Rapid and parallel chromosomal number reductions in muntjac deer inferred from mitochondrial DNA phylogeny

Muntjac deer (Muntiacinae, Cervidae) are of great interest in evolutionary studies because of their dramatic chromosome variations and recent discoveries of several new species. In this paper, we analyze the evolution of karyotypes of muntjac deer in the context of a phylogeny which is based on 1,844-bp mitochondrial DNA sequences of seven generally recognized species in the muntjac subfamily. The phylogenetic results support the hypothesis that karyotypic evolution in muntjac deer has proceeded via reduction in diploid number. However, the reduction in number is not always linear, i.e., not strictly following the order: 46-->14/13-->8/9-->6/7. For example, Muntiacus muntjak (2n = 6/7) shares a common ancestor with Muntiacus feae (2n = 13/14), which indicates that its karyotype was derived in parallel with M. feae's from an ancestral karyotype of 2n >/= 13/14. The newly discovered giant muntjac (Muntiacus vuquangensis) may represent another parallel reduction lineage from the ancestral 2n = 46 karyotype. Our phylogenetic results indicate that the giant muntjac is relatively closer to Muntiacus reevesi than to other muntjacs and may be placed in the genus Muntiacus Analyses of sequence divergence reveal that the rate of change in chromosome number in muntjac deer is one of the fastest in vertebrates. Within the muntjac subfamily, the fastest evolutionary rate is found in the Fea's lineage, in which two species with different karyotypes diverged in around 0.5 Myr.     

Comparative sequence analyses reveal sites of ancestral chromosomal fusions in the Indian muntjac genome

Background

Indian muntjac (Muntiacus muntjak vaginalis) has an extreme mammalian karyotype, with only six and seven chromosomes in the female and male, respectively. Chinese muntjac (Muntiacus reevesi) has a more typical mammalian karyotype, with 46 chromosomes in both sexes. Despite this disparity, the two muntjac species are morphologically similar and can even interbreed to produce viable (albeit sterile) offspring. Previous studies have suggested that a series of telocentric chromosome fusion events involving telomeric and/or satellite repeats led to the extant Indian muntjac karyotype.

Results

We used a comparative mapping and sequencing approach to characterize the sites of ancestral chromosomal fusions in the Indian muntjac genome. Specifically, we screened an Indian muntjac bacterial artificial-chromosome library with a telomere repeat-specific probe. Isolated clones found by fluorescence in situ hybridization to map to interstitial regions on Indian muntjac chromosomes were further characterized, with a subset then subjected to shotgun sequencing. Subsequently, we isolated and sequenced overlapping clones extending from the ends of some of these initial clones; we also generated orthologous sequence from isolated Chinese muntjac clones. The generated Indian muntjac sequence has been analyzed for the juxtaposition of telomeric and satellite repeats and for synteny relationships relative to other mammalian genomes, including the Chinese muntjac.

Conclusions

The generated sequence data and comparative analyses provide a detailed genomic context for seven ancestral chromosome fusion sites in the Indian muntjac genome, which further supports the telocentric fusion model for the events leading to the unusual karyotypic differences among muntjac species.     

Identification of black muntjac (Muntiacus crinifrons) in Tibet, China, by cytochrome b analysis

Full cytochrome b gene sequence of mtDNA was used to identify and analyze four skin samples collected from Tibet, China in this research. By searching for highly similar sequences (megablast) on NCBI, we found all four samples have the highest similarities with the published sequence: AY239042 of the black muntjac (Muntiacus crinifrons). By comparing our sequences to those available on GenBank, all four samples were identified as the black muntjac (Muntiacus crinifrons) by high sequence similarity. We therefore record two new localities for the black muntjac and it is a new distribution in Tibet and hope this study will not only promote more advanced researches on the evolution and phylogeny about this species, but also enhance the conservation work for this species and local biodiversity.     

Comparative gene mapping in the species Muntiacus muntjac.

An extreme case of chromosomal evolution is presented by the two muntjac species Muntiacus muntjac (Indian muntjac, 2n = 6 [females], 7 [males]) and M. reevesi (Chinese muntjac, 2n = 46). Despite disparate karyotypes, these phenotypically similar species produce viable hybrid offspring, indicating a high degree of DNA-level conservation and genetic relatedness. As a first step toward development of a comparative gene map, several Indian muntjac homologs of known human type I anchor loci were mapped. Using flow-sorted, chromosome-specific Southern hybridization techniques, homologs of the protein kinase C beta polypeptide (PRKCB1) and the DNA repair genes ERCC2 and XRCC1 have been assigned to Indian muntjac chromosome 2. The male-specific ZFY gene was presumptively mapped to Indian muntjac chromosome Y2. Ultimate generation of a comparative physical map of both Indian and Chinese muntjac chromosomes will prove invaluable in the study of mammalian karyotype evolution.     

Compound kinetochores of the Indian muntjac

The chromosomes of the Indian muntjac (Muntiacus muntjak vaginalis) are unique among mammals due to their low diploid number (2N=6, 7) and large size. It has been proposed that the karyotype of this small Asiatic deer evolved from a related deer the Chinese muntjac (Muntiacus reevesi) with a diploid chromosome number of 2n= 46 consisting of small telocentric chromosomes. In this study we utilized a kinetochore-specific antiserum derived from human patients with the autoimmune disease scleroderma CREST as an immunofluorescent probe to examine kinetochores of the two muntjac species. Since CREST antiserum binds to kinetochores of mitotic chromosomes as well as prekinetochores in interphase nuclei, it was possible to identify and compare kinetochore morphology throughout the cell cycle. Our observations indicated that the kinetochores of the Indian muntjac are composed of a linear beadlike array of smaller subunits that become revealed during interphase. The kinetochores of the Chinese muntjac consisted of minute fluorescent dots located at the tips of the 46 telocentric chromosomes. During interphase, however, the kinetochores of the Chinese muntjac clustered into small aggregates reminiscent of the beadlike arrays seen in the Indian muntjac. Morphometric measurements of fluorescence indicated an equivalent amount of stained material in the two species. Our observations indicate that the kinetochores of the Indian muntjac are compound structures composed of linear arrays of smaller units the size of the individual kinetochores seen on metaphase chromosomes of the Chinese muntjac. Our study supports the notion that the kinetochores of the Indian muntjac evolved by linear fusion of unit kinetochores of the Chinese muntjac. Moreover, it is concluded that the evolution of compound kinetochores may have been facilitated by the nonrandom aggregation of interphase kinetochores in the nuclei of the ancestral species.     

黑麂MHC - DQA2 基因多态性

利用牛特异性扩增DQA2 第2 外显子的嵌套引物,对黑麂基因组DNA 进行PCR 扩增和克隆测序,基于该
序列设计出黑麂DQA2 基因第2 外显子特异性引物。利用该引物,通过PCR - SSCP 以及克隆测序技术,从40 个
黑麂样品中获得4 个不同的DQA2 等位基因。没有一个个体同时具有2 个以上的等位基因,所有序列均不含插入
或缺失突变,不含终止密码子,因此,本研究所扩增的DQA2 基因可能是表达的单基因座位。抗原结合区(Peptide
binding region,PBR)非同义替换率(dn)显著大于同义替换率(ds) (P <0.05),暗示该座位曾经历过明
显的正选择作用;进一步利用CODEML 程序中的相关模型以及贝叶斯法检测出4 个受选择作用的氨基酸位点
(α11、α58、α62、α66),这4 个位点均位于PBR 区。基于NJ 法构建的部分偶蹄类DQA 外显子2 系统发生关系
显示,黑麂4 个DQA2 等位基因与牛、羊以及梅花鹿的DQA2 等位基因构成独立的进化枝,在该进化枝内,黑麂
DQA2 等位基因优先与牛DQA2 等位基因聚类,暗示黑麂DQA2 基因在进化过程中存在跨物种进化现象。上述结
果表明平衡选择是维持黑麂DQA2 基因多态性的主要机制。然而,本研究从40 个样品中仅检测出2 个杂合子,
黑麂DQA2 等位基因之间的频率存在显著差异,推测可能是所检测的样品来源于不同种群,由于华伦德效应
(The Whalund effect)导致杂合度降低,也不排除本文所设计的引物在PCR 扩增过程中存在无效等位基因。     

Chromosomes of the Indian Muntjac (Muntiacus muntjak): Comeback

Cell and Tissue Biology - The chromosomes of the Indian muntjac (Muntiacus muntjak, 2n = 6 in females and 2n = 7 in males) are described using the methods of G-, C-, CDAG-, and AgNOR-staining, and...     

Defining the orientation of the tandem fusions that occurred during the evolution of Indian muntjac chromosomes by BAC mapping

The Indian muntjac (Muntiacus muntjak vaginalis) has a karyotype of 2n=6 in the female and 7 in the male, the karyotypic evolution of which through extensive tandem fusions and several centric fusions has been well-documented by recent molecular cytogenetic studies. In an attempt to define the fusion orientations of conserved chromosomal segments and the molecular mechanisms underlying the tandem fusions, we have constructed a highly redundant (more than six times of whole genome coverage) bacterial artificial chromosome (BAC) library of Indian muntjac. The BAC library contains 124,800 clones with no chromosome bias and has an average insert DNA size of 120 kb. A total of 223 clones have been mapped by fluorescent in situ hybridization onto the chromosomes of both Indian muntjac and Chinese muntjac and a high-resolution comparative map has been established. Our mapping results demonstrate that all tandem fusions that occurred during the evolution of Indian muntjac karyotype from the acrocentric 2n=70 hypothetical ancestral karyotype are centromere–telomere (head–tail) fusions.     

麂亚科动物钾离子通道基因片段及其内含子 序列的克隆与进化分析

运用PCR扩增、T-A克隆、测序等技术,获得黑麂,小麂,赤麂和毛冠鹿4种麂亚科动物基因组DNA的钾离子通道部分序列。序列分析表明:麂属动物之间的外显子区域序列差异为0.90%～1.44%,毛冠鹿与麂属动物之间的差异为0.90%～1.26%,可见这一段序列的同源性较高。而内含子部分序列差异在麂属动物之间的差异为0～1.22%,毛冠鹿与麂属动物的差异为1.83%左右。由NJ法和最大简约法（Mp法）构建的进化树表明黑麂与赤麂亲缘关系较近,小麂与他们同为一属但关系较远,而毛冠鹿与它们之间的分化程度已达到属间水平。研究表明基因组的内含子序列能够较真实反映近缘动物之间的关系,是进行分子进化比较分析的较理想标记。 Abstract: In this study, partial fragments of potassium ion channel gene were amplified using the genomic DNA of muntjak, reevesi, crinifrons, and Elaphodus cephalophus. The PCR products were ligated to the plasmid of pMD18-T Vector by the method of direct T-A cloning. The positive clones were identified by colony PCR. The sequences of the recombinant clones were determined using M13-47/RV-M universal primers and aligned by the software CLUSTALW. The nucleotide divergences of exon were 0.90%-1.44% among three species of Muntiacus, 0.90%-1.26% between E. cephalophus and each of Muntiacus deer. In the nucleotide of intron there is 0%-1.22% difference among these muntjac deers, and the divergene reached about 1.83% between E. cephalophus and the three species of Muntiacus. Using the software of MEGA to analyse molecular phylogeny, Phylogenetic trees were constructed with neighbor-joining method and maximum parsimony method. The result showed Muntiacus, crinifrons is most closely related to muntjak, with reevesi as their sister species. E. cephalophus is in the other genus.     

合肥野生动物园黑麂的繁殖资料

合肥野生动物园自1978年开始进行黑麂(Muntiacus crinifrons)的饲养和繁殖,1989年第一胎圈养条件下繁殖的黑麂出生,到2001年底累计繁殖黑麂51头,繁殖种群正处迅速增长期。13年的繁殖资料统计结果表明,圈养条件下育龄母麂平均每12个月产一胎(多数在11—13个月,少数仅6—9个月),孕期240d左右,哺乳期2—3个月,少数母麂可产后发情,但极少有两年三次产仔现象。值得注意的是圈养条件下黑麂多在9—11月交配,4—7月产仔(80．39％)。圈养条件下黑麂幼年的死亡率较低(7．84％),成年黑麂多死于消化道及呼吸道感染等疾病(56％)。     

圈养条件下黑麂雄性标记行为与雌性化学信息存留位置的关系

化学物质被认为是哺乳动物标记行为信息传递的主要载体,而标记在鹿科动物的竞争雌性资源中具有重要的作用,因此本文重点对雄性黑麂的化学标记与雌性化学信息存留点的关系进行了行为学研究。观察发现面腺标记(利用额腺和眶下腺)是雌雄黑麂共有的化学标记行为,而行为性排尿和刨地行为(利用蹄腺)则仅为雄性具有的标记行为;躺卧不具有化学标记作用,而排粪在化学标记上的作用则难以确定。同时分析表明,雄性黑麂更倾向于在靠近雌性化学信息存留区的区域进行化学标记,且可能会因竞争压力的增大而扩大领域范围。     

Structural organization of chromosomes of the Indian muntjac (Muntiacus muntjak)

The identification, morphology, and banding pattern of the chromosomes of the Indian muntjac (Muntiacus muntjak) are described. A diagrammatic representation of the banding pattern as revealed by various techniques is presented following the nomenclature suggested by Paris Conference (1971) for human chromosomes. The Y2 chromosome and the neck of the X chromosome are late replicating based on observations made with the use of a bromodeoxuridine plus Giemsa technique. Most of the G-bands are early replicating, contrary to earlier findings based on autoradiography.     

High-density comparative BAC mapping in the black muntjac (Muntiacus crinifrons): molecular cytogenetic dissection of the origin of MCR 1p+4 in the X1X2Y1Y2Y3 sex chromosome system

The black muntjac (Muntiacus crinifrons, 2n = 8[female symbol]/9[male symbol]) is a critically endangered mammalian species that is confined to a narrow region of southeastern China. Male black muntjacs have an astonishing X1X2Y1Y2Y3 sex chromosome system, unparalleled in eutherian mammals, involving approximately half of the entire genome. A high-resolution comparative map between the black muntjac (M. crinifrons) and the Chinese muntjac (M. reevesi, 2n = 46) has been constructed based on the chromosomal localization of 304 clones from a genomic BAC (bacterial artificial chromosome) library of the Indian muntjac (M. muntjak vaginalis, 2n = 6[female symbol]/7[male symbol]). In addition to validating the chromosomal homologies between M. reevesi and M. crinifrons defined previously by chromosome painting, the comparative BAC map demonstrates that all tandem fusions that have occurred in the karyotypic evolution of M. crinifrons are centromere-telomere fusions. The map also allows for a more detailed reconstruction of the chromosomal rearrangements leading to this unique and complex sex chromosome system. Furthermore, we have identified 46 BAC clones that could be used to study the molecular evolution of the unique sex chromosomes of the male black muntjacs.     

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.     

宁夏兽类新纪录——小麂 (Muntiacus reevesi Ogilby, 1839)

During the camera-trapping survey at Wanghuannan, Erlonghe and Hongxia Forest Farms in the Liupan Mountain National Nature Reserve in Guyuan City, Ningxia Hui Autonomous Region, 49 independent detections of Reeve’s muntjac, Muntiacus reevesi were captured by eight infrared-triggered camera-traps with 274 photographs and 12 video clips from July, 2017 to August, 2018. The Reeve’s muntjac is an endemic species of China and mainly distributed in south China, such as Jiangxi, Guangdong, Guangxi, Hunan, Hubei, Yunnan, Sichuan, Guizhou and Taiwan Provinces, and also distributed in Shaanxi and Gansu Provinces adjacent to Ningxia Hui Autonomous Region. This species was for the first time recorded in Ningxia Hui Autonomous Region, so its distribution range has been expanded in China. This discovery has enriched the distribution information of Reeve’s muntjac. This discovery will also play an important role in the study of biodiversity and species integrity in the area of Liupan Mountain.     

Complete mitochondrial genome of the leaf muntjac (Muntiacus putaoensis) and phylogenetics of the genus Muntiacus

The leaf muntjac (Muntiacus putaoensis) is an endemic deer species found in the east transHimalayan region.In recent years,population numbers have decreased due to heavy hunting and habitat loss,and little genetic data exists for this species,thus our knowledge of distribution rangs and population sizes likewise remain limited.We obtained mtDNA genes and the complete mitochondrial genome sequence of M.putaoensis using PCR,followed by direct sequencing.The complete mitogenome sequence was determined as a circular 16 349 bp mitochondrial genome,containing 13 protein-coding genes,two rRNA genes,22 tRNA genes,and one control region,the gene composition and order of which were similar to most other vertebrates so far reported.Most mitochondrial genes,except for ND6 and eight tRNAs,were encoded on the heavy strand.The overall base composition of the heavy strand was 33.1％ A,29.3％ T,24.2％ C,and 13.4％ G,with a strong AT bias of 62.4％.There were seven regions of gene overlap totaling 95 bp and 11 intergenic spacer regions totaling 74 bp.Phylogenetic analyses (ML and BI) among the Muntiacus genus based on the sequenced of mitogenome and ND4L-ND4 supported M.putaoensis as a member of Muntiacus,most closely related to M.vuquangensis.However,when analyses based on cyt b included two more muntjacs,M.truongsonensis was most closely related to M.putaoensis rather than M.vuquangensis,and together with M.rooseveltorum,likely forming a M.rooseveltorum complex of the species.This study will help in the exploration of the evolutionary history and taxonomic status of the leaf muntjac,as well as its protection as a genetic resource.     

A simple statistical analysis of Indian muntjac Giemsa band patterns.

Indian Muntiacus muntjac G-banded chromosomes were used for computerized analysis for standardized karyotype generation. Individual chromosomes on high-contrast photographic negatives were scanned densitometrically. Alignment of each chromosome for analysis was achieved by locating predominant peaks as well as the centromere. This provided better alignment that the use of the chromosome-end locations. The standardized set was obtained by determing the root-mean-square average density along 10-20 homologous chromosomes. The resulting standard karyotype differs from those published earlier. Prophase chromosomes exhibited greater detail than more condensed metaphase chromosomes. Although Indian muntjac chromosomes were used as a model, the method of analysis should be readily adaptable for examining chromosomes of any origin. The analytic technique should be within the capabilities of the smallest cytogenetic laboratories.     

New material of Muntiacinae (Artiodactyla,Mammalia) from the Late Miocene of the northeastern Qinghai-Tibetan Plateau,China

Recent expeditions in the northeastern Qinghai-Tibetan Plateau have produced additional mammalian fossils from the Lower Baodean (equivalent to Vallesian, Late Miocene), and, significantly, a new large and the earliest muntjac, Muntiacus noringenensis sp. nov. It is phylogenetically closer to the Late Pliocene M. fenghoensis and extant M. vuqangensis than the other fossil and extant muntjacs are. The existence of this muntjac and other folivores in the Late Miocene Qaidam Basin suggests a forested period in the basin and the evolution from a forested and humid environment to a desert one today. Such a dramatic evolution in environments is an evidence of the effect of the uplift of the Qinghai-Tibetan Plateau and of its influence on continental climates.     

Electrical fusion of cells and nucleiin vitro: A preliminary note

The electrical fusion of male skin fibroblasts of the Indian muntjac (Muntiacus muntjak)in vitro is described. Fusion of cell nuclei in the resulting multinucleated giant cells was observed after application of up to ten additional DC pulses. Electrical fusion of bovine erythrocytes and alveolar macrophages is also described. Cell fusion can be obtained without the use of protease pretreatment, but the yield of fused cells is about 20 times lower than with prior use of proteases.