| Most species in the genus Neolucanus Thomson exhibit complicated diversification in their morphology.Intraspecifically,the sexual dimorphism,male polymorphism and different color pattern could be coexisted with wide variations.On the other hand,the inter-specifically external characters are often so highly similar that lack of reliable characters to distinguish them.As a result,the systematics of Neolucanus stag beetles has long been a problematic issue.Thus,it could be very useful to explore and solve this problem based on the study of molecular phylogeny.However,less study was focused on this issue in the world.With the Oriental Realm of China,there is quiet rich species diversity of Neolucanus.Unfortunately,the classification of most known species still needs to be clarified in the area.In order to solve these problems,the molecular phylogenetic study of Neolucanus from China was carried out based on the multi-genes sequence.In this study,from a total of 101 samples belonging to 26 species including the N.sinicus complex were sequenced.Also,the mitochondrial genomes were firstly studied using the next-generation sequencing to understanding the mitochondrial evolution of this genus.The main contributions of this work are as follows:(1)A great deal of multi-gene fragments were obtained:177 16S rDNA(16S ribosomal DNA)with a length of 862 bp;286 COI(cytochrome oxidase subunit I)with a length of 927 bp;241 Cytb(cytochrome b)with a length of 774 bp;113 28S rDNA(28S ribosomal DNA)with a length of 816 bp;99 WG(Wingless)with a length of 41 1bp;(2)The analysis of molecular phylogeny supports that the genus Neolucanus is monophyletic,and sister to the genus Odontolabis.Neolucanus stag beetle mainly separated into three clades in Chinese fauna:the Himalayan clade(HM),the Southwest clade(SW)and the Southeast clade(SE).Within the SE clade,4 subspecies of the N.sinicus complex also clusted into a subclade,in which the N.championi and N.opacus exhibits a nested structure,respectively.(3)The estimation of divergent time indicated that the recent ancestral time of the Neolucaus and Odontolabis is about the early Miocene(17.61-30.12 Mya);the species diversification of Neolucanus has a slow increase until about 5.0 Mya,after this period,the diversification increased sharply.(4)The complete mitochondrial genomes of 7 Neolucanus species were obtained with lengths ranging from 16,183 to 18,299 bp.The structure and composition were consistent with the typical insect mitochondrial genome.The phylogenetic analysis based on the mitogenomic data also supports that Neolucanus is monophyletic,which is consistent with the results inferred from the multi-genes.(5)The mitogenome sequences provided a powerful way to designed primers of gene fragements in Neolucanus,which successfully improved the data quality of COI.16S rDNA and Cytb genes.Our result could help to resolve the problem of poor primer that has long been obstructed the molecular study of this genus.In conclusion,Neolucanus stag beetle from Chinese fauna began to differentiate in the early Miocene,and then divided into three major clades:HM,SW and SE,with a rapid species divergence about occurred in the early Pliocene.These evolutionary processes may be related to a series of important geological events,ecological changes and climate oscillation in Eurasia.Within the N.sinicus complex,three species were recognized,i.e.,N.sinicus,N.oberthuri and N.championi,and the subspecies of N.opacus was proposed to be a junior synonym of N.championi.Also,the mitochondrial genomes through the next-generation sequencing improved a good approach to design the primer for Neolucanus,and thus help to improve the gene fragment amplification quality and obtaining high-quality genetic data.Our study could provide the genetic data for studying the systematics and evolution of Neolucanus,and also provided necessary genetic data for promoted an in-depth ecological conservation. |
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