The Composition And Diversity Of The Bumblebee (Hymenoptera, Apidae) Fauna Of Gansu, Northwest China

Bumblebees are important pollinators for agricultural and natural ecosystems. Gansu Province,China, is located in a transition zone between the Qinghai-Tibetan plateau, the Inner Mongolian plateau,the Loess plateau and the Qinling Mountains, with a region of very varied geomorphology andvegetation, so there is a broad diversity of habitats for bumblebees in Gansu. This variety makes Gansuone of the hotspots of greatest bumblebee diversity in the world. The problem is that the bumblebeefauna in the region has been insufficiently studied. In order to document the current status ofbumblebee diversity in Gansu, a systematic field survey of bumblebees was made in the10principalhabitats within Gansu between2007～2011.The Bombus fauna and species diversity of Gansu are described; microsatellite DNA markers areisolated from the most dominant bumblebee species Bombus pyrosoma in Gansu; and genetic diversityof B. pyrosoma is assessed by analysing microsatellite markers within16populations from all knownmain locations in this study. The principal results include:(1) The composition of bumblebee fauna in GansuA sample of7606bumblebee specimens was collected from180sites within Gansu between2007～2011and assigned to53species belonging to10of the15subgenera of the genus Bombus.Among these53species,2belong to the subgenus Mendacibombus(Md.),3to Subterraneobombus(St.),8to Megabombus(Mg.),8to Thoracobombus(Th.),9to Psithyrus(Ps.),7to Pyrobombus(Pr.),4toBombus s. str.(Bo.),4to Alpigenobombus(Ag.),6to Melanobombus(Ml.),2to Sibiricobombus(Sb.).13species (more than one fifth of the fauna) are recorded for the first time from Gansu: B.(Mg.)bicoloratus, B.(Th.) opulentus, B.(Th.) deuteronymus, B.(Ps.) turneri, B.(Ps.) expolitus, B.(Ps.)chinensis, B.(Ps.) coreanus, B.(Ps.) norvegicus, B.(Bo.) hypocrita s.l., B.(Ag.) breviceps, B.(Ag.)grahami, B.(Ml.) festivus and B.(Sb.) asiaticus. I provide distribution maps and describe speciesabundance and list the food plants used in Gansu. The main regional components of Gansu bumblebeesare Oriental (45.3%) and Oriental+Palaearctic (52.8%).The fauna of Gansu is transitional between thesouthern Sichuan-Chongqing Region fauna and the northern Shanxi-Hebei-Beijing-Tianjin Regionfauna, although it is more similar in composition to the southern Sichuan-Chongqing fauna.(2) Species diversity of bumblebees within GansuBumblebees have been found to be distributed widely in7of the10habitats within Gansu butabsent from the Tengger desert, Hexi corridor gobi and dry Beishan mountains. The bumblebee speciesdiversity index (H′) ranges from0.3805to1.2238in the7habitats, and can be ranked fromQinghai-Tibetan plateau meadows in the southwest> Wuqiaoling mountain forests in the west> Wetriver valley mountain forests in the south> Qilianshan mountain meadows in the northwest> WestQinling mountain forests in the southeast> Loess plateau scrub in the east> Altyn mountain-Kumtagdesert edge scrub in the northwest. The evenness index (Jsw) was highest (0.8113) in Wuqiaoling mountain forests and lowest (0.6751) in West Qinling mountain forests. The relative abundances of thebumblebee species differ among the53species. B.(Ml.) pyrosoma (23.2%) is especially abundant inGansu and accounted more than one fifth of the total. Species including B.(Bo.) ignitus (9.8%), B.(Bo.)hypocrita (6.0%), B.(Ml.) rufofasciatus (5.0%), and B.(Pr.) picipes (4.8%) are also abundant in Gansu.At the opposite extreme, some species, such as all of the species of the subgenus Psithyrus, and some ofthe characteristically southern species, such as B.(Pr.) flavescens and B.(Ag.) grahami and B.(Mg.)bicoloratus, and some of the characteristically Qinghai-Tibetan species such as B.(St.) difficillimus andB.(Mg.) religiosus and B.(Mg.) supremus, are generally rare in Gansu with less than10individuals inmy samples, so that their relative abundances are all less than0.1%. Specimens are recorded here fromsites at elevations between719～4011m, from the low Baishuijiang river valley to the high Zhaganameadows of the Qinghai-Tibetan plateau. The largest number of bumblebee individuals is recorded atelevations between1500～2000m, while the largest number of species is recorded at elevations between2500～3000m. B.(St.) difficillimus (3656m), B.(Ps.) expolitus (3524m), B.(Ps.) rupestris (3454m),B.(Ml.) ladakhensis (3376m) and B.(St.) personatus (3362m) are the5species with the highestrecorded mean elevations, while B.(Ag.) breviceps (944m), B.(Mg.) bicoloratus (1110m), B.(Pr.)flavescens (1122m), B.(Mg.) koreanus (1164m) and B.(Th.) opulentus (1181m) are the5with thelowest mean elevations.(3) Isolation of microsatellite markers from B. pyrosomaBombus pyrosoma, is the most dominant bumblebee species in Gansu and an endemic to China,plays a vital role as one of the most abundant pollinators for many wild flowers and crops in NorthChina. Isolation and characterization of microsatellite markers within this population is made for thefirst time in order to support investigations into its genetic structure. Magnetic bead-based enrichmentand PCR were used to isolate microsatellite markers from B. pyrosoma. The species specificity of theisolated primers was confirmed through tests with7other Chinese bumblebee species of the samesubgenus, Melanobombus. The results show that four dinucleotide probes have different hybridizationrates to the microsatellite DNA sequence. The highest hybridization rate (82%) was obtained with theprobe TC, the moderate hybridization rate (28%) with TG, and the lowest hybridization rates (0%) withAT and GC. For31polymorphic microsatellite loci isolated from B. pyrosoma, four types ofmicrosatellite can be found by sequence analysis, i.e.18perfect types (58.1%),1compound perfect type(3.2%),10imperfect types (32.3%) and2compound imperfect types (6.4%). The repeat unit of themicrosatellite core sequence is different in each probe. The highest repeat unit for TC and TG probes is28and15respectively, and the lowest repeat unit for TC and TG probes is7and11respectively. Theprimer specificity test on31primer pairs showed that products can be amplified from all7bumblebeespecies with26primer pairs, the other5primer pairs were found to be specific for some of the7species,of which1primer (BPM5) was found to be specific for B. pyrosoma. These novel microsatellitemarkers will be useful not only for future studies on the genetic structure and molecular evolution ofthis endemic species, but also for population genetic studies of other Melanobombus species.(4) Genetic diversity of B. pyrosoma based on microsatellite markers In order to investigate the genetic diversity of B. pyrosoma, an assessment is made using the12microsatellite DNA markers in the16B. pyrosoma populations from all known main locations. Withthis, results of the experiment confirmed that178alleles were obtained from the microsatellite DNAmarkers analysis. Consequently, the highest allele number (18alleles) was from locus BPM9andBPM35and the lowest allele number (only5alleles) was from locus BPM17. The mean number ofeffective allele (Ne), the mean genetic heterozygosity (H) and the mean polymorphism informationcontent (PIC) of the16populations is4.377,0.7040and0.6680respectively. Within these populations,the GSMJ population has the highest of the3indices, with5.3967,0.7809and0.7450respectively,while the CQCK population has the lowest, with3.1260,0.6138and0.5690respectively. This showsthat the heterozygosity and genetic diversity of B. pyrosoma are high. The GSMJ population (GansuMaijishan National Forest Park) has the richest genetic diversity and it should be an important priorityarea for conservation of this species. The heterozygosity at the total population level (Fit), theheterozygosity at the individual population level (Fis), and the coefficient of genetic differentiation (Fst)of the16populations is0.121,0.060and0.064respectively. It shows that the genetic differentiation ofthe whole population is not high and the mutation of B.pyrosoma comes mainly from the corepopulation. A combination analysis of Phylogeny, PCA and STRUCTURE show a pattern of geneticvariation that is structured geographically into two groups. The CQCK population located in theDabashan mountains forms one distinctive group, while the remaining15populations form anothergroup. The latter group also can be divided into two groups, one in the Luliangshanmountains-Taihangshan mountains-Tanshan mountains-Neimenggu plateau, the other group in theLoess plateau-Qinling mountains-Baishuijiang river valley-Eastern edge of Qinghai-Tibetan plateau.Compared with NJ dendrogram, UPGMA based on Da data worked well for analysis of B. pyrosomapopulations when considering its geographical distribution and evolution. No divergence from theL-shape distribution was detected by Bottleneck analysis among the16populations. However,heterozygosity excess and/or heterozygosity deficiency were detected in all16populations. This showsthat B. pyrosoma populations have undergone a genetic bottleneck in their history, although, thebottleneck effect is smaller or perhaps acted on just part of the population.This study will be useful not only for future studies of the status of Chinese bumblebee diversityand the genetic structure of their populations, but will also be a resource for bumblebee conservationand in applications for agricultural pollination.