甘肃熊蜂区系组成与多样性分析
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摘要
熊蜂属（Bombus）昆虫是一类重要的传粉昆虫，在维护自然生态系统平衡和农业生产中发挥着十分重要的作用。甘肃省位于青藏高原、蒙古高原、黄土高原和秦岭山地交汇的地带，境内地形、植被和栖息环境的多样性，使甘肃成为全球熊蜂多样性研究的热点区域之一。由于缺乏对甘肃熊蜂区系的系统研究，甘肃熊蜂多样性特点不详。为了探究甘肃熊蜂多样性现状，作者于2007～2011年连续5年，对甘肃不同生态区的熊蜂资源进行了系统调查。根据所采集的标本，分析了甘肃熊蜂区系组成和物种多样性现状；又以甘肃优势种类火红熊蜂为材料，进行了熊蜂微卫星标记的筛选；最后，基于微卫星标记分析了火红熊蜂主要分布区16个地理种群的遗传多样性特点。取得如下研究结果：
（1）甘肃熊蜂区系组成
2007～2011年，在甘肃180个分布点共采集熊蜂属昆虫标本7606头，隶属于10亚属53种，其中假熊蜂亚属Mendacibombus（Md.）2种，地熊蜂亚属Subterraneobombus（St.）3种，巨熊蜂亚属Megabombus（Mg.）8种，胸熊蜂亚属Thoracobombus（Th.）8种，拟熊蜂亚属Psithyrus（Ps.）9种，火熊蜂亚属Pyrobombus（Pr.）7种，真熊蜂亚属Bombus s. str.（Bo.）4种，阿熊蜂亚属Alpigenobombus（Ag.）4种，黑熊蜂亚属Melanobombus（Ml.）6种，西熊蜂亚属Sibiricobombus（Sb.）2种。在甘肃53种熊蜂中，其中13种熊蜂为甘肃新纪录种，分别为二色熊蜂B.(Mg.) bicoloratus、富丽熊蜂B.(Th.) opulentus、德熊蜂B.(Th.) deuteronymus、图拟熊蜂B.(Ps.) turneri、探索拟熊蜂B.(Ps.)expolitus、中华拟熊蜂B.(Ps.) chinensis、科尔拟熊蜂B.(Ps.) coreanus、挪威拟熊蜂B.(Ps.) norvegicus、小峰熊蜂B.(Bo.) hypocrita s.l.、短头熊蜂B.(Ag.) breviceps、灰熊蜂B.(Ag.) grahami、白背熊蜂B.(Ml.)festivus和亚西伯熊蜂B.(Sb.) asiaticus。本文首次系统报道了这53种熊蜂的区系分布和采访植物特性。甘肃熊蜂区系成份主要以东洋区种（45.3%）、东洋区和古北区共有种（52.8%）为主，介于川渝地区向华北地区过渡的中间阶段，与川渝地区熊蜂的种类相似度比较接近，与华北地区的种类相似度相距较远。
（2）甘肃熊蜂物种多样性分析
在甘肃10个生态区，除北部腾格里沙漠、西北部河西走廊戈壁地带和干燥北山3个生态区未发现有熊蜂分布外，其他7个生态区均有熊蜂分布。甘肃7个生态区的熊蜂物种多样性指数（H′）范围为0.3805~1.2238，由高到低的顺序依次为：西南部青藏高原牧场、西部乌鞘岭山地林区、南部白水江河谷山地林区、西北部祁连山牧场、东南部西秦岭山地林区、东部黄土高原灌丛和西北部阿尔金山-库姆塔格沙漠边缘灌丛。甘肃熊蜂物种均匀度指数（J_(sw)）西部乌鞘岭山地最高（0.8113），东南部西秦岭山地最低（0.6751）。西南部青藏高原和西部乌鞘岭山地的物种相似度最高（60.98%），南部亚热带湿润河谷山地林区和西北部干燥阿尔金山-库姆塔格沙漠边缘灌丛未见共同种。甘肃53种熊蜂中，火红熊蜂B.(Ml.) pyrosoma的物种相对多度最高（23.2%），占甘肃熊蜂总体数量的五分之一以上，另外，红光熊蜂B.(Bo.) ignitus（9.8%）、小峰熊蜂B.(Bo.) hypocrita s.l.（6.0%）、红束熊蜂B.(Ml.) rufofasciatus（5.0%）和重黄熊蜂B.(Pr.) picipes（4.8%）在甘肃分布也较丰富；相反，另外一些种类，如拟熊蜂亚属（Ps.）的所有种类，还有一些典型的南方种类如黄熊蜂B.(Pr.) flavescens、灰熊蜂B.(Ag.) grahami、二色熊蜂B.(Mg.) bicoloratus）和一些青藏高原种类如猛熊蜂B.(St.) difficillimus、圣熊蜂B.(Mg.) religiosus、越熊蜂B.(Mg.) supremus在甘肃境内非常稀有，其标本数量均在10头以下，其物种多度均在0.1%以内。甘肃熊蜂的垂直分布范围为海拔719~4011m，其中海拔1500~2000m范围内数量最多，海拔2500~3000m范围内种类最丰富；猛熊蜂B.(St.) difficillimus（3656m）、探索拟熊蜂B.(Ps.) expolitus（3524m）、石拟熊蜂B.(Ps.) rupestris（3454m）、拉达克熊蜂B.(Ml.) ladakhensis（3376m）和伪猛熊蜂B.(St.) personatus（3362m）位居平均海拔高度的前5位，短头熊蜂B.(Ag.) breviceps（944m）、二色熊蜂B.(Mg.) bicoloratus（1110m）、黄熊蜂B.(Pr.) flavescens（1122m）、朝鲜熊蜂B.(Mg.) koreanus（1164m）和富丽熊蜂B.(Th.)opulentus（1181m）位居平均海拔高度的末5位。
（3）火红熊蜂微卫星标记的筛选
火红熊蜂是甘肃最优势的熊蜂种类，也是中国特有的熊蜂资源，在中国北方地区分布十分丰富，是众多野生植物和农作物的重要传粉昆虫。为了探究火红熊蜂的分子遗传结构，首次进行了该种熊蜂微卫星标记的筛选。本研究采用生物素-磁珠吸附分离法从火红熊蜂基因组中筛选微卫星标记，并选取火红熊蜂同一亚属黑熊蜂亚属Melanobombus其他7种熊蜂进行了这些标记的种特异性验证。不同核苷酸探针与磁珠杂交产生的微卫星标记的阳性克隆率存在很大差异，TC探针的微卫星阳性克隆率最高为82%；TG探针次之，为28%；AT和GC探针分离未获得微卫星标记克隆。根据获得的阳性克隆的序列设计引物进行筛选验证，共获得31对熊蜂微卫星序列特异性引物。31对微卫星引物分为4种类型，其中完美型18个，占58.1%；混合完美型1个，占3.2%；非完美型10个，占32.3%；混合非完美型2个，占6.4%。不同核苷酸探针的微卫星核心序列重复数不同，TC探针和TG探针的微卫星核心序列最高重复数分别为28和15个，最低重复数分别为7和11个。31对微卫星引物在黑熊蜂亚属其他7种熊蜂上的检测结果表明，有26对引物可以扩增所测试的7种熊蜂，其余5对引物只能扩增部分熊蜂种类，其中BPM5是火红熊蜂种特异性引物。本研究从火红熊蜂基因组中筛选的微卫星标记，不仅可用于火红熊蜂遗传结构与分子进化等方面的研究，而且还可用于黑熊蜂亚属其他种类的遗传特性分析。
（4）火红熊蜂遗传多样性分析
为了探明火红熊蜂的遗传多样性特点，本研究基于12个微卫星标记首次分析了火红熊蜂主要分布区16个地理种群的遗传多样性水平。12个微卫星标记共获得等位基因数为178个，其中BPM9和BPM35等位基因数最高均为18个，BPM17等位基因数最低为5个。火红熊蜂16个地理种群的平均有效等位基因数（Ne）、平均遗传杂合度（He）和平均多态信息含量（PIC）分别为4.377、0.7040和0.6680。甘肃麦积种群的Ne、He、PIC3个指标最高，分别为5.3967、0.7809和0.7455；重庆城口种群的最低，分别为3.1260、0.6138和0.5693。说明火红熊蜂表现出较高的杂合度和丰富的遗传多样性。甘肃麦积种群的遗传多样性最高，甘肃麦积山理应成为该种熊蜂资源保护的重要区域。火红熊蜂16个地理种群的总近交系数（Fit）为0.121（P<0.001），种群内近交系数（Fis）为0.060（P<0.001），种群间遗传分化系数（Fst）为0.064（P<0.001），显示火红熊蜂种群发生了中等程度的遗传分化，其变异来源于种群间和种群内个体间。结合种群聚类树、主成分分析和祖先假设模型聚类分析，火红熊蜂16个地理种群分为2个分支：大巴山的重庆城口种群单独为一分支，其他15个地理种群聚为另一分支，这一大分支又可分为吕梁山-太行山-燕山-蒙古高原分支和黄土高原-秦岭-陇南河谷山地-青藏高原东缘分支2个小分支。相比于邻接法（NJ）聚类分析，基于Nei遗传距离Da的非加权配对算术平均法（UPGMA）更能显示火红熊蜂的地理分布特征及其演化关系。瓶颈效应检测发现火红熊蜂16个地理种群均未出现基因频率偏离L-shape分布，但16个种群中都有不同程度的杂合子缺失或过剩现象，说明火红熊蜂在历史演化过程中发生过遗传瓶颈，但这种瓶颈效应可能不太严重，或者仅对局部种群产生影响。
本研究探明了甘肃熊蜂区系组成、物种多样性现状及其优势种类的遗传多样性特点。研究结果为进一步探究中国熊蜂多样性现状及其遗传背景奠定了基础，也为中国熊蜂资源保护与农业授粉利用提供了理论依据。
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 Gansu
A 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 Gansu
Bumblebees 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. pyrosoma
Bombus 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.
（1）甘肃熊蜂区系组成
2007～2011年，在甘肃180个分布点共采集熊蜂属昆虫标本7606头，隶属于10亚属53种，其中假熊蜂亚属Mendacibombus（Md.）2种，地熊蜂亚属Subterraneobombus（St.）3种，巨熊蜂亚属Megabombus（Mg.）8种，胸熊蜂亚属Thoracobombus（Th.）8种，拟熊蜂亚属Psithyrus（Ps.）9种，火熊蜂亚属Pyrobombus（Pr.）7种，真熊蜂亚属Bombus s. str.（Bo.）4种，阿熊蜂亚属Alpigenobombus（Ag.）4种，黑熊蜂亚属Melanobombus（Ml.）6种，西熊蜂亚属Sibiricobombus（Sb.）2种。在甘肃53种熊蜂中，其中13种熊蜂为甘肃新纪录种，分别为二色熊蜂B.(Mg.) bicoloratus、富丽熊蜂B.(Th.) opulentus、德熊蜂B.(Th.) deuteronymus、图拟熊蜂B.(Ps.) turneri、探索拟熊蜂B.(Ps.)expolitus、中华拟熊蜂B.(Ps.) chinensis、科尔拟熊蜂B.(Ps.) coreanus、挪威拟熊蜂B.(Ps.) norvegicus、小峰熊蜂B.(Bo.) hypocrita s.l.、短头熊蜂B.(Ag.) breviceps、灰熊蜂B.(Ag.) grahami、白背熊蜂B.(Ml.)festivus和亚西伯熊蜂B.(Sb.) asiaticus。本文首次系统报道了这53种熊蜂的区系分布和采访植物特性。甘肃熊蜂区系成份主要以东洋区种（45.3%）、东洋区和古北区共有种（52.8%）为主，介于川渝地区向华北地区过渡的中间阶段，与川渝地区熊蜂的种类相似度比较接近，与华北地区的种类相似度相距较远。
（2）甘肃熊蜂物种多样性分析
在甘肃10个生态区，除北部腾格里沙漠、西北部河西走廊戈壁地带和干燥北山3个生态区未发现有熊蜂分布外，其他7个生态区均有熊蜂分布。甘肃7个生态区的熊蜂物种多样性指数（H′）范围为0.3805~1.2238，由高到低的顺序依次为：西南部青藏高原牧场、西部乌鞘岭山地林区、南部白水江河谷山地林区、西北部祁连山牧场、东南部西秦岭山地林区、东部黄土高原灌丛和西北部阿尔金山-库姆塔格沙漠边缘灌丛。甘肃熊蜂物种均匀度指数（J_(sw)）西部乌鞘岭山地最高（0.8113），东南部西秦岭山地最低（0.6751）。西南部青藏高原和西部乌鞘岭山地的物种相似度最高（60.98%），南部亚热带湿润河谷山地林区和西北部干燥阿尔金山-库姆塔格沙漠边缘灌丛未见共同种。甘肃53种熊蜂中，火红熊蜂B.(Ml.) pyrosoma的物种相对多度最高（23.2%），占甘肃熊蜂总体数量的五分之一以上，另外，红光熊蜂B.(Bo.) ignitus（9.8%）、小峰熊蜂B.(Bo.) hypocrita s.l.（6.0%）、红束熊蜂B.(Ml.) rufofasciatus（5.0%）和重黄熊蜂B.(Pr.) picipes（4.8%）在甘肃分布也较丰富；相反，另外一些种类，如拟熊蜂亚属（Ps.）的所有种类，还有一些典型的南方种类如黄熊蜂B.(Pr.) flavescens、灰熊蜂B.(Ag.) grahami、二色熊蜂B.(Mg.) bicoloratus）和一些青藏高原种类如猛熊蜂B.(St.) difficillimus、圣熊蜂B.(Mg.) religiosus、越熊蜂B.(Mg.) supremus在甘肃境内非常稀有，其标本数量均在10头以下，其物种多度均在0.1%以内。甘肃熊蜂的垂直分布范围为海拔719~4011m，其中海拔1500~2000m范围内数量最多，海拔2500~3000m范围内种类最丰富；猛熊蜂B.(St.) difficillimus（3656m）、探索拟熊蜂B.(Ps.) expolitus（3524m）、石拟熊蜂B.(Ps.) rupestris（3454m）、拉达克熊蜂B.(Ml.) ladakhensis（3376m）和伪猛熊蜂B.(St.) personatus（3362m）位居平均海拔高度的前5位，短头熊蜂B.(Ag.) breviceps（944m）、二色熊蜂B.(Mg.) bicoloratus（1110m）、黄熊蜂B.(Pr.) flavescens（1122m）、朝鲜熊蜂B.(Mg.) koreanus（1164m）和富丽熊蜂B.(Th.)opulentus（1181m）位居平均海拔高度的末5位。
（3）火红熊蜂微卫星标记的筛选
火红熊蜂是甘肃最优势的熊蜂种类，也是中国特有的熊蜂资源，在中国北方地区分布十分丰富，是众多野生植物和农作物的重要传粉昆虫。为了探究火红熊蜂的分子遗传结构，首次进行了该种熊蜂微卫星标记的筛选。本研究采用生物素-磁珠吸附分离法从火红熊蜂基因组中筛选微卫星标记，并选取火红熊蜂同一亚属黑熊蜂亚属Melanobombus其他7种熊蜂进行了这些标记的种特异性验证。不同核苷酸探针与磁珠杂交产生的微卫星标记的阳性克隆率存在很大差异，TC探针的微卫星阳性克隆率最高为82%；TG探针次之，为28%；AT和GC探针分离未获得微卫星标记克隆。根据获得的阳性克隆的序列设计引物进行筛选验证，共获得31对熊蜂微卫星序列特异性引物。31对微卫星引物分为4种类型，其中完美型18个，占58.1%；混合完美型1个，占3.2%；非完美型10个，占32.3%；混合非完美型2个，占6.4%。不同核苷酸探针的微卫星核心序列重复数不同，TC探针和TG探针的微卫星核心序列最高重复数分别为28和15个，最低重复数分别为7和11个。31对微卫星引物在黑熊蜂亚属其他7种熊蜂上的检测结果表明，有26对引物可以扩增所测试的7种熊蜂，其余5对引物只能扩增部分熊蜂种类，其中BPM5是火红熊蜂种特异性引物。本研究从火红熊蜂基因组中筛选的微卫星标记，不仅可用于火红熊蜂遗传结构与分子进化等方面的研究，而且还可用于黑熊蜂亚属其他种类的遗传特性分析。
（4）火红熊蜂遗传多样性分析
为了探明火红熊蜂的遗传多样性特点，本研究基于12个微卫星标记首次分析了火红熊蜂主要分布区16个地理种群的遗传多样性水平。12个微卫星标记共获得等位基因数为178个，其中BPM9和BPM35等位基因数最高均为18个，BPM17等位基因数最低为5个。火红熊蜂16个地理种群的平均有效等位基因数（Ne）、平均遗传杂合度（He）和平均多态信息含量（PIC）分别为4.377、0.7040和0.6680。甘肃麦积种群的Ne、He、PIC3个指标最高，分别为5.3967、0.7809和0.7455；重庆城口种群的最低，分别为3.1260、0.6138和0.5693。说明火红熊蜂表现出较高的杂合度和丰富的遗传多样性。甘肃麦积种群的遗传多样性最高，甘肃麦积山理应成为该种熊蜂资源保护的重要区域。火红熊蜂16个地理种群的总近交系数（Fit）为0.121（P<0.001），种群内近交系数（Fis）为0.060（P<0.001），种群间遗传分化系数（Fst）为0.064（P<0.001），显示火红熊蜂种群发生了中等程度的遗传分化，其变异来源于种群间和种群内个体间。结合种群聚类树、主成分分析和祖先假设模型聚类分析，火红熊蜂16个地理种群分为2个分支：大巴山的重庆城口种群单独为一分支，其他15个地理种群聚为另一分支，这一大分支又可分为吕梁山-太行山-燕山-蒙古高原分支和黄土高原-秦岭-陇南河谷山地-青藏高原东缘分支2个小分支。相比于邻接法（NJ）聚类分析，基于Nei遗传距离Da的非加权配对算术平均法（UPGMA）更能显示火红熊蜂的地理分布特征及其演化关系。瓶颈效应检测发现火红熊蜂16个地理种群均未出现基因频率偏离L-shape分布，但16个种群中都有不同程度的杂合子缺失或过剩现象，说明火红熊蜂在历史演化过程中发生过遗传瓶颈，但这种瓶颈效应可能不太严重，或者仅对局部种群产生影响。
本研究探明了甘肃熊蜂区系组成、物种多样性现状及其优势种类的遗传多样性特点。研究结果为进一步探究中国熊蜂多样性现状及其遗传背景奠定了基础，也为中国熊蜂资源保护与农业授粉利用提供了理论依据。
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 Gansu
A 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 Gansu
Bumblebees 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. pyrosoma
Bombus 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.
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