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 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, 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 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 . 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: , Brachidontes, Byssogerdius, Southeast Asia,

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 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, The number involved can range from a few to hundreds Lamarck, 1799, Röding, 1798, and of individuals. In the latter case, extensive mats 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 , 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 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 were obtained from better documented. It was first described from the the shores of Singapore, Thailand, Indonesia and and subsequently reported to be Taiwan. They were examined carefully under the common in (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 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

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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. To ensure extension at 72°C for 3 min. maximum binding of extracted DNA to the spin For the internal transcribed spacer 1 (ITS1), an columns, the initial precipitated DNA was passed approximately 420 bp region was amplified using through the column twice. Spin columns were ITS1A-sal: 5’-AAAAAGCTTTTGTACACAC- washed with their associated wash buffers twice to CGCCCGTCGC-3’ and ITS1B-sal: 5’-AGCTT- remove contaminants. At the final elution step of GCTGCGTTCTTCATCGA-3’ (Pleyte et al. 1992) pure genomic DNA, the eluant was allowed to with the following thermal profile: initial denaturation incubate in the column at 50-70°C (depending on at 94°C for 1.5 min, followed by 35 cycles the kit) to maximise the amount of DNA dissolved of denaturation at 94°C for 1.5 min, annealing at into the elution buffer. Extracted genomic DNA 65.2°C for 1 min, extension at 72°C for 2 min, was quantified using a NanoDrop™ Lite (Thermo followed by a final extension at 72°C for 7 min Scientific) spectrophotometer and/or a Qubit™ (Pleyte et al. 1992). 3 fluorometer (Invitrogen). Aliquots of genomic For the D1 expansion region of the 28S rRNA DNA were taken and diluted to a concentration of gene (D1R), an approximately 350 bp region was 1 ng/µl. Concentrated genomic DNA was stored amplified using LSU5b: 5’-ACCCGCTGAAYT- frozen at -20°C for future use. TAAGCA-3’ (McArthur and Koop 1999) and D1R: Extracted genomic DNA was amplified through 5’-AACTCTCTCMTTCARAGTTC-3’ (Colgan et PCR in a T100 thermal cycler (Bio-Rad) or a al. 2003) with the following thermal profile: initial ProFlex™ 3 x 32-well PCR System (Applied denaturation at 95°C for 5 min, 49°C for 45 s and Biosystems). 72°C for 1 min, followed by 34 cycles of denaturation For the mitochondrial gene cytochrome c oxidase at 95°C for 30 s, annealing at 52°C for 45 s, extension subunit 1 (mt-CO1), an approximately 700 base at 72°C for 1 min, and a final cycle at 95°C for 30 s, pair (bp) region was amplified using universal 52°C for 45 s, extension at 72°C for 5 min. primers LCO 1490: 5’-GGTCAACAAATCATA- The histone H3 gene (H3) was amplified using AAGATATTGG-3’ and HCO: 2198 5’-TA- H3af: 5’- ATGGCTCG- TACCAAGCAGACVGC- AACTTCAGGGTGACCAAAAAATCA-3’ designed ‘3’ and H3ar: 5’- ATATCCTTRGG- CATRATRGT- by Folmer et al. (1994), with the following thermal GAC -3’ with the following thermal profile: initial profile: initial denaturation at 95°C for 3 min, denaturation at 94°C for 3 min, followed by 30 followed by 35 cycles of denaturing at 95°C for cycles of denaturation at 95°C for 30 s, annealing 1 min, annealing at 45.5°C for 1 min, extension at at 49°C for 90 s, extension at 72°C for 120 s, and a 72°C for 1.5 min; and a final extension at 72°C for final extension at 72°C for 5 min. 7 min (Alves et al. 2012). The 16S ribosomal RNA gene (16S) was amplified Should amplification with the Folmer primers using 16SarL: 5’-CGC CTG TTT ATC AAA AAC fail, samples were amplified using the Colgan primers AT-3’ and 16SbrH:5’-CCG GTC TGA ACT CAG COXAF: 5'-CWAATCAYAAAGATATTGGAAC- ATC ACG T-3’ with the following thermal profile: 3’and COXAR: 5'-AATATAWACTTCWGGGT- initial denaturation at 94°C for 5 min, followed by GACC-3' (Colgan et al. 2001) with the following 35 cycles of denaturation at 94°C for 30 s, annealing 80

Phuket mar. biol. Cent. Res. Bull. at 52°C for 40 s, extension at 72°C for 45 s, and a had to be constructed using composites from final extension at 72°C for 5 min. different studies. Maximum likelihood trees using the In general, each PCR contained 5 µl of 1 ng/µl Tamura-Nei model were also generated in MEGA DNA, 1 µl of each primer (10 µM), 15 µl of GoTaq X for 1000 bootstrap replicates, with 95% site Green PCR Master Mix (Promega) or exTEN 2X coverage cut-off for gaps and missing data. PCR master mix (First Base) in a 30 µl reaction Calculations of pairwise distances, between-group volume, with the remainder made up of nuclease- mean distances, net between-group mean distances free water and other PCR additives (e.g., DMSO, and within-group mean distances were also conducted BSA) as necessary. in MEGA X. Bayesian analysis were conducted in MrBayes Gel analysis and PCR purification. PCR (Huelsenbeck and Ronquist 2001) using the GTR products were then analyzed on a 1% agarose gel substitution model partitioned by the different genes, stained with SYBR Safe DNA Gel Stain or FloroSafe with gamma-distributed rate variation across sites DNA Stain (First Base). Gel electrophoresis was and a proportion of invariable sites. All parameters carried out in standard 1X TAE buffer for 1 hour were unlinked between partitions. Samples were run and visualised using the Dark reader DR46B independently twice for a minimum of two million Transilluminator (Clare Chemical Research). generations until posterior probability convergence Gel bands of appropriate sequence length and as confirmed in Tracer v1.7 (Rambaut et al. 2018), quantity were then purified using the Qiagen Gel with a sampling frequency of every 500 generations Purification kit according to manufacturer’s and a diagnostic frequency of every 5,000 generations, instructions. Alternatively, aliquots of amplified with one cold and three heated chains, and a relative DNA were analyzed on 1% agarose gel, with the burn-in fraction of 0.25. Independent runs were not remainder purified using the QIAquick PCR combined due to the similarity of tree topology and Purification kit according to manufacturer’s posterior probability values. The resultant trees were instructions. Purified samples were sequenced processed in FigTree v. 1.4.4 (Rambaut 2018) with bidirectionally using the BigDye Terminator tree annotations added in GNU Image Manipulation sequencing system (Applied Biosystems) by First Program (GIMP). Base Asia. The Thai words used on specimen lables and in the text are Kho (= Island). Abbreviations used Data analysis. Sequences were filtered by trace in the text: BMNH – Natural History Museum, scores, QV20+ and contiguous read length in London; LV – left valve; MNHN – Museum Sequence Scanner Software 2 (Applied Biosystems). National d’Histoire Naturelle, Paris; MZB – Museum Sequences were trimmed, edited and aligned in Zoologicum Bogoriense, Cibinong, Java, Indonesia; Aliview (Larsson 2014) using MUSCLE, with PMBC – Phuket Marine Biological Center; RV – right alignment being conducted with respect to codons valve; SL – shell length; ZRC – Zoological Reference for CO1 and H3. Collection, Lee Kong Chian Natural History Museum, All sequences obtained in this study were National University of Singapore; ZSI – Zoological deposited in Genbank (Appendix 1). Reference Survey of India, Kolkata. sequences are obtained from NCBI Genbank (Appendix 2). For the concatenated dataset, reference RESULTS sequences were chosen in accordance with the sequences used in Liu et al. (2018) and Morton et Phylogenetic analysis (Fig. 1). Maximum likelihood al. (2020a; 2020b). (ML) and Bayesian inference (BI) analyses for Sequences were concatenated using Sequence mtCO1, mt16S, ITS1, 28S D1R and H3 were able Matrix (Vaidya et al. 2011). Preliminary phylogenetic to consistently, strongly and independently resolve trees of reference sequences were generated in Byssogerdius (i.e., ‘Brachidontes striatulus’ and MEGA X (Kumar et al. 2018) to determine if there ‘Brachidontes setiger’; see Systematic Description were any aberrant sequences due to DUI or section below) as monophyletic and separate from misidentification, as some concatenated sequences members of Brachidontes. In addition, with the 81

Taxonomic re-description and relationships of two mat-forming mussels exception of 28S D1R, the other genes were able to probabilities (PP) and bootstrap values (BS) resolve Byssogerdius as consisting of two distinct supporting the separation of the two species species. However, individual gene phylogenies within Byssogerdius (PP value of 1, BS value of presented an inconsistent picture of the position 99) as well as its monophyly as a genus relative to of various genera relative to one another, and thus other Mytilidae (PP value of 1, BS value of 100). concatenated datasets of various genes were made Concatenation generally increased support for less to resolve this issue. In most cases, ML analyses well-supported branches in the individual gene gave lower support values than BI. trees. All concatenated datasets had strong support Despite the differing evolutionary histories for the hypothesis presented above, albeit with and mutation rates between nuclear and mitochondrial some differences in support for certain branches. genes, we concatenated mtCO1, ITS1, 28S D1R and We also examined pairwise differences within H3 into a single dataset for phylogenetic analysis. and between the two putative species of Byssogerdius, The topology of the tree (Fig. 1A) generated from and between Byssogerdius and other genera in this dataset matched that of a dataset containing the Mytilidae, in order to determine if Byssogerdius latter 3 nuclear genes only, with the exception of striatulus and B. subsulcatus were sufficiently higher support values for all branches. different to be separate species, and Byssogerdius While ITS1 (de Souza et al. 2015) and 28S D1R a separate genus. Pairwise differences within both (Trovant et al. 2013; Colgan and da Costa 2013) Byssogerdius species were under 1%, within the have been used in several studies on Mytilidae, there acceptable threshold for species. Pairwise differences was a general lack of good alignments for the ITS1 between both Byssogerdius species were either and 28S D1R sequences obtained in this study with similar to or lower than that in other reference genera. those in reference databases. Thus, a concatenated Using the mt CO1 dataset as an example, intra- tree of mtCO1 and H3 (Fig. 1B) was also generated genus pairwise differences for Byssogerdius were with the addition of reference sequences from about 0.02, compared to 0.26 for , and 0.35 published literature and reference databases. for . Additional barcode data for Byssogerdius for Byssogerdius was found to be sufficiently the 18S and 28S regions used in Liu et al. (2018) different from its sister groups in the subfamily and Morton et al. (2020a; 2020b) are needed to Mytilinae to at least be its own genus, although constrain the relationships between Byssogerdius whether it is different enough to be part of another and other Mytilidae. Despite the smaller number subfamily altogether is uncertain. Inter-genus of mt16S sequences obtained in this study, we pairwise differences between Byssogerdius and generated a tree based on a concatenated dataset of other mytilid genera ranged from 0.288 to 0.322 the two mitochondrial genes, mtCO1 and mt16S within Clade B (following Morton et al. 2020a) (Fig. 1C), which once again showed identical of Mytilidae, while pairwise differences between topology to the tree from the 4-gene dataset (Fig. genera in this clade ranged from 0.187 between 1A) and the nuclear gene-only dataset (Fig. 1D). and Mytilus, which are within the same Support values were significantly higher for some subfamily, to 0.348 between Trichomya and branches, such as for the relative position of . Musculinae compared to Mytilinae and Byssogerdius. Pairwise differences between known subfamilies The further addition of H3 data to the mtCO1 + 16S in this clade ranged from 0.293 between Musculinae dataset (Fig. 1E) had support values more similar and Mytilinae, to 0.323 between Septiferinae and to the 4-gene dataset, being slightly higher for Mytilinae. Given that the pairwise difference between some groups, and lower for the relative position of Byssogerdius and Mytilus is lower than this value Musculinae. (0.231), Byssogerdius fits the threshold to qualify Overall, all concatenated datasets generated as a member of Mytilinae, although subfamily phylogenies that were well supported for most taxa assignments in Mytilidae should be taken with and their tree topologies were nearly identical. The caution. 4-gene dataset showed especially high posterior 82

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Taxonomic re-description and relationships of two mat-forming mussels B 84

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Taxonomic re-description and relationships of two mat-forming mussels D 86

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Taxonomic re-description and relationships of two mat-forming mussels , ) . (2020a) Crassostrea et al ; mytilid taxa in gen. nov., and Byssogerdius striatulus Byssogerdius ” (= Xenostrobus Byssogerdius . (2020a in text; reversed with ) (see also Appendices 1 and 2 and et al Modiolus Brachidontes striatulus . Byssogerdius subsulcatus Byssogerdius ” (= F . (2018). B. Concatenated Bayesian trees based on mtCOI and nuclear and mtCOI on based trees Bayesian Concatenated B. (2018). . F Isognomon . Phylogenetic relationships within et al and B. setiger Figure 1 Figure ITS1, mtCO1, of analysis sequence Concatenated A. Mytilidae: other amongst 28S D1R and H3 genes from “ and “ for shells). 3 Posterior probabilities (left and 2 Figs. and details sequence for number) and of (right number) nodes values maximum likelihood bootstrap are shown. The tree is rooted by of Mytilidae, as A shown in Morton Clade Clade B in Allfigure). tree annotations are modified after Morton Liu and H3 genes; C. mtCOI and mt16S genes; D. Nuclear genes and (ITS1, H3) only; E. 28S mtCOI, mt16S D1R and nuclear H3 genes; mtCOI F, gene only. Posterior probabilities (former number) and maximum likelihood bootstrap values (latter number) of major nodes are shown. Bootstrap values are shown for major branches on the mtCOI gene tree where topologies are congruent non-mytilidsby rooted were Trees approaches. two the between Pinna 88

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Figure 2. Shells of sequenced specimens of “Brachidontes striatulus” (= Byssogerdius striatulus): A–D, L–M: Ao Kung Krabaen, Chanthaburi Province, Thailand; A. B1T, SL=25.2 mm; B. B2T, SL=33.6 mm; C. B3T, SL=24.1 mm; D. B4T, SL=27.3 mm; L, BS TKK0719A, SL=22.3 mm; M, BS TKK0719B, SL=21.6 mm; E–I, Siglap Canal, East Coast Park, Singapore; E. B2SG, SL=22.0 mm; F. BSS1, SL=19.7 mm; G. BSS2, SL=20.5 mm; H. BSS3, SL=14.1 mm; I. BSS6, SL=17.4 mm; J–K: Pantai Badur, Madura, East Java, Indonesia; J. BST I1, SL=17.2 mm; K. BST I2, SL=22.6 mm; N, O: Ko Sirey, Phuket, Thailand; N. BST SW1, SL=24.5 mm; O. BST TSW1, SL=27.3 mm; P: Kuraburi, Ranong Province, Thailand; BST TSW2, SL=16.3 mm. See Appendix 1 for corresponding voucher specimen reference numbers associated with the DNA codes used above. 89

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Figure 3. Shells of sequenced specimens of “Brachidontes setiger” (= Byssogerdius subsulcatus): A–B: Penghu, Taiwan; A, 18BTW2, SL=17.7 mm; B, 18BTW3, SL=21.1 mm; C–F: Ko Sirey, Phuket, Thailand; C. BST SW2, SL=20.0 mm; D. BST SW3, SL=18.7 mm; E. BST SW4, SL=22.7 mm; F. BST SW5, SL=18.8 mm; G: Siglap Canal, East Coast Park, Singapore; BSS 7, SL=18.7 mm; H–I: Pattaya, Thailand; H. BST P0719, SL=21.4 mm; I. BST P0719B, SL=22.6 mm; J: Kho Kut, Trat, Thailand; B1TT, SL=26.1 mm. See Appendix 1 for corresponding voucher specimen reference numbers associated with the DNA codes used above.

Systematic description region. Shell microstructure: Outer shell layer under periostracum comprises a sublayer of a thin Order calcitic homogeneous crust overlying an aragonitic Superfamily Rafinesque, 1815 lower sublayer of irregular prisms. The middle Family Mytilidae Rafinesque, 1815 layer with aragonitic sheet nacre is usually the Byssogerdius new genus thickest layer. The innermost aragonitic nacreous shell layer comprises variably developed irregular Type species. Modiola striatula Hanley, 1843 here simple prismatic structure. Anatomy: Posterior designated. mantle edges bear numerous branched finger- like processes (guard papillae) densely distributed Diagnosis. Shell: length to 36 mm, mytiliform, along the margin of the inhalant siphon. The plicate umbones subterminal, surface with fine radial ribs. organ (=organ of Sabatier) is found along the base Hinge plate wedge shaped, broader posteriorly. of the posterior half of the outer demibranch on Crenulations present along almost the entire each side of the . An inner plicate organ is shell margin except along part of the ventral absent. 90

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Remarks. The genus of the type species was Ramakrishna and Dey 2010, p. 80–81 and pl. IV, initially assigned to Modiola by Hanley (1843), in figs. 13–14. line with its possession of subterminal umbones, Gregariella striatula - Nielsen 1976, p. 3 and fig. but was later placed in the genus Brachidontes (as 11; - Tantanasiriwong 1979, p. 5; - Morris and Pur- Brachydontes Jukes-Browne, 1905) for having chon 1981, p. 323. radial ribs on its external surfaces and denticles on Brachidontes emarginatus - Lee and Morton 1985, the inner dorsal margins of the shell. Molecular p. 53 and pl. 1C; - Nguyen 2001, p. 412, fig. phylogenetic analysis (see above, this study) Brachidontes striatulus - Wang 1997, pp. 82–84, however has shown that Modiola striatula is neither fig. 32 and pl. 4, fig. 2; - Morton and Tan 2006, related to Modiolus nor Brachidontes, and hence fig. 2; - Higo et al. 2001, p. 148, fig. B112; - Xu we propose a new genus here to accommodate this and Zhang 2008, p. 48, fig. 121 and p. 49; - Huber and one other species (see below). 2010, p. 118, fig. and p. 551; - Kurozumi 2017, pp. 517 and 1173, pl. 473, fig. 10 (in error as sturiatu- Etymology. Name derived from the Latin ‘’ lus); - Subba Rao 2017, p. 82 and pl. 15, fig. 64 for thread and ‘gerdius’ for weaver, in reference to (in part). the mat-making habits of its members. The noun is Brachiodontes sp. - Lozouet and Plaziat 2008, masculine in gender. plate 3, figs. 5–6.

Byssogerdius striatulus (Hanley, 1843) new Diagnosis. Shell generally elongate and often combination arcuate ventrally but can vary considerably in Figs. 1, 2, 4, 5, 9C, 10 and 11 outline. Periostracum olive-brown to dark brown dorsally and often lighter coloured ventrally. A Modiola striatula Hanley 1843: p. 241 and pl. 24, few to several dark brown (often nearly black) fig. 29 (BMNH 1965106; type locality: Batangas, radial lines or rays are usually present. The number Philippines); - Reeve 1858, vol. 10, species 59; pl. of such rays can differ between the left and right 10, fig. 72; - Annandale and Kemp 1916, pp. 360– valves. On each valve, the width of these 362, pl. 15, figs. 7–18. pigmented lines usually increases from the umbonal Modiola emarginatus Benson in Reeve, 1858: vol. region to the posterior. Shell surface whitish under 10, species 60; pl. 10, fig. 73 (BMNH 1965115; periostracum. type locality: unknown). Modiola emarginata - Küster and Clessin 1889, p. Type Material Examined. Three syntype lots of 120, species 44; pl. 25, figs. 7–8. Modiola striatula Hanley in the type collection Brachyodontes striatulus - Jukes-Browne 1905, p. of the section of the Natural History 223. Museum in London (NHM). The first lot (BMNH Modiola granolirata Sowerby, 1909: p. 201, fig., 1907.10.28.54) comprises a single intact specimen (holotype BMNH 1909.10.19.73; type locality: (Fig. 4A, B) measuring 35 mm in length. Externally Bay of Manila). both valves are pigmented almost uniformly brown Brachyodontes (Hormomya) emarginatus - Lynge, and no radial pigment rays were observed, as illustrated 1909, p. 135. in Hanley (1856, pl. 24, fig. 29). Some 70 radial Modiola celator Preston, 1911: p. 41, fig. 5 (length ribs cross the surface in the posterior half of the 7 mm) Indian Museum (ZSI) registration no. shell, and a further 25 are present anteriorly ventral M.5248/1, Puri Beach, Orissa. to the umbo. The label accompanying it states that Modiolaria (Brachydontes) striatula - Leschke, it is the ‘figured syntype’. The second syntype lot 1911, species 97 (list). (BMNH 1965106) consists of three intact specimens, Brachydontes striatulus - Lamy 1936, pp. 162–163. one measuring 34 mm and the other two 35 mm Brachydontes emarginatus var. eudeli Jousseaume each in shell length. All three specimens bear in Lamy, 1936: pp. 163–164 (MNHN). several radial stripes which are pigmented dark Modiolus striatulus - Subba Rao and Mookher- brown over a yellowish-brown periostracum (Fig. jee 1975, p. 172–173; - Morton 1977, figs. 1–2; 4C, D). The label accompanying the lot indicates - Subba Rao et al. 1992, p. 221 and pl. 16, figs. ‘close’, referring to the closeness of the syntypes 2, 4; - Subba Rao et al. 1995, p. 413–414 (key); - to Hanley’s description. The third lot (BMNH 91

Taxonomic re-description and relationships of two mat-forming mussels

1985010) also contains three specimens, of which Two possible syntypes of Modiola emarginatus the first (1985010/1) is not Modiola striatula but Reeve, 1858 (BMNH 1965115) are in the type an undetermined Modiolus species. The remaining collection of the NHM in London. Both are intact two syntype specimens, accompanied by a separate specimens. The larger, measuring 34 mm in length label (BMNH 1985010/2, 3) measure 35 mm (Fig. (Fig. 4G, H) has an intact olive-brown to brown 4E, F) and 22 mm respectively. The label indicates periostracum that is slightly darker on the ventral that the specimens were originally from the Leeds half of the shell. Some 60 radial ribs are present City Museum (see also Coan and Kabat 2012). over the dorsal and posterior ¾ of the shell, and a

Figure 4. Byssogerdius striatulus (Hanley, 1843). A–B. Modiola striatula Hanley, 1843, figured syntype, BMNH 1907.10.28.54, type locality: Batangas, Philippines; SL=35.2 mm; C–D. Modiola striatula Hanley, 1843, 1 of 3 syntypes, BMNH1965106, type locality: Batangas, Philippines; SL=35.0 mm; E–F. Modiola striatula Hanley, 1843, 1 of 2 syntypes, BMNH 1985010/2,3, type locality: Batangas, Philippines; SL=34.5 mm; G–H. Modiola emarginata Benson in Reeve, 1858, figured syntype BMNH 1965115, type locality: unknown; SL=34.1 mm; I–J. Modiola emarginata Benson in Reeve, 1858, syntype BMNH 1965115, type locality: unknown; SL=30.0 mm; K–L. Modiola celator Preston, 1911, holotype, ZSI M5241/1, type locality: Puri Beach, Orissa, India; SL=7.0 mm; M–N. Modiola granolirata Sowerby, 1909, holotype BMNH 1909.10.19.73, type locality Manila, Philippines; SL=24.7 mm; O–P. Brachydontes striatulus var. eudeli Jousseaume, 1936, 1 of 4 syntypes, MNHN-IM-2000-35845, type locality: Karikal, India; SL=31.2 mm. 92

Phuket mar. biol. Cent. Res. Bull. further 18 ribs anteriorly. In some areas, the ribs multiple images of the small (SL=7.0 mm), possibly are granulated where the commarginal growth juvenile shell, there are at least 22 radial ribs over lines intersect with them. The smaller specimen, the posterior half of the shell and another 23 on measuring 30 mm in shell length (Fig. 4I–J) is the anterior region. A paratype (not examined; narrower in profile with about 40 radial ribs over NMW.1955.158.15873) from the type locality is the posterior region of the shell, and about 23 in the Melville-Tomlin collection at the National anteriorly. The shell has a slight greenish tinge and Museum of Wales in Cardiff (UK). there is an indistinct dark brown radial band just Likewise, only the images of one of the four dorsal to the keel. syntypes lodged at the Muséum Nationale The holotype of Modiola celator Preston, 1911, d’Histoire Naturelle in Paris of Brachydontes ZSI M5241/1 (Fig. 4I–J) in the Zoological Survey of emarginatus var. eudeli Jousseaume, 1936 (MNHN- India was not physically examined but based on IM-2000-35845; SL=31.2 mm, Fig. 4O–P) were

Figure 5. Byssogerdius striatulus (Hanley, 1843). A–B. Cardwell, Queensland, Australia; SL=25.0 mm (BMNH); C–D. Cape Rachado, Malaysia; SL=26.1 mm (BMNH); E–F. BMNH 1984026, Sam Mun Tsui, Tolo Harbour, Hong Kong; SL=19.6 mm (in Lee and Morton 1985 p. 53 and pl. 1C, as Brachidontes emarginatus and referenced as BMNH 1984060); G–H. Ratnagiri, Maharashtra, India; SL=35.2 mm (BMNH); I–J. Solander Bridge, Dar-es-Salaam, Tanzania; SL=32.3 mm (BMNH); K–L. Myitkaya Creek etc., Bassein River, Myanmar; SL=13.5 mm (BMNH); M–N. Kolkata, India; SL=27.3 mm (BMNH); O. BMNH 20010211, Calcutta Docks, Bhut Ghat, Kolkata, India (as Brachidontes striatulus; collected and determined by B.S. Morton; see Morton 1977); P–Q. Chennai (Madras), India; SL=23.5 mm (P), 21.0 mm (Q) (BMNH); R–U. Kolkata, India; SL=25.1 mm (R), 27.2 mm (S), 23.4 mm (T), 24.4 mm (U) (all BMNH). 93

Taxonomic re-description and relationships of two mat-forming mussels studied. There are at least 40 radial ribs present the anterior third of the shell, where these ribs are over the posterior region of the shell, and about 13 weak, much reduced, obsolete or absent altogether anteriorly. The shell is distinctly greenish over the and the periostracum (if present) is also the darkest. posterior half of the shell, whilst the antero-ventral Up to six dark brown pigment lines or stripes may and dorsal regions are dark brown. be present on the posterior half of the shell and these are usually parallel to (but not always) the Shell (Figs. 2, 4–5). Length to 36 mm, mytiliform, radial ribs. The number of such pigment bands may equivalve, inequilateral, elongate, inflated. Outline be different between right and left valves of any of shell highly variable. The valves are widest individual. These pigment bands also vary in width slightly anterior to the halfway mark along a line and can occupy 1–10 ribs wide but may also be drawn from the anterior to the posterior extremities discontinuous. Anteriorly, similar pigment bands of the shell. Periostracum greenish olive-brown (about 2–4 in number) can sometimes be observed. to dark brown dorsally and yellow brown to dark In the same anterior region of the shell, ten or more brown ventrally below the keel. Umbones subterminal. closely set, short radial ribs emanate from the umbonal Fine, flattened radial ribs (approx. width 0.2 mm) region towards the antero-ventral edge. The dorso- are usually present over the almost entire external posterior region of the shell is sometimes covered surface of the valves except on the ventral half of with numerous byssal hairs, particularly in smaller

Figure 6. Species previously synonymized with Byssogerdius striatulus. A–B. Modiola annandalei Preston, 1911 paratype, BMNH 1910.12.13.17, type locality: Rambha, Chilka Lake, India; SL=18.5 mm; C–D. Modiola chilkaensis Preston, 1910 paratype, BMNH 1910.12.13.12, type locality: Rambha, Chilka Lake, India; SL=9 mm; E–F. Modiola cochinensis Preston, 1909, holotype Zoological Survey of India M.4612/I, type locality: Cochin, India; SL=10.5 mm; G–H. Modiola jenkinsi Preston, 1910 holotype, Zoological Survey of India M4426/1, Manikpatna, Chilka Lake, India; I–J. Modiola taprobanensis Preston, 1915, 1 of 3 paratypes, BMNH 910.12.13.18-20, type locality: Ceylon; SL=19 mm; K–L. Brachidontes virgiliae Barnard, 1964, possible syntype, BMNH 1964258, type locality: Keurbooms River, Cape Province, South Africa; SL=15.2 mm. 94

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(typically < 20 mm SL) individuals, but nearly all subcutaneous yellow pigment underneath around individuals examined possess them to a greater or the orifice. Anteriorly, the inner and outer labial lesser extent. palps are about the same size, approximately ¼ Byssal hairs when present are olive-greenish length of ctenidium; labial palps each with about yellow, simple, straight or curved and tapering to 40 folds. The organ of Sabatier (= plicate organ) a point, 1–2 mm in length and about 60 µm in is found along the base of the posterior half of the diameter at mid-region. Recently laid hairs, which outer demibranch on each side of the animal. An are lighter coloured, are nearer the posterior shell inner plicate organ is absent. The pericardial complex edge especially in larger individuals. Byssal threads (pericardium, ventricle and auricles) is located emanating from the base of foot are very fine and anterior to the posterior byssal retractor muscle extremely numerous. They are used to form mats block and directly ventral to the posterior half of incorporating shells, coral rubble, pebbles, stones the hinge plate. and sand grains. The stomach (see Dinamani 1967) lies dorsal The interior of the shell is thinly nacreous, to and between the pair of anterior foot retractor with a translucent shell layer overlying a purplish muscles. The intestine extends posteriorly along pigmented region underneath that is often darkest the right mid-dorsal region of the animal below the along the region corresponding to the area dorsal heart and makes a sharp upward turn at the dorsal to the keel of each valve, as well as to the pigment hump of the posterior adductor muscle. It then bands (if present) visible on the external surface. extends anteriorly along the left side of the animal Ventral to the keel towards the anterior end, the below the rectum until it reaches the left side of interior of the shell is light coloured. The inside the stomach, where it makes a turn again to extend margins anterior and posterior to the hinge bear backwards as the rectum through the heart and over small teeth, numbering up to 10 along the dorsal the posterior adductor muscle. The anus is simple. shell margin. About the same number or fewer dysodont teeth, which are largest in size towards Distribution (Figs. 10–11) the anterior tip of the shell, are also seen along the . The species is sometimes present in the inside of the anterior shell margin. Small teeth also intertidal zone of estuaries embedded in byssal occur underneath the ligament. Along the posterior mats that incorporate mud, sand and shell fragments inside edge of the shell, numerous crenulations (see Fig. 11). They can also occur singly, nestling corresponding in position to the termination of the amongst oysters or , preferring brackish radial ribs on the shell external surface are also water conditions. Byssal hairs are generally lacking present. Small, shallow pits occur on the inside but may be present in low density on the dorso- surface of the anterior half of the shell. The posterior posterior region of their valves. adductor muscle scar can appear separate or fused with the posterior byssal retractor scars. The area Geographical range. Indo-Pacific, ranging from of the anterior adductor muscle scar is about a third East Africa through to Indonesia and northern of the posterior adductor scar. Australia (Fig. 10). Morton and Tan (2006) earlier reported Byssogerdius (as Brachidontes) striatulus to Shell microstructure. See description for Byssogerdius be an alien species in Singapore, with the assumption subsulcatus and Fig. 8. that it originated from India. However, its geographical distribution appears to be across the entire Indo-west Animal (Fig. 9C). Foot large, vermiform, yellow. Pacific region, and the Indian form of this species Posterior mantle edges bear numerous branched is somewhat different in external appearance to those finger-like processes (guard papillae; see also currently occurring in Singapore. This brings to Audino et al. 2020) along the margin of the inhalant doubt the notion that the species was introduced siphon which are translucent white with yellow to Singapore. subcutaneous pigment aggregations. Towards the bases of the papillae, brown to black surface pigment Records. Tanzania: Zanzibar India: Chilka Lake, forms a narrow band along the outer and inner Kolkata, Pt. Canning (BMNH); Thailand: Ko regions of the inhalant siphon. The exhalant siphon Sirey, Phuket (ZRC); Ao Kungkrabaen, Chanthaburi is also outlined by black surface pigment and Province (ZRC); Malaysia: Cape Rachado 95

Taxonomic re-description and relationships of two mat-forming mussels

(BMNH); Singapore: East Coast Park (ZRC); Airway and Dey 2010; Huber 2010; Subba Rao 2017, as Buoy off Tanah Merah (ZRC); Indonesia: Madura, Brachidontes). However, Modiola cochinensis E. Java (MZB; ZRC); Philippines: Batangas (type Preston, 1909 (Fig. 6E–F) and M. taprobanensis locality of striatulus); China: Hainan Id (Wang 1997); Preston, 1915 (Fig. 6I–J) are unlikely to be synonyms Hong Kong (BMNH); Japan: Okinawa (Kurozumi of striatulus. The shell surface of the holotype of 2017); Australia: Cardwell, Queensland (BMNH). M. cochinensis (ZSI M4612/1) from Cochin, India has no radial sculpture whatsoever and is dominated Remarks. A number of species from the Indian by commarginal growth lines. However, the interior subcontinent, particularly those proposed by H. B. dorsal and anterior margins bear several crenulations. Preston (see Fig. 6 and Table 1), have been It is probably a distinct species with close affinities to synonymised with Byssogerdius striatulus (see Arcuatula. In the case of M. taprobanensis (‘co-types’; Annandale and Kemp 1916, as Modiola; Ramakrishna BMNH 1910.12.13.18-20) from Ceylon, apart

Table 1. Comparison of conchological characters between Byssogerdius striatulus and six other species previously synonymised with it (Annandale and Kemp 1916; Kilburn and Rippey 1982). Type material associated with these six species are shown in Figure 6.

Species / striatulus annandalei chilkaensis cochinensis jenkinsi taprobanensis virgiliae character Hanley, Preston, Preston, Preston 1909 Preston, Preston, 1915 Bernard, 1843 1911 1911 1910 1964 shell length 35.3 (M) 18.5 9 (paratype, 8.2 (M); 10.5 12.5 19 (paratype, 15.2 (mm): M: (paratype, M); 17.5 (holotype, P) (holotype, M); 18 (syntype, measured in M); 19.75 (holotype, M); 13.5 (holotype, P) M) this study; P: (holotype, P) (holotype, by original P) P) author

external brown or reddish reddish mottled dark mostly dark brown brown colour dark brown, brown to brown and light dark green, throughout or mixed with sometimes orange and green brown lighter dark greenish green with radial anteriorly, anteriorly; green at brown dorsally stripes patterned; green posterior and light dark green posteriorly region with brown ventral posteriorly pale brown to keel radial lines

position of sub- sub- sub- sub- sub- terminal terminal, umbones terminal terminal terminal terminal terminal black in coloration

radial ribs present present present none present none present over almost only ventral only ventral only over almost entire shell to umbo to umbo ventral to entire shell surface and at and at umbo and surface dorsal and dorsal and at dorsal posterior posterior margins margins margins

crenulations yes yes yes yes yes no yes inside anterior margin and posterior to hinge plate 96

Phuket mar. biol. Cent. Res. Bull. from having terminal umbones, the shell surface is - Küster and Clessin 1889, p. 113, sp. 32; pl. 28, also smooth without any trace of radial ribs, but the figs. 7–8. interior margins are similarly devoid of crenulations. Brachydontes (Hormomya) setiger - Lamy 1936, p. Lamy (1936) in fact regarded this species as 191 (not setiger Dunker, 1857). belonging to the genus . Gregariella striatulus - Nielsen 1976, p. 3 and fig. 11. Three other species, M. annandalei Preston, Gregariella subsulcata - Arnaud and Thomassin 1911 (Fig. 6A–B), M. chilkaensis Preston, 1910 1990, p. 335, 338. (Fig. 6C–D) and M. jenkinsi Preston, 1910 (Fig. Gregariella sp. - Oliver 1992, p. 51 and pl. 5 fig. 6G–H) are here also suggested to be distinct from 8a–b. striatulus. Although all of them clearly possess Brachidontes setiger - Kuroda 1932, p. 130; - Kira subterminal umbones, and the presence of anterior 1965, p. 127 and pl. 46, fig. 2; - Wang 2004, p. 232 radial ribs and crenulations on the inside margins of fig. 42 (map) and pl. 123A (shell); - Xu and Zhang the shell are as in typical striatulus, radial ribs are 2008, p. 48, fig. 122 and p. 49; - Huber 2010, p. only present over the dorsal and posterior margins 118, fig.; - Poppe 2010, p. 510, pl. 946, figs. 3–4; of the shells in M. annandalei and M. chilkaensis. - Kurozumi 2017, pp. 517 and 1173–1174, pl. 473, In M. jenkinsi, most of the surface of the shell is fig. 11 (notsetiger Dunker, 1857). smooth, except along its dorsal margin. The general lack of radial ribs in the central region Diagnosis. Shell somewhat inflated, periostracum of the valves, together with the presence reddish- orange to olive- to dark brown dorsally and often brown markings over the shell surfaces of these lighter coloured ventrally; pigmented radial stripes three species seem to warrant their separation from absent. Shell surface whitish under periostracum. striatulus. However, there are clearly shells with intermediate features from the east coast of India Type Material Examined. A total of four syntypes (e.g., Figs. 4O–P; 5M–O) and further studies are of Volsella subsulcata Dunker, 1857 (BMNH needed to determine their status (see also Discussion 1967569) are present in the type collection of the below). Mollusca section of the Natural History Museum Kilburn and Rippey (1982) suggested that the in London. The measured syntype (BMNH South African Brachidontes virgiliae (Barnard, 1967569/1) comprises an intact specimen (Fig. 1964) (Fig. 6K–L; Table 1) could be a synonym 7E– F) has a shell length of 25.8 mm. The remaining of striatulus. However, careful examination of three syntypes are smaller in size (shell lengths a syntype of B. virgiliae deposited in the Natural 21.4, 24.0 and 24.6 mm). All bear simple, tapering History Museum in London (BMNH 1964258) byssal hairs at the posterior region of the valves. revealed the position of its umbones to be terminal. The umbones are also distinctively dark coloured, Shell (Fig. 7E–J). Length to 30 mm, mytiliform, which is not the case in other striatulus specimens equivalve, inequilateral, somewhat inflated, anterior so far examined. We regard the two species as and posterior ends rounded. The valves are widest distinct. about midway between their anterior and posterior We tentatively regard Sowerby’s granolirata ends. Periostracum orange- or olive-brown dorsally (Fig. 4M–N) from the Philippines as a synonym and yellow brown ventrally, sometimes worn in of B. striatulus. Given its rather inflated umbonal some areas to show a whitish shell surface underneath. region however, it remains possible that it is more Umbones subterminal. Fine, flattened radial ribs allied to Gregariella (see Remarks under subsulcata (approx. width 0.2 mm) are present over the entire below). external surface of the valves except on the ventral half of the anterior third of the shell, where these Byssogerdius subsulcatus (Dunker, 1857) new ribs are weak, much reduced, obsolete or absent combination altogether and the periostracum (if present) is also Figs. 1, 3, 7E–J, 8, 9A–B, 10, 11 the darkest. At the anterior region of the shell, ten or more (up to 20) closely set, short radial ribs Volsella subsulcata Dunker, 1857: p. 364 (type locality: emanate from the umbonal region towards the Manila, Philippines) (syntypes BMNH 1967569/1–4). antero-ventral edge. Dorso-posterior region of the Modiola subsulcata - Reeve 1857, pl. 8, fig. 47; shell is sometimes covered with numerous byssal 97

Taxonomic re-description and relationships of two mat-forming mussels hairs, particularly in smaller (typically < 20 mm coloured. The inside margins anterior and posterior SL) individuals, but nearly all individuals examined to the hinge bear small teeth, numbering up to 10 possess them to a greater or lesser extent. Byssal along the dorsal shell margin. About the same num- hairs olive-greenish yellow, simple, straight or ber or fewer, which are largest in size towards the curved and tapering to a point, 1–4 mm in length anterior tip of the shell, are also seen along the on and about 60 µm in diameter at mid-region. Recently inside of the anterior shell margin. Small teeth also laid hairs are nearer the posterior shell edge especially occur underneath the ligament. Along the posterior in larger individuals. Recently laid hairs are also inside edge of the shell, numerous crenulations lighter coloured. Byssal threads emanating from corresponding in position to the termination of the the base of foot are very fine, extremely numerous radial ribs on the shell external surface are also and form mats incorporating shells, coral rubble, present. The posterior adductor muscle scar is stones, pebbles, sand grains etc. continuous with the posterior byssal retractor The interior of the shell is thinly nacreous, scars. Anterior adductor muscle scar is about half with a translucent shell layer overlying a purplish the size of the posterior. Small, shallow pits occur pigmented region underneath that is often darkest the inside surface of the anterior half of the shell. along the region corresponding to the area dorsal to the keel of each valve. Ventral to the keel towards Shell microstructure (Fig. 8): Outer shell layer under the anterior end, the interior of the shell is light periostracum comprises a sublayer of a very thin

Figure 7. Gregariella setigera (Dunker, 1857) (A–D) and Byssogerdius subsulcatus (Dunker, 1857) (E–J). A–B. Volsella setigera Dunker, 1857 figured syntype, BMNH 1965107, type locality: unknown; SL=27.4 mm; C. Volsella setigera Dunker, 1857 measured syntype, BMNH 1965108/1, type locality: unknown; SL=25.8 mm; D. Volsella setigera Dunker, 1857, syntype BMNH1965108/2, type locality: unknown, SL=27.3 mm; E–F. Volsella subsulcata Dunker, 1857 measured syntype BMNH 1967569/1, type locality: Manila, Philippines, SL=25.9 mm; G–H. Pattaya, Thailand; SL=25.9 mm; I. Lamu, Kenya; SL=15.8 mm (BMNH); J. Musamba Cha, Shimoni, Kenya; SL=16.4 mm (BMNH). 98

Phuket mar. biol. Cent. Res. Bull.

(<20 µm) possibly calcitic homogeneous crust embedded with mud, sand and shell fragments in overlying an aragonitic lower sublayer of irregular the intertidal to subtidal zones, but they can also prisms (see also Owada and Hoeksema 2011). The occur singly nestling amongst oysters or barnacles, middle layer with aragonitic sheet nacre is thickest. preferring full salinity conditions. Byssal hairs are The innermost aragonitic nacreous shell layer is typically present on the dorso-posterior region of variably developed, sometimes having more than a their valves. The hairs trap silt and sediment which single layer of irregular simple prismatic-structure may help to camouflage the posterior mantle and nacre. siphonal tissue when the animal is agape.

Animal (Fig. 9A–B). See description for B. striatulus. Geographical range. Indo-Pacific, ranging from east Africa to possibly Fiji; southern Japan may be Distribution (Figs. 10–11) its northern limit. The species has not been reported Habitat. The species often occurs in byssal mats from Australia.

Figure 8. Byssogerdius subsulcatus: shell microstructure. A. Transverse section of shell showing periostracum (p), aragonitic layers a1 (irregular prisms), a2 (sheet nacre) and a3 (simple prismatic nacre). Scale bar 100 µm. B. Transverse section, detail showing possible thin calcitic shell interface immediately below periostracum; abbreviations as in A. Scale bar 20 µm. C. External surface view of shell showing calcitic layer (c) under periostracum (p). Scale bar 100 µm. 99

Taxonomic re-description and relationships of two mat-forming mussels

Figure 9. Byssogerdius: external anatomy. A. Right valve of B. subsulcatus from Pattaya, Thailand removed to show preserved animal from right side. B. Living B. subsulcatus from Pattaya, Thailand seen from the ventral side with fully extended posterior mantle margin showing numerous branched guard papillae. C. Dissected posterior mantle margin of B. striatulus from Siglap, Singapore. Abbreviations used: aa, anterior adductor muscle; an, anus; apr, anterior pedal retractor muscle; b, byssus; bg, byssal gland; bs, base of siphon; f, foot; id, inner demibranch; it, intestine; im, inner mantle lobe; k, kidney; lb, labial palp; mo, mouth; ms, mesosoma; od, outer demibranch; om, outer mantle lobe; pa, posterior adductor muscle; ppr, posterior pedal retractor muscles. 100

Phuket mar. biol. Cent. Res. Bull.

is neither allied to Byssogerdius nor Brachidontes Records. China: Hainan Island (BMNH); Kenya: but is a species of Gregariella. There are three Shimoni (BMNH Acc. 2224); Malaysia: Cape specimens in two syntype lots of Volsella setigera Rachado (BMNH Acc. 2264); Indonesia: Pulau Dunker, 1857 in the type collection of the Mollusca Berang off Pulau Lingga, Riau Province (ZRC); section of the Natural History Museum in London. Philippines: Luzon, Manila (type locality of The first lot (BMNH 1965107) comprises a single subsulcata); Taiwan: Penghu (Pescadores Ids) intact specimen (Fig. 7A–B) measuring 27.5 mm (ZRC); Thailand: Ko Sirey, Phuket, Thailand in length. The label accompanying it states that it (PMBC; ZRC); Pattaya, Thailand (ZRC); Kho is the ‘figured syntype’. The second syntype lot Mak, Gulf of Thailand (PMBC; ZRC). (BMNH 1965108) consists of two intact specimens (Fig. 7C–D). One intact specimen (1965108/1) Remarks. Dunker (1857) in describing some 14 with the valves glued together is labelled as the species of Volsella, distinguished two species of measured syntype (SL=25.8 mm). The other specimen shells with byssal hairs on their posterior regions: (1965108/2), also intact but valves separated, has subsulcata and setigera. He regarded subsulcata a shell length of 27.3 mm. The type specimens of to be intermediate in form between setigera and setigera are all characterized by an inflated umbonal another species sulcata Lamarck, although he did region that is distinctly raised dorsally above the not elaborate. Reeve (1858), Küster & Clessin hinge plate (compare Fig. 7A, C with 7E; also 7B (1889) and Lamy (1936; 1937) also treated them with 7F). This feature is shared by most, if not all as separate species. Lamy (1919; 1937) assigned species currently placed in the genus Gregariella subsulcata to Modiolaria (Gregariella) based on (see e.g., Palazzi 1981; Steger et al. 2018 fig. 5; Jousseaume’s material from the Red Sea and Cosel and Gofas 2019) including the type species, accompanying notes. However, subsequent authors, G. petagnae (Scacchi, 1832) (see Mienis 1973). possibly starting with Kuroda (1932), synonymized However, the straight ventral outline and the distinct subsulcata and setigera, and the latter species began subterminal umbones as seen in the type material to be used for Indo-Pacific forms of Brachidontes of setigera resemble the West African species G. with byssal hairs. Here we suggest that Dunker’s Volsella setigera

Figure 10. Geographical distribution of Byssogerdius striatulus (black circles) and B. subsulcatus (white circles), based on material examined in this study and on verifiable literature records (Kurozumi 2017; Wang 1997). Grey circles represent a distinct form of B. striatulus with a greenish periostracum. Base map modified from www.freevectormaps.com. 101

Taxonomic re-description and relationships of two mat-forming mussels

Figure 11 (Right). Byssal mats made by Byssogerdius species. A. Tidal monsoon canal, Siglap, Singapore, April 2019. Byssal mats (arrows) of B. striatulus and B. subsulcatus on sloping wall and floor, density 122.5±64.8 indiv. 100 cm-2 (n=4); B. Close-up of part of byssal mat of B. striatulus; C. Bed of B. subsulcatus, density 72.2±21.9 indiv. 100 cm-2 (n=10), depth 8 m between Kho Kradad and Kho Mak, Trat, Gulf of Thailand, September 2015; D. Close-up of part of byssal mat of B. subsulcatus; E. Rocky shore, Ko Sirey, Phuket, Thailand with byssal mats (arrows) of mixed populations of B. striatulus and B. subsulcatus, February 2019; F. Close-up of part of byssal mat with living individuals of Byssogerdius. 102

Phuket mar. biol. Cent. Res. Bull. multistriata (Smith, 1872) (see Smith 1872 pl. genus is to species of Brachidontes, although this 75, fig. 4). In contrast, the umbonal region of the observation is based on a single gene (Fig. 1F). valves of Byssogerdius (and Brachidontes) species Most Gregariella species (including the type species, remain small and located close to the hinge line as G. petagnae; see Mienis 1973; Cretella et al. 2004) viewed when the valves are flipped to show their are distributed in the and the internal surface. This is consistent in the shells of Atlantic coast of Africa. More than a few non-boring all sequenced specimens of subsulcatus (Fig. 3) as Gregariella species share similar mat-forming, well as the type specimen (Fig. 7F–G). gregarious habits (see e.g., Cosel and Gofas 2019) Byssogerdius subsulcatus has also been previously with Byssogerdius. Thus there is a distinct possibility that referred to as Brachidontes emarginata but it is Byssogerdius is the Indo-Pacific ecological equivalent suggested here that emarginata is a synonym of of Gregariella in the Atlantic Ocean, but proof of Byssogerdius striatulus, based on the range of shell this hypothesis awaits further analysis. variation displayed by sequenced specimens of B. While our analyses of molecular sequences have striatulus observed in this study (see above). demonstrated the considerable phenotypic plasticity of Byssogerdius striatulus and B. subsulcatus, we DISCUSSION were unable to obtain tissue samples from what appears to be a subgroup of B. striatulus centred This study clearly shows that two common in brackish water of the east and northeast mat-forming Indo-Pacific mussels, widely known regions of India. They are characterised by having a previously as Brachidontes striatulus and B. setiger wide range of shell outlines and shapes, but generally in the literature, are indeed distinct species, but do possess a greenish periostracum marked by a not belong to the genus Brachidontes. Genetically combination of regular and irregular reddish-brown (Fig. 1) they are well-defined, and morphologically markings. The radial ribs are generally wider, almost they are separable but are sufficiently similar to be forming flattened costulae in some cases, but these assigned to the same genus. Based on our results occur mainly towards the dorsal and posterior shell of phylogenetic analyses using both mitochon- margins, and the central region of the valve surface drial and nuclear genes, we have proposed a new is often smooth and devoid of radial grooves altogether. genus Byssogerdius to accommodate the two closely Their inclusion in B. striatulus is therefore tentative related species. After careful examination of type here, and they may prove to belong to a different material, we also suggest that the species name species. Observations of these animals made by subsulcatus is a more appropriate name instead of Morton (1977) in Calcutta showed that the pericardial setiger for the Indo-Pacific species treated here. This is complex is located dorsal to the posterior byssal because the types of subsulcatus do indeed resemble retractor muscles (category 3 of Morton 2015). closely the material examined and sequenced in this This is not the case for the striatulus specimens study. The types of setiger actually bear strong resem- obtained from Singapore and Thailand in this blance to species of Gregariella, which possess an study, where the heart is positioned anterior to the inflated and dorsally raised umbonal region that byssal retractor muscle block (category 2 of Morton is not observed in subsulcatus. It is interesting that 2015). both striatulus and subsulcatus have previously The concatenated datasets for mt CO1, mt 16S and been assigned to Gregariella (e.g., Lamy 1937; H3 (Fig. 1B–E) consistently showed Byssogerdius to Nielsen 1976; Morris and Purchon 1981; Oliver be a well-supported sister group to a clade consisting 1992) on account of their subterminal umbones and of the subfamily Mytilinae and the species Trichomya the presence of fine radial ribs and byssal hairs. hirsuta, which is nominally part of the subfamily We were unable to determine the phylogenetic Septiferinae, but has been shown to form a clade position of Gregariella in our concatenated trees with the Mytilinae in several phylogenetic studies as their sequences are not available. Nevertheless, (Liu et al. 2018; Morton et al. 2020a). The sister based solely on the COI gene of G. coralliophaga, group of the clade consisting of Byssogerdius and a somewhat aberrant member of the genus together putative Mytilinae is the Musculinae (inclusive with G. coarctata in view of their coral boring habits of Arcuatula, which is traditionally placed in the (Morton 1982; Kleemann 1986), Gregariella appears Crenellinae). However, support for the position of to be more related to Byssogerdius than the latter Musculinae to this clade in the mt CO1 + H3 dataset 103

Taxonomic re-description and relationships of two mat-forming mussels

(Fig. 1B) and the mt CO1 + mt 16S (Fig. 1C) dataset ACKNOWLEDGEMENTS is not particularly high, with posterior probability values between 0.81 to 0.85. On the other hand, It is a privilege and pleasure to be invited to both the ITS1 + 28S + H3 and mt CO1 + mt 16S + contribute an article towards this volume in honour of H3 datasets (Fig. 1D–E) provide strong support for the late Professor Jørgen Hylleberg, and we thank this, with posterior probability values of between Dr. Kathe Jensen for this unique opportunity to do 0.96–1. Nevertheless, Musculinae is a well-supported so. Through the Tropical Marine Mollusc Programme sister group to Mytilinae in other studies (Kartavtsev (TMMP), Jørgen was instrumental in bringing et al. 2018; Liu et al. 2018; Lee et al. 2019; Zhang together malacologists from many countries et al. 2019; Morton et al. 2020a), and it is safe to through projects, workshops and conferences hosted say that Mytilinae is the sister group to Byssogerdius, in India and Southeast Asia, for which we are all assuming Trichomya hirsuta as part of Mytilinae, grateful. We are also indebted to Mr. Supasit and together with Boonphienphol and Mr. Amonthep Khemto as part of Musculinae (Fig. 1B–F; see also Lubośny (Phuket Marine Biological Laboratory, Thailand), et al. 2020b). Ms. Shermaine Than (Republic Polytechnic, Singapore), In comparison with other radially sculptured as well as Drs. Jaruwat Nabhitabhata (Prince of mussels, the two species of Byssogerdius possess a Songkhla University, Thailand) and Fred Wells thin, possibly calcitic layer just beneath the (Curtin University, Australia) for their cheerful periostracum, a feature also seen in and assistance collecting mussels in the field. In Taiwan, some species of Brachidontes, Gregariella, and Prof. Liu Li-lian, Ms. Kang Dun-ru and Ms. Kuo Modiolus (Carter et al. 1990). While Gregariella Mong-ying (National Sun Yat-sen University, may be more related to Byssogerdius, the other Kaohsiung) rendered much needed help both in the genera are not, and the presence of calcite under laboratory and during field collection. Excellent the periostracum probably evolved independently digitalized images of type specimens deposited in different mytilid genera. Similarly, the presence in the Zoological Survey of India (Kolkata) were of only an outer plicate gland at the base of the provided by Dr. Basudev Tripathy, while those in outer demibranch of their ctenidia in both the Muséum National d’Histoire Naturelle (Paris) Byssogerdius striatulus and B. subsulcatus is were made available by Ms. Virginie Heros, Drs. shared by and Xenostrobus Philippe Maastrati and Pierre Louzuet. We are very securis (Thomsen et al. 2018), but this anatomical grateful for all their kind efforts. Shell sections for feature appears to have developed independently in SEM were prepared by Ms. Sharmaine Lee (Republic the two mytilid clades (i.e., clades A and B of Polytechnic). Many thanks are also due to Drs. Morton et al. 2020a; see Fig. 1) as well. The position Tom White and John Taylor at the Mollusca of the pericardial complex relative to the posterior Section, Natural History Museum (London) for byssal retractor muscles has also been suggested facilitating access to the bivalve collection in their as an informative phylogenetic character (Morton care. Helpful comments from Dr. P. Graham Oliver 2015). The pericardial complex of Byssogerdius lies and an anonymous reviewer improved the manuscript. in front of the posterior byssal retractors (i.e., category We acknowledge the generous support provided 2 of Morton 2015). This condition is shared by by the Singapore National Researach Foundation members of the Mytilinae as well as Brachidontes, (NRF) Marine Science Research and Development which presumably allows the shell to assume a Programme (MSRDP project P25). The study was lower dorso-ventral profile and may be related to conducted at the St John’s Island National Marine their mat-forming/nest-building habits (Morton 2015). Laboratory, a national infrastructure under the NRF.

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Manuscript received: 29 October 2020 Accepted: 4 December 2020 110

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Appendix 1. List of mussel specimens and gene sequences obtained in this study with associated GenBank accession numbers used in the phylogenetic analysis. See also Figs. 2 and 3 for images of shells from which tissue was sampled to obtain the DNA sequences. Voucher specimens (valves and tissue) were deposited in the Zoological Reference Collection (ZRC), Lee Kong Chian Natural History Museum, National University of Singapore.

Species Locality DNA code mtCOI mt16S ITS1 28S D1R H3 voucher specimen no. (prefix ZRC. MOL.) “Brachidontes Siglap, B1SG - - MW300114 MW295363 MW298135 20426 striatulus” Singapore (=Byssogerdius striatulus) B2SG MW217294 - MW300113 MW295362 MW298132 20427 BSS6 MW217296 - MW300115 MW295367 MW298142 20428 B14 MW300842 - - - - - C11 MW300844 - - - - - C13 MW305243 - - - - - D31 MW305240 - - - - - D42 MW305241 - - - - - D44 MW305244 - - - - - D51 MW305242 - - - - - Ao Kung B1T MW217293 - MW300118 MW295370 MW298137 20429 Krabaen, Chanthaburi, Gulf of Thailand B2T MW300843 - MW300119 MW295366 MW298136 20430 B3T MW300841 - MW300117 MW295371 MW298138 20431 B4T MW217291 - MW300116 MW295368 MW298131 20432 BS TKK MW217292 - MW300123 MW295379 MW298133 20433 0719A BS TKK MW217290 - MW300124 MW295372 MW298134 20434 0719B Pantai BST I1 MW217295 - MW300125 MW 295364 - 20435 Badur, Madura, E. Java, Indonesia BST I2 MW217297 - MW298143 20436 Ko Sirey, BS TSW1 - MW300101 MW300112 MW295381 MW298139 20437 Phuket, Thailand BST TSW1 MW217288 MW300100 MW300121 MW295378 MW298141 20438 Kuraburi, BST TSW2 MW217287 MW300102 MW300120 MW295377 MW298130 20439 Ranong Province, Thailand 111

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Species Locality DNA code mtCOI mt16S ITS1 28S D1R H3 voucher specimen no. (prefix ZRC. MOL.) BST TSW3 - - MW300122 MW295369 MW298140 20440 "Brachidontes Penghu, 18BTW2S MW217285 - MW300111 MW295380 MW298126 20441 setiger" Taiwan (=Byssogerdius subsulcatus) 18BTW3S MW217286 - MW300127 MW295387 MW298122 20442 Siglap, BSS1 MW217278 - MW300137 MW295383 MW298123 20443 Singapore BSS2 MW217281 - MW300136 MW295385 MW298121 20444 BSS3 MW217282 - MW300135 MW295376 MW298128 20445 BSS7 MW300839 - MW300138 MW295384 - 20446 Pattaya, BS TP MW217275 - MW300130 MW295388 MW298125 20447 Thailand 0719 BS TP MW217277 - MW300131 MW295389 - 20448 0719B Ko Sirey, BS TSW2 MW217283 MW300097 MW300132 MW295375 MW298129 20449 Phuket, Thailand BS TSW3 MW217274 MW300098 MW300129 MW295374 MW298127 20450

BS TSW4 MW217276 MW300099 MW300128 MW295373 - 20451 BS TSW5 MW217284 - MW300133 MW295382 - 20452 off Kho B1TT MW300840 - MW300134 MW295386 MW298124 20453 Kut, Trat, Thailand Modiolus Pak Bala, MMT1 MW300838 - MW300107 MW295390 MW298144 20454 modulaides Satun, (=metcalfei) Thailand Arcuatula Kenjeran, AM3I MW290540 - MW300108 MW295359 MW298149 20455 senhousia Surabaya, Indonesia Brachidontes cf. Changi, B1 MW300686 MW300103 MW300140 MW259392 MW298148 20456 mutabilis Singapore Brachidontes Fukuoka, HMJ1 MW300846 MW300105 MW300141 MW295393 MW298147 20457 mutabilis Japan Brachidontes Zanzibar, BV TZ MW300847 MW300104 MW300139 MW295391 MW298145 20458 variabilis Tanzania 0419 Mytilisepta Fukuoka, MSV NJ MW302342 MW300106 MW298150 20459 virgata Japan 0519 Mytilisepta Clearwater SMHK1V MW300845 - - - - 20460 virgata Bay, Hong Kong Xenostrobus Shing Mun X1HK MW293749 - MW300110 MW259361 MW298146 20461 securis River, Hong Kong 112

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Appendix 2. List of reference sequences from NCBI GenBank used to construct the trees shown in Fig. 1. For the concatenated dataset, reference sequences were chosen in accordance with the sequences used in Liu et al. (2018) and Morton et al. (2020a; 2020b). * see Appendix 1.

Species mt CO1 mt 16S ITS1 28S D1R H3 Reference Arcuatula senhousia (see * GQ472179.1 * * * Liu et al. also Appendix 1) 2011 ater JF301757.1 - - - - Van Pelt et al. 2015 azoricus AY649795 KF611758.1 - - KF720621 Jones et al. 2006; Thubaut et al. 2013 Brachidontes adamsianus AY825173.1 - - - - Trovant et al. 2016 Brachidontes darwinianus KT318209.1 - - - - Trovant et al. 2016 Brachidontes exustus KY454043.1; KX713196.1 - - - Combosch AY825212.1 et al. 2017; Trovant et al. 2016 Brachidontes granulatus KT318187.1 - - - - Trovant et al. 2016 AY825221.1 - - - - Lee and Foighil 2005 Brachidontes mutabilis (see KY081293.1; GQ472178.1 * * * Liu et also Appendix 1) al. 2018 (COI, as B. sinensis); Liu and Li (16S, unpubl.) MF345922.1 DQ833531.1 - - - Amor 2017, COI, unpubl.); Terranova et al. 2007 Brachidontes puniceus HM999775.1 - - - - see Cunha et al. 2011 Brachidontes purpuratus KY454047.1 KF159849.1 - - - Garćia- Souto et al. 2017; Vargas et al. 2015 Brachidontes rodriguezii KC844468.1 - - - - Trovant et al. 2013 Brachidontes rostratus KJ453834.1 - - - - Trovant et al. 2015 Brachidontes semilaevis AY825114.1 - - - - Lee and Foighil 2005 Brachidontes variabilis (see DQ836021.1 DQ836016.1 * * KY081374 Terranova also Appendix 1) et al. 2007; Liu et al. 2018 113

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Species mt CO1 mt 16S ITS1 28S D1R H3 Reference chorus MT103131.1 - - - - Oyarzún et al. 2020 Crassostrea angulata KY081307 AJ553901.1 - - KY081392 Boudry et al. 2003; Liu et al. 2018 Crenomytilus grayanus MN119672.1 AF317547.1 - - - see Lee et al. 2019 Geukensia demissa MN449488.1 U68772.1 - - - Hoeh et al. 1997; Lubośny et al. 2020a Geukensia granosissima AY621920.1 - - - - Lee and Foighil 2004 Gregariella coralliophaga NC044129.1 - - - - see Lee et al. 2019 modiolaeformis KT216482.1 KF611772.1 - - KF720635.1 Thubaut et al. 2013; Laming et al. 2015 recurvum AY621932.1 KT959476.1 - - - Lee and Foighil 2004 Isognomon ephippium KY081310 KY081325.1 - - KY081394 Liu et al. 2018 Leiosolenus lima KY081294; KY081316.1 - - KY081378; Liu et al. KY081295 KY081379 2018 Limnoperna fortunei AB520627.1 JQ267790.1 - - KY568785.1 Fujinaga et al. (unpubl.); Kartavtsev et al. (unpubl.); Liu et al. 2018 lithophaga AF120644 JF496757 - - - Giribet and Wheeler 2002 Modiolus modiolus MH242852.1; KX901458.1; - - KF720595 Plazzi et FJ890501 KF611732 al. 2011; Thubaut et al. 2013; Robicheau et al. 2017 Musculus costulatus MT012814.1 - - - - Almada et al. (unpubl.) Musculus discors MG935083.1 KX447416.1 - - - Gusman et al. 2016; Lundin (unpubl.) MG935283.1 - - - - Lundin (unpubl.) Mytella charruana MF075128.1 - - - - Calazans et al. (unpubl.) 114

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Species mt CO1 mt 16S ITS1 28S D1R H3 Reference minimus DQ836022.1 DQ836017.1 - - - Terranova et al. 2007 Mytilaster solisianus KT318262.1 - - - - Trovant et al. 2016 Mytilisepta virgata (see also AB076941.1 GQ472166 - - - Matsumoto Appendix 1) (unpubl.); Liu et al. (unpubl.) Mytilus californianus KF931757.1 U68771.1 - - AY267745.1 Hoeh et al. 1997; Eirin-Lopez et al. 2004; Crego- Prieto et al. 2015 - GQ472148.1 - - - Liu et al. 2011 Mytilus edulis KC429095 KX446919.1 - - KC429166 Sharma et al. 2013; Robicheau et al. 2017 Mytilus galloprovincialis KP976302.1 GQ472152.1 - - KY568777.1 Liu et al. 2011; Shen et al. 2016; Kartavtsev et al. (unpubl.) KF643956.1 GQ455403.1 - - AY267747.1 Eirin-Lopez et al. 2004; Westfall et al. 2010; Layton et al. 2014 Perna perna KU743163.1 DQ923881.1 - - - Weber et al. 2009; Gardner et al. 2016; Perna viridis KY081304; GQ472162.1 - - KY568781.1 Liu et al. KY081305 2011; 2018; Kartavtsev et al. (unpubl.) Pinna muricata KY081311 KY081326.1 - - KY081395 Liu et KY081326 al. 2018; Morton et al. 2020a Semimytilus algosus MT103137.1 - - - - Oyarzún et al. 2020 Septifer excisus KY081306 GQ472173.1 - - KY081390 Liu et al. 2011; Morton et al. 2020a Sinomytilus harmandi MK642885 MK629443 MK629448 MK642882 Morton et al. 2020b 115

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Species mt CO1 mt 16S ITS1 28S D1R H3 Reference Trichomya hirsuta GQ480305.1 GQ472163.1 - - MF320284.1 Liu et al. 2011; 2018; see also Colgan 2018 Xenostrobus atratus AB298599.1 GQ472184.1 - - - Kimura in Pascual et al. 2009; Liu et al. 2011 Xenostrobus securis (see * AB372227.1 * * * Tanaka also Appendix 1) et al. (unpubl.) 116

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During excursion tip of the 6th TMMP Workshop, Tamilnadu, India, 1995

During excursion tip of the 9th TMMP Workshop, Borobudur, Indonesia, 1998