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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 relationships 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 hybridization 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 allegedly 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 pronounced 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. [ABSTRACT FROM AUTHOR]

The Asian box turtle genus Cuora currently comprises 13 species with a wide distribution in Southeast Asia, including China and the islands of Indonesia and Philippines. The populations of these species are rapidly declining due to human pressure, including pollution, habitat loss, and harvesting for food consumption. Notably, the IUCN Red List identifies almost all species of the genus Cuora as Endangered (EN) or Critically Endangered (CR). In this study, we explore the karyotypes of 10 Cuora species with conventional (Giemsa staining, C-banding, karyogram reconstruction) and molecular cytogenetic methods (in situ hybridization with probes for rDNA loci and telomeric repeats). Our study reveals a diploid chromosome number of 2n = 52 chromosomes in all studied species, with karyotypes of similar chromosomal morphology. In all examined species, rDNA loci are detected at a single medium-sized chromosome pair and the telomeric repeats are restricted to the expected terminal position across all chromosomes. In contrast to a previous report, sex chromosomes are neither detected in Cuoragalbinifrons nor in any other species. Therefore, we assume that these turtles have either environmental sex determination or genotypic sex determination with poorly differentiated sex chromosomes. The conservation of genome organization could explain the numerous observed cases of interspecific hybridization both within the genus Cuora and across geoemydid turtles. [ABSTRACT FROM AUTHOR]