Molecular Phylogeny Of Lucanus Scopoli(Coleoptera:Lucanidae)

Lucanus,as a type genus of Lucanidae(Coleoptera),is a typical representative of stag beetles.At present,there are about 110 known species(subspecies)in the world,with the exception of a few species distributed in the Palaearctic and Nearc tic sector,mostly in the Oriental.It has developed and striking mandible,obvious dimo rphism and male polymorphism.Since the age of Darwin,it is a good material to study the formation and differentiation of species,the mechanism of sexual selection and the evolution of phenotypes.Because of the complex morphology of the genus,the phylogenetic relationship and species definition of subgenera,species and subspecies based on morphological data are more prominent and lack of molecular systematics to discuss and solve these problems.This study selected 62 typical species(subspecies)of Lucanus and 44 specimens of th eL.maculifemoratus complex and 3 mitochondrial genes(COI,16S rDNA,Cytb)and 3 nuclear genes(Wingless,ITS2,EF-la)were used as molecular markers,counterpoint,statistical base content etc.Based on methods and techniques of molecular phylogenetics to reconstruct system relationship and estimated of the time of differentiation of the genera.Finally,we get the following results and conclusions:(1)Through total DNA extraction,PCR amplification and sequencing,we obtai ned the effective molecular data as follow:123 COI gene sequences,131 16S rDNA g ene sequences,84 Cytb gene sequences,111 sequences of Wingless,100 of EF-la and 101 sequences of ITS.(2)Phylogenetic analysis of genera showed that:Lucanus(Lucanus)Scopoli.Lucanus(Pseudolucanus)Hope&Westwood,Lucanus(Eolucanus)Kurosowa and NoesolucanusAraya formed a monophyletic clade.Among them,the representative species of L.(Lucanus),L.(Eolucanus)and Noesolucanus were clustered into one branch respectively,showing distinct monophyletic characteristics.The species of L.(Eolucanus)Kurosowa clustered into a clade and presented sister group relationship with Noesolucanus.But L.(Pseudolucanus)itself was not monophyletic,The pattern species L.(P.)capreolus and 5 other representative species and the species of L.(Lucanus)are mosaic.However,because of the limited sample size of the subgenus,it is suggested that it still be used as an effective subgenus and its classification status needs further study.The genetic distance between L.(Lucanus)and L.(Eolucanus)is close to the average genetic distance among the genera,which further supports their monophyletic and elevate the classification of them to the independent genus.(3)Using 3 methods(ABGD,GMYC and PTP)to identify in 44 samples for 4 taxa of L.maculifemoratus complex.Among them,the ABGD method had a serious over-classification of species,almost every geographic population was defined as one species;PTP had only a slight over-classification in the populations ofL.m.boileaui,and the definition of GMYC resulted in a reasonable division of different geographical populations.The genetic distance between 4 taxa is 0.068-0.163,which is lower than the average genetic distance between the species of Lucanus.Based on the results of the 3 methods and genetic distance,we suggest that these 4 taxa are subspecies relationships:L.maculifemoratus maculifemoratus,L.maculifemoratus dybowskyi,L.maculifemoratus taiwanus and L.maculifemoratus boileaui.In addition,the genetic distance of L.m.adachii and L.m.maculifemoratus is only 0.020,and the external morphology and anatomical structure of the genitalia are very similar of them and there is no significant geographic isolation.So L.m.adachii should be a synonym of L.m.maculifemoratus.(4)The divergence time based on Beast analysis showed that:the time of divergence between((Lucanus+Eolucanus)+Pseudolucanus)+Noseolucanus and other beetles was in the Middle Eocene(46.66Mya,95%confidence interval:36.81?63.1Mya).Among them,Noseolucanus and Pseudolucanus were first differentiated They were more primitive and they were in the roots of the tree.Eolucanus differentiated in the early Oligocene(36.34Mya)and formed an independent branch in 33.08Mya(95%confidence interval:16.44?39.45Mya).The Lucanus(s.s.)formed at about 35.75Mya(95%confidence interval:33.76?37.68Mya)and the differentiation time of L.maculifeoratus complex was 15.09Mya(95%confidence interval:7.22?23.8Mya).