mtDNA confirms the presence of Moschus leucogaster (Ruminantia, Moschidae) in Gaurishankar Conservation Area, Nepal

mtDNA confirms the presence of Moschus leucogaster (Ruminantia, Moschidae) in Gaurishankar Conservation Area, Nepal. Musk deer (genus Moschus), an endangered mammal, is not only of great concern for its conservation, but it is also of great interest to understand its taxonomic and phylogenetic associations in Nepal. The aim of this study was to identify the taxonomic status of musk deer in Gaurishankar Conservation Area (GCA) using mitochondrial genomic data of cytochrome b (370 bps) through phylogenetic analysis of all the species of musk deer. The results showed that the species found in GCA is confirmed as Himalayan musk deer Moschus leucogaster, further expanding its distributional range in Nepal.


Introduction
With the advent of biotechnology, genetic information can be obtained through animals' degraded remains such as bones, dried skins, feces and fossils (Ramón-Laca et al., 2015;Silva et al., 2015). Genetic information can be accrued from DNA contained with these animal specimens. DNA test is the most popular method for identification of species. In this system, DNA sequence of mitochrondrial cytochrome b (cyt-b) gene has been widely used for species' identifications (Khatiwada et al., 2015). Cyt-b is probably the best-known mitochondrial gene with respect to function and structure of its protein product (Esposti et al., 1993). It is used as a valuable tool for the construction of the evolutionary relationship among population, species, and higher taxa (Harrison, 1989;Su et al., 1999) since it contains both slowly and rapidly evolving codon positions, as well as more conservative and more variable regions or domains (Meyer and Wilson, 1990;Irwin et al., 1991;Cantatore et al., 1994;Farias et al., 2001).
Musk deer (genus Moschus, Linnaeus, 1758) are widely distributed in the sub-alpine and alpine vegetation (2,500 to 4,500 m) of the Himalayan region of Nepal (Kattel, 1992). All the species of musk deer belongs to order Cetartiodactyla of family Moschidae (IUCN, 2013). Although many morphological studies have been conducted on the taxonomy of this group, there are still controversies regarding the number of its species and sub-species and the phylogenetic relationship among them (Groves et al., 1995). Seven species within the genus Moschus are recognized in the world. They include Anhui musk deer (M. anhuiensis Wang et al., 1982), Kashmir musk deer (M. cupreus Grubb, 1982), Siberian musk deer (M. moschiferus Linnaeus, 1758), black musk deer (M. fuscus Li, 1981), Himalayan musk deer (M. leucogaster Hodgson, 1839), forest musk deer (M. berezovskii Flerov, 1929) and Alpine musk deer (M. chrysogaster Hodgson, 1839) (IUCN, 2013). Three species of musk deer ie M. chrysogaster, M. leucogaster and M. fuscus are said to be found in Nepal (Jnawali et al., 2011;Timmins and Duckworth, 2015;Wang and Harris, 2015;Harris, 2016) but their exact distribution and status are still dubious, and they all were considered as one species: M. chrysogaster before their taxonomic separation (Jnawali et al., 2011). Although all the three species of musk deer found in Nepal are categorized as Endangered by IUCN (IUCN, 2013), only M. chrysogaster is enlisted as a protected mammal by the National Park and Wildlife Conservation Act 1973 due to lack of documentation on the confirmation of species of musk deer (Jnawali et al., 2011). There are only a few studies on taxonomic and phylogenetic analysis of musk deer in the context of Nepal (Singh et al., 2019). The Gaurishankar Conservation Area (GCA) is the newly established conservation area where molecular studies confirming the species presence are crucial for its conservation and management in the future. In this study, we aimed to determine the taxonomic status of musk deer in the GCA using mitochondrial genomic data of cytochrome b through phylogenetic analysis of all the species of musk deer.

Study area
The GCA is located in central Nepal, encompassing Ramechhap, Dolakha and Sindhupalchok districts. It has an area of 2,179 km 2 ( fig. 1). It was declared the 'Conservation Area' in January 2010, and its management was entrusted for twenty years to the National Trust for Nature Conservation (NTNC) in July 2010 (DNPWC, 2011). It is located in the high mountain physiographic region of Nepal and consists of 35.38 % forestland, 9.76 % shrubland and 8.79 % grassland. It has 16 major vegetation types and great faunal diversity that includes 34 species of mammals, 16 species of fishes, 10 species of amphibians, 8 species of lizards, 14 species of snakes and 235 species of birds (DNPWC, 2013). Besides musk deer (Moschus spp.), endangered species found in the conservation area are snow leopard (Panthera uncia), clouded leopard (Neofelis lupus), leopard cat (Felis benghalensis), red panda (Ailurus fulgens), wolf (Canis lupus), and Chinese pangolin (Manis pentadactyla) (DNPWC, 2013;Shrestha and Meng, 2014).
Our study included the surrounding areas of Risan Gumbo Himal from Hum danda to Gumbo danda at Lapche area of Lamabagar VDC of Dolakha district which encompasses an elevation range of 3,500 to 4,200 m and lies between 28º 6' 7'' and 28º 7' 3'' N latitude and 86º 9' 59'' and 86º 10' 52'' E longitude. It has four types of vegetation: Betula forest mostly dominated by Betula utilis; mixed forest having a mixed species of Betula utilis, Abies spectabilis, Sorbus spp., Rhododendron campanulatum, Salix spp. and Juniperus indica; rhododendron forest mostly dominated by Rhododendron campanulatum; and alpine scrub mostly dominated by shrubby rhododendron species i.e., Rhododendron lepidotum, Rhododendron ciliatum and Rhododendron anthopogan.

Pellet sample collection
We opportunistically collected 39 fecal pellet samples (faeces) from the Lapche area of GCA ( fig. 1) between 2013 and 2014. These samples were preserved in 95 % ethanol for further molecular analysis. For each pellet sample, the sampling date, the location and geographical coordinates were recorded. The sample pellets were used for laboratory analysis for species' identification.

DNA extraction, PCR amplification and sequencing
A dry pellet from each sample was cut into smaller pieces using sterile scissors and tweezers and further processed for DNA extraction as recommended in the protocol by Qiagen QIAamp DNA Stool kit (Qiagen, Valencia, CA, USA). Polymerase chain reaction (PCR) was used to amplify the mitochondrial genes cytochrome b (cyt-b). The primers and PCR conditions were used as described by Pan et al. (2015). PCR products were visualized in 1.5 % agarose gel and positive PCR products were sequenced following bi-directional sequencing from a ABI 3100 automated sequencer.

Sequence analysis
All available nucleotide sequences of the cyt-b gene of Moschus species were downloaded from the NCBI GenBank database and the data source made available by Pan et al. (2015) and Singh et al. (2019) (table 1). The sequences of Alces alces americana, Ovis aries and Tragulus kanchil were also downloaded to be used as outgroups. Only 10 of the 39 pellet samples yielded a complete sequence. All nucleotide sequences were assembled by SeqMan and visually checked to determine the accuracy of the variables site identified by the program. All the sequences were then aligned with ClustalW in BIOEDIT Version 7.1.9 (Thompson et al., 1994) using the default settings. All the newly determined sequences were deposited in GenBank under accession numbers (MN720942-MN720951). Phylogenetic analysis was conducted using the maximum likelihood (ML) estimation. The maximum likelihood analysis was conducted in MEGA7 with 1,000 bootstraps (Kumar et al., 2016).

Results and discussion
Phylogenetic relationship of musk deer from GCA The aligned dataset of cyt-b sequence contained 370 bps including 69 variable sites and 51 parsimony informative sites (excluding outgroups). The phylogenetic relationships strongly support genus Moschus as a monophyletic clade with higher bootstrap supports (bootstrap = 99). Molecular data analysis suggested that the population of musk deer in Lapche, GCA, were genetically similar with M. leucogaster from western Nepal and Qinhai, Tibet, China and were nested together in a ML tree ( fig. 2). The uncorrected genetic divergence of the cyt-b gene sequences among the M. leucogaster population of Lapche, GCA, Manang, Nepal and Tibet, China ranged from 0.00 to 0.3 % (table 2) whereas relatively low genetic divergences between M. leucogaster and its closest relatives M. chrysogaster and M. fuscus were 1.4 % and 1.7 % respectively. The pellet samples used in this study confirmed the presence of Himalayan musk deer M. leucogaster in the GCA. Although three species of musk deer (Moschus fuscus, Moschus chrysogaster and Moschus leucogaster) are said to be distributed in Nepal (Timmins and Duckworth, 2015;Wang and Harris, 2015;Harris, 2016), most studies conducted in central and eastern Nepal regarding distribution, habitat ecology, latrines, associated plant composition and diversity, and gastro-intestinal parasites of musk deer have considered musk deer species as M. chrysogaster (Aryal et al., 2010;Aryal and Subedi, 2011;Subedi et al., 2012;Shrestha and Moe, 2015;Achhami et al., 2016). The potential misidentification of musk deer species is due to their secretive behaviour and similar morphological characteristics (Groves et al., 1995;Su et al., 2000;Guha et al., 2007). As musk deer are shy and nocturnal it is difficult to detect them in daytime (Green, 1986). Additionally, if they are seen in daytime they hide in the shrub understory, and even if they are encountered in forest and open areas they are visible only for a few seconds (Singh et al., 2019). Furthermore, it is not easy to identify the species of musk deer from their physical appearance and morphological