Field evaluation of mortality from hemolymph extraction as a source of DNA, and application to PCR-RFLP identification of threatened freshwater mussel species

Evolutionary convergence and plasticity of shell characters creates great confusion in freshwater mussel systematics and complicates field census efforts. Genetic identification offers a powerful alternative. But methods involving DNA sequencing are expensive and require tissue sampling, whose effects on survival and health of animals from natural populations have seldom been assessed. We used hemolymph sampling as a nonlethal source of tissue for DNA extraction, and developed genetic identification methods using Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis as an alternative for field surveys. We focused on two morphologically similar endemic species in Lake Waccamaw, North Carolina: the abundant Elliptico waccamawensis and the less common Lampsilis fullerkati. These served as models for surveyors who are often faced with conducting accurate census of cryptic rare species that co-occur with more common forms. Hemolymph-sampled and control individuals of these two species and Leptodea ochracea were caged together in enclosures in Lake Waccamaw. Eight-week survival was 100% and we detected no significant effect of hemolymph extraction on shell growth. PCR-RFLP analysis of the 16s rRNA gene reliably identified species and detected cases of morphological mis-identification, both in these collections and from belt transects in the lake. We also developed PCR-RFLP markers that distinguished pairs of cryptic taxa from surveys of streams in southeastern North Carolina. Our results show how nonlethal tissue sampling and PCR-RFLP assays designed for a regional fauna can be useful tools in freshwater mussel conservation programs.