Genetic Confirmation of Mya Japonica Jay, 1857 (Bivalvia: Myidae) in Tasmania, Australia: First Record of Any Species of Mya in the Southern Hemisphere

BioInvasions Records (2020) Volume 9, Issue 1: 103–108

CORRECTED PROOF

Rapid Communication Genetic confirmation of Mya japonica Jay, 1857 (Bivalvia: Myidae) in Tasmania, Australia: first record of any species of Mya in the southern hemisphere

Alison L. Dann1,*, Kevin Ellard1, Simon J. Grove2 and Richard C. Willan3 1Biosecurity Tasmania, Department of Primary Industries, Parks, Water and Environment, GPO Box 44, Hobart, Tasmania 7000, Australia 2Collections and Research Facility, Tasmania Museum and Art Gallery, 5 Winkleigh Place, Rosny, Tasmania 7018, Australia 3Museum and Art Gallery of the Northern Territory, GPO Box 4646, Darwin, Northern Territory 0801, Australia Author e-mails: [email protected] (AD), [email protected] (KE), [email protected] (SG), [email protected] (RW) *Corresponding author

Citation: Dann AL, Ellard K, Grove SJ, Willan RC (2020) Genetic confirmation of Abstract Mya japonica Jay, 1857 (Bivalvia: Myidae) in Tasmania, Australia: first The Japanese soft-shelled clam Mya japonica Jay, 1857 is native to the north- record of any species of Mya in the western Pacific Ocean but has invaded the northeastern Pacific Ocean, North southern hemisphere. BioInvasions Atlantic Ocean, Barents Sea and Mediterranean Sea. Here, we confirm its presence Records 9(1): 102–108, https://doi.org/10. in the southern hemisphere using mitochondrial COI, 16S rRNA and nuclear 28S 3391/bir.2020.9.1.14 rRNA gene sequences. These analyses permit us to distinguish it genetically from Received: 17 July 2019 its equally invasive congener, M. arenaria Linnaeus, 1758. We found M. japonica Accepted: 9 November 2019 to be well established in one location on the southeast coast of Tasmania. Published: 6 January 2020 Thematic editor: April Blakeslee Key words: Soft-shelled clam, cytochrome oxidase I, biosecurity, invasive species, first record, myid Copyright: © Dann et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0). Introduction OPEN ACCESS. The Japanese soft-shelled clam Mya japonica Jay, 1857 is a large bivalve native to muddy intertidal and shallow subtidal habitats in the temperate northwestern Pacific Ocean (Japan, China, Korean Peninsula and eastern Russia). It is conchologically very similar to M. arenaria Linnaeus, 1758, and has previously been considered synonymous with that species. Mya arenaria is considered native to the northwestern Atlantic Ocean, but was apparently introduced into northern European waters several hundred years ago (Strasser 1998) and, through subsequent human-mediated transplantation, has since colonised the Mediterranean and Black Seas. Genetic data support the hypothesis of post-Pleistocene colonisation of European shores from eastern North America (Cross et al. 2016; Lasota et al. 2016). Furthermore, Essink and Oost (2017) noted that M. arenaria became extinct in European coasts during the Pleistocene glaciation and was re-populated into northwestern European coasts from northeast Northern America pre-Columbus. The authors hypothesised that Viking visits to northeastern North America and Greenland facilitated the re- introduction. Mya arenaria was also accidentally introduced to California

Dann et al. (2020), BioInvasions Records 9(1): 102–108, https://doi.org/10.3391/bir.2020.9.1.14 103 Genetic confirmation of Mya japonica in Tasmania

via oyster seeding in the 1860s or 1870s, and either through natural spread or human-aided transplantation, has subsequently spread northwards as far as Alaska (Stearns 1881; Hanna 1966; Carlton 1992). Strasser (1998), in his review of M. arenaria occurrences, noted that no records of this species had ever been found in the southern hemisphere. Recent molecular analyses reinstated M. japonica as a valid species separate to M. arenaria and determined that it too had been introduced to the northeastern Pacific, most likely via either oyster seeding or ballast water from the northwestern Pacific, with confirmed records from two widely separated localities along the coast of British Columbia (Zhang et al. 2018). In the same publication, the authors also asserted that the two species could be separated based on subtle differences in shell morphology and sperm ultrastructure. The propensity for M. arenaria to colonise coasts beyond its native range and build up large densities led to the recognition of it (and, by extension, the newly reinstated M. japonica) as a potential marine pest should it ever be detected in southern Australian waters (Hayes and Sliwa 2003). Being a burrower, it has limited potential for spread through biofouling but may be spread in ballast water discharged from shipping. Both these species of Mya have the potential to compete with native species (of clams and other benthic invertebrates) for space and for food and often play a dominating role in local benthic communities (Carlton 1992; Strasser 1998). In this paper, we confirm the presence of M. japonica in Australian waters based on molecular evidence.

Materials and methods Specimen collection In May 2018 there was a significant storm event in Tasmania, delivering record rainfall to most of the southeast areas of the island, causing flash flooding in many catchments, including the Prosser River, at Orford (42.557°S; 147.867°E). The heavy runoff scoured the surface sediments from the mouth of the river and exposed a bed of deep-burrowing clams (Figure 1) never seen in this area previously. These specimens were immediately identified as belonging to Mya, a genus never previously recorded live from Australia or indeed the southern hemisphere (Grove et al. 2018).

Sequence analysis Four specimens of the Mya sp. were collected and stored in 70% ethanol until DNA extractions could be performed at Biosecurity Tasmania’s molecular diagnostic laboratory. These specimens were subsequently deposited in the mollusc collection in the Tasmanian Museum and Art Gallery under the registration numbers E58299–E58302. Extractions were performed using the Qiagen DNeasy Blood and Tissue kit following the manufacturer’s instructions.

Dann et al. (2020), BioInvasions Records 9(1): 102–108, https://doi.org/10.3391/bir.2020.9.1.14 104 Genetic confirmation of Mya japonica in Tasmania

Figure 1. Left photo: A shell of Mya japonica collected at Orford, Prosser River, Tasmania, May 2018. The length of the shell is 94 mm, Photo: Simon Grove. Right photo: Mya japonica in situ exposed at the mouth of the Prosser River, Orford, following a storm event, May 2018. Photo: John Bostock.

The Cytochrome Oxidase I (COI), 16S rRNA and 28S rRNA fragments were amplified by PCR following the method described by (Zhang et al. 2018) to distinguish between M. japonica and M. arenaria. PCR products were confirmed by 2% agarose gel electrophoresis stained with GelGreen™ Nucleic Acid Gel Stain (Biotum). Product sizes were checked by comparing amplified DNA to 5 μL of the Fast DNA Ladder (New England Biolabs Inc). The PCR product from each specimen was then sent to the Australian Genome Research Facility (AGRF) for Sanger sequencing. Both the forward and reverse sequences were quality checked and aligned with Geneious Prime (2019.1.2) (https://www.geneious.com), and sequence analysis performed using the BLAST algorithm in GenBank (Altschul et al. 1990). The COI sequences were aligned using the MAFFT alignment tool in Geneious (Katoh and Standley 2013). The alignment was put through MODELTEST to determine the best model for phylogenetic analysis (Posada and Crandall 1998) executed through http://hiv.lanl.gov/ content/sequence/findmodel/findmodel.html. The phylogenetic tree (Figure 2) was produced using MrBayes (HKY85 substitution model, 100,000 chain length, gamma rate variation, 25,000 Burn-in Length) (Ronquist and Huelsenbeck 2001) and compared to sequences from Zhang et al. (2018) (Table 1). All sequences were submitted to the NCBI database GenBank (https://www.ncbi.nlm.nih.gov/). The COI sequence and specimen data was deposited in BOLD (ISBMJ001-19) and all sequences were submitted to GenBank (28S – MK873083, 16S – MK873084, COI – MK873085).

Dann et al. (2020), BioInvasions Records 9(1): 102–108, https://doi.org/10.3391/bir.2020.9.1.14 105 Genetic confirmation of Mya japonica in Tasmania

Figure 2. MrBayes phylogenetic tree based on COI sequences (sample details in Table 1) and the specimens from Tasmania (pink text). Branch labels are posterior probability (i.e. the probability that a specific clade/branch will be found within the distribution of the Bayesian trees) and the scale bar is the number of substitutions per site. Mya uzenensis was used as the outgroup.

Results and discussion The sequence analysis confirmed the Mya specimens from Tasmania were M. japonica not M. arenaria; all four specimens had 100% similar sequences so counted as one finding. Separately the sequences matched M. japonica (99.8–98.5% for COI to KX534198 to KX534192 as compared to M. arenaria (87.7% KX534201), (100–98.4% for 16S rRNA to KX534167 to KX534175 as compared to M. arenaria (94.5% KX534176), however, 28S rRNA sequences were not able to distinguish M. japonica from M. arenaria (see Table 1 for accession numbers). This is the first detection of Mya japonica in the southern hemisphere, and a biosecurity response was initiated by Biosecurity Tasmania after reporting the detection to the Australian Consultative Committee on Introduced Marine Pest Emergencies. The response involved surveillance and implementation, establishment of population distribution and densities within this area, larval dispersal modelling, and research on population age structure based on shell size distributions and analyses of

Dann et al. (2020), BioInvasions Records 9(1): 102–108, https://doi.org/10.3391/bir.2020.9.1.14 106 Genetic confirmation of Mya japonica in Tasmania

Table 1. Species, voucher/specimen no., location, GenBank Accession numbers, and reference of sequences used for comparison in the MrBayes phylogenetic analysis based on COI sequences. GenBank/BOLD Species Voucher/Specimen No. Location Reference Accession Mya arenaria 1 Pechanga Bay, Barents Sea, Russia KX534201 Zhang et al. 2018 Mya arenaria 2 Pechanga Bay, Barents Sea, Russia KX534202 Mya arenaria Haplotype A NE Pacific/NW NE Atlantic HQ728053 Strasser and Barber 2009 Mya arenaria Haplotype B NE Pacific/NW NE Atlantic HQ728057 Mya arenaria Haplotype D NW Atlantic HQ728062 Mya arenaria Haplotype E NE Atlantic HQ728071 Mya arenaria Haplotype G NE Atlantic HQ728060 Mya arenaria Haplotype M NW Atlantic HQ728052 Mya arenaria Haplotype S NW Atlantic HQ728055 Mya arenaria Haplotype Z NW Atlantic HQ728067 Mya arenaria H2 Europe KX576718 Lasota et al. 2016 Mya arenaria H3 Europe KX576719 Mya arenaria H6 NE Pacific KX576722 Mya arenaria H7 NE Pacific KX576723 Mya arenaria H8 NW Atlantic KX576724 Mya arenaria H10 NW Atlantic KX576726 Mya arenaria H12 NW Atlantic KX576728 Mya arenaria H14 NW Atlantic KX576730 Mya arenaria H16 NW Atlantic KX576732 Mya arenaria MT01604 NE Atlantic KR084471 Barco et al. 2016 Mya arenaria MT01606 NE Atlantic KR084554 Mya arenaria 10BCMMOL-00276 NW Atlantic KF644013 Layton et al. 2014 Mya japonica 1 Qingdao Coast, Yellow Sea, China KX534192 Zhang et al. 2018 Mya japonica 2 Qingdao Coast, Yellow Sea, China KX534193 Mya japonica 3 Qingdao Coast, Yellow Sea, China KX534194 Mya japonica 4 Qingdao Coast, Yellow Sea, China KX534195 Mya japonica 5 Qingdao Coast, Yellow Sea, China KX534196 Mya japonica 6 Qingdao Coast, Yellow Sea, China KX534197 Mya japonica 7 Qingdao Coast, Yellow Sea, China KX534198 Mya japonica 8 Qingdao Coast, Yellow Sea, China KX534199 Mya japonica 9 Vostok Bay, Peter the Great Bay, Sea KX534200 of Japan, Russia Mya japonica PEB0093 British Columbia DFO097-11 Mya japonica CCDB22173-C05 British Columbia RBCMI219-14 Mya japonica 047 Russia MN119666 unpublished from GenBank Mya japonica 005 China MN119674 unpublished from GenBank Mya uzenesis MBM229025 Vostok Bay, Peter the Great Bay, Sea KX534204 Zhang et al. 2018 of Japan, Russia

growth patterns. These studies suggested that M. japonica has been present in the Prosser River for at least a decade due to shell size distribution, ships originating from the northern hemisphere not having docked in this area since that time and that in some areas they were established at very high densities (Grove et al. 2018). This distribution is suggestive of the species having arrived as larvae in ballast water from ships docking at the former export woodchip facility at the nearby international port of Triabunna (42.510°S; 147.914°E), but this hypothesis cannot be confirmed (Grove et al. 2018). While their habit of burrowing in fine sediment removes the clams from view on the surface, their impact on native Tasmanian benthic species has not yet been determined. At present, the known populations are restricted to a single bay system in the Prosser River, but the impact may become more apparent if populations increase within the bay or establish

Dann et al. (2020), BioInvasions Records 9(1): 102–108, https://doi.org/10.3391/bir.2020.9.1.14 107 Genetic confirmation of Mya japonica in Tasmania

elsewhere in Tasmania through natural larval spread. Establishment of this species in active port areas through domestic transport in ballast water would also represent a threat to other regions.

Acknowledgements

The authors would like to thank John Bostock, Andrew North and Bernadette Northeast for reporting the discoveries of this bivalve and assisting in collection, identification and photos. We would also like to thank Jeanette Jackson for submitting the shells found in 2013 to the Tasmanian Museum and Art Gallery. In addition, assistance in collecting specimens that was provided by Matt Pauza and Sue Robinson at Biosecurity Tasmania and staff at the Tasmanian Institute of Marine and Antarctic Studies is also very much appreciated. The comments of two referees greatly benefitted the final paper.

References

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. Journal of Molecular Biology 215: 403–410, https://doi.org/10.1016/S0022-2836(05)80360-2 Barco A, Raupach MJ, Laakmann S, Neumann H, Knebelsberger T (2016) Identification of North Sea molluscs with DNA barcoding. Molecular Ecology Resources 16: 288–297, https://doi.org/10.1111/1755-0998.12440 Carlton JT (1992) Introducted marine and estaurine mollusks of North America: an end-of-the- 20th-century perspective. Journal of Shellfish Research 11(2): 489–505 Cross ME, Bradley CR, Cross TF, Culloty S, Lynch S, McGinnity P, O’Riordan RM, Vartia S, Prodöhl PA (2016) Genetic evidence supports recolonisation by Mya arenaria of western Europe from North America. Marine Ecology Progress Series 549: 99–112, https://doi.org/ 10.3354/meps11672 Essink K, Oost AP (2017) How did Mya arenaria (Mollusca; Bivalvia) repopulate European waters in mediaeval times? Marine Biodiversity 49: 1−10, https://doi.org/10.1007/s12526-017- 0816-y Grove SJ, Willan R, Ellard K, Dann AL (2018) Detection of the invasive exotic Japanese softshell clam Mya japonica Jay, 1857 (Bivalvidae: Myidae) in Tasmania. The Malacological Society of Australasia Newsletter 167: 3–5 Hanna GD (1966) Introduced mollusks of western North America. Occasional Papers of the California Academy of Sciences 48: 1–108 Hayes KR, Sliwa C (2003) Identifying potential marine pests: a deductive approach applied to Australia. Marine Pollution Bulletin 46: 91–98, https://doi.org/10.1016/S0025-326X(02)00321-1 Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution 30: 772−780, https://doi.org/10.1093/molbev/mst010 Lasota R, Pierscieniak K, Garcia P, Simon-Bouhet B, Wolowicz M (2016) Large-scale mitochondrial COI gene sequence variability reflects the complex colonization history of the invasive soft-shell clam, Mya arenaria (L.) (Bivalvia). Estuarine, Coastal and Shelf Science 181: 256−265, https://doi.org/10.1016/j.ecss.2016.08.033 Layton KK, Martel AL, Hebert PD (2014) Patterns of DNA barcode variation in Canadian marine molluscs. PLoS ONE 9: e95003, https://doi.org/10.1371/journal.pone.0095003 Posada D, Crandall KA (1998) MODELTEST: testing the model of DNA substitution. Bioinformatics 14: 817–818, https://doi.org/10.1093/bioinformatics/14.9.817 Ronquist F, Huelsenbeck JP (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17: 754–755, https://doi.org/10.1093/bioinformatics/17.8.754 Stearns REC (1881) Mya arenaria in San Francisco Bay. The American Naturalist 15: 362–366, https://doi.org/10.1086/272816 Strasser CA, Barber PH (2009) Limited genetic variation and structure in softshell clams (Mya arenaria) across their native and introduced range. Conservation Genetics 10: 803–814, https://doi.org/10.1007/s10592-008-9641-y Strasser M (1998) Mya arenaria - an ancient invader of the North Sea coast. Helgoländer Meeresuntersuchungen 52: 309–324, https://doi.org/10.1007/BF02908905 Zhang J-L, Yurchenko OV, Lutaenko KA, Kalachev AV, Nekhaev IO, Aguilar R, Zhan Z, Ogburn MB (2018) A tale of two soft-shell clams: an integrative taxonomic analysis confirms Mya japonica as a valid species distinct from Mya arenaria (Bivalvia: Myidae). Zoological Journal of the Linnean Society 184: 605–622, https://doi.org/10.1093/zoolinnean/ zlx107

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