Genetic Diversity And Distribution Pattern Of Rana In Henan Province

Rana Linnaeus(1758),as the type genus of Ranidae,is widely distributed in Eurasia and northern America.There are 25 species of Rana in China.Henan Province is located in the transition zone between the Palaearctic and Oriental boundaries,and between central and northern China,spanning four major river systems including Haihe River,Yellow River,Huaihe River and Yangtze River.Numerous mountains like Taihang Mountain,Qinling Mountain,Funiu Mountain,Tongbai-Dabie Mountains are located respectively in the northwest,west,southwest and southeast of Henan Province.The Amphibian fauna composition in Henan Province is complicated,the classification and distribution of Rana species are controversial.The distribution range and limit of Rana chensinensis and Rana luanchuanensis are not clear,and the distribution of Rana zhenhaiensis and Rana culaiensis in Henan Province are controversial.In this study,genetic diversity and genetic differentiation of Rana in Henan Province were investigated by constructing phylogenetic tree based on 16 S r RNA gene,COI gene,and 12 S r RNA-ND2-Cytb combined gene,respectively.In order to reveal the species diversity,genetic diversity and distribution pattern of Rana in Henan Province,the comparative morphological methods including statistical analysis of 24 morphological measures and canonical discriminant analysis of Rana(No.135,?55,?80)were used to provide scientific support for the development of biodiversity conservation strategies.The main research results show that:(1)Phylogenetic tree based on mitochondrial gene haplotypes showed that Rana in Henan Province could be divided into five clades(I-V)with high support.Among them,clade I and haplotype of Rana kukunoris formed a sister group relationship;The haplotype of Rana chensinensis is embedded in clade II;Clade IV is closely related to the haplotype system of Rana zhenhaiensis and Rana culaiensis;Rana luanchuanensis shares haplotype H13 with clade V.(2)The minimum genetic distance between clade I and II(16S r RNA gene 1.4%,COI gene 6.3%)was much higher than that between Rana culaiensis and Rana zhenhaiensis(16S r RNA gene 0.5%,COI gene 3.3%);The genetic distance between Clades I,II and III was small,and the genetic distance between Clades IV and V was large,and Clades IV and V had large genetic differentiation with all clades,respectively.(3)The genetic distance among clade I and Rana chensinensis(16S r RNA gene 1.4%,COI gene5.4%)and Rana kukunoris(16S r RNA gene 1.2%,COI gene 5.5%)was greater than that between Rana culaiensis and Rana zhenhaiensis.The genetic distance between clade II and Rana chensinensis was 0.00(based on 16 S r RNA gene)and 0.2%(based on COI gene).The genetic distance between clade III and Rana chensinensis(16S r RNA gene 2.1%,COI gene 6.2%)was not only larger than that between Rana culaiensis and Rana zhenhaiensis,but also similar to that between Rana chensinensis and Rana kukunoris(16S r RNA gene 2.0%,COI gene 6.5%),similarly,the genetic distance between clade III and Rana kukunoris(16S r RNA gene 2.6%,COI gene 6.3%)was also larger than that between Rana culaiensis and Rana zhenhaiensis,and similar to that between Rana chensinensis and Rana kukunoris.The genetic distance between clade IV and Rana culaiensis(16S r RNA gene 0.2%,COI gene 0.4%)was smaller than that between Rana culaiensis and Rana zhenhaiensis,similarly,the genetic distance between clade IV and Rana zhenhaiensis(0.3% of 16 S r RNA gene and 3.0% of COI gene)was also smaller than that between Rana culaiensis and Rana zhenhaiensis.The genetic distance between clade V and Rana luanchuanensis was 0.1%.(4)Clade I is mainly distributed in the eastern part of Taihang Mountains and Qinling Mountains;clade II is mainly distributed in the western part of Taihang Mountains,Funiu Mountains and Qinling Mountains;clade III and clade IV are distributed along the Tongbai-Dabie Mountain line,reaching as far north as Nanzhao County on the southern of Funiu Mountain;clade V is distributed in Nanzhao County(the southern of Funiu Mountain),and Han Qiu Village,Luanchuan County(the Northern of Funiu Mountain),the type locality of Rana luanchuanensis,belong to the Yangtze River and Yellow River systems respectively.(5)Morphological discriminant analysis showed that 5 clades of Rana in Henan Province had high positive discrimination rate(male 98.8%,female 100%).The morphological characteristics of clade IV tended to be that of Rana zhenhaiensis.(6)The five clades of female individuals showed five independent morphological groups,while the five clades of male individuals showed better separation,and the center of gravity among these branches is far apart.Clade V had a large genetic distance from the remaining four clades and showed significant segregation of morphological features;In clade I and IV with greater genetic differentiation,the morphology of males overlapped more,while that of females separated farther;the degree of morphological divergence among clades I,II,and III corresponded to genetic differentiation.There is a significant difference between the morphological characteristics of Rana(clade II)in Hanqiu Village(North Slope of Funiu Mountain)in Luanchuan County,where is the type locality of Rana luanchuanensis,while the morphological characteristics of the population(clades V)in the south slope of Funiu Mountain are consistent with the original description of the Rana luanchuanensis.(7)Clade III has the largest individual,which is larger than clade I,II and IV without significant difference in sexual dimorphism,while the individuals of Clade V were smallest with significant dimorphism.Conclusions:(1)Rana in Henan Province is a species complex.The genetic differentiation and morphological variation of five clades have reached the species or subspecies level,including Rana chensinensis(clade II),Rana luanchuanensis(clade V),two hidden species or subspecies(clade I and III),and clade IV for which the taxonomic classification is still unclear.(2)Clade I distributed in the eastern part of the Taihang Mountains.The sensu stricto Rana chensinensis is distributed in the western part of the Taihang Mountains,Funiu Mountains and Qinling Mountains in Henan Province,and they are continuously distributed in the Qinling Mountains where the type locality of Rana chensinensis.Clade III and IV distributed along the Tongbai-Dabie Mountains,as far as the southern part of Funiu Mountain;the distribution range and pattern of Rana luanchuanensis(clade V)have yet to be verified.(3)The Rana chensinensis of Funiu Mountain in western Henan Province have the highest abundance.Except for the clade I,the other clades are distributed,and exhibited the transition of zoogeographical division.