The lemur leaf frog, Agalychnis lemur, is a species of tree frog from Costa Rica, Panama and Colombia which is listed as Critically Endangered by the IUCN (Solís et al. 2004). Populations of this species have recently seen drastic declines due to climate change and the adverse effects of the chytrid fungus Batrachochytrium dendrobatidis. Lemur leaf frogs are currently only known from two locations within Costa Rica, and animals from both these areas are known to have a common origin (García-Rodríguez et al. 2012). A recent mitochondrial DNA study has shown that the Costa Rican A. lemur are genetically distinct from those in Panama (Gray 2011), highlighting the need for further conservation to support the remaining populations in Costa Rica.

In 2001, a small number of animals from Costa Rica were brought into captivity to establish a captive breeding programme. Captive maintenance and breeding guidelines have since been developed. However, as with all other current amphibian captive breeding programmes, no details are available regarding the specific genetic relationship of founder individuals. Here we characterise a panel of 9 microsatellite markers for A. lemur. Data derived from these markers will identify current relationships within captive individuals in order to establish a genetically informed studbook. The microsatellites will also be available to assess genetic diversity within the wild populations.

DNA was extracted from frozen femur tissue of 12 deceased captive individuals using a phenol–chloroform protocol (Sambrook et al. 1989). 36 living individuals were mouth swabbed with Isohelix™ DNA buccal swabs and DNA extracted following the Qiagen® Gentra Puregene Buccal Cell Kit protocol. Tri- and tetranoculeotide loci were isolated commercially by Genoscreen. Out of 24 unique loci we selected 11 for PCR optimisation and polymorphism testing. PCRs were performed in a 10 µl reaction volume with the following components: 1 µl reaction buffer, 1.5 mM MgCl2, 4.35 µl molecular grade H2O, 1 µM of each primer, 0.25 mM of each dNTP and 1 unit of GoTaq® DNA Taq polymerase. The PCR profile for all loci is 10 min at 95 °C, 40 cycles comprising of 30 s at 55 °C, 30 s at 55 °C, 1 min at 72 °C, and a final extension of 10 min at 72 °C. Forward PCR primers were labelled with HEX, FAM or AT550, and PCR products were separated on a 3,130 Genetic Analyser (Applied Biosystems). Alleles were scored using Genemapper (Applied Biosystems). GENEPOP v4.0 (Rousset 2008, web-based version) was used for the quantification of Hardy–Weinberg and linkage disequilibria.

Out of the 11 loci, one failed to consistently amplify while another only successfully amplified DNA extracted from femur tissue. The remaining 9 loci were consistently scorable, displaying between 3 and 8 alleles per locus (Table 1). Six loci showed significant deviations from Hardy–Weinberg equilibrium (heterozygote excesses as well as deficits), and three pairs of loci were in significant linkage disequilibrium (Al1–Al6, Al1–Al18, Al4–Al6). We however argue that these deviations are not due to poor marker performance, but caused by the fact that the sampled captive individuals were housed in small groups of identical descent, resulting in non-random mating and the sampling of closely related individuals. The moderate number of alleles per locus further supports the existence of genetic bottlenecks in the wild and/or captivity.

Table 1 Details of nine polymorphic microsatellite loci characterised in A. lemur
Full size table

While the present study is limited by the sampling regime, we demonstrate the potential of the nine characterised loci for future conservation studies. We are currently using these markers to establish a genetically-informed studbook for the captive breeding of A. lemur—according to our knowledge the first for any captive endangered amphibian species. Given the conservation status of this species the need to maintain a high genetic variation while reducing the amount of inbreeding within the captive population may prove crucial for its future survival.