Diversity of the Southeast Asian leaf turtle genusCyclemys: how many leaves on its tree of life?

In the present study, we use mtDNA sequence data (cyt b gene) in combination with nuclear DNA sequences (C-mos, Rag2 genes, R35 intron), nuclear genomic fingerprints (ISSR) and morphological data to reveal species diversity within the Southeast Asian leaf turtle genus Cyclemys, a morphologically difficult group comprising cryptic species. Two morphologically distinct major groupings exist, a yellow-bellied species group with three taxa (Cyclemys atripons, C. dentata, C. pulchristriata) and a dark-bellied species group. The latter contains besides the morphologically variable C. oldhamii three additional new species (C. enigmatica n. sp., C. fusca n. sp., C. gemeli n. sp.). According to mtDNA data, C. fusca and C. gemeli constitute with high support the sister group of a clade comprising all other species, indicating that the dark-bellied species are not monophyletic, despite morphological similarity. mtDNA sequences of C. enigmatica, being highly distinct in nuclear genomic markers, do not differ from the sympatric C. dentata, suggesting that the original mitochondrial genome of C. enigmatica was lost due to introgressive hybridization. Morphological discrimination of Cyclemys species is possible using multivariate methods. However, gross morphology of most dark-bellied species on the one hand and of C. atripons and C. pulchristriata on the other is so similar that reliable species determination is only possible when genetic markers are used. The high diversity within Cyclemys requires revision of the IUCN Red List Categories for leaf turtles because the former assessment was based on the wrong assumption that in the entire range of the genus occurs only a single species.