Phylogeographical analysis shows the need to protect the wild yaks' last refuge in Nepal

The wild yak Bos mutus was believed to be regionally extinct in Nepal for decades until our team documented two individuals from Upper Humla, north‐western Nepal, in 2014. The International Union for Conservation of Nature (IUCN) seeks further evidence for the conclusive confirmation of that sighting. We conducted line transects and opportunistic sign surveys in the potential wild yak habitats of Humla, Dolpa, and Mustang districts between 2015 and 2017 and collected genetic samples (present and historic) of wild and domestic yaks Bos grunniens. We also sighted another wild yak in Upper Humla in 2015. Phylogenetic and haplotype network analyses based on mitochondrial D‐loop sequences (~450 bp) revealed that wild yaks in Humla share the haplotype with wild yaks from the north‐western region of the Qinghai‐Tibetan Plateau in China. While hybridization with domestic yaks is a major long‐term threat, illegal hunting for meat and trophy put the very small populations of wild yaks in Nepal at risk. Our study indicates that the unprotected habitat of Upper Humla is the last refuge for wild yaks in Nepal. We recommend wild yak conservation efforts in the country to focus on Upper Humla by (i) assigning a formal status of protected area to the region, (ii) raising awareness in the local communities for wild yak conservation, and (iii) providing support for adaptation of herding practice and pastureland use to ensure the viability of the population.     

西藏牦牛mtDNA D-loop区的遗传多样性及其遗传分化

通过测定和分析西藏11个牦牛类群114个个体的mtDNA D-loop区全序列,对西藏牦牛的遗传多样性、类群间的亲缘关系及其遗传分化进行了研究。结果表明:①西藏牦牛mtDNA D-loop区全序列长度为890—896 bp,4种核苷酸T、C、A、G的平均比例分别为28.5%、25.3%、32.4%、13.8%,西藏牦牛mtDNA D-loop区富含碱基A+T,表现出一定的碱基偏好性。②共检测到130个变异位点,占分析总位点数的14.33%;其中单一多态位点85个,占多态位点总数的65.38%,简约信息位点45个,占多态位点总数的34.62%。序列变异中碱基缺失、插入和碱基替换等均有,其中碱基替换变异类型中转换114次,颠换12次,在转换变异类型中以A/G、T/C为主,占95.61%,在颠换变异类型中以A/T为主,占75%。③在114个个体中鉴定出90种单倍型,单倍型多样性为0.981±0.008,核苷酸多样性为0.01056±0.00701,均说明西藏牦牛具有丰富的单倍型类型。④90种单倍型分为2个聚类簇(Ⅰ、Ⅱ),聚类簇Ⅰ包含80种单倍型,占全部单倍型的88.89%,涵盖本研究中所有的西藏牦牛类群;聚类簇Ⅱ中有10种单倍型,占单倍型总数的11.11%,涉及的类群有工布江达、帕里、丁青、巴青、江达、类乌齐、桑桑、桑日、斯布,说明西藏牦牛可能有2个母系起源。⑤西藏牦牛类群间核苷酸分歧度(Dxy)在0.503%—1.416%之间,聚类分析和AMOVA分析显示西藏牦牛可分为两大类,康布牦牛、嘉黎牦牛为一类,其余的牦牛类群为另一类。     

Structural Variants Selected during Yak Domestication Inferred from Long-Read Whole-Genome Sequencing

Structural variants (SVs) represent an important genetic resource for both natural and artificial selection. Here we present a chromosome-scale reference genome for domestic yak (Bos grunniens) that has longer contigs and scaffolds (N50 44.72 and 114.39 Mb, respectively) than reported for any other ruminant genome. We further obtained long-read resequencing data for 6 wild and 23 domestic yaks and constructed a genetic SV map of 372,220 SVs that covers the geographic range of the yaks. The majority of the SVs contains repetitive sequences and several are in or near genes. By comparing SVs in domestic and wild yaks, we identified genes that are predominantly related to the nervous system, behavior, immunity, and reproduction and may have been targeted by artificial selection during yak domestication. These findings provide new insights in the domestication of animals living at high altitude and highlight the importance of SVs in animal domestication.     

牦牛的分类学地位及起源研究:mtDNA D-loop序列的分析

牦牛的起源与属级分类学地位至今仍然存在一定的争议。我们测定了家养牦牛和野生牦牛线粒体控制区(D-loop)序列,并以此构建牦牛和牛属、野牛属、水牛属以及非洲水牛属相关种的系统发育树。研究结果表明线粒体D-loop区与Cytb基因序列在构建牛族的系统发育具有同样重要的价值。系统发育关系显示野牛属的灭绝种草原野牛与现存种美洲野牛先聚合为一单系群,然后再和牦牛形成一单系分支,表明牦牛与野牛属的草原野牛、美洲野牛亲缘关系最近,具有最近的共同祖先,而与牛属的其它亚洲物种亲缘关系较远。因此,本研究不支持将牦牛独立为牦牛属———Poephagus,牛属与野牛属在分类上也应合并为一个属。基于上述研究结果和化石证据,我们进一步对牦牛起源的历史背景进行了讨论,认为牦牛与野牛属的分化是由于第四纪气候变化在欧亚大陆发生的,野牛通过白令陆桥进入北美;冰期结束后,由于欧亚大陆其它地区温度升高,牦牛只能局限分布在较为寒冷的青藏高原;而野牛属在北美先后分化为草原野牛和美洲野牛,前者可能是后者的直接祖先。     

Prevalence,Associated Risk Factors,and Phylogenetic Analysis of Toxocara vitulorum Infection in Yaks on the Qinghai Tibetan Plateau,China

Toxocara vitulorum has been rarely reported in yaks at high altitudes and remote areas of Sichuan Province of Tibetan Plateau of China. The current study was designed to investigate the prevalence, associated risk factors, and phylogenetic characteristics of T. vitulorum in yak calves on the Qinghai Tibetan plateau. Fecal samples were collected from 891 yak calves and were examined for the presence of T. vitulorum eggs by the McMaster technique. A multivariable logistic regression model was employed to explore variables potentially associated with exposure to T. vitulorum infection. T. vitulorum specimens were collected from the feces of yaks in Hongyuan of Sichuan Province, China. DNA was extracted from ascaris. After PCR amplification, the sequencing of ND1 gene was carried out and phylogenetic analyses was performed by MEGA 6.0 software. The results showed that 64 (20.1%; 95% CI 15.8–24.9%), 75 (17.2; 13.8–21.1), 29 (40.9; 29.3–53.2), and 5 (7.6; 2.5–16.8) yak calves were detected out to excrete T. vitulorum eggs in yak calve feces in Qinghai, Tibet, Sichuan, and Gansu, respectively. The present study revealed that high infection and mortality by T. vitulorum is wildly spread on the Qinghai Tibetan plateau, China by fecal examination. Geographical origin, ages, and fecal consistencies are the risk factors associated with T. vitulorum prevalence by logistic regression analysis. Molecular detection and phylogenetic analysis of ND1 gene of T. vitulorum indicated that T. vitulorum in the yak calves on the Qinghai Tibetan plateau are homologous to preveiously studies reported.     

贺兰山牦牛冬春季的生境选择

在2009年12月－2010年1月和2010年4－5月,采用样线法结合直接观察法对贺兰山牦牛的冬春季生境选择进行了研究。结果表明,牦牛冬季对11种生境因子有选择性,偏爱山地针叶林带,海拔小于2 000 m,优势乔木为灰榆,坡度小于10,下坡位,距水源距离大于1 200 m,人为干扰距离2 000－4 000 m,隐蔽级大于70 %;春季牦牛对13种利用生境生态因子有选择性,偏爱于亚高山灌丛和草甸带,海拔大于3 000 m,乔木密度小于1株,乔木高度小于3 m,乔木距离大于3 m,灌木密度大于4 0株,灌木距离小于1 m,植被盖度大于7 0 %,上坡位,距水源的距离小于8 00 m,人为干扰距离大于4 000 m,隐蔽级大于7 0 %。冬春季牦牛在海拔、植被类型、地形特征、优势乔木、灌木种类、坡位、坡向、人为干扰距离、距水源距离上存在显著差异。主成分分析表明冬季第一主成分的贡献率21.100 %,其中绝对值较大的相关系数是乔木距离、优势乔木、乔木高度和乔木密度;春季第一主成分的贡献率是31.247 %,其中绝对值较大的相关系数是植被类型、海拔高度、地形特征和灌木密度。与其他分布地区的牦牛相比,贺兰山地区的牦牛能适应当地的地理特征和气候环境。     

羌塘、可可西里无人区野牦牛种群数量和分布现状

野牦牛(Bos grunniens)是青藏高原特有的大型有蹄类动物, 主要分布在西藏羌塘, 青海可可西里、三江源, 新疆阿尔金山等地的高海拔生境。我们于2012-2014年在西藏羌塘和青海可可西里对野牦牛进行了较为全面的实地调查, 并且估算西藏羌塘地区野牦牛种群在11,222-21,072头之间, 可可西里野牦牛种群数量在659-1,793头之间。据本次野外调查和历史文献资料显示, 自20世纪90年代起, 中国野牦牛种群数量逐年增长, 但是其分布区面积却逐步缩减, 目前仅分布在几个相对孤立且远离人类居住地的高寒区域。随着全球气候变化与青藏高原人类活动的加强,野牦牛的生存现状依然严峻, 我们仍需加强对青藏高原特有物种的保护。     

Phylogeographical analyses of domestic and wild yaks based on mitochondrial DNA: new data and reappraisal

Aim We aimed to examine the phylogeographical structure and demographic history of domestic and wild yaks (Bos grunniens) based on a wide range of samples and complete mitochondrial genomic sequences. Location The Qinghai‐Tibetan Plateau (QTP) of western China. Methods All available D‐loop sequences for 405 domesticated yaks and 47 wild yaks were examined, including new sequences from 96 domestic and 34 wild yaks. We further sequenced the complete mitochondrial genomes of 48 domesticated and 21 wild yaks. Phylogeographical analyses were performed using the mitochondrial D‐loop and the total genome datasets. Results We recovered a total of 123 haplotypes based on the D‐loop sequences in wild and domestic yaks. Phylogenetic analyses of this dataset and the mitochondrial genome data suggested three well‐supported and divergent lineages. Two lineages with six D‐loop haplogroups were recovered for all morphological breeds of domestic yaks across their distributions in the QTP, while one more lineage and more endemic haplogroups or haplotypes were found for wild yaks. Based on the mitochondrial genome data, the divergences of the three lineages were estimated to have occurred around 420,000 and 580,000 years ago, consistent with the geological records of two large glaciation events experienced in the QTP. Main conclusions There are distinct phylogeographical differences between wild and domestic yaks. However, there is no apparent geographical correlation between identified haplogroups and distributions of domestic yaks. Three differentiated lineages of yaks probably evolved allopatrically in different regions during the Pleistocene glaciation events, then reunited into a single gene pool during post‐glacial population expansion and migrations before the start of the domestication of yaks in the Holocene.     

From ‘third pole’ to north pole: a Himalayan origin for the arctic fox

The ‘third pole’ of the world is a fitting metaphor for the Himalayan–Tibetan Plateau, in allusion to its vast frozen terrain, rivalling the Arctic and Antarctic, at high altitude but low latitude. Living Tibetan and arctic mammals share adaptations to freezing temperatures such as long and thick winter fur in arctic muskox and Tibetan yak, and for carnivorans, a more predatory niche. Here, we report, to our knowledge, the first evolutionary link between an Early Pliocene (3.60–5.08 Myr ago) fox, Vulpes qiuzhudingi new species, from the Himalaya (Zanda Basin) and Kunlun Mountain (Kunlun Pass Basin) and the modern arctic fox Vulpes lagopus in the polar region. A highly hypercarnivorous dentition of the new fox bears a striking resemblance to that of V. lagopus and substantially predates the previous oldest records of the arctic fox by 3–4 Myr. The low latitude, high-altitude Tibetan Plateau is separated from the nearest modern arctic fox geographical range by at least 2000 km. The apparent connection between an ancestral high-elevation species and its modern polar descendant is consistent with our ‘Out-of-Tibet’ hypothesis postulating that high-altitude Tibet was a training ground for cold-environment adaptations well before the start of the Ice Age.     

青海省可可西里地区几种有蹄类动物的食物重叠初步分析

2005年7月和2006年1月,应用粪便显微组织分析法测定了可可西里地区的藏野驴、藏羚、藏原羚、野牦牛,以及家牦牛和藏羊在冷季(1月)和暖季(7月)的食物构成.用Schoener's Index计算了这些同域分布动物种间的食物重叠度.结果表明,藏野驴分别与藏羚羊、野牦牛、藏原羚在暖季的食物重叠度各为63.0%、48.4%和24.1%,在冷季的重叠度为别为71.6%、42.0%和11.4%;藏羚羊与野牦牛、藏原羚在暖季的食物重叠度分别为52.0%和33.4%,其在冷季的食物重叠度各为50.3%和29.3%;野牦牛与藏原羚在冷暖季的食物重叠度分别为13.1%和15.9%.在可可西里地区吲域分布野牛有蹄类动物种间,藏原羚与其他有蹄类动物食物重叠较少,而藏羚羊、藏野驴、野牦牛之间则存在不同程度的食物重叠,且随季节不同而变化,反映了这些动物之间复杂的竞争和共存关系.此外,家牦牛和藏羊均与这些野生有蹄类动物存在高度的食物重叠.     

Isolation and characterization of polymorphic microsatellites in the genome of Yak (Bos grunniens)

The researches on yak genetics and breeding were extremely restricted due to lacking of reliable DNA molecular markers. The microsatellites with repeat motif (AC)_n/(GT)_n in yak genome were enriched by Dynal magnetic beads and the gene libraries containing (AC)_n/(GT)_n were constructed. Among the 92 identified and sequenced positive clones, 40 contained perfect repeats (43.48 %), 41 contained imperfect repeats (44.57 %) and 11 contained compound repeats (11.96 %). As compared with the percentage of perfect repeats, no significant increases of imperfect repeats were observed in yak genome, which indicated that the level of adaptive evolution of the ability to repair damaged genomic DNA for yaks were high enough to endure the natural pressure of nucleotide substitution resulted from ultraviolet irradiation in high-altitude areas. Totally 19 polymorphic microsatellite loci were screened and genotyped on the basis of electropherograms on an ABI 310 Genetic Analyzer. All the loci exhibited moderate to high-level polymorphisms in a test population of Bos grunniens and the polymorphic information content ranged from 0.299 to 0.861 (mean 0.678). The newly isolated (AC)_n/(GT)_n repeats from yak genome will display their potential values in examining intra-population genetic structure and inter-population relationships, and also in investigating molecular markers for production and adaptive traits of individual/population.     

Domestication relaxed selective constraints on the yak mitochondrial genome

Wild yaks roaming in the high-altitude Qinghai-Tibetan plateau have to maintain high metabolic efficiency. However, domestic yaks do not require such high efficiency because of their limited activity. Hence, domestication may have caused the relaxation of selective constraints on the yak mitochondrial genome because mitochondrial mutations are extremely sensitive to energy-related selective pressures. We have tested this hypothesis by analyzing the mitochondrial genomes of 51 domestic yaks and 21 wild yaks. The results show that the ratio of nonsynonymous/synonymous substitutions in mitochondrial protein-coding genes is significantly higher in domestic yak lineages than those of wild yaks. This genetic difference suggests that the relaxation of selective constraints following the domestication in addition to bottlenecks has allowed faster accumulation of nonsilent substitutions in the yak mitochondrial genome, despite its short domestication history.     

Population history and genomic signatures for high-altitude adaptation in Tibetan pigs

Background

The Tibetan pig is one of domestic animals indigenous to the Qinghai-Tibet Plateau. Several geographically isolated pig populations are distributed throughout the Plateau. It remained an open question if these populations have experienced different demographic histories and have evolved independent adaptive loci for the harsh environment of the Plateau. To address these questions, we herein investigated ~ 40,000 genetic variants across the pig genome in a broad panel of 678 individuals from 5 Tibetan geographic populations and 34 lowland breeds.

Results

Using a series of population genetic analyses, we show that Tibetan pig populations have marked genetic differentiations. Tibetan pigs appear to be 3 independent populations corresponding to the Tibetan, Gansu and Sichuan & Yunnan locations. Each population is more genetically similar to its geographic neighbors than to any of the other Tibetan populations. By applying a locus-specific branch length test, we identified both population-specific and -shared candidate genes under selection in Tibetan pigs. These genes, such as PLA2G12A, RGCC, C9ORF3, GRIN2B, GRID1 and EPAS1, are involved in high-altitude physiology including angiogenesis, pulmonary hypertension, oxygen intake, defense response and erythropoiesis. A majority of these genes have not been implicated in previous studies of highlanders and high-altitude animals.

Conclusion

Tibetan pig populations have experienced substantial genetic differentiation. Historically, Tibetan pigs likely had admixture with neighboring lowland breeds. During the long history of colonization in the Plateau, Tibetan pigs have developed a complex biological adaptation mechanism that could be different from that of Tibetans and other animals. Different Tibetan pig populations appear to have both distinct and convergent adaptive loci for the harsh environment of the Plateau.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-834) contains supplementary material, which is available to authorized users.     

High-Altitude Adaptation of Yak Based on Genetic Variants and Activity of Lactate Dehydrogenase-1

This study investigates the molecular mechanism by which yaks (Bos grunniens) adapt to hypoxia based on lactate dehydrogenase (LDH). Three LDH1 variants of the yak were revealed in tissue extracts by electrophoresis, including LDH1-F, LDH1-M, and LDH1-S. Kinetic analysis using purified LDH1 variants showed that the yak LDH1-M variant exhibited a similar K _m (NADH) and the same mobility on a gel as bovine LDH1, and the LDH1-F variant showed significant differences in K _m values for NADH or pyruvate from the other two variants of yak LDH1 and bovine LDH1. Among the three muscles assayed, yak longissimus dorsi showed the highest LDH activity and the lowest malate dehydrogenase (MDH) activity; heart muscle was exactly the opposite. Our results suggest that the three LDH1 variants might play an important role in the adaptation to hypoxia.     

Genome‐wide variation within and between wild and domestic yak

The yak is one of the few animals that can thrive in the harsh environment of the Qinghai‐Tibetan Plateau and adjacent Alpine regions. Yak provides essential resources allowing Tibetans to live at high altitudes. However, genetic variation within and between wild and domestic yak remain unknown. Here, we present a genome‐wide study of the genetic variation within and between wild and domestic yak. Using next‐generation sequencing technology, we resequenced three wild and three domestic yak with a mean of fivefold coverage using our published domestic yak genome as a reference. We identified a total of 8.38 million SNPs (7.14 million novel), 383 241 InDels and 126 352 structural variants between the six yak. We observed higher linkage disequilibrium in domestic yak than in wild yak and a modest but distinct genetic divergence between these two groups. We further identified more than a thousand of potential selected regions (PSRs) for the three domestic yak by scanning the whole genome. These genomic resources can be further used to study genetic diversity and select superior breeds of yak and other bovid species.     

阿尔金山自然保护区基于野牦牛、藏野驴、藏羚羊适宜栖息地的生态容量估测

阿尔金山国家级自然保护区保护了以野牦牛(Bos mutus)、藏野驴(Equus kiang)、藏羚羊(Pantholops hodgsoni)为代表的青藏高原特有野生动物及其栖息地,但是近年来野生动物数量的快速增长引发了栖息地退化的问题,科学量化阿尔金山自然保护区各类栖息地对野牦牛、藏羚羊、藏野驴的生态容量,并提出相应的野生动物管理措施,是实现保护区可持续管理的根本途径。本文应用遥感技术和地面调查相结合的方法,系统分析了保护区内野牦牛、藏野驴和藏羚羊的栖息地需求,建立了植物生物量和NDVI的关系模型,结合三类野生动物的食性分析,估测了适宜栖息地(高寒草原、高寒荒漠草原、高寒草甸、高寒荒漠)为三类野生动物提供的可食植物量,推算了适宜栖息地和整个保护区可以承载三类野生动物的生态容量。结果表明:阿尔金山自然保护区内野牦牛、藏野驴和藏羚羊的适宜栖息地面积分别为31866.07、24035.51、24035.51 km~2,三类野生动物的适宜栖息地之间相互重叠,藏野驴和藏羚羊的适宜栖息地基本相同;全保护区内,高寒草原、高寒荒漠草原、高寒荒漠和高寒草甸分别提供了3944.91×10~4、3126.32×10~4、138.19×10~4、564.49×10~4kg可食植物量;结合三类野生动物的栖息地重叠程度及食物需求量分析,得出阿尔金山保护区的最大生态容量为野牦牛7951头/a、藏野驴6907头/a、藏羚羊27094只/a;结合三类野生动物对食物资源的占有率估计,得出阿尔金山三类野生动物的生态容量变幅为野牦牛3976—7156头/a,藏野驴3454—6216头/a、藏羚羊13547—24385只/a。根据阿尔金山自然保护区各类栖息地对三类野生动物的生态容量,提出适当控制藏野驴种群数量、增加藏羚羊种群数量的建议,以促进野生动物种群数量的持续增长和栖息地的有效保护。     

Rumen prokaryotic communities of ruminants under different feeding paradigms on the Qinghai-Tibetan Plateau

Yak and Tibetan sheep are the major indigenous ruminants on the Qinghai-Tibetan Plateau in China. The aim of this work was to study the differences in ruminal fermentation parameters and rumen prokaryotic community composition between hosts and feeding paradigms. The 16S rRNA genes targeting bacteria and archaea were sequenced using the MiSeq platform. The results showed that the prokaryotic community structure between yak and Tibetan sheep was significantly different (P < 0.01). A significant difference in structure was also found between groups of yaks barn fed with a total mixed ration (TMR) and those naturally grazing (NG) (P = 0.034), as well as for Tibetan sheep of the two groups (P = 0.026). The core prokaryotic populations that existed in the rumen mostly dominated the structure. There was an obvious correlation of the prokaryotic community composition at the phylum and genus levels with the host or the feeding pattern. In addition, Tibetan sheep showed significantly higher yields of volatile fatty acids (VFAs) than yak, as did the NG group compared with the TMR group. In conclusion, both the host and feeding pattern may influence rumen microbial ecology system, with host effects being more important than those of the feeding pattern.     

Associations of single nucleotide polymorphisms in candidate genes with the polled trait in Datong domestic yaks

The domestic yak (Bos grunniens) is an iconic symbol of animal husbandry at high altitudes. Yaks exhibit unique external characteristics including long hair and large horns. However, hornless yaks can be found in different breeds and different populations. The hornless trait is also known as polled, and the POLL locus has been fine‐mapped to chromosome 1 in cattle (Bos taurus), although the underlying genetic basis of the polled trait is still unclear in the yak. Thus, we performed an association study to identify the genetic polymorphisms responsible for the polled trait in the yak. Fifty polled Datong domestic yaks and 51 horned individuals were selected randomly from a huge herd and were used as the case and control groups respectively for the association analysis. Twelve genes located in the candidate region of the POLL locus in cattle were used as references to detect DNA polymorphisms related to yak polledness, which were analyzed by sequencing and a high‐resolution melting test. We applied Fisher's exact test and haplotype analysis to show that a 147‐kb segment that included three protein‐coding genes C1H21orf62, GCFC1 and SYNJ1 was the most likely location of the POLL mutation in domestic yaks.