ИСТИНА

The genus Ophryophryne joins around 5 known species of small mountain megophryid toads, inhabiting mountain forests of Indochina (Vietnam, Laos and Cambodia), southern China and northern Thailand. Morphologically, diagnostics of Ophryophryne species is difficult and application of molecular phylogenetic methods is needed to understand the real diversity and evolutionary history of this group. In the present study we examined 1 mtDNA fragment and 3 nuclear DNA genes to clarify phylogenetic relationships, diversity and taxonomy of this genus. We collected tissue samples of Ophryophryne in Northern, Central and Southern Vietnam and Laos in 2009-2015. DNA isolation, PCR and sequencing was conducted following standard protocols. For assessment of the genus diversity we sequenced a 16S rRNA mtDNA fragment (ca. 520 bp) for 123 samples collected from southern China and all across Vietnam to Laos and Cambodia, representing the almost entire distribution rage of the genus (with exception of northern Thailand) and all currently recognized species of Ophryophryne. For resolving phylogenetic relationships among Ophryophryne we additionally sequenced a 12S rRNA - 16S rRNA mtDNA fragment (total length up to 2676 bp) and three nuclear gene fragments: RAG-1 (606 bp), BDNF (693 bp) and TTN (909 bp) for 26 samples, representing main lineages within Ophryophyne. Phylogenetic analysis included 13 outgroup sequences of the genera Megophrys (s. lato), Brachytarsophrys, Leptobrachium and Leptolalax. We performed Bayesian (BI, Mr. Bayes), Maximum Likelihood (ML; Treefinder) and Maximum Parsimony (PAUP) phylogenetic analyses. The resulting phylogeny is well-resolved, altogether 15 mtDNA lineages are recognized within Ophryophryne. The monophyly of the genus Ophryophryne is well-supported; however the group is clearly placed within the genus Megophrys s. lato rendering the latter paraphyletic. Within Ophryophryne two major clades are supported: the first clade joins taxa from southernmost part of the Annamite (Truong Son) mountains (the Lang Bian Plateau), the second clade joins all other species from central and northern Indochina. Our data confirm validity of a poorly-known species O. poilani from Kon Tum Plateau in central Vietnam and also indicate high unrecognized diversity within Ophryophryne with several candidate species recovered. We will discuss the present taxonomy and distribution of Ophryophryne and biogeography of Indochina in scope of our data.