mtDNA indicates profound population structure in Indian tiger (Panthera tigris tigris)

We analyzed mtDNA polymorphisms (parts of control region, ND5, ND2, Cytb, 12S, together 902 bp) in 59 scat and 18 tissue samples from 13 Indian populations of the critically endangered Indian tiger (Panthera tigris tigris), along with zoo animals as reference. Northern tiger populations exhibit two unique haplotypes suggesting genetic isolation. Western populations from Sariska (extinct in 2004) and Ranthambore are genetically similar, such that Ranthambore could serve as a source for reintroduction in Sariska. Zoo populations maintain mitochondrial lineages that are rare or absent in the wild.     

圈养东北虎血液生理参数的测定

东北虎(Panthera tigris altaica)是一种淑临灭绝的食肉动物,现已被列入IUCN濒危物种红色名录(Goodrich et al.,2015)。东北虎主要分布于俄罗斯远东地区、中国东北地区和朝鲜的北部(Kitchener et al.,2017)。根据中俄边境地区的野生东北虎数量调查统计,该地区生存着成年老虎39只和幼虎22只(Matiukhina et al., 2016)。     

西藏墨脱县孟加拉虎种群数量调查

孟加拉虎曾在西藏墨脱县各乡镇均有分布记录,但自2002年后该地区未见确认报道。2013—2018年间,我们利用红外相机技术、信息网络收集法和足迹鉴定法调查了墨脱县孟加拉虎的9个潜在分布区域。结果显示,墨脱县仅存1~3只孟加拉虎非定居个体,仅在旱季(每年10月至来年3月)游荡活动于背崩乡、墨脱镇雅鲁藏布江南岸区,以及格当乡金珠藏布南岸区。该区域内孟加拉虎的保护前景不容乐观,其原因是多方面的。本文提出如下建议以加强针对野生孟加拉虎的保护:(1)继续加强虎的基础性监测;(2)加强针对偷盗猎活动的野外巡护与执法管理;(3)加强对原始森林的保护;(4)改善原住民生计,加强宣传教育,以减少对自然资源的消耗;(5)恢复与建立景观廊道,改善栖息地连通性,以促进虎的自然扩散;(6)加强不同机构与参与方之间的合作研究和技术交流。     

Incorporation of mitochondrial fragments in the nuclear genome (Numts) of the longhorned beetle Monochamus galloprovincialis (Coleoptera, Cerambycidae)

A mitochondrial DNA (mtDNA) study, based on 43 European populations (33 of them sampled in France) of Monochamus galloprovincialis , vector of the pinewood nematode, and 14 populations of its sister species Monochamus sutor was realized. Sequencing of 792 bp of the cytochrome oxidase I (COI) and 521 bp of the COII genes revealed numerous ambiguities on multiple nucleotide sites for half of M. galloprovincialis specimens studied (44.8%). Hypotheses of heteroplasmy and pseudogenes ( Numts ) were examined. The mtDNA isolation by alkaline lysis and cloning (for three successfully used individuals) both support the hypothesis that the ambiguous sequences amplified were not of mtDNA nature and validate the presence of Numts in the nuclear genome of M. galloprovincialis . Multiple copies of mtDNA-like sequences were found paralogous to COI, tRNA leucin and COII regions. Phenetic analysis placed different recently diverged mtDNA-like sequences as a close relative of mtDNA sequences, and grouped 10 closely related mtDNA-like sequences as a more basal clade, closer to ancestral states and to M. sutor . This result supports that this nuclear family of pseudogenes arose independently of the other events and may represent mitochondrial haplotypes sampled from M. galloprovincialis ancestral populations. This is the first time that Numts are proved for a longhorned beetle, whereas no Numts were found within its sister species M. sutor. The incorporation mechanism of Numts in unknown for M. galloprovincialis , however, excess of ambiguous sites corresponding to synonymous mutations placed on third codon position as well as the absence of Numts in M. sutor , conducted to the hypothesis of a recent transfer of these Numts in the nuclear genome of M. galloprovincialis .     

Twenty-one novel tri- and tetranucleotide microsatellite loci for the Amur tiger (Panthera tigris altaica)

Amur tiger is the largest subspecies of tiger in the world and his conservation has also received much attention. In this study, we isolated and characterized twenty-one tri- and tetranucleotide microsatellite markers from this species. The number of alleles for each locus ranged from two to nine in a group of 60 individuals and the observed and expected heterozygosities were 0.333–0.917 and 0.302–0.822, respectively. The overall discrimination power and exclusion probabilities in parentage and paternity testing for these markers were 1.00, 0.9947 and 0.9999, respectively, indicating high-resolution power of microsatellite markers.     

Cell cycle analysis and interspecies nuclear transfer of in vitro cultured skin fibroblasts of the Siberian tiger (Panthera tigris Altaica)

The present study was conducted to examine the effect of cell culture conditions, antioxidants, protease inhibitors (PI), and different levels of dimethylsulfoxide (DMSO) for the promotion of synchronization of different cell cycles of Siberian tiger skin fibroblasts. We also compared the ability of somatic cell nuclei of the Siberian tiger in pig cytoplasts and to support early development after reconstruction. Cell cycle synchronization between nuclear donor and recipient cells is considered to be one of the most crucial factors for successful cloning. Five experiments were performed each with a one-way completely randomized design involving three replicates of all treatments. Least significant difference (LSD) was used to determine variation among treatment groups. Experiment I focused in the effects of cycling, serum starved and fully confluent stages of Siberian tiger cells on different cell cycles. In Experiment II, the effects of different antioxidants like beta-Mercaptoethanol (beta-ME, 10 microM), cysteine (2 mM), and glutathione (2 mM) were examined after cells were fully confluent without serum starvation for 4 hr. In Experiment III, three PI, namely 6-dimethylaminopurine (6-DMAP, 2 mM), cycloheximide (7.5 microg/ml) and cytochalasin B (7.5 microg/ml) were used in the sane manner as in Experiment II. In Experiment IV, different levels of DMSO at 0%, 0.5%, 1.0%, and 2.5% were tested on different cell cycle stages of Siberian tiger examined by Flowcytometry (FACS). In Experiment I, 67.2% of the Siberian tiger skin fibroblasts reached the G0/G1 stage (2C DNA content) in fully confluent conditions which was more than the cycling (49.8%) and serum starved (SS) medium (65.5%; P < 0.05). Among the chemically treated group, glutathione (72.6%) and cycloheximide (71.3%) had little bit better results for the synchronization of G0 + G1 phases than serum starved and fully confluent. After nuclear transfer we did not see any significant differences on the development of tiger-porcine reconstructed embryos at cycling, SS and fully confluent. Data indicate that prolonged culture of cells in the absence of serum as well as using different chemicals for this experiment does not imply a shift in the percentage of cells that enter G0/G1 and that confluency is sufficient to induce quiescence. This finding can be beneficial in nuclear transfer programs in Siberian tiger, because there are negative effects, such as apoptosis associated with serum starvation.     

Fourteen new di- and tetranucleotide microsatellite loci for the critically endangered Indian tiger (Panthera tigris tigris)

We describe 11 dinucleotide and three tetranucleotide microsatellite loci for the critically endangered Indian tiger, Panthera tigris tigris. All of them were polymorphic with four to nine alleles per locus and an observed heterozygosity between 0.13 and 1.0. All primers also amplify microsatellite loci in leopard, Panthera pardus, and 12 primer pairs yielded reproducible results in domestic cat, Felis catus. These new microsatellites specifically developed for Indian tiger - in combination with those already available - comprise a reasonable number of loci to genetically analyse wild and captive populations of this illustrative species and might allow for recognition of individual tigers.     

圈养华南虎的个性评估及其性别差异

个性研究在动物生态研究和迁地保护中发挥着重要作用.华南虎(Panthera tigris amoyensis)作为最濒危的虎亚种,目前仍然缺乏对其个性特征的研究探索.本文通过个性特征主观评价法对6只圈养华南虎进行个性评估,并对比不同性别的华南虎个性特征的差异,使用新异物体测验进一步验证个性特征主观评价法的结果.最终提取...     

Development and cross‐species amplification of 18 microsatellite markers in the Sumatran tiger (Panthera tigris sumatrae)

Eighteen polymorphic microsatellite loci from the highly endangered Sumatran tiger (Panthera tigris sumatrae) were isolated and characterized. Upon polymerase chain reaction amplification, 16 of these markers produced a single, sharp band in all three tiger and 10 non‐tiger felid species examined. Of the two remaining loci, 6HDZ057 and 6HDZ635 failed to amplify genomic DNA from puma (Felis concolor) and cheetah (Acinonyx jubatus), respectively. The amplification of these markers across four genera is an indication of their usefulness for population genetics studies and conservation work in a wide range of felid species.     

笼养和半散放东北虎行为时间分配及活动规律差异的比较

1998年 3月～ 2 0 0 1年 4月 ,在哈尔滨动物园和黑龙江东北虎林园采用全事件取样法、扫描取样法和目标取样法 ,对笼养和半散放东北虎的行为时间分配和活动规律进行了比较研究。结果表明 ,在时间分配方面 ,笼养东北虎的休息远远高于半散放东北虎 ,而摄食、站立和其它行为则明显低于半散放东北虎 ,笼养和半散放东北虎的运动较为接近。在活动规律方面 ,虽然笼养和半散放东北虎均有 2个运动的高峰期 ,但笼养东北虎的相对集中且变化幅度较大 ;半散放东北虎的休息有 1个明显的高峰期和 2个不明显的高峰期 ,与笼养东北虎的 1个高峰期有很大不同 ;笼养东北虎的摄食仅出现在 16∶0 0～ 17∶0 0 ,而半散放东北虎则在 1d中均有发生 ,且在 9∶0 0～ 11∶0 0和 14∶0 0～ 16∶0 0有 2个高峰期 ;笼养和半散放东北虎的站立和其它行为变化规律较为相似 ,但半散放东北虎的发生强度要远远高于笼养东北虎。通过对笼养和半散放东北虎各种行为进行的配对T检验 ,发现二者的休息、摄食、站立和其它行为存在极显著差异 (P<0 0 0 1)。     

基于MaxEnt模型的小兴安岭东北虎潜在生境适宜性评价

小兴安岭是东北虎的历史分布区之一,近年来东北虎数次重返小兴安岭,预示了小兴安岭东北虎种群恢复的可能性。为了探明小兴安岭作为东北虎栖息地的适宜程度,本文以我国小兴安岭及俄罗斯联邦阿穆尔州、犹太自治州为整体研究区域,利用该区域内东北虎出现点数据,采用Maxent模型,以植被、气候、地形、积雪4类环境数据为基础,分析自然环境条件下小兴安岭东北虎潜在生境的适宜性及空间分布。结果显示：小兴安岭东北虎潜在适宜生境面积为0.96×10⁴~1.03 ×10⁴ km²,主要位于小兴安岭北部和东部,中部、西部和东南部有少量分散适宜生境;次适宜生境面积为2.46×10⁴~1.76 ×10⁴ km²,主要位于适宜生境周边区域;叶灌层差异、蒸散量、归一化植被指数、叶面积指数等植被相关因素及降水季节性、最冷季降水量等气候因素是影响东北虎栖息地适宜程度的主要环境特征变量。小兴安岭仍具备东北虎种群生存的自然条件。     

Use of oligonucleotide fingerprinting and faecal DNA in identifying the distribution of the Chinese tiger (Panthera tigris amoyensis Hilzheimer)

Chinese tiger (Panthera tigris amoyensis Hilzheimer) was once distributed in the Zhejiang Province, China, but there have not been any tiger sightings in this province since 1985. Fortunately, within the boundaries of the Fengyangshan-baishanzu State Natural Reserve, gamekeepers have found tiger footprints and tiger faeces many times since 1998. However, these tracks may have been left by the leopard or clouded leopard, which leave similar tracks. The presence of the Chinese tiger in Zhejiang Province was demonstrated using DNA fingerprinting and faecal DNA. The study not only revealed a new habitat for Chinese tigers, Zhejiang Province, but also provided an effective scatology method to identify the distribution of tigers and other wild felines.     

华南虎、东北虎、孟加拉虎的D-100p和ND5序列及其在系统进化分析中的应用

本文采用PCR和质粒克隆测序方法,获得了华南虎线粒体D-loop区的480bp序列和东北虎、孟加拉虎线粒体D-loop区的503bp序列;同时还获得了这三个虎亚种和金钱豹线粒体ND5基因5’端309bp的部分序列。根据D-loop序列分析,华南虎与孟加拉虎、东北虎的平均距离(p-distance)分别为0．11088和O．11087,而东北虎与孟加拉虎间的平均距离为0．00994;根据ND5序列分析,华南虎与孟加拉虎、东北虎的平均距离分别为0．11434和0．11758,而东北虎与孟加拉虎间的平均距离为0．00324。三个虎亚种的mtDNA D-loop和ND5序列比较表明,华南虎是这三个虎亚种中最为古老的亚种。     

东北虎雌性生殖系统的组织学

对10月份意外死亡的2例东北虎（Panthera tigris altaica)雌性生殖系统进行了组织学研究。卵巢基质中分布着一定数量的有腔卵泡及闭锁卵泡。间质腺不发达,在闭锁卵泡上皮样细胞增殖,形成一定量的间质腺细胞。输卵管漏半斗部至峡部黏膜上皮由柱状逐渐转化炎立方,黏膜形成许多皱褶,皱褶以漏斗部和壶腹部最多,子宫内膜上皮为单层立方上皮。6岁个体子宫腺上皮为单层柱状上皮;2岁个体在子宫角处的子宫腺上皮为单层柱状上皮,在子宫体外多为单层立方上皮,子宫腺密度从卵巢端到子宫体端呈阶梯下降,阴道黏膜上皮大多为2－3层的复层扁平上皮。     

笼养东北虎雌雄行为差异的比较

1998年4月－2001年3月在哈尔滨动物园,采用瞬时扫描取样法和全事件取样法对5只笼养东北虎（3雌2雄）的昼夜行为时间分配及活动规律进行了研究。结果表明：东北虎的睡眠、卧息、运动和其他行为在时间分配上两性存在一定差异,即雌性的睡眠和卧息时间比雄性多,雌性的运动和其他行为时间比雄性少;但摄食行为的时间分配相差不大。两性日活动规律的差异为雄性的睡眠高峰主要在夜晚,卧息较雌性少且相对集中,运动出现和结束的时间较雌性延迟约1h。对两性及不同个体行为时间分配的单因素方差分析发现,不同个体间仅运动存在显著差异（P＜0.05);两性间睡眠、卧息、运动和其他行为均存在显著差异（P＜0.05)。笼养东北虎两性行为的差异可能与其野外的行为习惯有关,而这些行为习惯又与其担当的性别角色有紧密联系。     

笼养东北虎行为的时间分配

1998 年4 月至2001 年3 月, 采用瞬时扫描取样法和全事件取样法对哈尔滨动物园的4 只笼养东北虎的活动规律进行了研究。结果表明, 笼养东北虎在全年的昼夜行为时间分配表现为睡眠所占比值最多; 其次是卧息和活动; 摄食和其它行为(包括饮水、排尿、排粪、修饰、嗅闻、嬉戏、站立、直立、发声等) 最少。在不同季节笼养东北虎的活动变化规律基本相似, 一昼夜的睡眠、运动和卧息均有两个高峰期, 但高峰期出现和持续的时间有所差异。睡眠的高峰期在10 : 00～14 : 00 和20 : 00 至次日06 : 00 , 活动的高峰期在05 : 00～10 : 00 和14 : 00～17 : 00 , 卧息的高峰期在05 : 00～10 : 00 和13 : 00～20 : 00 , 摄食仅在16 : 00～20 : 00 有一个高峰期。冬季(12～2 月) 与春(3～5 月) 、夏(6～8 月) 、秋季(9～11 月) 相比, 东北虎在白昼睡眠的时间显著减少,夜晚则显著增多。     

Evolution of mitochondrial and ribosomal gene sequences in anophelinae (Diptera: Culicidae): implications for phylogeny reconstruction

In this study, two mitochondrial genes, cyt b and ND5, and the D2 expansion segment of the 28S nuclear ribosomal gene were used to reconstruct a phylogeny of the mosquito subfamily Anophelinae. The ingroup consisted of all three genera of Anophelinae and five of six subgenera of Anopheles. Six genera of Culicinae were used as the outgroup. Extreme conservation at the protein level coupled with rapid saturation of synonymous positions probably accounted for the lack of meaningful phylogenetic signal in the cyt b gene. In contrast, abundant variation at all codon positions of the ND5 gene allowed recovery of the basal and most of the recent relationships. Phylogenetic analysis of D2 produced results consistent with those of ND5. Combined analysis indicated well-supported monophyletic Anophelinae (with Chagasia basal), Anopheles + Bironella, and subgeneric clades within the genus Anopheles. Moreover, subgenera Nyssorhynchus and Kerteszia were supported as a monophyletic lineage. The Kishino-Hasegawa test could not reject the monophyly of Anopheles, whereas the recently proposed hypothesis of close affinity of Bironella to the subgenus Anopheles was rejected by the analyses of ND5 and combined data sets. The lack of resolution of Bironella and Anopheles clades, or basal relationships among subgeneric clades within Anopheles, suggests their rapid diversification. Recovery of relationships consistent with morphology and previous molecular studies provides evidence of substantial phylogenetic signal in D2 and ND5 genes at levels of divergence from closely related species to subfamily in mosquitoes.     

半散养东北虎繁殖期PAE编码行为谱的构建

2006—2007年,采用全事件记录法、目标动物取样法和行为取样法对泰山东北虎园半散养东北虎繁殖期的各种行为进行了观察。基于以"姿势-动作-环境"(posture-act-environment,PAE)为轴心,以行为生态功能为依据的PAE编码系统,对半散养东北虎的行为进行分类和系统编码并构建PAE行为谱。研究结果区分和定义了14种姿势、47种动作和98种行为型;并将所区分、命名和编码的所有行为型归类于摄食、排遗、调温、发情、交配、分娩、冲突、育幼、通讯、聚群、休息、运动和杂类等13类;由此构建了基于PAE编码系统的半散养东北虎首个繁殖期行为谱。