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.


Genetic similarities and Phylogenetic analysis
For all the nucleotide sequences (Cytochrome-b and 16s rRNA), the MegAlign module of the DNASTAR software (DNASTAR laser gene) were used for alignment. CLUSTAL W method was employed for the alignment of all the sequences. For multiple assessments, the CLUSTAL W algorithms were used. For similarities of nucleotide sequence, the section of sequence distance in DNASTAR was used. The residue substitutions windows in this software show a table to demonstrate several residue substitutions predicted to have occurred to give rise to the differences of sequence in the current alignment (Abdoli et al., 2018).
For selected species of muntjac, the phylogenetic tree was created using the maximum livelihood method on MEGA7 software (Kumar et al., 2016). The phylogenetic relationship between all the aligned sequences was demonstrated in the phylogenetic tree. The bootstraps consensus tree inferred from 1000 replicates.

Introduction
Muntjac is commonly known as deer and recognized for their beauty and grace. Male muntjac deer has bony antlers that annually molt while females lack antlers (Wikipidia, 2007a, b). Muntjac deer is belonging to the genus Muntiacus, family Cervidae, and order Artiodactyla and of the class Mammalia (Wikipidia, 2007a, b). They are classified into nine species, which are Muntiacus feae, Muntiacus gongshanensis, Muntiacus crinifrons, Muntiacus reevesi, Muntiacus putaoensis, and Muntiacus rooseveltorum (Giao et al., 1998;Wang and Lan, 2000;Shi and Cai-Xia, 1988;Nowak and Paradiso, 1991;Amato et al., 1999). The most primitive species of order Artiodactyla is the Barking deer and it is also known as the ancestor of the living families of artiodactyls (Dubost, 1971;Godina et al., 1962;Colbert, 1969;Barrette, 1977). The most strange fact about Barking deer is they contain the smallest number of diploid chromosomes (male contain seven and female contain six) yet described in any other mammal (Wurster and Benirschke, 1970).
The muntjac deer and their nine classified species are distributed in most regions of all around the world. They are distributed in tropical and subtropical deciduous forests, scrub forests, savannas, and grasslands. Their altitudes ranging from sea level to 3000 m and prefer the habitat of diverse woodland like large areas of woodland which are near to pasture land. They are found in Asia, Europe, northern Africa, and South and North America. They are native to South East Asia including India and Srilanka to Southern China, Bangladesh, Taiwan, Japan, and Indonesian islands (Wikipidia, 2007a, b). This study aimed to identify phylogenetic similarities among major muntjac species (Muntiacus spp.).
Generally, phylogenetic studies help to determine the relationships among genes or species, however, they can also give valuable insights into species' migration, changes in demographic patterns, and causes of viral infection (Yang and Rannala, 2012). In phylogenetic analysis, the genomic materials are used and achieved from chromosomal DNA which is present in the nucleus, and additional chromosomal DNA, present in eukaryotes organelles like chloroplasts and mitochondria (Abdoli et al., 2018). For the phylogenetic relationships concerning ancient divergences, sequences of complete mitochondrial DNA (mtDNA) and nuclear ribosomal rRNA gene, have been used (Zardoya and Meyer, 1996;Van de Peer and Wachter, 1997;Naylor and Brown, 1998;Mindell and Honeycutt, 1990;Abouheif et al., 1998;Zardoya et al., 1998). Single mtDNA genes were used most of the time during research studies to find out the population and taxonomic relationships (Tsigenopoulos and Berrebi, 2000;Rocha-Olivares et al., 1999, reviewed in Meyer, 1993Lovejoy and Araújo, 2000). The mitochondrion organelle contains genetic information that can help in phylogenetic analyses (Moritz et al., 1987).

Material and Methods
The phylogenetic analyses of the seven major muntjac species including Indian muntjac (Muntiacus muntjak), 3/6 Genetic similarities and phylogenetic analysis of Muntiacus spp.
pruinosus and Ursus arctos shown in Table 1 and The phylogenetic tree of muntjac species (16s rRNA gene) shows the main two clusters, the one including Muntiacus putaoensis, Muntiacus truongsonensisMuntiacus rooseveltorum, and Muntiacus muntjak, and the second one including Muntiacus crinifrons and Muntiacus vuquangensis (Figure 1). While using cytochrome b genes shows that the Muntiacus muntjak and Muntiacus truongsonensis are clustered in the same group (see Figure 2). The Muntiacus reevesi exist separately in the phylogenetic tree (see Figure 1). The evolutionary history was inferred by using the Maximum Likelihood method based on the General Time Reversible model. The tree with the highest log likelihood (16s rRNA = -990.44, Cyt-b = -3254.06) is shown. The percentage of the tree in which the associated taxa clustered together is shown next to the branches. İnitial tree for the heuristic search were obtained automatically by applying Neighbor Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with lengths measured in the number of substitutions per site. The analysis involved 8 nucleotide sequence. Codon positions included were 1 st +2 nd +3 rd +Noncoding. All positions containing gaps and missing data were eliminated. There was a total of 436 (16s rRNA) 1127 (Cty-b) and positions in the final dataset. Evolutionary analyses were conducted in MEGA7.
The evolutionary history was inferred by using the Maximum Likelihood method based on the General Time Reversible model. The tree with the highest log likelihood  (-3254.06) is shown. The percentage of the tree in which the associated taxa clustered together is shown next to the branches. The initial tree for the heuristic search was obtained automatically by applying Neighbor Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach and then selecting the topology with a superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 8 nucleotide sequence. Codon positions included were 1 st +2 nd +3 rd +Noncoding. All positions containing gaps and missing data were eliminated. There was a total of 1127 positions in the final dataset. Evolutionary analyses were conducted in MEGA7.

Discussion
The mitochondrial genome in animals contains 37 genes, of which, 2 are ribosomal RNA encoding genes while 13 genes encode respiratory chain proteins (Boore, 1999). The phylogenetic analyses of genus Muntiacus have long been studied (James et al., 2008;Timmins and Duckworth, 2016). The complete mitochondrial genome of Muntiacus putaoensis was similar to other species of muntjac deer (Wu and Fang, 2005;Shi et al., 2003;Zhang et al., 2004;Hassanin et al., 2012). In this study, the similarities among nucleotide sequences (16s rRNA genome), the Muntiacus truongsonensis, and Muntiacus rooseveltorum had the highest similarities (99.2%), and the lowest similarities (96.8%) among Muntiacus crinifrons and Muntiacus putaoensi. The phylogenetic tree of muntjac species (16s rRNA gene) shows the main two clusters, the one including Muntiacus putaoensis, Muntiacus truongsonensis Muntiacus rooseveltorum, and Muntiacus muntjak, and the second one including Muntiacus crinifrons and Muntiacus vuquangensis. These results reconfirm the results of James et al. (2008) using the 16s rRNA genome, where they indicated that the Muntiacus putaoensis formed a nested clade with Muntiacus truongsonensis and Muntiacus rooseveltorum due to minor variation in their sequences. Amato et al. (1999) also confirmed these results based on collective analysis of 4 gene regions, showed that the Muntiacus putaoensis is a sister taxon of the Muntiacus truongsonensis and Muntiacus rooseveltorum.
The phylogenetic analysis of five species of the Muntiacus genus using cytochrome b genes was studied by Li et al. (2017). The Cytochrome b genes of Muntiacus spp. (Including Muntiacus putaoensis, Muntiacus truongsonensis, Muntiacus rooseveltorum, and Muntiacus vuquangensis) were also discussed by Le et al. (2014). James et al. (2008) also used the Cty b gene of five species of Muntiacinae subfamily. The phylogenetic tree in this study of muntjac species using cytochrome b genes shows that the Muntiacus muntjak and Muntiacus truongsonensis are clustered in the same group and are closely related having 100% similarities in their sequences. The result contradicts the results of Li et al. (2017), which indicates that Muntiacus truongsonensis was closely related to Muntiacus putaoensis based on cytochrome b genome sequences. TSIGENOPOULOS, C.S. and BERREBI, P., 2000