Phuket Mar. Biol. Cent. Res. Bull.78: 77–115 (2021) DOI: 10.14456/Pmbcrb.2021.7 TAXONOMIC RE-DESCRIPTION and RELATIONSHIPS OF
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Phuket mar. biol. Cent. Res. Bull.78: 77–115 (2021) DOI: 10.14456/pmbcrb.2021.7 TAXONOMIC RE-DESCRIPTION AND RELATIONSHIPS OF TWO MAT-FORMING MUSSELS FROM THE INDO-PACIFIC REGION, WITH A PROPOSED NEW GENUS Koh-Siang Tan1*, Samuel Hui-Min Tan1, Kitithorn Sanpanich2, Teerapong Duangdee3 and Reni Ambarwati4 1St John’s Island National Marine Laboratory, Tropical Marine Science Institute, National University of Singapore, Singapore 119227 2Institute of Marine Science, Burapha University, Muang District, Chon Buri 20131, Thailand 3Faculty of Fisheries, Kasetsart University, Chatuchak, Bangkok 10900, Thailand 4Faculty of Mathematics and Natural Sciences, Surabaya State University, East Java, Indonesia *Corresponding author: [email protected] ABSTRACT: Two common Indo-Pacific mat-forming mussels with subterminal umbones, widely known in the literature as Brachidontes setiger (Dunker, 1857) and B. striatulus (Hanley, 1843), are re-described and distinguished on morphological and molecular grounds. Overlapping intraspecific variation in the shells of both species has resulted in several synonyms that in turn have led to confusion over their identities, even though representative shell forms of the two congeners appear distinct. Typical B. setiger of authors has a somewhat inflated shell that is dorso-ventrally wide with a uniformly yellowish orange to brown periostracum usually covered partially by byssal hairs over the posterior half of the shell. This is in contrast to the narrowly elongate, dorso-ventrally compressed shell of a typical B. striatulus, which usually has up to six black or dark brown stripes of differing widths radiating from near the umbones towards the posterior region over an otherwise yellow to dark brown periostracum devoid of byssal hairs. However, these traits varied considerably between individuals even within populations in some cases. Our study showed that the two species are indeed closely related. Both are capable of secreting and depositing byssal hairs on their shells, as well as living gregariously in byssal mats. Mixed species stands were also observed in Singapore and Phuket, Thailand. The shell microstructure and anatomy of the two species were practically indistinguishable. Nevertheless, pair-wise genetic distances between the two species ranged between 0.06 to 0.07, based on concatenated mitochondrial (COI, 16S) and nuclear (ITS1, 28S, H3) gene trees. These phylogenetic trees also suggested that the two species are not closely related to other members of Brachidontes. A new genus Byssogerdius is proposed to accommodate them. Further, the syntypes of B. setiger appear to be a Mediterranean and/or West African species of Gregariella. The correct name for the Indo-Pacific species is thus suggested to be Byssogerdius subsulcatus (Dunker, 1857), a species that Dunker himself considered distinct from B. setiger. Keywords: Mytilidae, Brachidontes, Byssogerdius, Southeast Asia, taxonomy INTRODUCTION some 30 species distributed worldwide in tropical and temperate latitudes, there are also species that Amongst mussels, a number of marine and form aggregates which are nestled in mats. These estuarine species in the genera Arcuatula Jousseaume mats are formed by byssal threads that entrap in Lamy, 1919 (= Musculista Yamamoto and Habe, sediment, sand grains as well as biogenic material. 1958), Gregariella Monterosato, 1883, Modiolus The number involved can range from a few to hundreds Lamarck, 1799, Musculus Röding, 1798, and of individuals. In the latter case, extensive mats Mytella Soot-Ryen, 1955 are known to live either often cover many square meters over natural or singly or gregariously in byssal nests (Merrill and man-made substrata. Such mats presumably offer Turner 1963; Morton 1974; 1980; 1982; Willan thermal insulation for the inhabitants during low 1985; Slack-Smith and Brearley 1987; Lim et al. tide, whilst protecting them from desiccation and 2018). In the genus Brachidontes, which comprises possibly from predation as well. In Musculus discors, 78 Phuket mar. biol. Cent. Res. Bull. eggs are deposited along the inner lining of the two species. Clearly further clarification is needed byssal nest which serves to protect the larvae during to determine the taxonomic status of the two species. development (Merrill and Turner 1963). We provide here for the first time a detailed In Southeast Asia, a small number of mussel taxonomic re-description of two common, widespread species construct and live in such mats, amongst but hitherto poorly documented mat-forming which two species, often referred to as Brachidontes Brachidontes-like species distributed in the Indo-West setiger (Dunker, 1857) and B. striatulus (Hanley, Pacific region, with an assessment of their phylogenetic 1843), can be common in the tidal zone. The former relationships based on molecular data. species is poorly known, although recorded in regional species lists (e.g., Lynge 1909 for Gulf of MATERIALS AND METHODS Thailand; Kuroda 1941 for Taiwan; Kira 1965 for Japan; Wang 1997 for China). The latter species is Morphology. Living animals were obtained from better documented. It was first described from the the shores of Singapore, Thailand, Indonesia and Philippines and subsequently reported to be Taiwan. They were examined carefully under the common in India (Annandale and Kemp 1916; stereomicroscope as far as possible while they Morton 1977; Subba Rao 2017) and southern China were active, before preserving individuals in either (Wang 1997). Its morphology and ecology were absolute ethanol for DNA extraction (see Appendix described in detail by Morton (1977). It was also 1) or in 5% formalin in seawater for anatomical reported to be a possible invasive species in Singapore observations later in the laboratory. Shells and the (Morton and Tan 2006). animals within were routinely examined under the As part of a wider taxonomic study of stereomicroscope, and photographs were taken using Brachidontes species in the Indo-west Pacific region, a digital camera fitted with a macro lens. Shell the two species were examined to evaluate their images were isolated from the background using relationships with other congeners. In the process Photoshop. In addition to specimens collected of collecting and studying living material as well during 2017–2019, museum material from the as comparing them with relevant type specimens in Natural History Museum, London, and reference museums, we realised that there was overlapping specimens at the Phuket Marine Biological Center intraspecific variation in the shells of both species. in Phuket, Thailand were also examined. Several synonyms in the literature attest to this, Shell microstructure was determined by examining which in turn have led to confusion in their identities, cut shell surfaces under the scanning electron even though representative shell forms of the two microscope (SEM). The surfaces were prepared by congeners appear distinct. Typical B. setiger occur embedding clean, dry valves in epoxy resin (Struer from the low intertidal to subtidal zones at full Epofix), which were then cut using a low-speed salinity. It has a somewhat inflated shell that is dorso- saw (Buehler IsoMet). The resulting block with the ventrally wide with a uniformly yellow-orange desired cut shell surface was ground and polished periostracum usually covered partially by byssal by hand over lapping film (3M) through a series hairs. This is in contrast to the narrowly elongate, of descending particle sizes to 3 µm grade, after dorso-ventrally compressed shell of typical B. which the polished surface was rinsed gently in striatulus, which live in estuarine conditions. The distilled water and dried. A few drops of dilute 2% shell of this species has up to six black or dark HCl v/v was then applied briefly (about 5–10s) to brown stripes of different widths radiating from etch the cut surface before it was rinsed in distilled near the umbones towards the posterior region over water, dried and sputter coated (JEOL 3000FC) an otherwise dark or yellow-brown periostracum with platinum for viewing under the SEM (JEOL devoid of byssal hairs. However, these traits varied 6010/LVPlus). To determine the presence or absence considerably between individuals even within of a calcitic shell layer, the etched surface of a second populations in some cases. Such populations included specimen was treated with Feigl’s stain (Feigl and shells that were intermediate in shape, colour, and Leitmeier 1933; Gobac et al. 2009) before viewing pattern, which made identification difficult. This was it in the SEM and compared with an unstained cut exacerbated by equivocal results initially obtained surface. Terminology of shell microstructure follows after molecular sequencing of mitochondrial and Carter (1990). nuclear genes from individuals representing the 79 Taxonomic re-description and relationships of two mat-forming mussels DNA extraction and analysis thermal profile: initial denaturation at 95°C for 5 Tissue processing and PCR. Tissues were processed min, followed by 35 cycles of denaturation at 94°C using the Blood and Tissue Kit (Qiagen). Alternatively, for 3 min, annealing at 48°C for 1 min, extension difficult specimens were processed using the at 72°C for 1 min; and a final extension at 72°C for E.Z.N.A.® Mollusc DNA Kit (Omega-Biotek). 7 min (Colgan et al. 2003). To avoid the problem of Doubly Uniparental Finally, should the Colgan primers fail, a Inheritance (DUI) present in some mytilids, care 313bp subset of the mt-CO1 gene was amplified was taken to sample only somatic tissue, usually with mICOIintF: 5’-GGWACWGGWTGAACW- the foot or part of the posterior adductor muscle, GTWTAYCCYCC-3’ and jgHCO2198: 5’-TAIA- for DNA extraction. Tissue meant for DNA extraction CYTCIGGRTGICCRAARAAYCA-3’ (Leray et was stored in absolute ethanol at -20°C until use. al. 2013); with the following thermal profile: initial Dissected tissue was mostly processed according denaturation at min at 95°C for 3 min; 35 cycles of to manufacturer’s instructions, with several denaturation at 95°C for 1 min, annealing at 45°C modifications. Samples were incubated as long for 1 min, extension at 72°C for 1 min; and a final as necessary for complete digestion.