安徽麝线粒体DNA细胞色素b基因全长序列分析

自80年代安徽麝[Moschus (moschiferus/berezovskii)anhuiensis]被发现以来,其分类地位一直众说纷纭。在本研究中,我们对安徽麝模式皮张标本进行了线粒体DNA细胞色素b基因全长序列分析。研究结果表明,安徽麝同麝属中其他种的遗传分化已经相当明显.分子系统学的分析表明,安徽麝是一个单系群,它同麝属其他种的DNA序列差异已达到种间分化的程度。因此,线粒体DNA序列的证据支持将安徽麝列为麝属中一有效种（Moschus anhuiensis),而不是前人认为的原麝或林麝的亚种。     

Ecological correlates of Himalayan musk deer Moschus leucogaster

Himalayan musk deer (Moschus leucogaster; hereafter musk deer) are endangered as a result of poaching and habitat loss. The species is nocturnal, crepuscular, and elusive, making direct observation of habitat use and behavior difficult. However, musk deer establish and repeatedly use the same latrines for defecation. To quantify musk deer habitat correlates, we used observational spatial data based on presence–absence of musk deer latrines, as well as a range of fine spatial‐scale ecological covariates. To determine presence–absence of musk deer, we exhaustively searched randomly selected forest trails using a 20‐m belt transect in different study sites within the Neshyang Valley in the Annapurna Conservation Area. In a subsequent way, study sites were classified as habitat or nonhabitat for musk deer. A total of 252 plots, 20 × 20 m, were systematically established every 100 m along 51 transects (each ~0.5 km long) laid out at different elevations to record a range of ecological habitat variables. We used mixed‐effect models and principal component analysis to characterize relationships between deer presence–absence data and habitat variables. We confirmed musk deer use latrines in forests located at higher elevations (3,200–4,200 m) throughout multiple seasons and years. Himalayan birch (Betula utilis) dominated forest, mixed Himalayan fir (Abies spectabilis), and birch forest were preferred over pure Himalayan fir and blue pine (Pinus wallichiana) forest. Greater crown cover and shrub diversity were associated with the presence of musk deer whereas tree height, diameter, and diversity were weakly correlated. Topographical attributes including aspect, elevation, distance to water source, and slope were also discriminated by musk deer. Over‐ and understory forest management can be used to protect forests likely to have musk deer as predicted by the models to ensure long‐term conservation of this rare deer.     

Implication of musk deer (Moschus spp.) depletion from hunter reports and dung transect data in northwest Yunnan,China

Musk deer (Moschus spp.) have been over-exploited leading to steep population declines. The demand for musk for its use in medicine and perfumery industries poses the biggest threat to musk deer. The present study aimed to assess the depletion level of musk deer over time in northwest Yunnan by using hunter reports and dung transect data. Hunters were asked to recall musk gland catch-effort changes between the early 1990s and over two years (January 2009 to December 2010). Surveys using dung transect methodology were conducted on musk deer population in poaching and non-poaching sites. The results obtained revealed that the hunting frequency is constant in the two periods. Annual musk gland catch per hunter significantly declined from 7.4 (SD = 1.8) in the early 1990s to 0.7 (SD = 0.9) over the 2009–2010 period. A significantly higher proportion of hunters used snares over the 2009–2010 period (74%) than in the early 1990s (18%). The majority of the informants (70%) reported that the average distance from the village at which hunters operated has increased over the later two year period. The mean dung encounter rate values in the non-poaching site were about six times higher than that in poaching sites. Hunter responses were consistent with dung transect data that suggest musk deer population is seriously compromised throughout its range in northwest Yunnan. The present study has shown that catch-effort data from hunter reports can provide pertinent information about the current hunting practices and problems of prey depletion and for design of conservation interventions for hunted species.     

Phylogeography and population expansion of Western Himalayan highly-erratically migratory painted lady butterfly (Lepidoptera: Nymphalidae: Vanessa cardui)

Understanding population genetics, genetic diversity and the migration pattern of ecologically important species is crucial for effective conservation plans. We studied mitochondrial cytochrome c oxidase subunit-I (COI) gene variation in painted lady butterfly (Vanessa cardui) from different altitudinal gradients of Uttarakhand (Western Himalayas), India. We sequenced COI) gene of 91 samples and 22 COI sequences retrieved from GenBank as representatives of different localities for comparative analysis. We observed a total of 16 haplotypes, of which seven haplotypes were observed in Uttarakhand with intra-species sequence divergence ranges from 0.001 to 0.009. Haplotype (Hd) and nucleotide diversity (π) were comparatively low and averaging 0.635 and 0.002, respectively. The neutrality test, mismatch distribution graph and observed pattern of diversity indices (Hd and π) revealed population expansion. It was further confirmed by the Median Joining Network, which exhibited a star-like topology from the core haplotype, while the Bayesian phylogenetic tree displayed a mixing of haplotypes from different localities to an identical clade. Haplotype organization in the Bayesian tree and Median Joining Network was not clustered as per their topographical affinity, indicating incomplete lineage sorting and late species population development or colonization in the novel area.     

Six microsatellite loci in forest musk deer,Moschus berezovskii

Through an improved enrichment protocol, 20 microsatellite loci were isolated and characterized in an endangered animal, forest musk deer (Moschus berezovskii). In which, six loci showed high polymorphism with five to 13 alleles in 48 sampled individuals. The observed and expected heterozygosities were 0.362–0.955 and 0.784–0.906, respectively. The average polymorphism information content (PIC) was 0.8108 in these six microsatellites and they could be the valuable genetic markers for further investigation of forest musk deer species.     

林麝及马麝SRY基因片段克隆及其在系统进化分析中的应用

根据已报道的偶蹄目动物并参照人和狗SRY基因碱基序列设计一对简并引物,以林麝(Moschus berezovskii)及马麝(M．chrysogaster)基因组DNA为模板,通过PCR扩增出SRY基因CDS区。克隆的林麝及马麝SRY基因片段经测序,其长度为684bp。从父亲遗传角度出发,利用GenBank中收录的18种偶蹄目SRY基因,用邻位相接法(NJ)和最大简约法(MP)分别构建了鹿科、牛科和麝科的系统进化树。聚类树显示麝形成一个单系,支持麝作为独立一科的观点。     

圈养林麝春季活动节律及时间分配

2006 年春季,使用红外夜间观察记录仪,对崇明东平麝场12 只成年林麝(8 雌,4 雄)的行为进行了24 h昼夜连续观察。发现春季林麝的行为有37 种,可归并为休息、摄食、运动、社会、维持和排遗6 大行为类型,据此,建构了春季林麝的行为谱。根据各行为持续时间比率和频次分析表明,林麝的主要行为为休息;雌麝的摄食行为所占的比率高于雄麝,这是由于春季雌麝妊娠的能量需求较高所致。活动节律呈典型的晨昏双峰型,清晨(4:00 ～ 6:00)和傍晚(17:00 ～ 19:00)为活动高峰;夜间活动性显著高于白天。     

Phylogenetic Study of Complete Cytochrome b Genes in Musk Deer (Genus Moschus) Using Museum Samples

As an endangered animal group, musk deer (genus Moschus) are not only a great concern of wildlife conservation, but also of special interest to evolutionary studies due to long-standing arguments on the taxonomic and phylogenetic associations in this group. Using museum samples, we sequenced complete mitochondrial cytochrome b genes (1140 bp) of all suggested species of musk deer in order to reconstruct their phylogenetic history through molecular information. Our results showed that the cytochrome b gene tree is rather robust and concurred for all the algorithms employed (parsimony, maximum likelihood, and distance methods). Further, the relative rate test indicated a constant sequence substitution rate among all the species, permitting the dating of divergence events by molecular clock. According to the molecular topology, M. moschiferus branched off the earliest from a common ancestor of musk deer (about 700,000 years ago); then followed the bifurcation forming the M. berezovskii lineage and the lineage clustering M. fuscus, M. chrysogaster, and M. leucogaster (around 370,000 years before present). Interestingly, the most recent speciation event in musk deer happened rather recently (140,000 years ago), which might have resulted from the diversified habitats and geographic barriers in southwest China caused by gigantic movements of the Qinghai-Tibetan Plateau in history. Combining the data of current distributions, fossil records, and molecular data of this study, we suggest that the historical dispersion of musk deer might be from north to south in China. Additionally, in our further analyses involving other pecora species, musk deer was strongly supported as a monophyletic group and a valid family in Artiodactyla, closely related to Cervidae.     

Molecular polymorphism of MHC-DRB gene and genetic diversity analysis of captive forest musk deer (Moschus berezovskii)

The major histocompatibility complex (MHC) is one of the most important elements in immune system for nearly all vertebrates, and is thought to be essential for an organism to recognize foreign molecules. In this study, we investigated the MHC variation in 51 forest musk deer (Moschus berezovskii) collected from three captive populations in Sichuan Province, China. Seventeen haplotypes were isolated from the 51 samples. A total of 51 mutation sites were identified and yielded a nucleotide diversity of 0.056. These haplotype sequences possessed 83 putative amino acid sites, including 24 PBR sites (peptide binding region) and 59 non-PBR sites. Out of 24 PBR sites, 15 codons showed variation (0.625), while 12 codons showed variation (0.203) in 59 non-PBR sites. Non-synonymous substitutions primarily occurred in PBR, with analyses suggesting that the Mobe-DRB gene had undergone strong positive selection during their evolution. Compared with that of some other endangered species, the forest musk deer had relatively high level of MHC diversity. Our results suggested that the MHC diversity characteristic of captive forest musk deer populations might be not responsible for their high morbidity of abscess disease.     

林麝(Moschus berezovskii)研究概况和进展

林麝（Moschus berezovskii）是国家一级保护动物,已被列入CITES附录Ⅰ中,由于连续多年的恶性捕杀,加上国际高价走私,使中国的野生林麝资源遭到严重破坏。作者根据大量文献资料结合自己的研究,从林麝的分类地位、地理分布、组织解剖、种群生态和人工饲养等几个方面较为全面地总结了国内林麝研究的概况和进展,指出了林麝种群研究中存在的一些问题以及今后的研究方向。     

The olfactory subgenome and specific odor recognition in forest musk deer

Olfactory receptors (ORs) are encoded by OR genes. The OR genes in forest musk deer (Moschus berezovskii), which rely on olfaction for reproductive and social communication, are poorly understood. In this study, we analyzed the genome sequence of the forest musk deer to obtain its olfactory subgenome and compared it to other species. A total of 1378 OR‐related sequences were detected in the forest musk deer genome including 864 functional genes, 366 pseudogenes and 148 partial genes. These OR genes were classified into Class I and Class II and were further classified into 18 families and 244 subfamilies through sequence identity. Comparative analyses of the OR genes’ protein sequences in species from different orders (forest musk deer, human, mouse and dog) showed that 12 clusters were specific to forest musk deer. However, when compared to other Artiodactyl species (i.e. cattle, yak and pig) only two clusters were specific to forest musk deer. The odor identification potential of the OR genes in the forest musk deer was focused mainly on floral, woody, lemon, sweet and fatty odors. We also found that OR genes specific to forest musk deer were involved in the identification of spearmint and caraway. Our work is the first genome‐wide analysis of OR genes in forest musk deer. These findings will assist with better understanding the relationship between behavior and olfaction in the forest musk deer and the characteristics of the olfactory subgenome in Artiodactyl mammals.     

马麝诸种群地理分化初步探讨

马麝是我国的一种重要经济动物, 是古北界特有种, 主要分布在我国青藏高原及其临近地区。为保护物种多样性, 本文利用分子生物学技术在分子水平探讨了马麝各地理种群之间遗传多样性问题。用线粒体细胞色素b(Cytochrome b) 的DNA 序列进行系统进化分析显示: 马麝四川种群、甘肃种群和西藏两种群在大约100 万年前具有共同祖先。四川种群最先分化出来, 甘肃马麝与西藏马麝的关系最近, 四川马麝与西藏马麝、甘肃马麝间的关系较远, 甘肃马麝与四川马麝约在80 万年前分化, 而甘肃与西藏察隅和类乌齐两种群马麝间分化时间则分别为34万年和57 万年前, 四川马麝与西藏察隅和类乌齐两种群马麝分化时间在90 万年和100 万年前。结果初步表明分布于甘肃、四川和西藏的马麝种群可能为不同的亚种。     

圈养马麝的个性分类

本研究在甘肃兴隆山国家级自然保护区马麝繁育场展开,分别于2012年7月和2013年8月,针对374只圈养马麝,采用基于量表的个性评价法进行评价调查。结果表明:可建立包含17个个性项目的圈养马麝个性量表,因子分析显示每个项目公因子方差和载荷均大于0. 4,各因子累计贡献率为60. 33%,表明此量表具有较好的结构效度。此外Cronbach’sα系数为0. 787,表明量表具有较高的内部一致性信度,量表设计合理;因子分析和聚类分析结果显示,可将圈养马麝划分为五大个性维度:侵略性、社会性、刻板性、温顺性、活跃性。研究发现,圈养马麝个性维度与野生个体的个性类型划分存在差异,主要是受圈养条件和人为干扰等影响。圈养马麝个性量表和五大个性维度的建立对指导马麝繁育管理具有重要实践价值,填补了马麝个性研究的空缺,并为进一步进行麝类动物个性研究奠定了基础。另外,此类个性层次研究方法具有较好区分度,可为其他动物个性的初期研究提供参考。     

圈养林麝社会等级与粪样类固醇激素水平的关系

动物社群的社会等级可对个体的资源分配、社群稳定及个体的行为对策产生重要影响,圈养动物的类固醇激素水平与饲养管理有关,可反映动物的社群紧张水平。深入理解圈养动物的社会等级与其类固醇激素水平的关系是进行濒危动物迁地保育和成功驯养的基础。2018年6月15日—8月15日,用焦点取样法对四川马尔康林麝繁育场的25头林麝进行了冲突行为取样及社会等级计算,采用放射免疫分析法检测了同期粪样的类固醇激素水平,分析了林麝社会等级与粪样皮质醇、睾酮及雌二醇水平的关系。结果表明:低等级雌麝的雌二醇水平(289.037±59.710 pg·g^-1,n=11)显著高于高等级雌麝(45.670±27.283pg·g^-1,n=6)(P<0.05),低等级雄麝的睾酮水平(3.863±1.538 ng·g^-1,n=3)和高等级雄麝(8.017±1.295 ng·g^-1,n=5)无显著差异(P>0.05);低等级雄麝(37.891±7.564 ng·g^-1,n=3)和雌麝(37.262±1.544 ng·g^-1,n=11)的皮质醇水平与高等级雄麝(29.947±2.441 ng·g^-1,n=5)及雌麝(37.478±4.628 ng·g^-1,n=6)间的差异不显著(P>0.05)。不区分性别,低等级林麝的皮质醇水平(37.397±1.826 ng·g^-1,n=14)和高等级个体(34.055±2.886ng·g^-1,n=11)间无显著差异(P>0.05)。研究结果表明,圈养林麝雌体的社会等级与其粪样雌二醇水平呈负相关,低等级雌麝的粪样雌二醇水平显著高于高等级雌麝。在麝类驯养实践中,可监测雌麝的社会等级和粪样雌二醇水平变化,预测雌麝的行为健康及繁殖成效。     

Molecular evidence that Langeronia macrocirra and Langeronia cf. parva (Trematoda: Pleurogenidae) parasites of anurans from Mexico are conspecific

The genus Langeronia parasitizing the intestine of several species of anurans is distributed from North to Central America. We identified Langeronia macrocirra and Langeronia cf. parva from the same host and localities, and present here new data not applicable about their tegumental surface by scanning electron microscopy. We compared sequences of the rDNA ITS2 region and mtDNA cox1 gene for the two morphotypes. ITS2 exhibited a high degree of conservation. Phylogenetic reconstruction using cox1 revealed three clades (I, II, and III), which did not correspond to a previous identification or host. Little divergence was found within clades: sequences were identical in clade I, whereas clade II had 0.27% and clade III had 1.08%. Inter-clade divergence reached 8.69% (I vs. III). This pattern of genetic divergence indicated that both taxa probably belong to the same species, so we posit that the morphological changes could be correlated with development. Increasing sample size and geographical coverage will contribute to the taxonomy of the genus based on morphological and molecular evidence, and will open tracks toward the use of DNA barcodes to the genus in Mexico.     

New insights into the karyotypic relationships of Chinese muntjac (Muntiacus reevesi), forest musk deer (Moschus berezovskii) and gayal (Bos frontalis)

To investigate the karyotypic relationships between Chinese muntjac (Muntiacus reevesi), forest musk deer (Moschus berezovskii) and gayal (Bos frontalis), a complete set of Chinese muntjac chromosome-specific painting probes has been assigned to G-banded chromosomes of these three species. Sixteen autosomal probes (i.e. 6-10, 12-22) of the Chinese muntjac each delineated one pair of conserved segments in the forest musk deer and gayal, respectively. The remaining six autosomal probes (1-5, and 11) each delineated two to five pairs of conserved segments. In total, the 22 autosomal painting probes of Chinese muntjac delineated 33 and 34 conserved chromosomal segments in the genomes of forest musk deer and gayal, respectively. The combined analysis of comparative chromosome painting and G-band comparison reveals that most interspecific homologous segments show a high degree of conservation in G-banding patterns. Eleven chromosome fissions and five chromosome fusions differentiate the karyotypes of Chinese muntjac and forest musk deer; twelve chromosome fissions and six fusions are required to convert the Chinese muntjac karyotype to that of gayal; one chromosome fission and one fusion separate the forest musk deer and gayal. The musk deer has retained a highly conserved karyotype that closely resembles the proposed ancestral pecoran karyotype but shares none of the rearrangements characteristic for the Cervidae and Bovidae. Our results substantiate that chromosomes 1-5 and 11 of Chinese muntjac originated through exclusive centromere-to-telomere fusions of ancestral acrocentric chromosomes.     

圈养马麝刻板行为表达与粪样类固醇激素的关系

2017年7月1日-8月31日及2018年6月1日-7月31日,在甘肃兴隆山保护区马麝繁育中心,采用焦点取样法和连续记录法进行了圈养马麝的刻板行为取样,采集同期粪样,并用放射免疫分析法（RIA）检测粪样中肾上腺皮质醇、睾酮及雌二醇激素的水平,分析了圈养马麝刻板行为表达与上述3种激素水平的关系。结果显示,展现刻板行为的圈养马麝的皮质醇水平（111.099 ± 16.231）ng/g略高于无刻板行为表达的马麝（95.640± 9.738） ng/g,差异未达显著（P> 0.05）;展现刻板行为雄麝的睾酮水平（135.900± 21.582）ng/g略高于无刻板行为的雄麝（108.182 ± 9.689） ng/g,差异也不显著（P> 0.05）;展现刻板行为雌麝的雌二醇水平（0.445 ± 0.116）ng/g显著低于无刻板行为雌麝（10.843 ± 1.142）ng/g（P< 0.05）。研究结果表明,圈养雄性马麝的刻板行为表达与其类固醇激素水平不相关;而雌麝的刻板行为表达与雌二醇分泌显著负相关,这与其繁殖及健康状况有关。在麝类驯养实践中,可将粪样类固醇激素水平（尤其是雌二醇）作为其受胁迫水平及行为健康的监测指标。     

应用流式细胞术分析林麝外周血 CD4+、CD8+ T淋巴细胞

通过对圈养林麝(Moschusberezovskii)外周血淋巴细胞CD4~+、CD8~+亚群的检测,探讨林麝细胞免疫功能状态,并探索应用流式细胞仪分析其淋巴细胞亚群的方法,为研究林麝重大疾病的病理机制及诊断方法提供科学依据。本研究选取健康林麝和患呼吸道疾病林麝各5头,以双色流式细胞术检测其外周血淋巴细胞CD4~+、CD8~+亚群的含量,并进行比较。结果显示,羊源CD4、CD8的流式荧光抗体能够标记林麝细胞并有效检测;患病林麝与健康林麝相比,外周血CD4~+细胞含量无差异(P 0.05),CD8~+细胞含量则显著降低(P 0.01),CD4~+/CD8~+比值显著增高(P 0.01)。结果表明,患呼吸系统炎性疾病的林麝其外周血淋巴细胞CD8~+亚群变化显著,检测淋巴细胞亚群对林麝疾病的诊断有重要意义。     

Isolation and characterization of microsatellite loci from forest musk deer (Moschus berezovskii)

This study reported the isolation and characterization of eight polymorphic microsatellite loci in endangered forest musk deer Moschus berezovskii. An improved enrichment protocol was used to isolate microsatellites, and polymorphism was explored with samples from wild musk deer population collected in Miyalo of Sichuan Province in China. Approximately 70% of clones from the genomic library constructed in current study contained dinucleotide (AC) repeats. Eight microsatellite loci amplified were highly polymorphic within forest musk deer population. The number of alleles per locus ranged from 6 to 14, and the observed and expected heterozygosities ranged from 0.41 approximately 1.0 and from 0.8 approximately 0.9, respectively. The average polymorphic information content (PIC) value for these markers was 0.82. This demonstrated that the eight microsatellite loci developed here are highly polymorphic, and can be used as genetic markers for further investigation of musk deer. Also, the results showed that the musk deer distributed in Miyalo had a relatively higher level of genetic variation.     

Genetic diversity of captive forest musk deer (Moschus berezovskii) inferred from the mitochondrial DNA control region

Forest musk deer ( Moschus berezovskii ) were once distributed widely in China. However, wild populations have declined dramatically because of poaching and habitat loss. Captive breeding populations have been established for several decades, but the genetic backgrounds of most captive populations were unclear and the population sizes increased very slowly. To provide useful information for conservation and management of this species, we investigated the genetic diversity and population structure of forest musk deer by analysing a 582-bp fragment of the mitochondrial DNA (mtDNA) control region (CR) in three captive breeding populations in Sichuan Province, China. Ninety-four variable sites and 27 haplotypes were observed in 109 individuals, and the nucleotide and haplotype diversities were relatively high compared with those of other endangered mammals. Of the three investigated populations, the Maerkang population had the highest nucleotide diversity ( π  = 0.0568), haplotype diversity ( h  =   0.836) and average intra-population genetic distance (0.062). The analysis of molecular variance demonstrated that most variation occurred within samples and that there was significant differentiation of the three populations. Estimates of gene flow indicated that there were few genetic exchanges among the three populations. Building pedigree records and increasing gene flow between populations will be helpful for conserving these populations and this species.