Reading the signs: Camera-trapping provides new insights on scent marking in the large-antlered muntjac (Muntiacus vuquangensis).

We present evidence of scent marking in the large-antlered muntjac (Muntiacus vuquangensis). Given the importance of scent marking in individual recognition among ungulates, this behavior may serve to communicate the fitness cost of antagonistic interactions among rival males and could serve as a mechanism for mate assessment among females.

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

Abstract 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.

Resumo 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.

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.

Morphological, Phaneroptic, Habitat and Population Description of Three Muntjac Species in a Tibetan Nature Reserve.

Researchers have proposed a variety of classification schemes for the species in the genus Muntiacus (Artiodactyla: Cervidae) based on morphological, molecular, and other evidence, but disputes remain. The Tibetan Yarlung Zangbo Grand Canyon National Nature Reserve in the Eastern Himalayas is an area with a rich diversity of muntjac species. The habitats of many species overlap in this area, but systematic research in this area is lacking. To clarify the species, population and habitat size of muntjac species in the study area, we used camera-traps to monitor muntjacs in the nature reserve from 2013 to 2021 and described and compared morphological characteristics of the muntjac species. Subsequently, we used the MaxEnt model to simulate the habitats of the muntjac species and the Random Encounter Model to estimate the population density and numbers of muntjacs. Three muntjac species were found in the area, namely Muntiacus vaginalis (n = 7788 ± 3866), Muntiacus gongshanensis (n = 6673 ± 2121), and Muntiacus feae (n = 3142 ± 942). The red muntjac has the largest habitat area, the highest population density, and largest size, followed by Gongshan muntjac and Fea's muntjac. This study provides basic data for improving the background knowledge of the animal diversity in the Eastern Himalayan biodiversity hotspot, as well as detailed information and references required by wildlife workers for species identification.

Augmin-dependent microtubule self-organization drives kinetochore fiber maturation in mammals.

Chromosome segregation in mammals relies on the maturation of a thick bundle of kinetochore-attached microtubules known as k-fiber. How k-fibers mature from initial kinetochore microtubule attachments remains a fundamental question. By combining molecular perturbations and phenotypic analyses in Indian muntjac fibroblasts containing the lowest known diploid chromosome number in mammals (2N = 6) and distinctively large kinetochores, with fixed/live-cell super-resolution coherent-hybrid stimulated emission depletion (CH-STED) nanoscopy and laser microsurgery, we demonstrate a key role for augmin in kinetochore microtubule self-organization and maturation, regardless of pioneer centrosomal microtubules. In doing so, augmin promotes kinetochore and interpolar microtubule turnover and poleward flux. Tracking of microtubule growth events within individual k-fibers reveals a wide angular dispersion, consistent with augmin-mediated branched microtubule nucleation. Augmin depletion reduces the frequency of kinetochore microtubule growth events and hampers efficient repair after acute k-fiber injury by laser microsurgery. Together, these findings underscore the contribution of augmin-mediated microtubule amplification for k-fiber self-organization and maturation in mammals.

Bushmeat Species Identification: Recombinase Polymerase Amplification (RPA) Combined with Lateral Flow (LF) Strip for Identification of Formosan Reeves' Muntjac (Muntiacus reevesi micrurus).

The identification of animal species of meat in meat products is of great concern for various reasons, such as public health, religious beliefs, food allergies, legal perspectives, and bushmeat control. In this study, we developed a new technique to identify Formosan Reeves' muntjac in meat using recombinase polymerase amplification (RPA) in combination with a lateral flow (LF) strip. The DNA extracted from a piece of Formosan Reeves' muntjac meat was amplified by a pair of specific primers based on its mitochondrial cytochrome b gene for 10 min at a constant temperature ranging from 30 to 45 °C using RPA. Using the specific probe added to the RPA reaction system, the amplified products were visualized on the LF strip within 5 min. The total operating time from quick DNA extraction to visualizing the result was approximately 30 min. The RPA-LF system we designed was efficient when using boiled, pan-fried, roasted, stir-fried, or stewed samples. The advantages of simple operation, speediness, and cost-effectiveness make our RPA-LF method a promising molecular detection tool for meat species identification of either raw or variously cooked Formosan Reeves' muntjac meat. It is also possible to apply this method to identify the meat of other wildlife sources.

Associations between Japanese spotted fever (JSF) cases and wildlife distribution on the Boso Peninsula, Central Japan (2006-2017).

Populations of large mammals have been dramatically increasing in Japan, resulting in damage to agriculture, forestry, and ecosystems. However, their effects on tick-borne diseases have been poorly studied. Here, we focused on the relationship between Japanese spotted fever (JSF), a tick-borne disease caused by Rickettsia japonica, and populations of large mammals. To explore factors that affected the area in which JSF cases occur, we used generalized linear mixed models (GLMMs). We demonstrated that the expansion of the area of JSF occurrence can be predicted by deer density and geographical factors, which is likely due to differences in landscape structure. However, the associated models have limitations because of the lack of information about the distribution of vectors and reservoirs. To reduce the risk of humans contracting JSF, potential reservoirs should be confirmed.

Weissella muntiaci sp. nov., isolated from faeces of Formosan barking deer (Muntiacus reevesi).

A Gram-stain-positive strain, 8 H-2T, was isolated from faeces of Reeves' muntjac (Muntiacus reevesi) barking deer in Taiwan. Cells of the strain were short rod-shaped, non-motile, non-haemolytic, asporogenous, facultatively anaerobic, heterofermentative and did not exhibit catalase and oxidase activities. Comparative analyses of 16S rRNA, pheS and dnaA gene sequences demonstrated that the novel strain was a member of the genus Weissella. On the basis of 16S rRNA gene sequence similarities, the type strains of Weissella oryzae (99.2 %), Weissella confusa (97.8 %), Weissella cibaria (97.6 %) and Weissella soli (97.3 %) were the closest neighbours to strain 8 H-2T. The concatenated housekeeping gene sequence (pheS and dnaA) similarities of 8 H-2T to closely related type strains were 72.5-84.9 %, respectively. The genomic DNA G+C content was 40.5 mol%. The average nucleotide identity and digital DNA-DNA hybridization values with these type strains were 70.2-75.4% and 25.1-30.1 %, respectively. Phenotypic and genotypic test results demonstrated that strain 8 H-2T represents a novel species belonging to the genus Weissella, for which the name Weissella muntiaci sp. nov. is proposed. The type strain is 8 H-2T (=BCRC 81133T=NBRC 113537T).

Establishment and characterization of a fibroblast cell line from postmortem skin of an adult Chinese muntjac (Muntiacus reevesi).

Isolation and culture of somatic cells from animals especially endangered species have raised great concerns as it is being an effective and convenient way to preserve genetic materials for future studies. As a species native to China, Chinese muntjac (Muntiacus reevesi) is listed as a beneficial species with economic and scientific research values. To our knowledge, however, there have been no published reports on somatic cell preservation of this species to date. To conserve biological resources for sustainability of Chinese muntjacs' genetic diversity, we established a fibroblast cell line from the postmortem ear skin of an adult male Chinese muntjac. The cultured cells were adherent to the plastic and showed an elongated, thin, and spindle-like shape. Moreover, they were FSP1- and VIM-positive characterizing them to be fibroblastic. No microorganisms (bacteria, fungi, or mycoplasmas) were detected throughout the whole study. Cell viability was high although it declined somehow after passaging. The population doubling time was 21.28 h according to the growth curve. Chromosome analysis revealed that the established fibroblast cell line contained 23 pairs of chromosomes, one pair of which was sex chromosomes (XY). Mitochondrial cytochrome C oxidase I gene of cultured cells shared 98.32% identity with those of Muntiacus reevesi registered in GenBank, which verified the cell line was derived from Muntiacus reevesi. In conclusion, we propagated and characterized fibroblast cells from a Chinese muntjac. We believe that this somatic cell line could facilitate animal cloning and breeding studies and become a useful in vitro model to address genetic questions.

Functional Dissection of Mitosis Using Immortalized Fibroblasts from the Indian Muntjac, a Placental Mammal with Only Three Chromosomes.

During cell division in eukaryotes a microtubule-based network undergoes drastic changes and remodeling to assemble a mitotic spindle competent to segregate chromosomes. Several model systems have been widely used to dissect the molecular and structural mechanisms behind mitotic spindle assembly and function. These include budding and fission yeasts, which are ideal for genetic and molecular approaches, but show limitations in high-resolution live-cell imaging, while being evolutionarily distant from humans. On the other hand, systems that were historically used for their exceptional properties for live-cell imaging of mitosis (e.g., newt lung cells and Haemanthus endosperm cells) lack the necessary genomic tools for molecular studies. In a CRISPR-Cas9 era, human cultured cells have conquered the privilege to be positioned among the most powerful genetically manipulatable systems, but their high chromosome number remains a significant bottleneck for the molecular dissection of mitosis in mammals. We believe that we can significantly broaden this scenario by establishing a unique placental mammal model system that combines the powerful genetic tools and low chromosome number of fission yeast and Drosophila melanogaster, with the exceptional cytological features of a rat kangaroo cell. This system is based on hTERT-immortalized fibroblasts from a female Indian muntjac, a placental mammal with the lowest known chromosome number (n = 3). Here we describe a series of methodologies established in our laboratory for the study of mitosis in Indian muntjac. These include standard techniques such as immunofluorescence, western blotting, and FISH, but also several state-of-the-art methodologies, including live-cell imaging, cell confinement, RNAi, super-resolution STED microscopy, and laser microsurgery.

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.

Estimating densities of large herbivores in tropical forests: Rigorous evaluation of a dung-based method.

When sighting-based surveys to estimate population densities of large herbivores in tropical dense forests are not practical or affordable, surveys that rely on animal dung are sometimes used. This study tested one such dung-based method by deriving population densities from observed dung densities of six large herbivores (chital, elephant, gaur, muntjac, sambar, and wild pig) in two habitats, dry deciduous forests (DDF) and moist deciduous forests (MDF), within Nagarahole National Park, southern India. Using the program DUNGSURV, dung pile counts, decay rates estimated from field experiments, and defecation rates derived from literature were analyzed together by a model that allows for random events affecting dung decay. Densities of chital were the highest, followed by sambar. Wild pig densities were similar in the two habitats, sambar densities were higher in DDF, and densities of the other species were higher in MDF than in DDF. We compared DUNGSURV estimates with densities estimated using distance sampling in the same season. DUNGSURV estimates were substantially higher for all species in both habitats. These differences highlight the challenges that researchers face in computing unbiased estimates of dung decay rates and in relying on defecation rates from literature. Besides the elephant, this study is the first to rigorously test the efficacy of using a dung-based approach to estimate densities of large herbivore species in Asia, and based on this evaluation, we provide specific recommendations to address issues that require careful consideration before observed dung densities are used to derive animal densities. Our results underline the need for an experimental study of a known population in a fenced reserve to validate the true potential of using dung-based approaches to estimate population densities.

Chromosome Segregation Is Biased by Kinetochore Size.

Chromosome missegregation during mitosis or meiosis is a hallmark of cancer and the main cause of prenatal death in humans. The gain or loss of specific chromosomes is thought to be random, with cell viability being essentially determined by selection. Several established pathways including centrosome amplification, sister-chromatid cohesion defects, or a compromised spindle assembly checkpoint can lead to chromosome missegregation. However, how specific intrinsic features of the kinetochore-the critical chromosomal interface with spindle microtubules-impact chromosome segregation remains poorly understood. Here we used the unique cytological attributes of female Indian muntjac, the mammal with the lowest known chromosome number (2n = 6), to characterize and track individual chromosomes with distinct kinetochore size throughout mitosis. We show that centromere and kinetochore functional layers scale proportionally with centromere size. Measurement of intra-kinetochore distances, serial-section electron microscopy, and RNAi against key kinetochore proteins confirmed a standard structural and functional organization of the Indian muntjac kinetochores and revealed that microtubule binding capacity scales with kinetochore size. Surprisingly, we found that chromosome segregation in this species is not random. Chromosomes with larger kinetochores bi-oriented more efficiently and showed a 2-fold bias to congress to the equator in a motor-independent manner. Despite robust correction mechanisms during unperturbed mitosis, chromosomes with larger kinetochores were also strongly biased to establish erroneous merotelic attachments and missegregate during anaphase. This bias was impervious to the experimental attenuation of polar ejection forces on chromosome arms by RNAi against the chromokinesin Kif4a. Thus, kinetochore size is an important determinant of chromosome segregation fidelity.

The complete mitochondrial genome of Fea's muntjac (Muntiacus feae Thomas and Doria, 1889) with phylogenetic analysis†.

The complete mitochondrial genome (mitogenome) of the rare Fea's muntjac (Muntiacus feae) was sequenced (GenBank accession nos. MG857662-MG857664). The mitogenome was found to be 16,355 bp in length with base compositions of 33.16% A, 24.59% C, 13.46% G, and 28.78% T and a GC content of 38.06%. The genome is comprised of 13 protein-coding genes (PCGs), 22 transfer RNA genes, 2 ribosomal RNA genes, and a control region (D-loop). Phylogenetic analysis revealed that Fea's muntjac is more closely related to Black muntjac (M. cronifrons) than to Red muntjac (M. muntjak). These data will be useful for further studies on the genetic diversity and molecular phylogenetic relationship of the genus Muntiacus.

Detection of a novel gammaherpesvirus (genus Rhadinovirus) in wild muntjac deer in Northern Ireland.

This study represents the initial part of an investigation into the potential for non-native, wild, free-living muntjac deer (Muntiacus reevesi) to carry viruses that could be a threat to livestock. A degenerate PCR assay was used to screen a range of tissues from muntjac deer culled in Northern Ireland for the presence of herpesviral nucleic acids. This was followed by sequencing of PCR amplicons and phylogenetic analysis. We report the detection of a novel gammaherpesvirus most closely related to a type 2 ruminant rhadinovirus from mule deer. It remains to be determined if this new virus is pathogenic to deer or presents a risk to food security through the susceptibility of domestic livestock.

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.

MANAGEMENT OF A REEVE'S MUNTJAC ( MUNTIACUS REEVESI) WITH SEIZURES USING LEVETIRACETAM.

This report describes the diagnosis and management of idiopathic epilepsy in a 4-yr-old intact female Reeve's muntjac ( Muntiacus reevesi). The patient was initially witnessed to have isolated paroxysmal events consistent with epileptic seizures (altered consciousness, lateral recumbency, tonic/clonic movement of limbs) lasting less than 3 min with an immediate return to normal consciousness. The seizure frequency increased to >3 seizures within 24 hr and phenobarbital 3 mg/kg orally every 12 hr was started. Because of continued epileptic seizures and low serum phenobarbital levels, the dose was increased until significant elevations of aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were detected. Levetiracetam 40 mg/kg orally every 12 hr was initiated and the phenobarbital was weaned and discontinued. One breakthrough seizure has been witnessed in the 10 mo since starting levetiracetam.

Molecular evidence for piroplasms in wild Reeves' muntjac (Muntiacus reevesi) in China.

DNA from liver samples of 17 free-ranging wild Reeves' muntjac (Muntiacus reevesi) was used for PCR amplification of piropalsm 18S rRNA gene. Of 17 samples, 14 (82.4%) showed a specific PCR product which were cloned and sequenced. BLAST analysis of the sequences obtained showed similarities to Babesia sp., Theileria capreoli, Theileria uilenbergi and Theileria sp. BO302-SE. Phylogenetic analysis showed that the Babesia sp. detected in the present study was distantly separated from known Babesia species of wild and domestic animals. Six sequences showed 100% similarity to T. capreoli while five sequences were separated from all known Theileria species and constituted an independent clade with Theileria sp. BO302-SE derived from roe deer in Italy; two sequences were close to T. uilenbergi with 97% similarity. This is the first description of hemoparasite infection in free-ranging wild Reeves' muntjac in China. Our results indicate that wild Reeves' muntjac may play an important reservoir role for hemoparasites.