Molecular phylogeny of musk deer: a genomic view with mitochondrial 16S rRNA and cytochrome b gene

The phylogenetic status of the infra order Pecora is controversial, even though it is supported by paleontological, morphological, and molecular evidence. We analyzed two mitochondrial genes (i.e., 16S rRNA and cytochrome b) to resolve the phylogenetic position of pecoran species, i.e., the Bovidae, Cervidae, and Moschidae endemic to the Indian subcontinent. We used phylogenetic analysis based on different algorithms, including neighbor joining, maximum parsimony, Bayesian inference, maximum likelihood, minimum evolution, median joining network, along with multidimensional scaling, and DNA word analysis. Our results established the basal position of Tragulidae and the monophyly of the infra order Pecora within the Suborder Ruminantia. Our results also demonstrated that Bovidae, Cervidae, and Moschidae are allied with the placement of musk deer as more closely related to bovids than to cervids. Molecular dating based on sequence analysis shows that the radiation of Pecora occurred during the early Oligocene and that the majority of the pecoran families radiated and dispersed rapidly during the Oligocene/Miocene transition.     

Molecular and morphological phylogenies of ruminantia and the alternative position of the moschidae

The ruminants constitute the largest group of ungulates, with >190 species, and its distribution is widespread throughout all continents except Australia and Antarctica. Six families are traditionally recognized within the suborder Ruminantia: Antilocapridae (pronghorns), Bovidae (cattle, sheep, and antelopes), Cervidae (deer), Giraffidae (giraffes and okapis), Moschidae (musk deer), and Tragulidae (chevrotains). The interrelationships of the families have been an area of controversy among morphology, palaeontology, and molecular studies, and almost all possible evolutionary scenarios have been proposed in the literature. We analyzed a large DNA data set (5,322 nucleotides) for 23 species including both mitochondrial (cytochrome b, 12S ribosomal RNA (rRNA), and 16S rRNA) and nuclear (kappa-casein, cytochrome P-450, lactoferrin, and alpha-lactalbumin) markers. Our results show that the family Tragulidae occupies a basal position with respect to all other ruminant families, confirming the traditional view that separates Tragulina and Pecora. Within the pecorans, Antilocapridae and Giraffidae emerge first, and the families Bovidae, Moschidae, and Cervidae are allied, with the unexpected placement of Moschus close to bovids rather than to cervids. We used these molecular results to assess the homoplastic evolution of morphological characters within the Ruminantia. A Bayesian relaxed molecular clock approach based on the continuous autocorrelation of evolutionary rates along branches was applied to estimate the divergence ages between the major clades of ruminants. The evolutionary radiation of Pecora occurred at the Early/Late Oligocene transition, and Pecoran families diversified and dispersed rapidly during the Early and Middle Miocene. We propose a biogeographic scenario to explain the extraordinary expansion of this group during the Cenozoic era.     

New ruminant hosts and wider geographic range identified for Babesia odocoilei (Emerson and Wright 1970)

Babesia odocoilei was found to infect two previously unknown host species, desert bighorn sheep (Ovis canadensis nelsoni) and musk oxen (Ovibos moschatus), both of which are members of the family Bovidae. Previously, B. odocoilei has been reported in only Cervidae hosts. New geographic regions where B. odocoilei infections have not been reported previously include Pennsylvania and New York, where fatal babesiosis has occurred in reindeer (Rangifer tarandus tarandus); New Hampshire, where elk (Cervus elaphus canadensis) have been affected; and California, home of the infected desert bighorn sheep. Infection with B. odocoilei in these hosts was confirmed by parasite small subunit ribosomal RNA gene sequence analysis. A serosurvey for B. odocoilei antibody activity in New Hampshire showed prevalence rates of 100% at two elk farms and 12% at another farm. Control of potential vector ticks, Ixodes scapularis, especially when translocating livestock, is imperative to prevent outbreaks of babesiosis in managed herds of potential host species.     

Antibodies to ruminant alpha-herpesviruses and pestiviruses in Norwegian cervids

A serologic survey revealed that Norwegian populations of free-ranging reindeer (Rangifer tarandus tarandus), roe deer (Capreolus capreolus), red deer (Cervus elaphus), and moose (Alces alces) have been exposed to alpha-herpesviruses and pestiviruses. A total of 3,796 serum samples collected during the period 1993-2000 were tested in a neutralization test for antibodies against bovine herpesvirus 1 (BHV-1) or cervid herpesvirus 2 (CerHV-2), and 3,897 samples were tested by a neutralization test and/or enzyme-linked immunosorbent assay for antibodies against bovine viral diarrhea virus (BVDV). Antibodies against alpha-herpesvirus were found in 28.5% of reindeer, 3.0% of roe deer, and 0.5% of red deer, while all moose samples were negative. In reindeer, the prevalence of seropositive animals increased with age and was higher in males than females. Antibodies against BVDV were detected in 12.3% of roe deer, 4.2% of reindeer, 2.0% of moose and 1.1% of red deer. The results indicate that both alpha-herpesvirus and pestivirus are endemic in reindeer and pestivirus is endemic in roe deer in Norway. The viruses may be specific cervid strains. Seropositive red deer and moose may have become exposed as a result of contact with other ruminant species.     

Insight into gene evolution within Cervidae and Bovidae through genetic variation in MHC-DQA in the black muntjac (Muntiacus crinifrons)

The critical role that the major histocompatibility complex plays in the immune recognition of parasites and pathogens makes its evolutionary dynamics exceptionally relevant to ecology, population biology, and conservation studies. The black muntjac is a rare deer endemic to a small mountainous region in eastern China. We found that this species has two DQA loci through cDNA expression and sequence variation analysis. The level of variation at both DQA loci was found to be extremely low (three alleles for DQA1 and four alleles for DQA2), possibly because of past bottlenecks and the species' relatively solitary behavior pattern. The ratio of d(N)/d(S) in the putative peptide binding region of the DQA2 locus (13.36, P = 0.012) was significantly larger than one but not that of DQA1 (0.94, P = 0.95), suggesting strong positive selection at the DQA2 but not at the DQA1 locus. This difference might reflect different sets of evolutionary selection pressures acting on the two loci. The phylogenetic tree showed that DQA1 alleles from two species of Cervidae and two of Bovidae grouped together, as did the DQA2 alleles. However, different genes from the four species were located in separate branches. These results lead us to suggest that these DQA alleles are derived from primordial DQA genes from a common ancestor and are maintained in Cervidae and Bovidae since their divergence around 25.5-27.8 million years ago.     

Mitochondrial DNA and microsatellite DNA variation in domestic reindeer (Rangifer tarandus tarandus) and relationships with wild caribou (Rangifer tarandus granti, Rangifer tarandus groenlandicus, and Rangifer tarandus caribou)

Reindeer (Rangifer tarandus tarandus) in Alaska are semidomestic livestock descended from 1280 animals introduced from Siberia, Russia, approximately 100 years ago. Genetic variation at 18 microsatellite DNA loci and the cytochrome b gene of mitochondrial DNA (mtDNA) was quantified in reindeer from Alaska, Siberia (Russia), and Scandinavia and compared with wild North American caribou. Mean sequence divergence among 15 mtDNA haplotypes in reindeer was 0.007 substitutions per nucleotide site, and reindeer mtDNA is polyphyletic with caribou mtDNA. Microsatellite allele and mtDNA haplotype frequencies are similar between Alaskan and Russian reindeer and differentiated between these and Scandinavian reindeer. The frequencies of microsatellite alleles and mtDNA haplotypes are different in reindeer and wild caribou (Rangifer tarandus granti, Rangifer tarandus groenlandicus, and Rangifer tarandus caribou). Alaskan reindeer have maintained a genetic variation comparable to that in Russia and differentiated from that of wild caribou, >100 years after their introduction to Alaska.     

Molecular phylogeny of deer (Cervidae: Artiodactyla)

Sequences of mitochondrial genes 12S and 16S rRNA (2 445 bp) and the region of the nuclear beta-spectrin gene (828 bp) were analyzed in members of the family Cervidae and in other artiodactyls. Several molecular synapomorphies characteristic both of Cervidae and musk deer have been found. According to our data, Cervidae is a sister clade to Bovidae, which are very close to Moschidae. The family Giraffidae is exterior to this common clade, while Antilocapridae occupies a more basal position. The family Cervidae proper splits into three clades including the genera Cervus and Muntiacus (1), Capreolus, Hydropotes, Alces (2), and Rangifer, Odocoileus, and the remaining genera (3). In general, our phylogenetic reconstructions conform to the results of earlier molecular genetic studies, but substantially differ from the traditional taxonomy of Ruminantia.     

基于Cyt b基因探讨羚羊亚科原羚属系统发生关系

原羚属物种在羚羊亚科中的分类地位尚存在很多争议。本文测定了原羚属的黄羊和藏原羚细胞色素b基因全序列(1140bp),并与牛科其它属31个种的同源序列进行比较,对其碱基组成变异情况及核苷酸序列差异进行了分析。基于细胞色素b基因全序列,用简约法(MP)、邻接法(NJ)和似然法(ML)构建了系统进化树。结果表明：黄羊和藏原羚的序列差异为3．78％,颠换数目近乎为0,其突变远未饱和;原羚属内黄羊和藏原羚为不同种,单系发生;原羚属与赛加羚羊属、犬羚属及跳羚属等并系发生,原羚属隶属于羚羊亚科,应为独立属;羚羊亚科组成属间多为并系起源。根据序列差异值2％／百万年的细胞色素6分子钟,推测黄羊和藏原羚分歧时间大约为1～2百万年;原羚属与羚羊亚科其它属分歧时间大约在5．7～8百万年。     

Phylogenetic relationships among deer in China derived from mitochondrial DNA cytochromeb sequences

The phylogenetic relationships of Cervidae and Moschidae were examined using partial sequence data of mitochondrial DNA (mtDNA) cytochromeb. Ten new sequences were obtained for six species of Cervidae and Moschidae, and aligned with those previously reported for other deer species. Our results demonstrated that the phylogenetic status of the taxa inferred from molecular data was congruent with taxonomy based on morphological studies. Cervidae formed a monophyletic group that consists of four subfamilies: Cervinae, Muntiacinae, Hydropotinae, and Odocoileinae. Moschidae occurred at the base of the Cervidae clade. On the basis of molecular clocks for genetic distance, the divergence time of mtDNA haplotypes within the subfamily Cervinae, among subfamilies in Cervidae, and between Moschidae and Cervidae was estimated to date 2–7 MYA, 6–10 MYA and 8–13 MYA, respectively.     

An epizootic of besnoitiosis in captive caribou (Rangifer tarandus caribou), reindeer (Rangifer tarandus tarandus) and mule deer (Odocoileus hemionus hemionus)

Besnoitia sp. was diagnosed in two caribou (Rangifer tarandus caribou) which died of pneumonia at the Assiniboine Park Zoo (Winnipeg, Manitoba, Canada) in 1983. During the following 3 yr besnoitiosis spread to an isolated herd of caribou, to mule deer (Odocoileus hemionus hemionus) and to reindeer (Rangifer tarandus tarandus). Reduction of exposure to biting insects appears to have reduced the transmission of besnoitiosis within the reindeer herd. The morbidity rate was approximately 82% in caribou and 67% in mule deer over the age of 2 mo. Most animals with clinical signs were euthanized; this precluded an estimation of the disease-related mortality rate. Twenty-eight caribou, 10 mule deer and three reindeer have been euthanized or died as a result of this epidemic. Attempts to artificially transmit the disease to potentially susceptible intermediate and definitive hosts were unsuccessful.     

Serologic survey for antibodies against Mycobacterium a vium subsp. paratuberculosis in free-ranging cervids from Norway

Affinity between protein-G and immunoglobulins from red deer (Cervus elaphus), moose (Alces alces), roe deer (Capreolus capreolus), and reindeer (Rangifer tarandus tarandus) was tested in a competition binding assay. Sera from red deer, reindeer, and moose inhibited the assay less than sera from cattle (less affinity), whereas sera from roe deer showed a slightly higher affinity to protein-G than did sera from cattle. The conclusion was made that protein-G could be used instead of anti-species antibodies for these cervid species, where the aim of the screening was to look for exposure or lack of exposure to mycobacteria in the tested populations. Serologic screening of 1,373 free-ranging cervids for antibodies against Mycobacterium avium subsp. paratuberculosis was conducted. All sera were tested by a protein-G-based antigen-absorbed enzyme-linked immunosorbent assay (ELISA). Seropositive moose (10/537; 1.9%), red deer (14/371; 3.8%), roe deer (6/49; 12.2%), and semidomesticated reindeer (11/325; 3.4%) were found, whereas wild reindeer (n = 91) were seronegative. In addition, the red deer sera were tested with a commercial ELISA, by which two animals tested positive and nine were suspicious of having M. avium subsp. paratuberculosis antibodies. Tissue samples and feces from 10 moose originating from a population with a clustering of seropositive animals were investigated by histology and bacteriology with negative results. Paratuberculosis has never been diagnosed in free-ranging or farmed cervid species in Norway. Thus, further studies are indicated to prove that the present findings reflect an infection with M. avium subsp. paratuberculosis.     

Examination of hemiplasy, homoplasy and phylogenetic discordance in chromosomal evolution of the Bovidae

Robertsonian chromosomal fusions predominate in shaping the genomes of many species of Bovidae. These and other cytogenetic data (from 52 taxa representing 51 species and 9 tribes of Bovidae) were (i) examined for usefulness in defining phylogenetic relationships and (ii) subsequently mapped to a consensus tree based on mitochondrial and nuclear DNA gene sequences with divergence dates of the corresponding species calculated from cytochrome b sequences. This permitted persistence time estimates for the various rearrangements. The chromosomal data resulted in an unsupported higher-level topology, but with recognition of the monophyly of some genera and tribes within Bovidae. The distribution and temporal spread of character states on the species tree is suggestive of a restricted role for hemiplasy (the retention of an ancestral chromosomal polymorphism through multiple speciation events) and for introgression (resulting from secondary contact among taxa), processes that can potentially lead to phylogenetic discordance. We conclude that the most probable interpretation for these data is that genuine karyotypic homoplasy predominates, but that hemiplasy (and/or introgression) is a realistic hypothesis for the observed patterns of several shared characters in Bovidae.     

Conservation of repetitive DNA sequences in deer species studied by Southern blot transfer

Summary The Cervidae show one of the largest variations in chromosome number found within a mammalian family. The five species of the deer family which are the subject of this study vary in chromosome number from 2n=70 to 2n=6. Digestion with the restriction enzymes EcoRI, HpaII, HaeIII and MspI reveals that there is a series of highly repetitive sequences forming similar band patterns in the different species. To obtain information on the degree of homology among these conserved sequences we isolated a HpaII restriction fragment of approximately 990 base pairs from reindeer DNA. This DNA sequence was³²P-labelled and hybridized by the Southern blot technique to DNAs cleaved with HpaII and HaeIII from the reindeer and four other Cervidae species. Hybridization to specific restriction fragments was recorded in all species. The patterns of hybridization showed a higher degree of similarity between reindeer, elk and roe deer than between reindeer and the Asiatic species (fallow deer and muntjac). Homologies are still present between the highly repetitive sequences of the five species despite the drastic reorganization that led to a change in chromosome number from 6 to 70.     

Experimental oral transmission of chronic wasting disease to reindeer (Rangifer tarandus tarandus)

Chronic wasting disease (CWD), a transmissible spongiform encephalopathy of cervids, remains prevalent in North American elk, white-tailed deer and mule deer. A natural case of CWD in reindeer (Rangifer tarandus tarandus) has not been reported despite potential habitat overlap with CWD-infected deer or elk herds. This study investigates the experimental transmission of CWD from elk or white-tailed deer to reindeer by the oral route of inoculation. Ante-mortem testing of the three reindeer exposed to CWD from white-tailed deer identified the accumulation of pathological PrP (PrP(CWD)) in the recto-anal mucosa associated lymphoid tissue (RAMALT) of two reindeer at 13.4 months post-inoculation. Terminal CWD occurred in the two RAMALT-positive reindeer at 18.5 and 20 months post-inoculation while one other reindeer in the white-tailed deer CWD inoculum group and none of the 3 reindeer exposed to elk CWD developed disease. Tissue distribution analysis of PrP(CWD) in CWD-affected reindeer revealed widespread deposition in central and peripheral nervous systems, lymphoreticular tissues, the gastrointestinal tract, neuroendocrine tissues and cardiac muscle. Analysis of prion protein gene (PRNP) sequences in the 6 reindeer identified polymorphisms at residues 2 (V/M), 129 (G/S), 138 (S/N) and 169 (V/M). These findings demonstrate that (i) a sub-population of reindeer are susceptible to CWD by oral inoculation implicating the potential for transmission to other Rangifer species, and (ii) certain reindeer PRNP polymorphisms may be protective against CWD infection.     

DNA evidence that Ostertagia gruehneri and Ostertagia arctica (Nematoda: ostertagiinae) in reindeer from Norway and Svalbard are conspecific

DNA sequences of ITS-1 and ITS-2 of rDNA were determined for 16 individual adult males each of Ostertagia gruehneri and Ostertagia arctica from Svalbard reindeer (Rangifer tarandus platyrhynchus) and Eurasian tundra reindeer (R. t. tarandus). Each ITS was virtually identical in O. gruehneri and O. arctica and the three mixed bases detected were shared by both species. Our results strongly suggest that O. gruehneri and O. arctica are dimorphic males of the same species.     

Molecular phylogeny based on the κ-casein and cytochrome b sequences in the mammalian suborder Ruminantia

Nucleotide sequences for the -casein precursor proteins have been determined from the genomic DNAs or hair roots of the Ruminantia. The coding regions, exons 2, 3, and 4, were amplified separately via the three kinds of PCRs and then directly sequenced. The primers were designed from the sequence of bovine -casein gene; they were applicable for the amplification of the -casein genes from the 13 species in the Ruminantia except exon 2 of the lesser mouse deer. These results permitted an easy phylogenetic analysis based on the sequences of an autosomal gene. A phylogenetic tree was constructed from the mature K-casein sequences and compared with the tree of the cytochrome b genes which were sequenced from the same individuals. The Cervidae (sika deer, Cervus nippon) were separated from the branch of the Bovidae on the tree of -casein genes with a relatively high confidence level of the bootstrap analysis, but included in the branch of the Bovidae on the tree of cytochrome b genes. The -casein tree indicated a monophyly of the subfamily Caprinae, although the internal branches were uncertain in the Caprinae. The tree based on the nucleotide sequences of cytochrome b genes clearly showed the relationships of the closely related species in the genus Capricornis consisting of serow (C. smatorensis), Japanese serow (C. crispus), and Formosan serow (C. swinhoei). These results would be explained by the difference of resolving power between the -casein and the cytochrome b sequences. Correspondence to: K. Chikuni     

一种鹿科动物标本的分子鉴定

中国是世界上鹿科动物资源最丰富的国家之一,共有18种,分属獐亚科、麂亚科、鹿亚科和美洲鹿亚科,占全球鹿种的41.7％(盛和林等,1992).在皖南分布的鹿科动物有梅花鹿华南亚种(Cervus nippon pseudaxis)、黑麂(Muntiacus crinifrons)、黄麂(Muntiacus reevesi)和毛冠鹿(Elaphadus cephalophus)(王岐山,1990).2001年11月从泾县获得一具标本(图1-A,标本现保存于安徽师范大学标本馆),体形及大小似梅花鹿,身体背部毛棕褐色,无明显白斑,尾背面黑色、腹面纯白,但鹿角主干表面光滑,无眉叉及其它分叉.鹿角是鹿科动物所特有的结构,是区别于其它有蹄类的标志之一.     

Molecular phylogenetic relationships among seven Japanese species of Cercopithifilaria

DNA sequences from a portion of the mitochondrial COI gene were used to clarify phylogenetic relationships among Japanese species in the genus Cercopithifilaria. Sequences were determined from seven Japanese species, five (C. shohoi, C. multicauda, C. minuta, C. tumidicervicata and C. bulboidea) from the serow (Capricornis crispus F. Bovidae) and two (C. longa and C. crassa) from the sika deer (Cervus nippon nippon F. Cervidae). No base substitutions were observed between C. bulboidea and C. longa, suggesting that recent host switching of a lineage of C. bulboidea between bovid and cervid hosts gave rise to C. longa. In phylogenetic trees inferred using a variety of methods, the morphologically ancestral type, C. bulboidea, appeared as a derived species. C. multicauda was found to be basal in the analyses. It seems therefore that C. multicauda is the most primitive out of the seven species.     

Evolution and recombination of bovine DNA repeats

The history of the abundant repeat elements in the bovine genome has been studied by comparative hybridization and PCR. The Bov-A and Bov-B SINE elements both emerged just after the divergence of the Camelidae and the true ruminants. A 31-bp subrepeat motif in satellites of the Bovidae species cattle, sheep, and goat is also present in Cervidae (deer) and apparently predates the Bovidae. However, the other components of the bovine satellites were amplified after the divergence of the cattle and the Caprinae (sheep and goat). A 23-bp motif, which as subrepeat of two major satellites occupies 5% of the cattle genome, emerged only after the split of the water buffalo and other cattle species. During the evolution of the Bovidae the satellite repeat units were shaped by recombination events involving subrepeats, other satellite components, and SINE elements. Differences in restriction sites of homologous satellites indicate a continuing rapid horizontal spread of new sequence variants. Correspondence to: J.A. Lenstra     

Evaluation of the fluorescence polarization assay and comparison to other serological assays for detection of brucellosis in cervids

The complement fixation test (CFT), competitive enzyme immunoassay (CELISA), indirect enzyme immunoassay (IELISA) and fluorescence polarization assay (FPA) were evaluated for the detection of antibodies to Brucella abortus and Brucella suis biotype 4 in caribou (Rangifer tarandus caribou), elk (Cervus elapus), red deer (Cervus elapus), and reindeer (Rangifer tarandus tarandus). When combining the data the FPA and the CELISA were determined to be the most suitable tests for serodiagnosis of Cervidae. The overall actual sensitivity of the CFT and the IELISA was 100%. The overall actual sensitivity for the CELISA and FPA was 99%. The overall relative specificity of the CFT (including treatment of anti-complementary data as positive or negative for analysis), the CELISA, the IELISA and the FPA were 65%, 93%, 99%, 99%, and 99%, respectively. The specificities of the buffered plate agglutination test (BPAT), the CFT, the CELISA, the FPA and the IELISA for 55 elk vaccinated with B. abortus strain 19 and tested 4 mo post vaccination were 14%, 31%, 51%, 84%, and 2%, respectively. The FPA is the diagnostic test of choice because it has sensitivity and specificity values comparable to the CELISA; it has the capability to distinguish vaccinal antibody and antibody resulting from exposure to cross-reacting organisms such as Yersinia enterocolitica 0:9 from antibody to Brucella spp. in most cases; it is technically simple to do; it is adaptable to field use and it is relatively inexpensive.