基于线粒体Cyt b 和核基因ZFY 探讨羊族物种之间的系统发生关系

为了阐明羊族物种之间的系统发生关系并解决岩羊属中矮岩羊物种的有效性问题,本文测定了来自金沙江河谷地区栖息于林线以上岩羊和林线以下矮岩羊共226 份粪便DNA 样品的线粒体Cyt b 基因全序列(1 140 bp)和核基因ZFY 部分序列(612 bp),结合从GenBank 中检索到的羊族物种同源DNA 序列进行比较,利用最大简约法和最大似然法构建分子系统发育树,根据获得的拓扑结构初步探讨它们的系统进化关系。结果表明,绵羊属的绵羊与山羊属、塔尔羊属、岩羊属各物种亲缘关系最远,喜马拉雅塔尔羊和岩羊属、山羊属的亲缘关系最近。在进化树的岩羊属这一分支中,金沙江河谷地带岩羊和矮岩羊与内蒙古、青海、四川其它地理种群的岩羊聚为一支,同时分布在这一区域的部分岩羊和矮岩羊在Cyt b 基因和ZFY 基因单倍型上存在共享现象。历史上的气候事件可能造成金沙江河谷地带岩羊和矮岩羊种群之间相互迁移,偏雄性扩散促进了各地理种群之间的基因交流。因此不支持矮岩羊为独立的物种,建议将金沙江河谷地带的岩羊和矮岩羊都划分到岩羊四川亚种(Pseudois nayaur szechuanensis)。由于它们的形态和生态上存在一定的分化,建议将林线以下矮岩羊作为一个独立的管理单元进行保护与管理。     

从细胞色素b基因全序列差异分析岩羊和矮岩羊的系统进化关系

矮岩羊的分类地位从它被发现后就一直存在着争议。本文测定了分别来自四川和青海的岩羊(Pseudois nayaur)和矮岩羊(Pseudois schaeferi)共7个个体的Cyt b基因全序列(1140bp)，结合山羊(Capra hircus)、北山羊(Capra ibex)、绵羊(Ovis aries)和盘羊(Ovis ammon)同源DNA序列进行比较，分析了碱基组成和变异情况以及核苷酸序列差异。用简约法和距离距阵法构建了分子系统树，得到了相同的拓扑结构。结果显示：岩羊和矮岩羊互为单系群，有着较山羊、北山羊、绵羊和盘羊间更近的亲缘关系，它们之间的序列差异(Cyt b＝3．2％)低于牛科不同种间的序列差异水平(8．3％-12．1％)，明显高于种内个体的序列差异，而处于牛科亚种的序列差异范围内。因此，推测矮岩羊与岩羊之间的差异可能已经达到了亚种的水平。它们的分歧时间为l-3百万年[动物学报49(2)：198—204，2003]。     

雀形目15种鸟类Co Ⅰ与Cyt b基因序列的比较

对雀形目Passeriformes 6科15种鸟类线粒体DNA的Cyt b基因全序列(1 143 bp)和CoⅠ基因部分序列(1 176 bp)进行了比较,结果显示Cyt b和CoⅠ基因序列的变异位点分别为454个和366个,简约信息位点为337个和303个,而且CoⅠ基因比Cyt b基因略微保守,进化速率也较低.采用邻接法、最大简约法、最大似然法和贝叶斯法分别构建了CoⅠ和Cyt b基因两组数据集的分子系统发生树及其合一树,并对建树结果进行了比较分析.基于以上两点,本文认为CoⅠ基因比Cyt b基因更适合于确定雀形目科级阶元之间的系统发生关系,而且它也能够作为雀形目物种鉴定的分子标记,但在物种鉴定方面不如Cyt b基因稳定和准确.     

栗背短脚鹎的种群分化与遗传多样性

栗背短脚鹎(Hemixos castanonotus)是我国南方山区常见的杂食性鸟类。为探明其遗传多样性及分化现状,采用线粒体Cyt b基因和7个核基因非编码区片段作为分子标记,对分布于广东、广西、海南、贵州和江西五省(自治区)的栗背短脚鹎11个地理种群进行了遗传分化及遗传多样性研究。基于所获得的Cyt b基因866 bp和7个核基因内含子序列6 808 bp进行分析。结果显示,在Cyt b基因中,共检测到37个单倍型,共享单倍型占单倍型总数的35.6%,推测这些共享单倍型可能属于祖先单倍型。分子方差分析结果显示,遗传变异主要来源于种群内部(79.77%)。Tajima’s D和Fu’s Fs中性检验分析结果均支持栗背短脚鹎种群可能曾经历过种群扩张现象。基于7个核基因内含子联合序列的贝叶斯天际线(BSP)分析,推断其种群在大约5.3 ~ 3.7百万年前(Mya)和约0.7 ~ 0.3百万年前(Mya)发生过扩张。基于Cyt b基因的贝叶斯系统发育分析,11个地理种群共分为两支,一支为海南猴猕岭地理种群,属指名亚种(H. c. castanonotus),其他10个地理种群聚为另一支,属H. c. canipennis亚种,并且后者尚未形成显著的地理结构,单倍型网络图分析也获得相似的结果。本研究所用分子数据基本支持两个亚种的分化,对于存在争议的广西南部分布的指名亚种,其分子数据与形态学亚种归属不一致,有待更深入研究。     

基于DNA条形码和微卫星标记分析矮岩羊的进化地位

岩羊Pseudois nayaur是目前岩羊属Pseudois中承认的唯一物种,主要分布在青藏高原及其周边山脉地区,是该地区代表性的大型有蹄类动物。岩羊种内存在矮岩羊这一个形态特异的种群,其分类地位一直处在争议当中。本次研究从岩羊分布区域内搜集到26个岩羊样品,其中包括5个矮岩羊样品,选取了COⅠ基因DNA条形码标准区域和8个微卫星标记位点探讨了矮岩羊与岩羊不同地理种群的系统进化关系,同时分析了矮岩羊的进化地位。结果表明矮岩羊和岩羊间目前处于基因交流半隔离状态,不应该被分为2个不同的物种或者亚种,而矮岩羊应该被作为岩羊四川亚种P.n.szechuanensis中一个形态变异的特殊分类群。另外,结合生境选择和生态观察的结果推测矮岩羊目前可能已经处于单独进化的早期阶段。     

基于细胞色素b基因的中国岩羊不同地理种群遗传差异分析

为了揭示中国岩羊不同地理种群的遗传差异,探讨岩羊亚种分化的分子机制,采用中国岩羊不同地理种群的细胞色素b(Cyt b)基因的全序列,分析了碱基变异情况、遗传距离以及核苷酸序列差异。用最大似然法和贝叶斯法构建分子系统树并对获得的拓扑结构进行分析。结果发现,西藏亚种与四川亚种Cyt b基因平均序列差异为4.2%(±0.007),处于偶蹄目亚种的序列差异范围内,支持了目前对岩羊西藏亚种的分类地位。四川亚种内部各地理种群之间的遗传距离(0.033±0.0 111)与它们分别到西藏种群的遗传距离(0.042±0.007)差异不显著(t=1.824,P=0.084),说明四川亚种内部各地理种群间已经发生较显著的遗传分化。其中,四川、甘肃和青海种群亲缘关系较近,并与四川亚种内部的其它种群已产生了显著的遗传分化。因此认为四川亚种内部各地理种群的种下分化需要深入研究。     

金沙江河谷地区岩羊种群遗传多样性分析

岩羊为国家Ⅱ级保护动物。目前,由于人类活动范围日益扩大,其栖息地遭到了不同程度的破坏,岩羊种群数量和分布范围呈明显下降趋势。为了及时制定科学的保护策略必须了解它们的种群遗传结构。本文以粪便为研究材料,对来自金沙江河谷地区林线以上岩羊和林线以下矮个子岩羊共4个地理种群的169份有效样品进行线粒体CR全序列分析,共检测出210个变异位点,定义了4种单倍型,单倍型多样性为0.68,核苷酸多样性为0.0242,显示种群整体遗传多样性水平较低。基于最大简约法构建的系统发育树中,金沙江河谷地区的4个地理种群被划分到四川种群的2个亚分支中,但云南曲宗贡的部分岩羊和四川竹巴笼的矮个子岩羊单倍型存在共享现象。根据分子钟计算,林线上下岩羊分化时间在39~32万年前,在中更新世早中期(105~36万年前)气候影响下,导致岩羊在金沙江河谷高低海拔之间相互迁移。对于岩羊在历史上所表现出的潜在迁徙能力,我们建议将白马雪山自然保护区到竹巴笼自然保护区之间的金沙江河谷地区作为岩羊和矮个子岩羊的栖息地整体保护。     

从细胞色素b基因全序列探讨大额牛的分子系统发生

大额牛是一种半野生半家养的珍稀牛种, 有关其起源和系统地位一直存在争议。通过PCR扩增、测序等步骤共获得了11头大额牛细胞色素b(Cyt b)基因全序列(1 140 bp)。应用分析软件, 对大额牛11条Cyt b序列进行了分析, 并结合GenBank中牛属动物6个近缘种的同源序列, 以亚洲水牛(Bubalus bubalis)为外群, 分别采用邻接法(NJ)和最大简约法(MP)构建了牛属动物分子系统发育树。序列分析结果表明, 11条大额牛Cyt b序列1 140位点中, 共发现95个变异位点(占分析位点总数的8.33 %), 定义了6种单倍型, 表明大额牛群体的Cyt b基因遗传多态性比较丰富。构建的NJ和MP分子系统树均显示, 大额牛研究群体明显分为3支, 第1支与普通牛(Bos taurus)相聚, 第2支与瘤牛(Bos indicus)相聚, 第3支与印度野牛(Bos gaurus)相聚。系统发育分析表明, 大额牛很可能是印度野牛的家养型或驯化种, 我国大额牛群体可能曾受到其他牛种血缘的入侵。     

从线粒体Cyt b基因探讨红瘰疣螈物种地位的有效性

疣螈属的红瘰疣螈(Tylototriton shanjing)和棕黑疣螈(T.verrucosus)的物种界限一直不清楚。测定了来自中国西南地区14个地点的T.shanjing和T.verrucosus共40只标本的线粒体DNACytb基因(753bp)。结果表明:(1)用邻接法、最大简约法和贝叶斯法等3种系统发育分析法分别重建棕黑疣螈种组系统发育树的拓扑结构不支持T.shanjing是单系群;(2)T.shanjing与T.verrucsus的mtDNA Cytb序列差异平均值仅为1.2%,未达到种级水平。因此,全部T.shanjing样品都属于同一个物种,即T.verrucosus,不支持T.shanjing的物种地位,T.shanjing为T.verrucosus的同物异名,并建议恢复T.verrucosus的中文名红瘰疣螈。根据基于40个样品Cyt b基因序列的系统发育树和遗传变异以及地理分布,这些红瘰疣螈(T.verrucosus)样品聚为3支,即中国西南地区的红瘰疣螈可分为片马、滇中滇西和滇东南3个地理居群。     

岩羊不同种群细胞色素b基因序列差异的初步分析

测定了来自四川和宁夏两个种群的岩羊Pseudois nayaur共10个个体的线粒体DNA细胞色素b基因全序列(1140 bp),分析了碱基组成和变异情况以及核苷酸序列差异,计算了岩羊种群的核苷酸多样性,采用简约法和距离矩阵法构建了不同的分子系统树,得到了相似的拓扑结构,从分子水平初步探讨了岩羊不同种群的遗传结构.结果显示:岩羊整个群体的遗传多态性较高,四川种群的遗传变异水平较宁夏种群的高;同时,四川种群岩羊个体之间的平均序列差异为2.15,宁夏种群岩羊个体之间的平均序列差异为0.71,四川种群和宁夏种群之间的序列差异为2.61,这反映了四川种群和宁夏种群种群内的差异以及四川种群和宁夏种群种群间的差异都属于个体间的变化;然而四川种群的个体11(Si11)与该种群内的其它个体的平均序列差异和平均Kimura两参数距离远高于它与宁夏种群个体间的相应数值,存在着较为明显的分歧.形成这一现象的原因可能要涉及到岩羊在中国的地理演化和扩散问题.     

Molecular evidence for the subspecific differentiation of blue sheep (Pseudois nayaur) and polyphyletic origin of dwarf blue sheep (Pseudois schaeferi)

Blue sheep (Pseudois nayaur), a Central Asian ungulate with restricted geographic distribution, exhibits unclear variation in morphology and phylogeographic structure. The composition of species and subspecies in the genus Pseudois is controversial, particularly with respect to the taxonomic designation of geographically restricted populations. Here, 26 specimens including 5 dwarf blue sheep (Pseudois schaeferi), which were collected from a broad geographic region in China, were analyzed for 2 mitochondrial DNA fragments (cytochrome b and control region sequences). In a pattern consistent with geographically defined subspecies, we found three deeply divergent mitochondrial lineages restricted to different geographic regions. The currently designated two subspecies of blue sheep, Pseudois nayaur nayaur and Pseudois nayaur szechuanensis, were recognized in the phylogenetic trees. In addition, the Helan Mountain population showed distinct genetic characteristics from other geographic populations, and thus should be classified as a new subspecies. In contrast, dwarf blue sheep clustered closely with some blue sheep from Sichuan Province in the phylogenetic trees. Therefore, dwarf blue sheep appear to be a subset of Pseudois nayaur szechuanensis. After considering both population genetic information and molecular clock analysis, we obtained some relevant molecular phylogeographic information concerning the historical biogeography of blue sheep. These results also indicate that western Sichuan was a potential refugium for blue sheep during the Quaternary period.     

Molecular phylogeography and genetic differentiation of blue sheep Pseudois nayaurszechuanensis and Pseudois schaeferi in China

Within the genus Pseudois (Bovidae, Artiodactyla), two challenges are faced by taxonomists. First, the designation of the two subspecies of blue sheep, Pseudois nayaur nayaur and Pseudois nayaurszechuanensis, tends to be questionable with the accumulation of morphological and molecular data. Second, the taxonomic status of the dwarf blue sheep (Pseudois schaeferi) has been controversial since its discovery, and molecular analyses with relatively small sample sizes have not painted a clear picture. We collected samples and DNA sequence data from a broad geographic range in China to investigate the population structure of Pseudois nayaurszechuanensis and its phylogenetic relationship with Pseudois schaeferi. Employing mitochondrial cytochrome b and control region sequences, as well as the nuclear gene zona pellucida glycoprotein 3, further investigation was performed to extend upon previous studies. We found that the population of Helan Mountain of Ningxia Hui Autonomous Region has significantly differentiated from the populations in Qinghai, Gansu and Sichuan provinces, and may be classified as a new subspecies according to the 75% rule. The dwarf blue sheep was found to be some individuals of Pseudois nayaurszechuanensis with morphological variations and is not a species or subspecies, since they did not fall into a monophyletic group in the phylogenetic analyses with blue sheep. Gene flow between dwarf blue sheep and common blue sheep might have occurred in different areas of western Sichuan, which would result in the paraphyly of dwarf blue sheep.     

羚牛的遗传多样性及其种群遗传结构分析

羚牛是亚洲大陆一种特有的大型珍稀动物,目前正面临着栖息地丧失、片段化和人类活动的严重威胁。为了有效地保护这种濒危动物,全面了解羚牛的种群结构、进化历史和整个分布区内遗传多样性的分布是至关重要的。本研究以mtDNA D-loop330bp基因片段为分子标记,比较分析了来自陕西秦岭、甘肃南部、四川岷山、邛崃山和云南贡山的40个样品的序列差异,根据分布特点将所采集到的羚牛分为3个地理单元,即秦岭、四川和云南。结果表明,在3个地理单元中存在4种单倍型,且地理单元间不存在共享单倍型,相互单倍型之间的平均序列差异为1.66％。进一步分析表明,3个地理单元间的基因流较低,存在着显的遗传分化 ,说明羚牛具有明显的系统地理分布格局。同时提出应将分布于秦岭山区、唐家河青川地区、天全以及云南贡山地区作为独立的管理单元分别加以保护。     

Mitochondrial phylogeography and genetic diversity of Tibetan gazelle (Procapra picticaudata): implications for conservation

The Tibetan gazelle (Procapra picticaudata) is a threatened species and distributed on the Qinghai-Tibet Plateau of China (Qinghai Province, Tibet Autonomous Region and the adjacent Gansu Province, Sichuan Province, and Xinjiang Uigur Autonomous Region). Small peripheral populations of Tibetan gazelle were once found in northern Sikkim and Ladakh, but now these are close to extinction. To describe the evolutionary history and to assess the genetic diversity within this monotypic species and population structure among different geographic locations in China, we sequenced mitochondrial DNA from the control region (CR) and cytochrome (cyt) b gene for 46 individuals from 12 geographic localities in Qinghai, Tibet, Xinjiang, Gansu, and Sichuan. A total of 25 CR haplotypes and 16 cyt b haplotypes were identified from these gazelle samples. CR haplotype diversity (0.98+/-0.01) and nucleotide diversity (0.08+/-0.009) were both high. Phylogenetic trees indicate that the Tibetan gazelle in China can be divided into three main clades: Tibet, Sichuan (SCH) and Qinghai-Arjin Shan-Kekexili (QH-ARJ-KKXL). Analysis of molecular variance (AMOVA) and network analysis consistently support this geographic structure in both datasets. Significant differentiation between populations argues for the presence of management units (MUs). Such differentiation may reflect a geographic separation resulting from the uplift of the Qinghai-Tibet Plateau during the Late Pliocene and Pleistocene. Mismatch distribution analysis implies that Tibetan gazelle has undergone complex population changes. We suggest that the present population structure has resulted from habitat fragmentation during the recent glacial period on the Qinghai-Tibet Plateau and population expansion from glacial refugia after the glacial period. It is likely that the present populations of Tibetan gazelle exhibit a pattern reminiscent of several bottlenecks and expansions in the recent past.     

从细胞色素b 基因全序列分析岩羊和山羊、绵羊的系统发生关系

本文测定了来自四川和青海岩羊的细胞色素b 基因全序列(1 140 bp) , 结合从GenBank 中检索获得的山羊、北山羊、绵羊和盘羊4 个近缘种细胞色素b 核苷酸同源区序列进行比较, 分析了碱基组成和变异情况以及核苷酸序列差异。分别采用简约法和距离矩阵法构建分子系统树, 得到了基本相同的拓扑结构, 从分子水平初步探讨了岩羊的系统起源问题。岩羊与山羊属的山羊、北山羊有着比绵羊属的绵羊、盘羊更近的亲缘关系。岩羊与山羊、北山羊的分歧时间大约在3～6 百万年, 而与盘羊、绵羊的分歧时间大约在6～8 百万年。     

Phylogenetic structure of Zacco platypus (Teleostei, Cyprinidae) populations on the upper and middle Chang Jiang (=Yangtze) drainage inferred from cytochrome b sequences

We examined the genetic structure and phylogenetic relationships of some Chinese populations from the Chang Jiang (=Yangtze) drainage of the cyprinid Zacco platypus. We sequenced the complete mitochondrial cytochrome b gene of 64 individuals from 6 upper and middle tributaries of the Sichuan and Hunan Provinces to assess their population structure and systematics. The combined analyses of the phylogenetic information and the population structure suggested that Chinese Z. platypus consist of four distinct mtDNA lineages which exhibit high genetic variation and haplotypic diversity (Zacco A-D). The high molecular divergence observed among Zacco A-D mtDNA lineages (TrN+I (0.76) distance, mean 8.9%+/-1.7%) and their phylogeographic structure indicate that all four lineages have evolved independently. Analysis of molecular variance (AMOVA) indicates that most of the genetic variation observed is found among the four Zacco mtDNA lineages (thetaCT = 0.94) suggesting restricted gene flow among the Chang Jiang populations. Long-term interruption of gene flow was also evidenced by thetaST values higher than 0.9 that could be favoured by the discontinuous distributions of the lineages inhabiting upper (Sichuan Province) and middle (Hunan Province) Chang Jiang tributaries. The significant correlation between the geographic and genetic distances provide support for the importance of geographic discontinuity in shaping the Zacco genetic structure. Nested clade analysis (NCA) results were congruent with phylogenetic relationships recovered and confirm the genetic distinctiveness of four independent Zacco groups. These four groups correspond to the four Zacco A-D mtDNA lineages recovered in the phylogeny and were defined by nucleotide synapomorphies permitting bootstrapped and Bayesian confidence of 95% or greater. The high level of mitochondrial sequence divergence separating all Zacco mtDNA lineages suggested that the Z. platypus populations from the Chang Jiang drainage probably correspond to four different species.     

Molecular phylogenetics of Triculine snails (Gastropoda: Pomatiopsidae) from southern China

Triculine (Gastropoda: Rissooidea: Pomatiopsidae) snails are involved in the transmission of schistosomiasis and paragonimiasis; their distributions are mainly across southeastern Asia and southern China. In the present investigation, partial sequences of COI, 16S, and 28S were examined to infer the phylogenetic relationships among the species rich and poorly understood gastropod. Samples were collected from 12 geographic locations in six provinces of southern China. Several methods such as maximum parsimony, maximum likelihood and distance analysis were used in phylogenetic analyses among these taxa. The resultant phylogenetic trees showed a similar topology irrespective of the phylogenetic methods used. The taxa fell into two clades, with those from Fujian, Guangxi, and Zhejiang Provinces in one clade and those from Hunan, Sichuan and Hubei in the other. Among the taxa in Hubei Province, five formed a monophyletic clade, but Tricula sp. H-SHY fell into a sister clade of Tricula hortensis of Sichuan, whilst Tricula hongshanensis formed a single clade. Sister taxa Tricula pingi and Tricula hsiangi formed well-supported clade within almost all the trees. These results, while preliminary, represent the first attempt to reconstruct a phylogeny for Triculinae across China.