Kleemann, 2002; Hoeksema and Gittenberger, 2008), which presently are considered eight genera together (Gittenberger et al., 2011). Leiosolenus punctatus (Kleemann & Hoeksema, 2002), exclusively recorded from Fungiidae, and Leiosolenus laevigatus (Quoy & Gaimard, 1835), infesting many species of living corals, bore into six species of Fungiidae and nine species of five scleractinian families, respectively (Kleemann, 1980; Kleemann and Hoeksema, 2002). Consequently, among the Mytilidae, F. eilatensis has the largest number of confirmed host coral species and the largest number of hosts belonging to a single coral family, which comprises at least 50 species (listed by Gittenberger et al., 2011).

Research on the evolution of symbiosis between the monospecific genus Fungiacava and the Fungiidae is relevant to the understanding of the complexity of interspecific associations in coral reef ecosystems, for which phylogeny reconstructions of both the Mytilidae and the Fungiidae are necessary. Although phylogeny reconstructions of fungiid corals are available (Hoeksema, 1989, 1991a, 1993; Gittenberger et al., 2011), the phylogenetic position of F. eilatensis is not yet fully investigated. In addition, the classification of F. eilatensis is not consistent among researchers. For example, Goreau et al. (1969) described F. eilatensis as a species of the subfamily Crenellinae based on the shell form, whereas others treated Fungiacava as belonging to the subfamily Lithophaginae based on the mode of life, along with other genera of boring mussels (Morton, 1990). Therefore, the phylogenetic position of F. eilatensis needs to be analysed on the basis of morphological characters and DNA sequences that strongly reflect the mytilid phylogeny.

It is known that shell microstructure characters are generally consistent within each family. In the Mytilidae, however, the shell microstructure characters are relatively variable (Carter, 1990; Kobayashi, 1998, 2004; Taylor et al., 1969, 1973). This variation may support the classification of F. eilatensis. Furthermore, the 18s rRNA sequences of many species were investigated and deposited in the DNA Data Bank of Japan. The accumulated data are useful to reconstruct the phylogenetic position of F. eilatensis. In the present study, 18s rRNA sequences and the shell microstructures of ten and 12 mytilid species, respectively, were investigated in order to reconstruct the phylogenetic position of F. eilatensis and to classify the genus Fungiacava in relation to other mytilids.

Material and methods


Analysis of molecular phylogeny

Specimens of F. eilatensis were obtained from two specimens of the mushroom coral Sandalolitha robusta (Quelch, 1886) collected at Ternate, northern Molluccas, Indonesia (October 2009). After sampling, the specimens were directly fixed in 95% ethanol. Total DNA was extracted from the fixed soft tissue by using DNeasy Blood & Tissue Kit (QIAGEN). The sequence encoding the 18s rRNA was amplified using Premix Ex Taq and the polymerase chain reaction (PCR) thermal cycler PERSONAL (TaKaRa). The PCR amplification conditions consisted of denaturation (94°C and 30 s), annealing (58°C and 30 s), extension (72°C and 1 min) and 30 cycles. Used primers are given in Table 1. The PCR products were purified using ExoSAP-IT (USB Corporation), and the sequencing reaction was performed using BigDye Terminator v3.1 Cycle Sequencing Kit (ABI PRISM). The determination of DNA sequence was carried out using 3130 Genetic Analyzer (ABI PRISM).

FIG2

Table 1. Amplification and sequencing primers for 18s rRNA sequence. 22F and 1789R were used to amplify in the PCR.

The sequence data were aligned using ClustalW version 1.8 (Thompson et al., 1997); gaps and sites without confirmed homology were excluded from the analyses. Thirteen species belonging to nine families that are related to Mytilidae were used as outgroups. A molecular phylogenetic tree was constructed using both the maximum likelihood and Bayesian methods, and a strict consensus tree was computed from the two trees. The model for maximum likelihood analysis was determined using Modeltest version 3.7 (Posada and Crandall, 1998), and the tree was constructed using the likelihood ratchet algorithm (Vos, 2003), which was run 2000 times by using PAUP* version 4.0b10 (Swofford, 1997); the bootstrap replication number was 1000 times. The model for Bayesian analysis was determined using Mrmodeltest version 2.3 (Posada and Buckley, 2004), and the tree was constructed using MrBayes version 3.1.2 (Ronquist and Huelsenbeck, 2003). This program was run for 5,000,000 generations, with sampling every 1000th generation. The 18s rRNA-encoding sequences of 10 species that were determined in this study were registered in the DNA Data Bank of Japan. Species names and accession numbers of the analysed samples are given in Table 2.

FIG2

Table 2. Species of bivalves used for molecular phylogenetic analysis.

Scanning electron microscope observations

After removal of soft tissues, the shells were cleaned and desiccated, and mechanically fragmented with a hammer. The fracture surfaces were coated with platinum by using ion coater JFC-1600 (JEOL) and observed under a scanning electron microscope (SEM) JSM-T20 (JEOL). The shell microstructure was identified on the basis of the features described by Taylor et al. (1969, 1973) and Carter (1990). Species names and sampling localities of the observed samples are given in Table 3.

FIG2

Table 3. Sampling localities and patterns of shell microstructures. The symbol ? indicates species described by Owada (2009). FP: fibrous prism structure, Ho: homogeneous structure, ISP: irregular simple prism structure, ISpP: irregular spherulitic prism structure, SN: sheet nacreous structure.

Results


Molecular phylogeny

The 18s rRNA-encoding sequences consisted of 1721-1742 bp long. After alignment, the analysed sequences were 1681 bp long, with 361 variable sites. In the maximum likelihood analysis, the TrNef + I + G model was selected by Modeltest, and the likelihood index (-ln L) of the constructed tree was 6968.1327. In the Bayesian method, the SYM + I + G model was selected by Mrmodeltest. A strict consensus tree was computed from these 2 trees (Fig. 1).

FIG2

Fig. 1. The strict consensus tree of the mytilid genera composed from the 2 trees constructed using the maximum likelihood and Bayesian methods. The nodes on which the topologies of the 2 trees were not concordant are indicated as polytomies. The mark • on a node indicates bootstrap value > 50% and Bayesian posterior probability > 0.90 for the respective monophyletic clade. The branch length was calculated using the maximum likelihood method.

The strict consensus tree topology indicates that the phylogenetic relationships between subfamilies are not clear although the Mytilidae appears to be monophyletic (bootstrap value > 50% and Bayesian posterior probability > 0.90). Lithophaga, Modiolus, and Mytilus are monophyletic, but Idas, Leiosolenus, and Musculus not. Furthermore, the groups [Adipicola + Bathymodiolus + Benthomodiolus + Gigantidas + Idas + Myrina + Tamu], [Brachidontes + Geukensia + Hormomya], [Fungiacava + Leiosolenus], and [Musculista + Musculus + Mytilus + Septifer] also exhibit monophyly. In the present tree, F. eilatensis is a sister taxon of Leiosolenus simplex.

Shell microstructure

Shells of all of the observed species consisted of three layers: outer, middle, and inner shell layers. The outer shell layer was composed of either one or two of the following three shell microstructures: fibrous prism (FP) structure, homogeneous (Ho) structure, and irregular simple prism (ISP) structure (Fig. 2, Table 3). The ISP structure of the outer shell layer was consistently observed in Adula, Leiosolenus, Lithophaga, and Modiolus, though it was absent in some species of Leiosolenus and Modiolus. The middle shell layer was composed of the sheet nacreous (SN) structure in all observed species. The inner shell layer of most species was composed of the ISP structure, except in Hormomya mutabilis and Septifer bilocularis, in which the inner shell layer was composed of alternating ISP and SN structures. The ISP structure was absent in Mytilus galloprovincialis. The outer shell layer of F. eilatensis was Ho structure, and the shell microstructure of each layer was similar to that of each layer of Leiosolenus malaccanus and L. simplex (see Fig. 4-E in Owada, 2009).

FIG2

Fig. 2. Shell microstructures observed using SEM. The outer shell layer is partially shown. The arrows indicate the direction of the umbo. The scale bars indicate 10 μm. FP: fibrous prism structure, Ho: homogeneous structure, ISP: irregular simple prism structure, Pe: periostracum, SN: sheet nacreous structure. A: Adula aff. californiensis, B: Brachidontes setigera (Dunker, 1856), C: Fungiacava eilatensis, D: Hormomya mutabilis, E: Leiosolenus simplex, F: Modiolus auriculatus, G: Modiolus flavidus (Dunker, 1857), H: Modiolus philippinarum, I: Musculus cupreus, J: Musculus senhousia, K: Mytilus galloprovincialis, L: Septifer bilocularis.

Discussion


Phylogeny of Fungiacava and Leiosolenus

Fungiacava was included in the Leiosolenus clade of mussels that live as endosymbionts in living corals, and forms a sister taxon of L. simplex. Musculus and Musculista, which belong to the Crenellinae, did not form a clade with Fungiacava. Furthermore, the shell microstructure of F. eilatensis was more similar to that of L. malaccanus and L. simplex than that of Musculus cupreus and Musculista senhousia. These observations suggest that F. eilatensis has descended from a coral-boring Leiosolenus species.

The fossil record suggests that Leiosolenus dates back to the Jurassic and that the symbiosis between Leiosolenus and the corals was established since the Cretaceous (Kleemann, 1983, 1990, 1994, 1996). Fungiid corals appeared since the Paleocene, and the number of species has increased since the Miocene (Wells, 1966; Hoeksema, 1989). Furthermore, the symbiosis between Fungiacava and the fungiid corals was not established before the Pleistocene (Goreau et al., 1976). The time period of the establishment of symbiosis between Leiosolenus and fungiid corals is uncertain. However, one of the Leiosolenus lineages must have diverged into Fungiacava between the Paleocene, the period to which the earliest known presence of Fungiidae has been attributed, and the Pleistocene, the period from which the earliest specimens of Fungiacava has been recorded. Since Fungiacava is found in 14 mushroom coral species (Hoeksema and Kleemann, 2002) distributed over eight genera (Gittenber­ger et al., 2011), it is likely that the association between Fungiacava and the Fungiidae evolved early in this period, unless Fungiacava has only recently become a generalist with regard to its host preferences.

Juvenile free-living fungiids are attached to a hard substratum and break loose before they reach their adult stage (Hoeksema, 1989; Yamashiro and Yama­zato, 1996; Vizel et al., 2009). Their mode of life is quite different from that of the attached fungiid species (see e.g. Hoeksema, 2009) since they can move towards different types of substrata, including sand and silt (Hoeksema and Moka, 1989; Hoeksema and Koh, 2009). Of the 14 recorded mushroom coral hosts of F. eilatensis, only two species are unable to detach from the substratum. Because these attached species are closely related to free-living host species (Hoeksema, 1989, 1991a; Gittenberger et al., 2011), the original appearance of Fungiacava in the evolutionary history of the Fungiidae appears to be related to the detached growth form of mushroom corals. Owing to their mobility, mushroom corals may form mixed multi-species assemblages (Hoeksema, 1991b; Hoeksema and Koh, 2009; Hoeksema and Matthews, 2011), and the boring mussels move along with their hosts over the reef bottom. This may facilitate their infestation success, especially if the symbionts are not host-specific. On the other hand, it is remarkable that some mushroom coral species that are known to reach high population densities, such as Zoopilus echinatus Dana, 1846, Halomitra clavator Hoeksema, 1989, Heliofungia actiniformis (Quoy & Gaimard, 1833) and Heliofungia fralinae (Nemenzo, 1955) are not infested by Fungia­cava. Z. echinatus and H. clavator are thin mushroom corals, which can form large aggregations of loose fragments (Hoeksema and Gittenberger, 2010). They usually do not offer much space to boring organisms and when they break, endosymbionts may become more easily exposed to predators. Furthermore, the mushroom coral species H. actiniformis and H. fralinae both have fleshy polyps and may also form large aggregations (Hoeksema, 1989). They may reproduce asexually by growing many buds on vacated stalks in H. actiniformis (Hoeksema, 1989; Knittweis et al., 2009) or on dying parent corals in H. fralinae (Hoeksema, 2004). Mushroom corals with fleshy polyps may be difficult to infest by coral-boring mussels, and since live-coral-boring bivalves benefit from a long life span of their host corals for their own survival, corals that die easily are not a suitable habitat for them. Therefore, the success of the host selection for Fungiacava may be related to the special niche that they have entered with their free-living hosts.

Taxonomical position of Fungiacava

In the Mytilidae, both Leiosolenus and Lithophaga are characterized by an elongated cylindrical shell form (Newell, 1969). In addition, Leiosolenus is classified by the calcareous incrustations covering the periostracum, while Lithophaga lacks such incrustations (Oliver, 1992). The shell microstructure characters of the two genera are quite different (Table 3), and the genetic distance is also large (Owada, 2007, 2009). The shell form of Fungiacava looks like that of Solamen (Crenellinae) rather than that of the two aforementioned genera, and the calcareous incrustations are not observed on the periostracum (Goreau et al., 1969). However, the shell microstructure characters of Fungiacava are very similar to those of Leiosolenus (Table 3), and Fungiacava was included in the Leiosolenus clade of the present molecular phylogenetic tree (Fig. 1). Therefore, it would be adequate for Fungiacava to be treated as Leiosolenus, but its unique heart-shaped shell form is a reason to keep it a separate genus, as originally proposed by its authors (Goreau et al., 1968, 1969).

Acknowledgements


We would like to express our deep thanks to K. Kanazawa (Kanagawa University) for the support of this study. We are grateful to T. Haga (JAMSTEC) for donating samples and providing valuable comments. We are indebted to A. Hino (Kana­gawa University) for helping with the DNA sequencing experiments and S. Suzuki (Kanagawa University) for helping with the SEM observations. The field research at Ternate was sponsored by the Research Centre for Oceanography, Indonesian Institute of Sciences (PPO-LIPI). The research permit was issued by the Indonesian State Ministry of Research and Technology (RISTEK). We also thank to two anonymous reviewers whose comments and suggestions helped significantly improve the manuscript.

Received: 21 October 2010

Revised and accepted: 15 January 2011

Published online: 22 June 2011

Editor: M. Schilthuizen

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