DOI: 10.5061/dryad.0zpc866tx
title: Genetic differentiation and overexploitation history of the critically endangered Lehmann’s Poison Frog: Oophaga lehmanni

description: Species conservation with fragmented and endangered populations must be
 based on a prior and thorough knowledge of the structure and population
 dynamics. Oophaga lehmanni is a dendrobatid species endemic of Colombia
 and is restricted to its type locality. This species has a fragmented
 distribution and is considered as critically endangered mainly due to
 habitat destruction and overexploitation. Oophaga lehmanni exhibits
 phenotypic variation in the dorsal color pattern (red and yellow morphs).
 We reconstructed the overexploitation history that this species has faced
 in the last 40 years. In addition, we collected genetic and morphological
 data for the first time in natural populations to describe genetic
 diversity between and within populations, and to evaluate morphological
 and genetic differences between red and yellow morphs. Overexploitation
 data suggest that more than 80.000 (Min=60.047 - Max=102.236) frogs were
 extracted from the field in the last four decades, probably generating the
 local extirpation or population decline from the type locality. Genetic
 data showed reduced genetic diversity. Observed heterozygosity
 (mean±s.d.=0.599±0.165) is lower than expected (mean±s.d.=0.867± 0.082).
 We did not find differences in body size and heterozygosity between the
 two morphs; however, individuals analyzed were assigned to two genetic
 clusters, which corresponded to the O. lehmanni-yellow and O.
 lehmanni-red. In addition, FST (0.209) and Nei genetic distance (0.18)
 values indicated genetic differentiation between the two morphs;
 therefore, red and yellow morphs should be treated as independent
 management units. This information will help to define appropriate and
 long-term conservation units, as a useful tool to mitigate the extinction
 risk of this species.
description: We obtained
 genomic DNA from wild populations of <i>O. lehmanni-</i>red
 from the FCNP (N=54) and of <i>O. lehmanni-</i>yellow from
 areas near the type locality (N=15). We do not provide precise coordinates
 given the species' threat levels (Fig. 1A). Additionally, samples
 from <i>O. lehmanni </i>with yellow and red phenotypes were
 obtained from specimens kept in captivity at the Cali Zoo (N=15). These
 animals were confiscated between 2006 and 2009. We collected tissue
 samples through a noninvasive technique of mouth swabs (Goldberg et al.
 2003; Pidancier et al. 2003; Beebee 2008) for all individuals. In the
 field, samples were stored dry at room temperature. In the laboratory,
 these were stored at 4ºC until extraction. DNA was extracted and purified
 using the DNeasy tissue extraction kit (QIAGEN, Valencia, CA), following
 the manufacturer’s protocol. Extracted DNA samples were diluted to 12ng/µl
 and used as template in polymerase chain reactions (PCR) for ten di-,
 tri-, and tetranucleotide microsatellite loci previously designed: Dpum
 110, Dpum 14, Dpum 63, Dpum 44 (Wang and Summers 2009) and Oop C11, Oop
 F1, Oop B9, Oop G5, Oop H5, Oop E3 (Hauswaldt et al. 2009).
 Microsatellites are suitable to identify genetic differences between
 individuals and populations, and these have been widely used in
 conservation genetics (Bruford and Wayne 1993; Jarne and Lagoda 1996).
 Forward primers for each PCR amplification were labeled with a 5′-
 fluorescent tag (6-FAM, NED, VIC, or PET) for later visualization. We
 amplified the loci individually and confirmed the PCR amplification using
 an agarose gel electrophoresis for all samples. The PCR reactions were
 performed using this thermal cycling program: following an initial
 denaturation at 95ºC for 3 min, 35 cycles were run with 95ºC for 30 s,
 54ºC for 30s and 72ºC for 4s. This was followed by a 6-min extension at
 72ºC and 25 min incubation at 60ºC. This amplification cycling was used
 for seven markers (Oop B9, Oop G5, Oop H5, Oop E3, Dpum 14, Dpum 63, and
 Dpum 44). For Dpum 110 annealing temperatures were at 58°C and Oop C11 and
 Oop F1 amplified at 64°C. For genotyping, we performed a multiplex of
 three markers. Fragments were sized with LIZ- 500 size standard and then
 scored genotypes using GeneMapper V4.1 (Applied Biosystems). We repeated
 the scoring procedure two times on all samples to check that alleles were
 assigned appropriately. PCR products were run on an ABI3500 Genetic
 Analyzer (Applied Bio-systems) in the Sequentiation Center at the
 Universidad de los Andes.
description: missing values = 0