Molecular differentiation and conservation of the Indochinese box turtles Cuora galbinifrons, Cuora bourreti, and Cuora picturata

The Asian box turtles of the Cuora galbinifrons complex (C. galbinifrons, C. bourreti, and C. picturata) rank among the most endangered chelonian species in the world. Despite several previous studies, the phylogenetic relation-ships and species boundaries of this complex remain a matter for dispute due to a shortage of field-collected samples for genetic validation, observed discordance between mitochondrial and nuclear markers, and reported intergradation zones combined with a strong tendency of hybridization in the genus. Here, we re-investigate the relationships and potential hy-bridization between the species of the C. galbinifrons complex based on the most comprehensive dataset to date consisting of 394 morphologically identified specimens (136 C. galbinifrons, 200 C. bourreti, 49 C. picturata, and nine individuals al-legedly from Hainan). The turtles mainly came from assurance colonies as well as from zoological and private collections across the USA and Europe. Bayesian and Maximum Likelihood analyses of a concatenated mitochondrial dataset (COI and ND4 plus adjacent tRNA genes) yielded almost identical topologies, supporting three major clades corresponding to C. galbinifrons, C. bourreti, and C. picturata, respectively. In accordance with previous studies, C. bourreti represented the sister clade to C. galbinifrons and these two clades together were sister to C. picturata. Haplotype networks revealed pro-nounced mitochondrial divergences between the taxa. STRUCTURE and PCA analyses using 12 microsatellite loci also confirmed three distinct clusters that are in agreement with the recognized species. Only a few specimens with admixed ancestry (hybrids) or mismatched mitochondrial identity were revealed, suggesting extremely limited gene flow among the three species. However, this pattern could also reflect the separate captive management of the individual taxa and an underrepresentation of geographic contact zones in our sampling.