Intraspecific genetic variation in the common midwife toad (Alytes obstetricans): subspecies assignment using mitochondrial and microsatellite markers

The common midwife toad (Alytes obstetricans), widely distributed in the northern half of the Iberian Peninsula and part of Western Europe, is currently subdivided into four subspecies: A. o. obstetricans, A. o. boscai, A. o. pertinax and A. o. almogavarii. However, the delimitation of these subspecies and their ranges are still under discussion because strong discordances have been found between morphological and molecular data, and especially among different genetic markers. Here, we screen a set of novel microsatellite loci and mtDNA sequences of A. obstetricans populations representative of all currently recognized subspecies to investigate the correspondence between genetic groupings inferred from clustering analysis of microsatellite genotypes and the described subspecies and test whether patterns of mtDNA variation are concordant with those genetic clusters. Our results confirm previous expectations of extremely high intraspecific diversity in A. obstetricans in Iberia. Analyses of microsatellite and mtDNA data were concordant in recovering five well-defined groups, of which three correspond to previously defined subspecies, while the two additional clusters correspond to populations of subspecies A. o. boscai separated by the Douro River. Our results suggest the occurrence of two distinct genetic units within A. o. boscai that likely result from a long independent evolutionary history, thus deserving special attention from a conservation point of view.This work was financed through the Programa Operacional Factores de Competitividade (COMPETE) and by national funds from Fundaçao para a Ciência e a Tecnologia (FCT), through the research projects PTDC/BIABEC/105083/2008 and PTDC/BIA-BEC/099915/2008 to FS and HG and grants CGL2008-04271-C02-01/BOS and CGL2011-28300 (Ministerio de Ciencia e Innovacion, Ministerio de Economía y Competitividad, Spain, and FEDER) and PPII10-0097- 4200 (Junta de Comunidades de Castilla la Mancha) to IMS. FS and HG are supported by a postdoctoral grant from FCT (SFRH⁄BPD⁄87721⁄2012 and SFRH/BPD/26555/2006, respectively), and BMC is supported by a PhD grant from FCT (SFRH/BD/60305/2009). IMS was supported by a ‘Ramon y Cajal’ postdoctoral fellowship funded by the Spanish Ministerio de Ciencia e Innovacion and the Universidad de Castilla la Mancha and is currently funded by Project “Biodiversity, Ecology and Global Change” cofinanced by North Portugal Regional Operational Programme 2007/2013 (ON.2–O Novo Norte), under the National Strategic ReferenceFramework (NSRF), through the European Regional Development Fund (ERDF), and JGR is supported by the Consejo Superior de Investigaciones Cientıficas of Spain (CSIC) and the European Social Fund (ESF) (JAE-pre-PhD fellowship).Peer Reviewe