Molecular Phylogeny Of The Genus Odorrana Inferred From Mitochondrial And Multiple Nuclear Genes

The genus Odorrrana,belonging to the family Ranidae of Anura in the class Amphibia,lives in swift boulder-strewn streams in the mountains of the sounthern China,the Ryukyu islands,India northeast and southeast Asia,and it is endemic to Asia.This genus includes 58 species with 36 species in China,and it is evolutionarily significant during the transition from Rana to Hylorana in the family Ranidae,and reconstructing their phylogeny,origin and the history of dispersal has been the interests of phylogenetic studies for Amphibia.However,there is still some controversy about their taxonomic position,species constitution,the validity of different species complex and their phylogenetic relationships due to the limited species and molecular markers used.Eighty-six percent representatives of the genus Odorrana species were collected by extensive sampling during the field survey and data collection from the Gene Bank.Phylogenetic relationships of the genus Odorrana was reconstructed using the Bayesian Inference and Maximum Likelihood based on the combined sequences of 16 053 bp of the mitochondrial 12S?16Sr RNA and 16 nuclear genes.In addition,the phylogeographic patterns of the odorous frogs were analyzed based on S-DIVA and BBM method in RASP to further explore the origin,diversification and the dispersal mechanism.Results:(1)The monophyly of the genus Odorrana was significantly supported and was divided into eight obvious clades(i.e.,Clades A-H).(2)Odorrana lipuensis was the earliest clade diversified within Odorrana and was denoted clade A.(3)Odorrana chapaensis and O.geminata are grouped and together constitutes clade B.(4)Clade C is composed with 13 odorous frogs which are mainly distributed in Yunnan-Guizhou Plateau.However,the phylogenetic position of the clade O.yizhangensis,O.lungshengensis and O.anlungensis(i.e.,Clades C1)is not stable.In the phylogenetic trees based on the mitochondrial and the combined mitochondrial + nuclear dataset it clustered with species of O.andersonii group(i.e.,Clades C2)and formed Clade C,whereas it clustered with the O.schmackeri group based on nuclear genes dataset.(5)Odorrana ishikawae from the Ryukyu Islands was included in the genus Odorranaand denoted clade D,but the support for its position was not significant.(6)The clade E included O.hainanensis,O.tiannanensis,O.fengkaiensis and representatives of O.schmackeri species complex from Qinling Mountain to Daba Mountain,Wushan Mountain and Southeast hills.But the interrelationships within the Clade E are not resolved.(7)Odorrana rotodora and O.zhaoi were included in the O.graminea species complex and they together formed clade F with high support.The O.graminea complex are widely distributed in the Hengduan Mountain,Qinling-Daba Mountains,Southeast hills and Indochina.(8)Odorrana tormota clustered with species of O.versabilis species complex and formed Clade distributed in Southeast hills(i.e.,Clade G)with high support.(9)Species from the Ryukyu Islands(exclusively of O.ishikawae)and two species from Taiwan(i.e.,O.swinhoana and O.taiwaniana)are formed a highly-supported Island Clade(i.e.,Clade H).(10)The genus Odorrana originated in 21.62 Ma B.P.(Early Miocene)and rapid diversification with the genus Odorrana occurred from 19.25 to 8.69 Ma B.P.(Middle and Late Miocene)with successive diversification from clade A to H.(11)The mainly speciation events occurred after the Pleistocene in Quaternary period(2.6 Ma),accounting for 60% of the 50 Odorrana species investigated in the present study;Speciation events were mainly occurred in the Yunnan-Guizhou Plateau with 10 new species,9 events occurred in the Southeast hills area,and five of seven species distributed in Taiwan and Ryukyu Islands diversified between 2.6 Ma to 1.6 Ma.Conclusions:(1)The genus Odorrana was divided into eight clades.(2)The Odorrana originated at Early Miocene and 8 clades diversified successively from Middle Miocene to Late Miocene.Rapid diversification and speciation events of the genus Odorrana occurred in Pleistocene.(3)The Yunnan-Guizhou Plateau was the origin center for the genus Odorrana,and they dispersed to Southeast hills area,Taiwan and Ryukyu Islands under the influence of the uplift of Qinghai-Tibet Plateau.With the formation of the Hengduan Mountains,the genus Odorrana dispersed to the North to the southern side of the Himalayas Mountains and areas of Qinling Mountain,Daba Mountain and Wushan Mountain,and to the South to theIndochina.(4)The uplift of Qinghai-Tibet Plateau,climate fluctuation during the Quaternary glacial and interglacial as well as the formation of the glacial refuge lead to the rapid diversification of the Odorrana species in Pleistocene.