Whole-genome sequencing of wild Siberian musk deer (Moschus moschiferus) provides insights into its genetic features.

BACKGROUND: Siberian musk deer, one of the seven species, is distributed in coniferous forests of Asia. Worldwide, the population size of Siberian musk deer is threatened by severe illegal poaching for commercially valuable musk and meat, habitat losses, and forest fire. At present, this species is categorized as Vulnerable on the IUCN Red List. However, the genetic information of Siberian musk deer is largely unexplored. RESULTS: Here, we produced 3.10 Gb draft assembly of wild Siberian musk deer with a contig N50 of 29,145 bp and a scaffold N50 of 7,955,248 bp. We annotated 19,363 protein-coding genes and estimated 44.44% of the genome to be repetitive. Our phylogenetic analysis reveals that wild Siberian musk deer is closer to Bovidae than to Cervidae. Comparative analyses showed that the genetic features of Siberian musk deer adapted in cold and high-altitude environments. We sequenced two additional genomes of Siberian musk deer constructed demographic history indicated that changes in effective population size corresponded with recent glacial epochs. Finally, we identified several candidate genes that may play a role in the musk secretion based on transcriptome analysis. CONCLUSIONS: Here, we present a high-quality draft genome of wild Siberian musk deer, which will provide a valuable genetic resource for further investigations of this economically important musk deer.

Complete mitogenome of Kashmir musk deer (Moschus cupreus) and its comparative phylogenetic relationships.

Kashmir musk deer, Moschus cupreus (KMD) is one the most threatened species endemic to the Himalayan region of Kashmir, Pakistan and Afghanistan. Herein, we have sequenced, annotated and characterized the complete mitogenome of M. cupreus. The investigation and comparison of the mitogenome provide crucial information for phylogenetic analysis to understand the evolutionary relationships. The mitogenome of KMD was 16,354 bp long, comprising 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs) and non-coding control region. Its composition was highly A+T biased 68.42%, and exhibited a positive AT-skew (0.082) and negative GC-skew (- 0.307). The phylogenetic analysis suggested that KMD was a primitive and extant species in the genus Moschus, whereas Alpine musk deer (M. chrysogaster) and Himalayan musk deer (M. leucogaster) were the closest relatives. It indicated the placement of M. cupreus within the monotypic family Moschidae of musk deer. Hence, it provides a better understanding of lineage identification and musk deer evolution for further research.

Shiga toxin-producing Escherichia coli isolates from red deer (Cervus elaphus), roe deer (Capreolus capreolus) and fallow deer (Dama dama) in Poland.

Shiga toxin-producing Escherichia (E.) coli (STEC) pathogens are responsible for the outbreaks of serious diseases in humans, including haemolytic uraemic syndrome (HUS), bloody diarrhoea (BD) and diarrhoea (D), and they pose a significant public health concern. Wild ruminants are an important environmental reservoir of foodborne pathogens that can cause serious illnesses in humans and contaminate fresh products. There is a general scarcity of published data about wildlife as a reservoir of foodborne pathogens in Poland, which is why the potential epidemiological risk associated with red deer, roe deer and fallow deer as reservoirs of STEC/AE-STEC strains was evaluated in this study. The aim of the study was to investigate the prevalence of STEC strains in red deer (Cervus elaphus), roe deer (Capreolus capreolus) and fallow deer (Dama dama) populations in north-eastern Poland, and to evaluate the potential health risk associated with wild ruminants carrying STEC/AE-STEC strains. We examined 252 rectal swabs obtained from 134 roe deer (Capreolus capreolus), 97 red deer (Cervus elaphus) and 21 fallow deer (Dama dama) in north-eastern Poland. The samples were enriched in modified buffered peptone water. Polymerase chain reaction (PCR) assays were conducted to determine the virulence profile of stx1, stx2 and eae or aggR genes, to identify the subtypes of stx1 and stx2 genes, and to perform O and H serotyping. E. coli O157:H7 isolates were detected in the rectal swabs collected from 1/134 roe deer (0.75%) and 4/97 red deer (4.1%), and they were not detected in fallow deer (Dama dama). The remaining E. coli serogroups, namely O26, O103, O111 and O145 that belong to the "top five" non-O157 serogroups, were detected in 15/134 roe deer (11.19%), 18/97 red deer (18.56%) and 2/21 fallow deer (9.52%). STEC/AE-STEC strains were detected in 33 roe deer isolates (24.63%), 21 red deer isolates (21.65%) and 2 fallow deer isolates (9.52%). According to the most recent FAO/WHO report, stx2a and eae genes are the primary virulence traits associated with HUS, and these genes were identified in one roe deer isolate and one red deer isolate. Stx2 was the predominant stx gene, and it was detected in 78.79% of roe deer and in 71.43% of red deer isolates. The results of this study confirmed that red deer and roe deer in north-eastern Poland are carriers of STEC/AE-STEC strains that are potentially pathogenic for humans. This is the first report documenting the virulence of STEC/AE-STEC strains from wild ruminants in Poland.

Evidence for adaptive introgression of exons across a hybrid swarm in deer.

BACKGROUND: Secondary contact between closely related lineages can result in a variety of outcomes, including hybridization, depending upon the strength of reproductive barriers. By examining the extent to which different parts of the genome introgress, it is possible to infer the strength of selection and gain insight into the evolutionary trajectory of lineages. Following secondary contact approximately 8000 years ago in the Pacific Northwest, mule deer (Odocoileus hemionus hemionus) and black-tailed deer (O. h. columbianus) formed a hybrid swarm along the Cascade mountain range despite substantial differences in body size (up to two times) and habitat preference. In this study, we examined genetic population structure, extent of introgression, and selection pressures in freely interbreeding populations of mule deer and black-tailed deer using mitochondrial DNA sequences, 9 microsatellite loci, and 95 SNPs from protein-coding genes. RESULTS: We observed bi-directional hybridization and classified approximately one third of the 172 individuals as hybrids, almost all of which were beyond the F1 generation. High genetic differentiation between black-tailed deer and mule deer at protein-coding genes suggests that there is positive divergent selection, though selection on these loci is relatively weak. Contrary to predictions, there was not greater selection on protein-coding genes thought to be associated with immune function and mate choice. Geographic cline analyses were consistent across genetic markers, suggesting long-term stability (over hundreds of generations), and indicated that the center of the hybrid swarm is 20-30 km to the east of the Cascades ridgeline, where there is a steep ecological transition from wet, forested habitat to dry, scrub habitat. CONCLUSIONS: Our data are consistent with a genetic boundary between mule deer and black-tailed deer that is porous but maintained by many loci under weak selection having a substantial cumulative effect. The absence of clear reproductive barriers and the consistent centering of geographic clines at a sharp ecotone suggests that ecology is a driver of hybrid swarm dynamics. Adaptive introgression in this study (and others) promotes gene flow and provides valuable insight into selection strength on specific genes and the evolutionary trajectory of hybridizing taxa.

Genome-wide study on genetic diversity and phylogeny of five species in the genus Cervus.

BACKGROUND: Previous investigations of phylogeny in Cervus recovered many clades without whole genomic support. METHODS: In this study, the genetic diversity and phylogeny of 5 species (21 subspecies/populations from C. unicolor, C. albirostris, C. nippon, C. elaphus and C. eldii) in the genus Cervus were analyzed using reduced-representation genome sequencing. RESULTS: A total of 197,543 SNPs were identified with an average sequencing depth of 16 x. A total of 21 SNP matrices for each subspecies/population and 1 matrix for individual analysis were constructed, respectively. Nucleotide diversity and heterozygosity analysis showed that all 21 subspecies/populations had different degrees of genetic diversity. C. eldii, C. unicolor and C. albirostris showed relatively high expected and observed heterozygosity, while observed heterozygosity in C. nippon was the lowest, indicating there was a certain degree of inbreeding rate in these subspecies/populations. Phylogenetic ML tree of all Cervus based on the 21 SNP matrices showed 5 robustly supported clades that clearly separate C. eldii, C. unicolor, C. albirostris, C. elaphus and C. nippon. Within C. elaphus clade, 4 subclades were well differentiated and statistically highly supported: C. elaphus (New Zealand), C. e. yarkandensis, C. c. canadensis and the other grouping the rest of C. canadensis from China. In the C. nippon clade, 2 well-distinct subclades corresponding to C. n. aplodontus and other C. nippon populations were separated. Phylogenetic reconstruction indicated that the first evolutionary event of the genus Cervus occurred approximately 7.4 millions of years ago. The split between C. elaphus and C. nippon could be estimated at around 3.6 millions of years ago. Phylogenetic ML tree of all samples based on individual SNP matrices, together with geographic distribution, have shown that there were 3 major subclades of C. elaphus and C. canadensis in China, namely C. e. yarkandensis (distributed in Tarim Basin), C. c. macneilli/C. c. kansuensis/C. c. alashanicus (distributed in middle west of China), and C. c. songaricus/C. c. sibiricus (distributed in northwest of China). Among them, C. e. yarkandensis was molecularly the most primitive subclade, with a differentiation dating back to 0.8-2.2 Myr ago. D statistical analysis showed that there was high probability of interspecific gene exchange between C. albirostris and C. eldii, C. albirostris and C. unicolor, C. nippon and C. unicolor, and there might be 2 migration events among 5 species in the genus Cervus. CONCLUSIONS: Our results provided new insight to the genetic diversity and phylogeny of Cervus deer. In view of the current status of these populations, their conservation category will need to be reassessed.

Temporal patterns of chronic wasting disease prion excretion in three cervid species.

Chronic wasting disease (CWD) is the only naturally occurring transmissible spongiform encephalopathy affecting free-ranging wildlife populations. Transmission of CWD occurs by direct contact or through contaminated environments; however, little is known about the temporal patterns of CWD prion excretion and shedding in wild cervids. We tested the urine and faeces of three species of captive cervids (elk, mule and white-tailed deer) at 6, 12, 18 and 24 months after oral inoculation to evaluate the temporal, species- and genotype-specific factors affecting the excretion of CWD prions. Although none of the animals exhibited clinical signs of CWD during the study, we determined that all three cervid species were excreting CWD prions by 6 months post-inoculation. Faecal samples were consistently positive for CWD prions for all three cervid species (88 %), and were more likely to be positive than urine samples (28 %). Cervids with genotypes encoding for the prion protein (PRNP) that were considered to be more susceptible to CWD were more likely to excrete CWD prions (94 %) than cervids with genotypes considered to be less susceptible (64 %). All cervids with CWD prions in their urine also had positive faeces (n=5), but the converse was not true. Our study is the first to demonstrate CWD prion excretion in urine by asymptomatic elk and mule deer. Our results indicate that the excretion of CWD prions in faeces and, to a lesser extent, urine may provide an important avenue for depositing prions in the environment.

[Identification of Cervus nippon, C.elaphus and their hybridize samples based on COI and SRY gene].

For rapid identification of Cervus nippon, C. elaphus and their hybridize samples, the specific PCR for mutual authentication of them was established based on the SNPs in COI and SRY sequence. C. nippon, C. elaphus and their hybridize samples were collected from different origins, total DNA of 24 identified samples were extracted, and the COI and SRY gene was seqenced. SNPs in the COI and SRY sequences of the samples were found by Clustul X 2.1 program. Primers for identifying C. nippon and C. elaphus were designed according to the SNP site, two multi-PCR reaction system were established to identify them. In addition, 24 samples which were randomly collected in different herbal medicine market were identified. The band special for C. nippon (232 bp)and band special for C. elaphus (518 bp) based on COI sequence,and the band special for C. nippon (803 bp)and band special for C. elaphus (425 bp) based on SRY sequence, were found using multi-PCR reaction, and three of the twenty-four samples were identified as the hybridize samples. The multi-PCR reaction system could be used to identify C. nippon, C. elaphus and their hybridize samples.

Contrasting origin of B chromosomes in two cervids (Siberian roe deer and grey brocket deer) unravelled by chromosome-specific DNA sequencing.

BACKGROUND: B chromosomes are dispensable and variable karyotypic elements found in some species of animals, plants and fungi. They often originate from duplications and translocations of host genomic regions or result from hybridization. In most species, little is known about their DNA content. Here we perform high-throughput sequencing and analysis of B chromosomes of roe deer and brocket deer, the only representatives of Cetartiodactyla known to have B chromosomes. RESULTS: In this study we developed an approach to identify genomic regions present on chromosomes by high-throughput sequencing of DNA generated from flow-sorted chromosomes using degenerate-oligonucleotide-primed PCR. Application of this method on small cattle autosomes revealed a previously described KIT gene region translocation associated with colour sidedness. Implementing this approach to B chromosomes from two cervid species, Siberian roe deer (Capreolus pygargus) and grey brocket deer (Mazama gouazoubira), revealed dramatically different genetic content: roe deer B chromosomes consisted of two duplicated genomic regions (a total of 1.42-1.98 Mbp) involving three genes, while grey brocket deer B chromosomes contained 26 duplicated regions (a total of 8.28-9.31 Mbp) with 34 complete and 21 partial genes, including KIT and RET protooncogenes, previously found on supernumerary chromosomes in canids. Sequence variation analysis of roe deer B chromosomes revealed a high frequency of mutations and increased heterozygosity due to either amplification within B chromosomes or divergence between different Bs. In contrast, grey brocket deer B chromosomes were found to be more homogeneous and resembled autosomes in patterns of sequence variation. Similar tendencies were observed in repetitive DNA composition. CONCLUSIONS: Our data demonstrate independent origins of B chromosomes in the grey brocket and roe deer. We hypothesize that the B chromosomes of these two cervid species represent different stages of B chromosome sequences evolution: probably nascent and similar to autosomal copies in brocket deer, highly derived in roe deer. Based on the presence of the same orthologous protooncogenes in canids and brocket deer Bs we argue that genomic regions involved in B chromosome formation are not random. In addition, our approach is also applicable to the characterization of other evolutionary and clinical rearrangements.

Variation in stability of elk and red deer populations with abiotic and biotic factors at the species-distribution scale.

Stability in population dynamics is an emergent property of the interaction between direct and delayed density dependence, the strengths of which vary with environmental covariates. Analysis of variation across populations in the strength of direct and delayed density dependence can reveal variation in stability properties of populations at the species level. We examined the stability properties of 22 elk/red deer populations in a two-stage analysis. First, we estimated direct and delayed density dependence applying an AR(2) model in a Bayesian hierarchical framework. Second, we plotted the coefficients of direct and delayed density dependence in the Royama parameter plane. We then used a hierarchical approach to test the significance of environmental covariates of direct and delayed density dependence. Three populations exhibited highly stable and convergent dynamics with strong direct, and weak delayed, density dependence. The remaining 19 populations exhibited more complex dynamics characterized by multi-annual fluctuations. Most (15 of 19) of these exhibited a combination of weak to moderate direct and delayed density dependence. Best-fit models included environmental covariates in 17 populations (77% of the total). Of these, interannual variation in growing-season primary productivity and interannual variation in winter temperature were the most common, performing as the best-fit covariate in six and five populations, respectively. Interannual variation in growing-season primary productivity was associated with the weakest combination of direct and delayed density dependence, while interannual variation in winter temperature was associated with the strongest combination of direct and delayed density dependence. These results accord with a classic theoretical prediction that environmental variability should weaken population stability. They furthermore suggest that two forms of environmental variability, one related to forage resources and the other related to abiotic conditions, both reduce stability, but in opposing fashion: one through weakened direct density dependence and the other through strengthened delayed density dependence. Importantly, however, no single abiotic or biotic environmental factor emerged as generally predictive of the strengths of direct or delayed density dependence, nor of the stability properties emerging from their interaction. Our results emphasize the challenges inherent to ascribing primacy to drivers of such parameters at the species level and distribution scale.

Genetic Structure and Effective Population Sizes in European Red Deer (Cervus elaphus) at a Continental Scale: Insights from Microsatellite DNA.

We analyzed more than 600 red deer (Cervus elaphus) from large parts of its European distribution range at 13 microsatellite loci, presenting the first continent-wide study of this species using nuclear markers. Populations were clearly differentiated (overall F ST = 0.166, Jost's D est = 0.385), and the BAPS clustering algorithm yielded mainly geographically limited and adjacent genetic units. When forced into only 3 genetic clusters our data set produced a very similar geographic pattern as previously found in mtDNA phylogeographic studies: a western group from Iberia to central and parts of Eastern Europe, an eastern group from the Balkans to Eastern Europe, and a third group including the threatened relict populations from Sardinia and Mesola in Italy. This result was also confirmed by a multivariate approach to analyzing our data set, a discriminant analysis of principal components. Calculations of genetic diversity and effective population sizes (linkage disequilibrium approach) yielded the lowest results for Italian (Sardinia, Mesola; N e between 2 and 8) and Scandinavian red deer, in line with known bottlenecks in these populations. Our study is the first to present comparative nuclear genetic data in red deer across Europe and may serve as a baseline for future analyses of genetic diversity and structuring in this widespread ungulate.

Development and Characterization of 15 Polymorphic Dinucleotide Microsatellite Markers for Tule Elk Using HiSeq3000.

The tule elk (Cervus elaphus nannodes) experienced a severe bottleneck in the 1800s, resulting in low genetic diversity. There is a need for high-resolution genetic assays that can be used to differentiate individual elk, including close relatives, with high confidence. An efficient assay requires multiple markers both polymorphic and that can be amplified in concert with other markers in multiplex reactions. To develop such markers, we employed 150-bp paired-end whole genome shotgun sequencing on an Illumina HiSeq3000 platform to discover dinucleotide microsatellite markers. After preliminary screening of these markers, we selected and screened 15 candidate loci and 5 existing tetra nucleotide markers in 56 tule elk. We combined these markers in 2 multiplex reactions and report primer concentrations and PCR conditions enabling their efficient amplification.

Growth in fossil and extant deer and implications for body size and life history evolution.

BACKGROUND: Body size variation within clades of mammals is widespread, but the developmental and life-history mechanisms by which this variation is achieved are poorly understood, especially in extinct forms. An illustrative case study is that of the dwarfed morphotypes of Candiacervus from the Pleistocene of Crete versus the giant deer Megaloceros giganteus, both in a clade together with Dama dama among extant species. Histological analyses of long bones and teeth in a phylogenetic context have been shown to provide reliable estimates of growth and life history patterns in extant and extinct mammals. RESULTS: Similarity of bone tissue types across the eight species examined indicates a comparable mode of growth in deer, with long bones mainly possessing primary plexiform fibrolamellar bone. Low absolute growth rates characterize dwarf Candiacervus sp. II and C. ropalophorus compared to Megaloceros giganteus displaying high rates, whereas Dama dama is characterized by intermediate to low growth rates. The lowest recorded rates are those of the Miocene small stem cervid Procervulus praelucidus. Skeletal maturity estimates indicate late attainment in sampled Candiacervus and Procervulus praelucidus. Tooth cementum analysis of first molars of two senile Megaloceros giganteus specimens revealed ages of 16 and 19 years whereas two old dwarf Candiacervus specimens gave ages of 12 and 18 years. CONCLUSIONS: There is a rich histological record of growth across deer species recorded in long bones and teeth, which can be used to understand ontogenetic patterns within species and phylogenetic ones across species. Growth rates sensu Sander & Tückmantel plotted against the anteroposterior bone diameter as a proxy for body mass indicate three groups: one with high growth rates including Megaloceros, Cervus, Alces, and Dama; an intermediate group with Capreolus and Muntiacus; and a group showing low growth rates, including dwarf Candiacervus and Procervulus. Dwarf Candiacervus, in an allometric context, show an extended lifespan compared to other deer of similar body size such as Mazama which has a maximum longevity of 12 years in the wild. Comparison with other clades of mammals reveals that changes in size and life history in evolution have occurred in parallel, with various modes of skeletal tissue modification.

A novel and rapid diagnostic method for discriminating between feces of sika deer and Japanese serow by loop-mediated isothermal amplification.

Severe damages to natural vegetation, agriculture, and forestry caused by overpopulation of sika deer (Cervus nippon) have markedly increased in Japan in recent years. To devise a population management plan of sika deer, information on the distribution and population size of the animal in each region is indispensable. An easy and effective method to obtain this information is to count the fecal pellets in the field. However, the habitat of sika deer in Japan overlaps that of Japanese serow (Capricornis crispus). Additionally, it is difficult to discriminate between the feces of both animals. Here, we present a rapid and precise diagnostic method for discriminating between the feces of sika deer and Japanese serow using loop-mediated isothermal amplification (LAMP) targeting cytochrome b gene in the mitochondrial DNA. Our results showed that the LAMP can discriminate between the feces of sika deer and Japanese serow, and the method is simpler and more sensitive than the conventional molecular diagnostic method. Since LAMP method does not require special skills for molecular biology techniques, even the field researchers who have never done a molecular experiment can easily carry out the protocol. In addition, the entire protocol, from DNA extraction from fecal pellet to identification of species, takes only about 75 min and does not require expensive equipment. Hence, this diagnostic method is simple, fast, and accessible to anyone. As such, the method can be a useful tool to estimate distribution and population size of sika deer.

Species cross-amplification, identification and genetic variation of 17 species of deer (Cervidae) with microsatellite and mitochondrial DNA from antlers.

Strong anthropogenic impact has caused 28 of the currently recognized 55 species of deer (Cervidae) to be listed on the IUCN Red List. Particular threats to vulnerable species include habitat deterioration and hybridization with alien, introduced species. The scarcity of many species has severely hampered genetic analyses of their populations, including the detection of loci for cross-species amplification. Because deer antlers are shed and re-grown annually, antlers offer the possibility for non-invasive genetic sampling of large individual numbers, and may provide material for reference genotyping from historical samples stored in zoos, museums and trophy collections of rare and extinct species/populations. In this paper, we report cross-species amplification of 19 nuclear microsatellite loci and the amplification of 16S mtDNA for barcoding from nearly a third of all deer species worldwide based on high quality DNA extracted from antler bone up to 40 years old. Phylogenetic analysis based on mtDNA of seventeen species and five subspecies corroborate previously published phylogenetic data, thus confirming the specific resolution of the DNA extraction methodology.

Molecular cloning and evolutionary analysis of captive forest musk deer bitter taste receptor gene T2R16.

Sensing bitter tastes is crucial for most animals because it can prevent them from ingesting harmful food. This process is mainly mediated by the bitter taste receptors (T2R) that are largely expressed in the taste buds. Previous studies have identified some T2R gene repertoires. Marked variation in repertoire size has been noted among species. However, research on T2Rs is still limited and the mechanisms underlying the evolution of vertebrate T2Rs remain poorly understood. In the present study, we analyzed the structure and features of the protein encoded by the forest musk deer (Moschus berezovskii) T2R16 and submitted the gene sequence to NCBI GenBank. The results showed that the full coding DNA sequence (CDS) of musk deer T2R16 (GenBank accession No. KP677279) was 906 bp, encoding 301 amino acids, which contained ATG start codon and TGA stop codon, with a calculated molecular weight of 35.03 kDa and an isoelectric point of 9.56. The T2R16 protein receptor had seven conserved transmembrane regions. Hydrophobicity analysis showed that most amino acid residues in T2R16 protein were hydrophobic, and the grand average of hydrophobicity (GRAVY) was 0.657. Phylogenetic analysis based on this gene revealed that forest musk deer had the closest association with sheep (Ovis aries), as compared to cow (Bos taurus), Tursiops truncatus, and other species, whereas it was genetically farthest from humans (Homo sapiens). We hope these results would complement the existing data on T2R16 and encourage further research in this respect.

[Research progress on molecular genetics of forest musk deer].

Forest musk deer is one of the large-scale farming musk deer animals with the largest population at the same time. The male musk deer can secrete valuable medicines, which has high medicinal and economic value. Due to the loss of habitat and indiscriminate hunting, the numbers of wild population specie and the distribution have been drastically reduced. Therefore, in-depth understanding of the molecular genetics progress of forest musk deer will pave a way for musk deer protection and breeding. In this review, the progress associated with the molecular marker, genetic classification, artificial breeding, musk secretion and disease in past decades were reviewed, in order to provide a theoretical basis for subsequent molecular genetic researches in forest musk deer.

[Molecular identification of hairy antler by analysis of high resolution melting].

High resolution melting (HRM) , an important technology for genotyping and mutation scanning, has broad prospects in the authenticity of traditional Chinese medicine. This paper selected universal CO I primers and used HRM to establish a new method for authenticity of Hairy Antler. PCR was conducted at the annealing temperature of 60 °C and 45 cycles. The range of the DNA template concentration, the primer concentration and the Mg2+ ion concentration were further optimized. The results showed that the Tm values of Cervus nippon were (81.96 ± 0.07), (84.51 ± 0.03) °C and Cervus elaphus was(82.58 ± 0.13), (85.95 ± 0.05) °C with 10-100 mg · L(-1) DNA template, 0.2 µLmol · L(-1) primer, 2.0 mmol · L(-1) Mg2+. This method can authenticate of hairy antler and is simple, fast, high-throughput, visualization.

The role of chromosome variation in the speciation of the red brocket deer complex: the study of reproductive isolation in females.

BACKGROUND: The red brocket deer, Mazama americana, has at least six distinct karyotypes in different regions of South America that suggest the existence of various species that are today all referred to as M. americana. From an evolutionary perspective, the red brockets are a relatively recent clade that has gone through intense diversification. This study sought to prove the existence of post-zygotic reproductive isolation in deer offspring between distinct chromosome lineages. To achieve this, inter-cytotype and intra-cytotype crosses were performed, which resulted in both F1 hybrid (n = 5) and pure offspring (n = 3) in captivity. RESULTS: F1 females were analyzed in terms of their karyotypes, ovarian histology, estrous cycles and in vitro embryo production. Pure females presented parameters that were similar to those previously reported for M. Americana; however, the parameters for hybrid females were different. Two hybrids were determined to be sterile, while the remaining hybrids presented characteristics of subfertility. CONCLUSIONS: The results support the existence of well-established reproductive isolation among the most distant karyotype lineages and elucidates the need to define all karyotype variants and their geographical ranges in order to define the number of species of red brocket.

Range-wide analysis of genetic structure in a widespread, highly mobile species (Odocoileus hemionus) reveals the importance of historical biogeography.

Highly mobile species that thrive in a wide range of habitats are expected to show little genetic differentiation across their range. A limited but growing number of studies have revealed that patterns of broad-scale genetic differentiation can and do emerge in vagile, continuously distributed species. However, these patterns are complex and often shaped by both historical and ecological factors. Comprehensive surveys of genetic variation at a broad scale and at high resolution are useful for detecting cryptic spatial genetic structure and for investigating the relative roles of historical and ecological processes in structuring widespread, highly mobile species. In this study, we analysed 10 microsatellite loci from over 1900 samples collected across the full range of mule deer (Odocoileus hemionus), one of the most widely distributed and abundant of all large mammal species in North America. Through both individual- and population-based analyses, we found evidence for three main genetic lineages, one corresponding to the 'mule deer' morphological type and two to the 'black-tailed deer' type. Historical biogeographic events likely are the primary drivers of genetic divergence in this species; boundaries of the three lineages correspond well with predictions based on Pleistocene glacial cycles, and substructure within each lineage demonstrates island vicariance. However, across large geographic areas, including the entire mule deer lineage, we found that genetic variation fit an isolation-by-distance pattern rather than discrete clusters. A lack of genetic structure across wide geographic areas of the continental west indicates that ecological processes have not resulted in restrictions to gene flow sufficient for spatial genetic structure to emerge. Our results have important implications for our understanding of evolutionary mechanisms of divergence, as well as for taxonomy, conservation and management.

Evolutionary neutrality of mtDNA introgression: evidence from complete mitogenome analysis in roe deer.

Introgressive hybridization offers a unique platform for studying the molecular basis of natural selection acting on mitogenomes. Most of the mtDNA protein-coding genes are extremely conserved; however, some of the observed variations have potentially adaptive significance. Here, we evaluated whether the evolution of mtDNA in closely related roe deer species affected by widespread mtDNA introgression is neutral or adaptive. We characterized and compared 16 complete mitogenomes of European (Capreolus capreolus) and Siberian (C. pygargus) roe deer, including four of Siberian origin introgressed into European species. The average sequence divergence of species-specific lineages was estimated at 2.8% and varied across gene classes. Only 21 of 315 fixed differences identified in protein-coding genes represented nonsynonymous changes. Only three of them were determined to have arisen in the C. pygargus lineage since the time to the most recent common ancestor (TMRCA) of both Capreolus species, reflecting a decelerated evolutionary ratio. The almost four-fold higher dN /dS ratio described for the European roe deer lineage is constrained by overall purifying selection, especially pronounced in the ND4 and ND5 genes. We suggest that the highly divergent C. capreolus lineage could have maintained a capability for genomic incorporation of the well-preserved and almost ancestral type of mtDNA present in C. pygargus. Our analyses did not indicate any signs of positive selection for Siberian roe deer mtDNA, suggesting that the present widespread introgression is evolutionarily neutral.