A dichotomous PCR–RFLP identification key for the freshwater mussels (Bivalvia: Unionida) of Peninsular Malaysia

Abstract

Accurate species identification is the basis for successful species conservation and research, but extensive phenotypic plasticity in freshwater mussel (Bivalvia: Unionida) species often leads to misidentification by morphology alone. Molecular techniques have proven a powerful alternative tool for identification of freshwater mussels in North America and Europe. Unfortunately, no such tools are currently available for tropical mussels, which are subject to particularly severe habitat alteration and destruction. We developed a PCR–RFLP key for the freshwater mussel fauna of Peninsular Malaysia, comprising nine native and one non-native species from seven genera and three unionid subfamilies (Gonideinae, Rectidentinae and Anodontinae). The key enables identification of each species in two to four single digestion steps using six restriction endonucleases. All species can be identified using a single PCR fragment of 374 bp length (Histone H3) with the exception of two Pseudodon species, which can be differentiated through amplification and subsequent digestion of a 709 bp CO1 gene fragment. Reliability of the key was tested with specimens from 46 populations sampled from 13 different river basins of Peninsular Malaysia.

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References

  1. Beggel S, Geist J (2015) Acute effects of salinity exposure on glochidia viability and host infection of the freshwater mussel Anodonta anatina (Linnaeus, 1758). Sci Total Environ 502:659–665. https://doi.org/10.1016/j.scitotenv.2014.09.067

    CAS  Article  PubMed  Google Scholar 

  2. Boyer SL, Howe AA, Juergens NW, Hove MC (2011) A DNA-barcoding approach to identifying juvenile freshwater mussels (Bivalvia: Unionidae) recovered from naturally infested fishes. J N Am Benthol Soc 30:182–194. https://doi.org/10.1899/10-004.1

    Article  Google Scholar 

  3. Campbell DC, Johnson PD, Williams JD et al (2008) Identification of “extinct” freshwater mussel species using DNA barcoding. Mol Ecol Resour 8:711–724. https://doi.org/10.1111/j.1755-0998.2008.02108.x

    CAS  Article  PubMed  Google Scholar 

  4. Cho A, Morris T, Wilson C, Freeland J (2016) Development of species-specific primers with potential for amplifying eDNA from imperilled freshwater unionid mussels. Genome. https://doi.org/10.1139/gen-2015-0196

    Article  PubMed  Google Scholar 

  5. Chowdhury GW, Zieritz A, Aldridge DC (2016) Ecosystem engineering by mussels supports biodiversity and water clarity in a heavily polluted lake in Dhaka, Bangladesh. Freshw Sci 35:188–199. https://doi.org/10.1086/684169

    Article  Google Scholar 

  6. Colgan DJ, Ponder WF, Eggler PE (2000) Gastropod evolutionary rates and phylogenetic relationships assessed using partial 28S rDNA and histone H3 sequences. Zool Scr 29:29–63. https://doi.org/10.1046/j.1463-6409.2000.00021.x

    Article  Google Scholar 

  7. de Bruyn M, Stelbrink B, Morley RJ et al (2014) Borneo and Indochina are major evolutionary hotspots for Southeast Asian biodiversity. Syst Biol 63:879–901. https://doi.org/10.1093/sysbio/syu047

    Article  PubMed  Google Scholar 

  8. Dudgeon D (2000) The ecology of tropical Asian rivers and streams in relation to biodiversity conservation. Annu Rev Ecol Syst 31:239–263. https://doi.org/10.1146/annurev.ecolsys.31.1.239 doi

    Article  Google Scholar 

  9. Elderkin CL (2009) Intragenomic variation in the rDNA internal transcribed spacer (ITS1) in the freshwater mussel Cumberlandia monodonta (Say, 1828). J Molluscan Stud 75:419–421. https://doi.org/10.1093/mollus/eyp043

    Article  Google Scholar 

  10. Folmer O, Black M, Hoeh W et al (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol 3:294–299

    CAS  PubMed  Google Scholar 

  11. Gerke N, Tiedemann R (2001) A PCR-based molecular identification key to the glochidia of European freshwater mussels (Unionidae). Conserv Genet 2:287–289

    CAS  Article  Google Scholar 

  12. Gustafson RG, Iwamoto EM (2005) A DNA-based identification key to pacific northwest freshwater mussel glochidia: importance to salmonid and mussel conservation. Northwest Sci 79:233–245

    CAS  Google Scholar 

  13. Haag WR, Williams JD (2014) Biodiversity on the brink: an assessment of conservation strategies for North American freshwater mussels. Hydrobiologia 735:45–60. https://doi.org/10.1007/s10750-013-1524-7

    Article  Google Scholar 

  14. Hartmann JT, Beggel S, Auerswald K et al (2016) Establishing mussel behavior as a biomarker in ecotoxicology. Aquat Toxicol 170:279–288. https://doi.org/10.1016/j.aquatox.2015.06.014

    CAS  Article  PubMed  Google Scholar 

  15. IUCN (2016) IUCN red list of threatened species. Version 2016-2. http://www.iucnredlist.org

  16. Källersjö M, von Proschwitz T, Lundberg S et al (2005) Evaluation of ITS rDNA as a complement to mitochondrial gene sequences for phylogenetic studies in freshwater mussels: an example using Unionidae from north-western Europe. Zool Scr 34:415–424

    Article  Google Scholar 

  17. Kneeland SC, Rhymer JM (2007) A molecular identification key for freshwater mussel glochidia encysted on naturally parasitized fish hosts in Maine, USA. J Molluscan Stud 73:279–282. https://doi.org/10.1093/mollus/eym017

    Article  Google Scholar 

  18. Kneeland SC, Rhymer JM (2008) Determination of fish host use by wild populations of rare freshwater mussels using a molecular identification key to identify-glochidia. J N Am Benthol Soc 27:150–160. https://doi.org/10.1899/07-036.1

    Article  Google Scholar 

  19. Lopes-Lima M, Froufe E, Do VT et al (2017) Phylogeny of the most species-rich freshwater bivalve family (Bivalvia: Unionida: Unionidae): defining modern subfamilies and tribes. Mol Phylogenet Evol 106:174–191. https://doi.org/10.1016/j.ympev.2016.08.021

    Article  PubMed  Google Scholar 

  20. Myers N, Mittermeier RA, Mittermeier CG et al (2000) Biodiversity hotspots for conservation priorities. Nature 403:853–858. https://doi.org/10.1038/35002501

    CAS  Article  PubMed  Google Scholar 

  21. Stoeckle BC, Kuehn R, Geist J (2016) Environmental DNA as a monitoring tool for the endangered freshwater pearl mussel (Margaritifera margaritifera L.): a substitute for classical monitoring approaches? Aquat Conserv Mar Freshw Ecosyst 26:1120–1129. https://doi.org/10.1002/aqc.2611

    Article  Google Scholar 

  22. Vannarattanarat S, Zieritz A, Kanchanaketu T et al (2014) Molecular identification of the economically important freshwater mussels (Mollusca-Bivalvia-Unionoida) of Thailand: developing species-specific markers from AFLPs. Anim Genet 45:235–239. https://doi.org/10.1111/age.12115

    CAS  Article  PubMed  Google Scholar 

  23. Vaughn CC (2018) Ecosystem services provided by freshwater mussels. Hydrobiologia 810:15–27

    Article  Google Scholar 

  24. Vesterlund S, Sorvari J, Vasemägi A (2014) Molecular identification of cryptic bumblebee species from degraded samples using PCR–RFLP approach. Mol Ecol Resour 14:122–126

    CAS  Article  Google Scholar 

  25. Wächtler K, Mansur MCD, Richter T (2001) Larval types and early postlarval biology in naiads (Unionoida). In: Bauer G, Wächtler K (eds) Ecology and evolution of the freshwater mussels Unionoida. Springer, Berlin, pp 93–125

    Google Scholar 

  26. White LR, McPheron BA, Stauffer JR (1994) Identification of fresh-water mussel glochidia on host fishes using restriction-fragment-length polymorphisms. Mol Ecol 3:183–185

    CAS  Article  Google Scholar 

  27. White LR, McPheron BA, Stauffer JR (1996) Molecular genetic identification tools for the unionids of French Creek, Pennsylvania. Malacologia 38:181–202

    Google Scholar 

  28. Zieritz A, Aldridge DC (2009) Identification of ecophenotypic trends within three European freshwater mussel species (Bivalvia: Unionoida) using traditional and modern morphometric techniques. Biol J Linn Soc 98:814–825. https://doi.org/10.1111/j.1095-8312.2009.01329.x

    Article  Google Scholar 

  29. Zieritz A, Aldridge DC (2011) Sexual, habitat-constrained and parasite-induced dimorphism in the shell of a freshwater mussel (Anodonta anatina, Unionidae). J Morphol 272:1365–1375. https://doi.org/10.1002/jmor.10990

    Article  PubMed  Google Scholar 

  30. Zieritz A, Lopes-Lima M (2018) Handbook and National Red-List of the Freshwater Mussels of Malaysia. IUCN

  31. Zieritz A, Hoffman JI, Amos W, Aldridge DC (2010) Phenotypic plasticity and genetic isolation-by-distance in the freshwater mussel Unio pictorum (Mollusca: Unionoida). Evol Ecol 24:923–938. https://doi.org/10.1007/s10682-009-9350-0

    Article  Google Scholar 

  32. Zieritz A, Gum B, Kuehn R, Geist J (2012) Identifying freshwater mussels (Unionoida) and parasitic glochidia larvae from host fish gills: a molecular key to the North and Central European species. Ecol Evol 2:740–750. https://doi.org/10.1002/ece3.220

    Article  PubMed  PubMed Central  Google Scholar 

  33. Zieritz A, Lopes-Lima M, Bogan AE et al (2016) Factors driving changes in freshwater mussel (Bivalvia, Unionida) diversity and distribution in Peninsular Malaysia. Sci Total Environ 571:1069–1078. https://doi.org/10.1016/j.scitotenv.2016.07.098

    CAS  Article  PubMed  Google Scholar 

  34. Zieritz A, Bogan AE, Froufe E et al (2018a) Diversity, biogeography and conservation of freshwater mussels (Bivalvia: Unionida) in East and Southeast Asia. Hydrobiologia 810:29–44. https://doi.org/10.1007/s10750-017-3104-8

    Article  Google Scholar 

  35. Zieritz A, Bogan AE, Rahim KAA et al (2018b) Changes and drivers of freshwater mussel diversity and distribution in northern Borneo. Biol Conserv 219:126–137

    Article  Google Scholar 

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Acknowledgements

This project was funded by the Ministry of Higher Education Research Grant FRGS/1/2015/WAB13/UNIM//1. We thank SNA Musa, SNK Che Samsuddin and SN Muhamad Nor for laboratory-work assistance.

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Correspondence to A. Zieritz.

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Razak, N.F.A., Supramaniam, C.V. & Zieritz, A. A dichotomous PCR–RFLP identification key for the freshwater mussels (Bivalvia: Unionida) of Peninsular Malaysia. Conservation Genet Resour 11, 457–464 (2019). https://doi.org/10.1007/s12686-018-1038-8

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Keywords

  • COI
  • Histone H3
  • Glochidia
  • Molecular identification
  • Molluscs
  • Unionidae