Species Delimitation And Phylogenetics Of Bombus Lucorum Complex In China (Hymenoptera: Apidae)

Bumble bees(Hymenoptera,Apidae,Bombus)represent an ideal biological system for studying cryptic speciation and biogeography because of their cold-adapted ability,high diversity or convergence in colour patterns,and wide geographic distribution.In recent years,with the development of molecular methods,DNA sequence has proven to be a suitable biological tool to obtain reliable results for identifying species.Considering the cephalic labial gland secretions(CLGS)produced by male bumble bees are species-specific,and thus have potential for recognising cryptic species and closely related species.Among 15 subgeneric system,most bumblebee species belong to the subgenus Bombus s.str.are regarded as taxonomically confused,with poorly defined cryptic species and species complex.One prime example is the B.lucorum complex.Although extensive studies have been conducted in Europe and North America,the taxonomy of this group within China is still uncertain.In this study,we investigated the species composition,morphological traits,semio-chemicals and phylogenetic relationships among Chinese B.lucorum cryptic species using a combination of DNA,morphological,and chemical data.The main results are as follows:(1)Analysis of Chinese B.lucorum complex based on mitochondrial COI barcodeswe evaluated extensive samples of B.lucorum cryptic species throughout China.These specimens have expanded our knowledge concerning the distribution and habitats of B.lucorum cryptic species.A total of 262 COI sequences were contracted to 114 unique haplotypes.Bayesian analysis(using BEAST and Mr Bayes)and results of two automated species delimitation methods(b GMYC and ABGD)clearly revealed that B.lucorum complex actually includes five species: B.terrestris,B.lucorum,B.cryptarum,B.longipennis and B.sp..In addition,our results show that B.lantschouensis and B.minshanensis(endemic to China)could be conspecific.(2)Analysis of Chinese B.lucorum complex based on morphometric comparisons The samples used for morphometric analysis were all female individuals.Four variables were measured:(Ⅰ)breadth of the collar on top of the thorax,(Ⅱ)length of collar below left tegula,(Ⅲ)length of collar below right tegula and(Ⅳ)wing shape.The results indicate that linear measurements related to collar were not suitable for species discrimination.However,most of the five taxa were well separated based on wing shape.We demonstrate that geometric morphometric analysing wing shape represent a promising approach for discriminating between morphologically cryptic taxa of the B.lucorum complex.(3)Analysis of Chinese B.lucorum complex based on CLGSThe CLGs of males were analyzed by gas chromatography-mass spectrometry(GC-MS).Atotal of60 compounds were detected.Cluster analysis indicated that B.lucorum complex could be divided into five groups.PERMANOVA pairwise comparison results show that in addition to B.cryptarum,B.sp.and other three cryptic species(B.terrestris,B.lucorum and B.longipennis)CLGS ingredients there are significant differences.The compounds Ethyl Oleate(Indval = 0.86)and(Z)-Tricos-9-ene(Indval =1.00)were identified as the indicator compounds of B.sp.,Ethyl oleate was also the main component of B.sp.,which could be used to distinguish B.sp.from B.cryptarum and the other three cryptic species.(4)Phylogenetics of Bombus lucorum complex in ChinaPhylogenetic analysis based on COI,16 S,PEPCK,and Opsin gene sequences showed that B.terrestris,B.lucorum,B.longipennis,B.cryptarum and B.sp.were all monophyletic groups with high degree of support.Among them,B.longipennis and B.lucorum were sister species,B.sp.and B.hypocrita were sister species.The results of divergences showed that the most recent common ancestor of the five monophyletic groups originated in the early Pliocene(5.1 Ma),and the divergences were as follows: B.lucorum(1.1 Ma),B.cryptarum(0.8 Ma),B.terrestris(0.7 Ma),B.longipennis(0.3 Ma),and B.sp.(0.3 Ma),respectively.B.sp.was found in Xinjiang,China,mainly distributed in the southern Tianshan Mountain hinterland of the alpine mountains,the altitude range of 1383-2569 m.We speculate that may be affected by global warming,Xinjiang extreme high temperature in summer and winter temperatures rise obviously,causing bumblebee this kind of cold-adapted insects migrate to higher latitudes and high altitude region,with field distribution between the subgroups of niche separation,thus form reproductive isolation and separation of the gene flow and cryptic speciation.