Identifying freshwater mussels (Unionoida) and parasitic glochidia larvae from host fish gills: a molecular key to the North and Central European species

Freshwater mussels (order Unionoida) represent one of the most severely endangered groups of animals due to habitat destruction, introduction of nonnative species, and loss of host fishes, which their larvae (glochidia) are obligate parasites on. Conservation efforts such as habitat restoration or restocking of host populations are currently hampered by difficulties in unionoid species identification by morphological means. Here we present the first complete molecular identification key for all seven indigenous North and Central European unionoid species and the nonnative Sinanodonta woodiana, facilitating quick, low-cost, and reliable identification of adult and larval specimens. Application of this restriction fragment length polymorphisms (RFLP) key resulted in 100% accurate assignment of 90 adult specimens from across the region by digestion of partial ITS-1 (where ITS is internal transcribed spacer) polymerase chain reaction (PCR) products in two to four single digestions with five restriction endonucleases. In addition, we provide protocols for quick and reliable extraction and amplification of larval mussel DNA from complete host fish gill arches. Our results indicate that this new method can be applied on infection rates as low as three glochidia per gill arch and enables, for the first time, comprehensive, large-scale assessments of the relative importance of different host species for given unionoid populations.