A PCR-RFLP method for stock assignment in the endemic Macaronesian gastropod, Patella candei d’Orbigny, and its potential for future conservation strategies

Abstract Patella candeid’Orbigny is an exploited limpetendemic to the archipelagosof the Azores, Madeira, Selvagens and Canaries (Macaronesia). Marked declines reported for the species likely reflect overexploitationand inadequacy of current management measureswith biodiversityfurther threatened by illegal harvesting and anthropogenic movement of limpetsamong archipelagos. Previous phylogeographic analyses of mtDNA COI sequence variation revealed that the (i) Azores, (ii) Madeira and (iii) Canaries and Selvagens represent three reciprocally monophyletic genetic stocks. The objective of this research was to identify diagnostic DNA sequence polymorphisms among the stocks that could be assayed by PCR-RFLP, thus permitting cost effective high throughput stock assignment. PCR-RFLP analysis of a COI fragment with two restriction enzymes( Taq I and Ava I) was sufficient to define diagnostic stock genotypes for the three stocks and permitted unambiguous stock assignment of a large sample of individuals (n = 952). This PCR-RFLP approach can be readily extended to discriminate other Patella stocks, through judicious selection of restriction enzymesand similar protocols and offers great potential for eco-certification associated practises.