Introgressive Hybridization and a Natural Barrier: Molecular Analyses Lay Out a Conservation Blueprint for the Critically Endangered Vietnamese Pond Turtle (Mauremys annamensis)

The critically endangered Vietnamese Pond Turtle (Mauremys annamensis), an endemic species occurring in a small lowland area in central Vietnam, has been virtually extirpated from its natural range. As a result, reintroduction of held individuals worldwide, especially from Europe, the United States, and Vietnam, will play a vital role in species recovery programs. Nevertheless, the discordance between different molecular markers in inferring the placements of two closely related species, M. annamensis and M. mutica, and the existence of two distinct mitochondrial lineages within M. annamensis, have hindered conservation strategies to properly maintain the genetic integrity of held populations to release individuals back to their natural habitat in the future. In this study, we sequenced 732 bps of a mitochondrial gene (ND4) and 1038 bps of an intron fragment of the RNA fingerprint protein 35 (R35) gene for 18 samples collected from the local trade in five provinces in Vietnam and 20 samples from founders in the Turtle Conservation Centre, Cuc Phuong National Park, Vietnam. DNA sequences analyzed by Bayesian Inference, Maximum Likelihood, and NeighborNet methods show that the Vietnamese Pond Turtle is a well-defined species and that the population of M. mutica from Hainan Island likely evolved through introgression between a lineage distantly related to true M. mutica and M. annamensis and a lineage closely related to the two mitochondrial haplotypes of M. annamensis. Interestingly, our analyses of samples collected from the local trade and those with known localities reveal that the two extant mitochondrial lineages within M. annamensis are geographically isolated and evolutionarily significant. Based upon the new findings, we recommend genetic screening for all held populations in Vietnam and elsewhere and that animals with different genetic histories be managed separately. In addition, it is important to use other molecular markers, such as microsatellites and single nucleotide polymorphisms (SNPs), to determine potential hybrids between the two mitochondrial haplotypes and remove them from conservation breeding programs before releasing the turtles back to their natural habitat.