Development of Molecular and Morphological Resources for the identification of and monitoring of “Look-Alike” Freshwater Mussel Species Belonging to the Genera Fusconaia and Pleurobema in the Green River, Kentucky

The Green River of central Kentucky is located in the Ohio River Basin and flows through the interior low plateau physiographic province (Benke and Cushing 2011). It is one of most biodiverse tributaries in the Ohio River system (Master et al. 1998). The upper part of the Green River is used for agriculture and has several impoundments, while the middle part of the river flows without impoundments for 100 miles through Mammoth Cave National Park and eight counties (Benke and Cushing 2011; Masters et al. 1999). The lower part of the Green river again has several dams and locks for coal transport. The principal sources of pollution affecting water quality and freshwater mussel habitat involve agriculture, which occurs throughout the Green River watershed. Biodiversity and water quality in the Mammoth Cave underground section of the Green River are affected by siltation (deforestation), inappropriate agriculture techniques, streamflow regulation, and sewage treatment (Masters et al. 1998). The prevention of introduced species, such the zebra mussel, Dreissena polymorpha, is of high concern in the Green River (Grabarkiewicz and Davis 2008; Masters et al. 1998).

The Green River, KY is characterized by high mussel biodiversity. In this system, there are approximately 71 mussel species (Benke and Cushing 2011; Grabarkiewicz and Davis 2008; Masters et al. 1998). Among them, are two species in the genus Fusconaia and 5 species in the genus Pleurobema occurring in the rivers (Grabarkiewicz and Davis 2008; Turgeon et al. 1998). Most are morphologically similar and difficult to distinguish even by experts. (Campbell and Lydeard, 2012; Campbell et al. 2005; Harper et al. 2000; Jones et al. 2015). Species of particular concern inhabiting the Green River are Rough Pigtoe (Pleurobema plenum, critically endangered), Clubshell (P. clava, critically endangered), Ohio Pigtoe (P. cordatum, near threatened), Pink Pigtoe (P. rubrum, near threatened), Round Pigtoe (P. sintoxia, least concern), Long-Solid (Fusconaia subrotunda, vulnerable), and Wabash Pigtoe (F. flava) (least concern) (Haag and Cicerello 2011; Master et al. 1998) (IUCN 2017). Early studies describing the species morphological characters (reproductive traits, shell morphology, and soft anatomy), while more recent studies have used genetic markers to identify species as well as management and evolutionarily significant units.

In these look-alike mussels, taxonomic characteristics useful for identification overlap due to the effect of environmental factors as well as individual age and size. There is a need for the development of a probabilistic dichotomous key for identification of these mussel species, as well as an assessment of effective population sizes (current and long-term). In order to meet these needs, we need to first correctly identify these mussel species and their biologically justified conservation units with the help of freshwater mussel experts and genetic analysis. After the correct identification, different morphological characteristics can be used properly for the development of dichotomous and photographic keys. Finally, the estimation of estimated population sizes with use of mitochondrial and microsatellite DNA markers will help to assess the current conservation status of these species and to identify of management units and development of management plans for the conservation of these look-alike species.

Objectives:

OBJECTIVE 1: Assessment of phylogenetic relationships among mussel species in the genera Fusconaia and Pleurobema in the Green River, KY

OBJECTIVE 2: Estimation genetic variability and effective sizes (Ne) of P. plenum, P. clava, P. cordatum, P. rubrum and P. sintoxia populations in the Green River, KY

OBJECTIVE 3: Quantification and test for morphological differences to develop and test morphology-based key among focal species from the genera Fusconaia and Pleurobema from the Green River, KY

References:

Campbell, D. C., J. M. Serb, J. E. Buhay, K. J. Roe, R. L. Minton, and C. Lydeard. 2005. Phylogeny of North American amblemines (Bivalvia, Unionoida): prodigious polyphyly proves pervasive across genera. Invertebrate Biology 124(2): 131-164.

Campbell, D. C., P. D. Johnson, J. D. Williams, A. K. Rindsberg, J. M. Serb, K. K. Small, C. Lydeard. 2008. Identification of ‘extinct’ freshwater mussel species using DNA barcoding. Molecular Ecology Resources 8(4): 711-724.

Benke, A. C., and C. E. Cushing. 2011. Rivers of North America. Academic Press.

Grabarkiewicz, J. D., and W. S. Davis. 2008. An introduction to freshwater fishes as biological indicators. US Environmental Protection Agency, Office of Environmental.

Haag, W. R., and R. R. Cicerello. 2016. A distributional atlas of the freshwater mussels of Kentucky. Scientific and Technical Series 8. Kentucky State Nature Preserves Commission, Frankfort, KY.

Harper, E. M., Taylor, J. D., and J. A. Crame, J. A. 2000. Unravelling the evolutionary biology of the Bivalvia: a multidisciplinary approach. Geological Society, London, Special Publications 177(1): 1-9.

Jones, J. W., N, Johnson, P. Grobler, D. Schilling, R. J. Neves, and E. M. Hallerman. 2015. Endangered rough pigtoe pearlymussel: assessment of phylogenetic status and genetic differentiation of two disjunct populations. Journal of Fish and Wildlife Management 6(2): 338-349.

Master, L. L., Flack, S. R., and B. A. Stein. 1998. Rivers of life: critical watersheds for protecting freshwater biodiversity (p. 71). Arlington, Virginia: Nature Conservancy.

Turgeon, D. D., Quinn, J. F., Bogan, A. E., Coan, E. V., Hochberg, F. G., Lyons, W. G., Mikkelsen, P. M., Neves, R. J., Roper, C. F., Rosenberg, G. and B. Roth. 1998. Common and scientific names of aquatic invertebrates from the United States and Canada: Mollusks

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