Hi-C sequencing unravels dynamic three-dimensional chromatin interactions in muntjac lineage: insights from chromosome fusions in Fea's muntjac genome.

Eukaryotes have varying numbers and structures of characteristic chromosomes across lineages or species. The evolutionary trajectory of species may have been affected by spontaneous genome rearrangements. Chromosome fusion drastically alters karyotypes. However, the mechanisms and consequences of chromosome fusions, particularly in muntjac species, are poorly understood. Recent research-based advancements in three-dimensional (3D) genomics, particularly high-throughput chromatin conformation capture (Hi-C) sequencing, have allowed for the identification of chromosome fusions and provided mechanistic insights into three muntjac species: Muntiacus muntjak, M. reevesi, and M. crinifrons. This study aimed to uncover potential genome rearrangement patterns in the threatened species Fea's muntjac (Muntiacus feae), which have not been previously examined for such characteristics. Deep Hi-C sequencing (31.42 × coverage) was performed to reveal the 3D chromatin architecture of the Fea's muntjac genome. Patterns of repeated chromosome fusions that were potentially mediated by high-abundance transposable elements were identified. Comparative Hi-C maps demonstrated linkage homology between the sex chromosomes in Fea's muntjac and autosomes in M. reevesi, indicating that fusions may have played a crucial role in the evolution of the sex chromosomes of the lineage. The species-level dynamics of topologically associated domains (TADs) suggest that TAD organization could be altered by differential chromosome interactions owing to repeated chromosome fusions. However, research on the effect of TADs on muntjac genome evolution is insufficient. This study generated Hi-C data for the Fea's muntjac, providing a genomic resource for future investigations of the evolutionary patterns of chromatin conformation at the chromosomal level.

Microtubule search-and-capture model evaluates the effect of chromosomal volume conservation on spindle assembly during mitosis.

Variation in the chromosome numbers can arise from the erroneous mitosis or fusion and fission of chromosomes. While the mitotic errors lead to an increase or decrease in the overall chromosomal substance in the daughter cells, fission and fusion keep this conserved. Variations in chromosome numbers are assumed to be a crucial driver of speciation. For example, the members of the muntjac species are known to have very different karyotypes with the chromosome numbers varying from 2n=70+3B in the brown brocket deer to 2n=46 in the Chinese muntjac and 2n=6/7 in the Indian muntjac. The chromosomal content in the nucleus of these closely related mammals is roughly the same and various chromosome fusion and fission pathways have been suggested as the evolution process of these karyotypes. Similar trends can also be found in lepidoptera and yeast species which show a wide variation of chromosome numbers. The effect of chromosome number variation on the spindle assembly time and accuracy is still not properly addressed. We computationally investigate the effect of conservation of the total chromosomal substance on the spindle assembly during prometaphase. Our results suggest that chromosomal fusion pathways aid the microtubule-driven search and capture of the kinetochore in cells with monocentric chromosomes. We further report a comparative analysis of the site and percentage of amphitelic captures, dependence on cell shape, and position of the kinetochore in respect to chromosomal volume partitioning.

Comparative studies of X chromosomes in Cervidae family.

The family Cervidae is the second most diverse in the infraorder Pecora and is characterized by variability in the diploid chromosome numbers among species. X chromosomes in Cervidae evolved through complex chromosomal rearrangements of conserved segments within the chromosome, changes in centromere position, heterochromatic variation, and X-autosomal translocations. The family Cervidae consists of two subfamilies: Cervinae and Capreolinae. Here we build a detailed X chromosome map with 29 cattle bacterial artificial chromosomes of representatives of both subfamilies: reindeer (Rangifer tarandus), gray brocket deer (Mazama gouazoubira), Chinese water deer (Hydropotes inermis) (Capreolinae); black muntjac (Muntiacus crinifrons), tufted deer (Elaphodus cephalophus), sika deer (Cervus nippon) and red deer (Cervus elaphus) (Cervinae). To track chromosomal rearrangements during Cervidae evolution, we summarized new data, and compared them with available X chromosomal maps and chromosome level assemblies of other species. We demonstrate the types of rearrangements that may have underlined the variability of Cervidae X chromosomes. We detected two types of cervine X chromosome-acrocentric and submetacentric. The acrocentric type is found in three independent deer lineages (subfamily Cervinae and in two Capreolinae tribes-Odocoileini and Capreolini). We show that chromosomal rearrangements on the X-chromosome in Cervidae occur at a higher frequency than in the entire Ruminantia lineage: the rate of rearrangements is 2 per 10 million years.

Genetic similarities and phylogenetic analysis of Muntjac (Muntiacus spp. ) by comparing the nucleotide sequence of 16S rRNA and cytochrome B genome / Semelhanças genéticas e análise filogenética de muntjac (Muntiacus spp. ) comparando a sequência de nucleótidos de rRNA 16S e genoma citocromo B

This study aimed to identify the phylogenetic similarities among the muntjac (Muntiacus spp.). The phylogenetic similarities among seven major muntjac species were studied by comparing the nucleotide sequence of 16s rRNA and cytochrome b genome. Nucleotide sequences, retrieved from NCBI databases were aligned by using DNASTAR software. A phylogenetic tree was created for the selected species of muntjac by using the maximum likelihood method on MEGA7 software. The results of nucleotide sequences (16s rRNA) showed phylogenetic similarities between, the M. truongsonensis and M. rooseveltorum had the highest (99.2%) while the lowest similarities (96.8%) found between M. crinifrons and M. putaoensi. While the results of nucleotide sequences (Cty b) showed the highest similarity (100%) between M. muntjak and M. truongsonensis and the lowest s (91.5%) among M. putaoensis and M. crinifrons. The phylogenetic tree of muntjac species (16s rRNA gene) shows the main two clusters, the one including M. putaoensis, M. truongsonensis, M. rooseveltorum, and M. muntjak, and the second one including M. crinifrons and M. vuquangensis. The M. reevesi exists separately in the phylogenetic tree. The phylogenetic tree of muntjac species using cytochrome b genes shows that the M. muntjak and M. truongsonensis are clustered in the same group.

Este estudo visou identificar as semelhanças filogenéticas entre os muntjac (Muntiacus spp.). As semelhanças filogenéticas entre sete grandes espécies muntjac foram estudadas comparando a sequência de nucleótidos de 16s rRNA e genoma citocromo b. As sequências de nucleótidos, obtidas a partir de bases de dados NCBI, foram alinhadas utilizando o software DNASTAR. Foi criada uma árvore filogenética para as espécies selecionadas de muntjac utilizando o método de probabilidade máxima no software MEGA7. Os resultados das sequências de nucleótidos (16s rRNA) mostraram semelhanças filogenéticas entre o M. truongsonensis e o M. rooseveltorum tiveram o maior número (99,2%) enquanto as semelhanças mais baixas (96,8%) encontradas entre M. crinifrons e M. putaoensi. Enquanto os resultados das sequências de nucleótidos (Cty-b) apresentaram a maior semelhança (100%) entre M. muntjak e M. truongsonensis e os mais baixos (91,5%) entre M. putaoensis e M. crinifrons. A árvore filogenética das espécies muntjac (gene rRNA 16s) mostra os dois principais aglomerados, o que inclui M. putaoensis, M. truongsonensis, M. rooseveltorum e M. muntjak, e o segundo incluindo M. crinifrons e M. vuquangensis. O M. reevesi existe separadamente na árvore filogenética. A árvore filogenética das espécies muntjac usando genes citocromo b mostra que os M. muntjak e M. truongsonensis estão agrupados no mesmo grupo.

Comparative Studies of Karyotypes in the Cervidae Family.

The family Cervidae is the second most diverse family in the infraorder Pecora and is characterized by a striking variability in the diploid chromosome numbers among species, ranging from 6 to 70. Chromosomal rearrangements in Cervidae have been studied in detail by chromosome painting. There are many comparative cytogenetic data for both subfamilies (Cervinae and Capreolinae) based on homologies with chromosomes of cattle and Chinese muntjac. Previously it was found that interchromosomal rearrangements are the major type of rearrangements occurring in the Cervidae family. Here, we build a detailed chromosome map of a female reindeer (Rangifer tarandus, 2n = 70, Capreolinae) and a female black muntjac (Muntiacus crinifrons, 2n = 8, Cervinae) with dromedary homologies to find out what other types of rearrangements may have underlined the variability of Cervidae karyotypes. To track chromosomal rearrangements and the distribution of nucleolus organizer regions not only during Cervidae but also Pecora evolution, we summarized new data and compared them with chromosomal maps of other already studied species. We discuss changes in the pecoran ancestral karyotype in the light of new painting data. We show that intrachromosomal rearrangements in autosomes of Cervidae are more frequent than previously thought: at least 13 inversions in evolutionary breakpoint regions were detected.

Molecular mechanisms and topological consequences of drastic chromosomal rearrangements of muntjac deer.

Muntjac deer have experienced drastic karyotype changes during their speciation, making it an ideal model for studying mechanisms and functional consequences of mammalian chromosome evolution. Here we generated chromosome-level genomes for Hydropotes inermis (2n = 70), Muntiacus reevesi (2n = 46), female and male M. crinifrons (2n = 8/9) and a contig-level genome for M. gongshanensis (2n = 8/9). These high-quality genomes combined with Hi-C data allowed us to reveal the evolution of 3D chromatin architectures during mammalian chromosome evolution. We find that the chromosome fusion events of muntjac species did not alter the A/B compartment structure and topologically associated domains near the fusion sites, but new chromatin interactions were gradually established across the fusion sites. The recently borne neo-Y chromosome of M. crinifrons, which underwent male-specific inversions, has dramatically restructured chromatin compartments, recapitulating the early evolution of canonical mammalian Y chromosomes. We also reveal that a complex structure containing unique centromeric satellite, truncated telomeric and palindrome repeats might have mediated muntjacs' recurrent chromosome fusions. These results provide insights into the recurrent chromosome tandem fusion in muntjacs, early evolution of mammalian sex chromosomes, and reveal how chromosome rearrangements can reshape the 3D chromatin regulatory conformations during species evolution.

Gene Expression Analysis with No Sequence Data: Study on Reeves's Muntjac (Muntiacus reevesi).

Despite scientific progress, the gene sequences for many species not commonly used in research have not yet been analyzed. This makes it difficult to carry out molecular studies on such animals, as the sequence of genes is the basic information used in many techniques. In this study, we attempt to design primers for a real-time PCR analysis, basing on a comparative analysis of selected gene sequences of species related to Reeves's muntjac (Muntiacus reevesi) and by identifying highly conservative regions. Results of PCR products sequencing and their alignment with the GenBank collection show that all selected primers gave products highly similar (> 90%) to the intended target (among compared species), which led us to the conclusion that our primers may be used for further analyses of gene expression.

Genetic similarities and phylogenetic analysis of Muntjac (Muntiacus spp.) by comparing the nucleotide sequence of 16S rRNA and cytochrome B genome.

This study aimed to identify the phylogenetic similarities among the muntjac (Muntiacus spp.). The phylogenetic similarities among seven major muntjac species were studied by comparing the nucleotide sequence of 16s rRNA and cytochrome b genome. Nucleotide sequences, retrieved from NCBI databases were aligned by using DNASTAR software. A phylogenetic tree was created for the selected species of muntjac by using the maximum likelihood method on MEGA7 software. The results of nucleotide sequences (16s rRNA) showed phylogenetic similarities between, the M. truongsonensis and M. rooseveltorum had the highest (99.2%) while the lowest similarities (96.8%) found between M. crinifrons and M. putaoensi. While the results of nucleotide sequences (Cty b) showed the highest similarity (100%) between M. muntjak and M. truongsonensis and the lowest s (91.5%) among M. putaoensis and M. crinifrons. The phylogenetic tree of muntjac species (16s rRNA gene) shows the main two clusters, the one including M. putaoensis, M. truongsonensis, M. rooseveltorum, and M. muntjak, and the second one including M. crinifrons and M. vuquangensis. The M. reevesi exists separately in the phylogenetic tree. The phylogenetic tree of muntjac species using cytochrome b genes shows that the M. muntjak and M. truongsonensis are clustered in the same group.

Molecular phylogeny of the genus Muntiacus with special emphasis on the phylogenetic position of Muntiacus gongshanensis.

Muntjac deer (Cervidae: Muntiacus) are often cited as an excellent model for the study of vertebrate evolution due to their fast rate of change in chromosome number among vertebrates. However, the phylogenetic relationships within Muntiacus generally, and the taxonomic status of Muntiacus gongshanensis specifically, remain unclear. Here, the phylogenetic relationships within Muntiacus were studied using mitochondrial genome (mitogenome) and cytochrome b (cyt b) segments. Our results recognize 12 species within Muntiacus and support the controversial species M. gongshanensis, M. putaoensis, and M. malabaricus. Furthermore, Bayesian inference (BI) and maximum-likelihood (ML) approaches revealed M. gongshanensis and M. crinifrons to be closely related species, with M. feae as their sister species, and M. putaoensis and M. truongsonensis to be closely related, with M. rooseveltorum as their sister species. The distribution range of M. gongshanensis was also confirmed in southwest China (Namdapha, Modong, Zayu and Gongshan) and northern Myanmar (Putao). The results of this study provide insight into the evolution of Muntiacus and further provide a molecular basis for the taxonomic evaluation of the genus in the future and fundamental data for the conservation of M. gongshanensis.

Analysis of muntjac deer genome and chromatin architecture reveals rapid karyotype evolution.

Closely related muntjac deer show striking karyotype differences. Here we describe chromosome-scale genome assemblies for Chinese and Indian muntjacs, Muntiacus reevesi (2n = 46) and Muntiacus muntjak vaginalis (2n = 6/7), and analyze their evolution and architecture. The genomes show extensive collinearity with each other and with other deer and cattle. We identified numerous fusion events unique to and shared by muntjacs relative to the cervid ancestor, confirming many cytogenetic observations with genome sequence. One of these M. muntjak fusions reversed an earlier fission in the cervid lineage. Comparative Hi-C analysis showed that the chromosome fusions on the M. muntjak lineage altered long-range, three-dimensional chromosome organization relative to M. reevesi in interphase nuclei including A/B compartment structure. This reshaping of multi-megabase contacts occurred without notable change in local chromatin compaction, even near fusion sites. A few genes involved in chromosome maintenance show evidence for rapid evolution, possibly associated with the dramatic changes in karyotype.

Effects of malleable kinetochore morphology on measurements of intrakinetochore tension.

The distance between fluorescent spots formed by various kinetochore proteins (delta) is commonly interpreted as a manifestation of intrakinetochore tension (IKT) caused by microtubule-mediated forces. However, large-scale changes of the kinetochore architecture (such as its shape or dimensions) may also contribute to the value of delta. To assess contributions of these non-elastic changes, we compare behaviour of delta values in human kinetochores with small yet mechanically malleable kinetochores against compound kinetochores in Indian muntjac (IM) cells whose architecture remains constant. Due to the micrometre-scale length of kinetochore plates in IM, their shape and orientation are discernible in conventional light microscopy, which enables precise measurements of IKT independent of contributions from changes in overall architecture of the organelle. We find that delta in IM kinetochores remains relatively constant when microtubule-mediated forces are suppressed by Taxol, but it prominently decreases upon detachment of microtubules. By contrast, large decreases of delta observed in Taxol-treated human cells coincide with prominent changes in length and curvature of the kinetochore plate. These observations, supported by computational modelling, suggest that at least 50% of the decrease in delta in human cells reflects malleable reorganization of kinetochore architecture rather than elastic recoil due to IKT.

A new viewpoint on antlers reveals the evolutionary history of deer (Cervidae, Mammalia).

Recent molecular phylogeny of deer revealed that the characters of antlers previously focused on are homoplasious, and antlers tend to be considered problematic for classification. However, we think antlers are important tools and reconsidered and analysed the characters and structures to use them for classification. This study developed a method to describe the branching structure of antlers by using antler grooves, which are formed on the antlers by growth, and then projecting the position of the branching directions of tines on the burr circumference. By making diagrams, comparing the branching structure interspecifically, homologous elements (tines, beams, and processes) of the antlers of 25 species of 16 genera were determined. Subsequently, ancestral state reconstruction was performed on the fixed molecular phylogenetic tree. It was revealed that Capreolinae and Cervini gained respective three-pointed antlers independently, and their subclades gained synapomorphous tines. We found new homologous and synapomorphous characters, as the antler of Eld's deer, which has been classified in Rucervus, is structurally close to that of Elaphurus rather than that of Rucervus, consistent with molecular phylogeny. The methods of this study will contribute to the understanding of the branching structure and phylogeny of fossil species and uncover the evolutionary history of Cervidae.

Complete mitochondrial genome of northern Indian red muntjac (Muntiacus vaginalis) and its phylogenetic analysis.

We report complete mitochondrial genome of Northern Indian red muntjac, Muntiacus vaginalis, and its phylogenetic inferences. Mitogenome composition was 16,352 bp in length and its overall base composition in the circular genome was A = 33.2%, T = 29.0%, C = 24.50% and G = 13.30%. It exhibited a typical mitogenome structure, including 22 transfer RNA genes, 13 protein-coding genes, two ribosomal RNA genes and a major non-coding control region (D-loop region). All the genes except ND6 and eight tRNA's were encoded on the heavy strand. Phylogenetic analyses showed that M. vaginalis is closely related to M. muntjak and formed a sister relationship with Elaphodus cephalophus. In view of the unclear distribution range and escalating habitat loss, it is important to identify its population genetic status. The complete mitogenome described in this study can be used in further phylogenetics, identification of extant maternal lineage, evolutionary significance unit and its genetic conservation.

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 trans-Himalayan 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.

Phylogeography of red muntjacs reveals three distinct mitochondrial lineages.

BACKGROUND: The members of the genus Muntiacus are of particular interest to evolutionary biologists due to their extreme chromosomal rearrangements and the ongoing discussions about the number of living species. Red muntjacs have the largest distribution of all muntjacs and were formerly considered as one species. Karyotype differences led to the provisional split between the Southern Red Muntjac (Muntiacus muntjak) and the Northern Red Muntjac (M. vaginalis), but uncertainties remain as, so far, no phylogenetic study has been conducted. Here, we analysed whole mitochondrial genomes of 59 archival and 16 contemporaneous samples to resolve uncertainties about their taxonomy and used red muntjacs as model for understanding the evolutionary history of other species in Southeast Asia. RESULTS: We found three distinct matrilineal groups of red muntjacs: Sri Lankan red muntjacs (including the Western Ghats) diverged first from other muntjacs about 1.5 Mya; later northern red muntjacs (including North India and Indochina) and southern red muntjacs (Sundaland) split around 1.12 Mya. The diversification of red muntjacs into these three main lineages was likely promoted by two Pleistocene barriers: one through the Indian subcontinent and one separating the Indochinese and Sundaic red muntjacs. Interestingly, we found a high level of gene flow within the populations of northern and southern red muntjacs, indicating gene flow between populations in Indochina and dispersal of red muntjacs over the exposed Sunda Shelf during the Last Glacial Maximum. CONCLUSIONS: Our results provide new insights into the evolution of species in South and Southeast Asia as we found clear genetic differentiation in a widespread and generalist species, corresponding to two known biogeographical barriers: The Isthmus of Kra and the central Indian dry zone. In addition, our molecular data support either the delineation of three monotypic species or three subspecies, but more importantly these data highlight the conservation importance of the Sri Lankan/South Indian red muntjac.

5-Methylcytosine-Rich Heterochromatin in the Indian Muntjac.

Two 5-methylcytosine (5-MeC)-rich heterochromatic regions were demonstrated in metaphase chromosomes of the Indian muntjac by indirect immunofluorescence using a monoclonal anti-5-MeC antibody. The metaphases were obtained from diploid and triploid cell lines. A major region is located in the 'neck' of the 3;X fusion chromosome and can be detected after denaturation of the chromosomal DNA with UV-light irradiation for 1 h. It is located exactly at the border of the X chromosome and the translocated autosome 3. A minor region is found in the centromeric region of the free autosome 3 after denaturing the chromosomal DNA for 3 h or longer. The structure and possible function of the major hypermethylated region as barrier against spreading of the X-inactivation process into the autosome 3 is discussed.

Isolation and characterization of polymorphic microsatellite loci of the Chinese muntjac (Muntiacus reevesi).

Eight polymorphic microsatellite markers for Muntiacus reevesi were identified and characterized in this study. The number of alleles per locus ranged from 3 to 10 across 24-48 samples. The loci showed expected and observed heterozygosities of 0.577-0.876 and 0.387-0.933, respectively, with an average polymorphic information content value of 0.682. These markers should be a useful tool for further population and conservation genetic studies of Muntiacus reevesi.

A novel chromatin tether domain controls topoisomerase IIα dynamics and mitotic chromosome formation.

DNA topoisomerase IIα (Topo IIα) is the target of an important class of anticancer drugs, but tumor cells can become resistant by reducing the association of the enzyme with chromosomes. Here we describe a critical mechanism of chromatin recruitment and exchange that relies on a novel chromatin tether (ChT) domain and mediates interaction with histone H3 and DNA. We show that the ChT domain controls the residence time of Topo IIα on chromatin in mitosis and is necessary for the formation of mitotic chromosomes. Our data suggest that the dynamics of Topo IIα on chromosomes are important for successful mitosis and implicate histone tail posttranslational modifications in regulating Topo IIα.

Mother to offspring transmission of chronic wasting disease in reeves' muntjac deer.

The horizontal transmission of prion diseases has been well characterized in bovine spongiform encephalopathy (BSE), chronic wasting disease (CWD) of deer and elk and scrapie of sheep, and has been regarded as the primary mode of transmission. Few studies have monitored the possibility of vertical transmission occurring within an infected mother during pregnancy. To study the potential for and pathway of vertical transmission of CWD in the native cervid species, we used a small cervid model-the polyestrous breeding, indoor maintainable, Reeves' muntjac deer-and determined that the susceptibility and pathogenesis of CWD in these deer reproduce that in native mule and white-tailed deer. Moreover, we demonstrate here that CWD prions are transmitted from doe to fawn. Maternal CWD infection also appears to result in lower percentage of live birth offspring. In addition, evolving evidence from protein misfolding cyclic amplification (PMCA) assays on fetal tissues suggest that covert prion infection occurs in utero. Overall, our findings demonstrate that transmission of prions from mother to offspring can occur, and may be underestimated for all prion diseases.

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.