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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

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.

Keywords:
barking deer; Muntjac; phylogenetic; 16S rRNA; Cty-b

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.

Palavras-chave:
veados ladrões; Muntjac; filogenético; 16S rRNA; Cty-b

1. 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, 2007aWIKIPIDIA, 2007a [viewed 30 January 2021]. Barking deer in Asia: wildlife parks in Asia [online]. Available from: www.wikipidia.com
www.wikipidia.com...
, bWIKIPIDIA, 2007b [viewed 30 January 2021]. Barking deer in Asia: wildlife parks in Asia. [online]. Available from: www.wikipidia.com
www.wikipidia.com...
). Muntjac deer is belonging to the genus Muntiacus, family Cervidae, and order Artiodactyla and of the class Mammalia (Wikipidia, 2007aWIKIPIDIA, 2007a [viewed 30 January 2021]. Barking deer in Asia: wildlife parks in Asia [online]. Available from: www.wikipidia.com
www.wikipidia.com...
, bWIKIPIDIA, 2007b [viewed 30 January 2021]. Barking deer in Asia: wildlife parks in Asia. [online]. Available from: www.wikipidia.com
www.wikipidia.com...
). They are classified into nine species, which are Muntiacus feae, Muntiacus gongshanensis, Muntiacus crinifrons, Muntiacus reevesi, Muntiacus putaoensis, and Muntiacus rooseveltorum (Giao et al., 1998GIAO, P.M., TUOC, D., DUNG, V.V., WIKRAMANAYAKE, E.D., AMATO, G., ARCTANDER, P. and MACKINNON, J.R., 1998. Description of Muntiacus truongsonensis, a new species of muntjac (Artiodactyla: Muntiacidae) from central Vietnam, and implications for conservation. Animal Conservation, vol. 1, no. 1, pp. 61-68.; Wang and Lan, 2000WANG, W. and LAN, H., 2000. Rapid and parallel chromosomal number reductions in muntjac deer inferred from mitochondrial DNA phylogeny. Molecular Biology and Evolution, vol. 17, no. 9, pp. 1326-1333. PMid:10958849.; Shi and Cai-Xia, 1988SHI, H.I. and CAI-XIA, M.A., 1988. A new karyotype of muntjac (Muntiacus sp.) from Gongshan county in China. Zoological Research, vol. 9, pp. 343-347.; Nowak and Paradiso, 1991NOWAK, R.M. and PARADISO, J.L., 1991. Mammals of the World. Baltimore: Johns Hopkins University Press, pp. 490-491.; Amato et al., 1999AMATO, G., EGAN, M.G. and RABINOWITZ, A., 1999. A new species of muntjac, Muntiacus putaoensis (Artiodactyla: Cervidae) from northern Myanmar. Animal Conservation, vol. 2, no. 1, pp. 1-7.). 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, 1971DUBOST, G., 1971. Observation ethologiques Sur le Muntjak (Muntiacus muntjak Zimmermann 1780 et M. reevesi Ogilby 1839) en captivite et semi-liberte. Zeitschrift für Tierpsychologie, vol. 28, pp. 387-427.; Godina et al., 1962GODINA, A.Y., GROMOYA, V.I., SOKOLOV, I.I., TRAFIMOV, B.A., FLEROV, K.K. and KHAVERSON, Y.I., 1962. Mammals, Order Artiodactyla. In: Y.A. ORLOV, ed. Fundamentals of Paleontology. Washington: Dept. of Commerce, pp. 467-572, vol. 8.; Colbert, 1969COLBERT, E.H., 1969. Evolution of vertebrates. New York: John Wiley & Sons, pp. 137-139.; Barrette, 1977BARRETTE, C., 1977. Some aspects of the behaviour of muntjacs in Wilpattu National Park. Mammalia, vol. 41, no. 1, pp. 1-34.). 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, 1970WURSTER, D.H. and BENIRSCHKE, K., 1970. Indian Momtjac, Muntiacus muntiak: a deer with a low diploid chromosome number. Science, vol. 168, no. 3937, pp. 1364-1366. PMid:5444269.).

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, 2007aWIKIPIDIA, 2007a [viewed 30 January 2021]. Barking deer in Asia: wildlife parks in Asia [online]. Available from: www.wikipidia.com
www.wikipidia.com...
, bWIKIPIDIA, 2007b [viewed 30 January 2021]. Barking deer in Asia: wildlife parks in Asia. [online]. Available from: www.wikipidia.com
www.wikipidia.com...
). 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, 2012YANG, Z. and RANNALA, B., 2012. Molecular phylogenetics: principles and practice. Nature Reviews. Genetics, vol. 13, no. 5, pp. 303-314. PMid:22456349.). 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., 2018ABDOLI, R., ZAMANI, P. and GHASEMI, M., 2018. Genetic similarities and phylogenetic analysis of human and farm animal species based on mitogenomic nucleotide sequences. Meta Gene, vol. 15, pp. 23-26.). For the phylogenetic relationships concerning ancient divergences, sequences of complete mitochondrial DNA (mtDNA) and nuclear ribosomal rRNA gene, have been used (Zardoya and Meyer, 1996ZARDOYA, R. and MEYER, A., 1996. Evolutionary relationships of the coelacanth, lungfishes, and tetrapods based on the 28S ribosomal RNA gene. Proceedings of the National Academy of Sciences of the United States of America, vol. 93, no. 11, pp. 5449-5454. PMid:8643595.; Van de Peer and Wachter, 1997VAN DE PEER, Y. and WACHTER, R., 1997. Evolutionary relationships among the eukaryotic crown taxa taking into account site-to-site rate variation in 18S rRNA. Journal of Molecular Evolution, vol. 45, no. 6, pp. 619-630.PMid:9419239.; Naylor and Brown, 1998NAYLOR, G.J. and BROWN, W.M., 1998. Amphioxus mitochondrial DNA, chordate phylogeny, and the limits of inference based on comparisons of sequences. Systematic Biology, vol. 47, no. 1, pp. 61-76. PMid:12064241.; Mindell and Honeycutt, 1990MINDELL, D.P. and HONEYCUTT, R.L., 1990. Ribosomal RNA in vertebrates: evolution and phylogenetic applications. Annual Review of Ecology and Systematics, vol. 21, no. 1, pp. 541-566.; Abouheif et al., 1998ABOUHEIF, E., ZARDOYA, R. and MEYER, A., 1998. Limitations of metazoan 18S rRNA sequence data: implications for reconstructing a phylogeny of the animal kingdom and inferring the reality of the Cambrian explosion. Journal of Molecular Evolution, vol. 47, no. 4, pp. 394-405. PMid:9767685.; Zardoya et al., 1998ZARDOYA, R., CAO, Y., HASEGAWA, M. and MEYER, A., 1998. Searching for the closest living relative (s) of tetrapods through evolutionary analyses of mitochondrial and nuclear data. Molecular Biology and Evolution, vol. 15, no. 5, pp. 506-517. PMid:10766578.). Single mtDNA genes were used most of the time during research studies to find out the population and taxonomic relationships (Tsigenopoulos and Berrebi, 2000TSIGENOPOULOS, C.S. and BERREBI, P., 2000. Molecular phylogeny of North Mediterranean freshwater barbs (genus Barbus: Cyprinidae) inferred from cytochrome b sequences: biogeographic and systematic implications. Molecular Phylogenetics and Evolution, vol. 14, no. 2, pp. 165-179. PMid:10679153.; Rocha-Olivares et al., 1999ROCHA-OLIVARES, A., ROSENBLATT, R.H. and VETTER, R.D., 1999. molecular evolution, systematics, and zoogeography of the rockfish subgenus Sebastomus (Sebastes, Scorpaenidae) based on mitochondrial cytochromeband control region sequences. Molecular Phylogenetics and Evolution, vol. 11, no. 3, pp. 441-458. PMid:10196084., reviewed in Meyer, 1993MEYER, A., 1993. Evolution in mitochondrial DNA in fishes. In: T.P. MOMMSEN and P.W. HOCHACHKA, eds. Molecular biology frontiers. Amsterdam: Elsevier, pp. 1-38. Biochemistry and Molecular Biology of Fishes, no. 2.; Lovejoy and Araújo, 2000LOVEJOY, N.R. and ARAÚJO, M.L.G., 2000. Molecular systematics, biogeography and population structure of Neotropical freshwater needlefishes of the genus Potamorrhaphis. Molecular Ecology, vol. 9, no. 3, pp. 259-268.PMid:10736024.). The mitochondrion organelle contains genetic information that can help in phylogenetic analyses (Moritz et al., 1987MORITZ, C.T.E.D., DOWLING, T.E. and BROWN, W.M., 1987. Evolution of animal mitochondrial DNA: relevance for population biology and systematics. Annual Review of Ecology and Systematics, vol. 18, no. 1, pp. 269-292.).

2. Material and Methods

The phylogenetic analyses of the seven major muntjac species including Indian muntjac (Muntiacus muntjak), Reeves’s muntjac (Muntiacus reevesi), Hairy fronted muntjac (Muntiacus crinifrons), Leaf muntjac (Muntiacus putaoensis), Gaint muntjac (Muntiacus vuquangensis), Roosevelt’s muntjac (Muntiacus rooseveltorum), and Troung Son muntjac (Muntiacus truongsonensis), were studied using 16s rRNA and cytochrome-b genomes.

2.1. Data description

Nucleotide sequence of cytochrome-b and 16s rRNA genes for seven muntjac species including Indian muntjac, Muntiacus muntjak (Cty-b = NCBI GeneBank AF042718.1, 1140 bp), (16s rRNA = NCBI GeneBank AF108039.1, 495bp); Reeves’s muntjac, Muntiacus reevesi (Cty-b = NCBI GeneBank AF042718.1, 1140 bp), (16s rRNA = NCBI GeneBank AF108037.1, 495bp); Hairy fronted muntjac, Muntiacus crinifrons (Cty-b = NCBI GeneBank DQ445735.1, 1140 bp), (16s rRNA = NCBI GeneBank AF108035.1, 495bp); Leaf muntjac, Muntiacus putaoensis (Cty-b = NCBI GeneBank KJ425280.1, 1140 bp), (16s rRNA = NCBI GeneBank AF108032.1, 495bp); Gaint muntjac, Muntiacus vuquangensis (Cty-b = NCBI GeneBank KJ425285.1, 1,140 bp), (16s rRNA = NCBI GeneBank AF108034.1, 495bp); Roosevelt’s muntjac, Muntiacus rooseveltorum (Cty-b = NCBI GeneBank KJ425282.1, 1140 bp), (16s rRNA = NCBI GeneBank AF108031.1, 495bp); Troung Son muntjac, Muntiacus truongsonensis (Cty-b = NCBI GeneBank KJ425277.1, 1140 bp), (16s rRNA = NCBI GeneBank AF108033.1, 495bp); and for out group Brown bear, Ursus arctos (Cty-b = NCBI Genebank HG008044.1, 1,140 bp) and Tibetan blue bear, Ursus arctos pruinosus (16s rRNA = NCBI Genebank MG131905.1, 495 bp) were retrived from NCBI databases.

2.2. 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., 2018ABDOLI, R., ZAMANI, P. and GHASEMI, M., 2018. Genetic similarities and phylogenetic analysis of human and farm animal species based on mitogenomic nucleotide sequences. Meta Gene, vol. 15, pp. 23-26.).

For selected species of muntjac, the phylogenetic tree was created using the maximum livelihood method on MEGA7 software (Kumar et al., 2016KUMAR, S., STECHER, G. and TAMURA, K., 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, vol. 33, no. 7, pp. 1870-1874. PMid:27004904.). The phylogenetic relationship between all the aligned sequences was demonstrated in the phylogenetic tree. The bootstraps consensus tree inferred from 1000 replicates.

3. Results

The consequences of nucleotide alignment analysis as similarities of sequences among 16s rRNA and cytochrome b of selected muntjac species (Muntiacus muntjak, Muntiacus reevesi, Muntiacus crinifrons, Muntiacus putaoensis, Muntiacus vuquangensis, Muntiacus rooseveltorum, Muntiacus truongsonensis) and out-group is Ursus arctos pruinosus and Ursus arctos shown in Table 1 and Table 2 respectively.

Table 1
Similarity and divergence percentage of the 16s rRNA of muntjac species.
Table 2
Similarity and divergence percentages of the cytochrome b of muntjac species.

The similarities among nucleotide sequences (16s rRNA), 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 Muntiacus reevesi had 97.8% similarities with Muntiacus crinifrons, 98.6% with Muntiacus muntjak, 97.2% Muntiacus putaoensis, 98.2% with Muntiacus vuquangensis, 98.4% Muntiacus rooseveltorum, 98.4% with Muntiacus truongsonensis. The Muntiacus crinifrons had 97.6% similarities with Muntiacus muntjak, 96.8% Muntiacus putaoensis, 98.0% with Muntiacus vuquangensis, 97.8% Muntiacus rooseveltorum, 97.8% with Muntiacus truongsonensis. The Muntiacus muntjak had 97.8% similarities with Muntiacus putaoensis, 98.0% with Muntiacus vuquangensis, 98.6% Muntiacus rooseveltorum, 98.6% with Muntiacus truongsonensis. The Muntiacus putaoensis had 97.8% similarities with Muntiacus vuquangensis, 98.0% Muntiacus rooseveltorum, 98.4% with Muntiacus truongsonensis. The Muntiacus vuquangensis had 98.2% similarities with Muntiacus rooseveltorum and Muntiacus truongsonensis. The Muntiacus rooseveltorumand Muntiacus truongsonensis had the highest similarities 99.2% (as shown in Table 1).

While the similarities among nucleotide sequences (Cty b), the highest similarities (100%) between Muntiacus muntjak and Muntiacus truongsonensis and the lowest similarities (91.5%) among Muntiacus putaoensis and Muntiacus crinifrons. The Muntiacus putaoensis had 94.1% similarities with Muntiacus vuquangensis, 91.5% with Muntiacus crinifrons, 96.8% Muntiacus muntjak, 92.8% with Muntiacus reevesi, 97.2% Muntiacus rooseveltorum, 96.8% with Muntiacus truongsonensis. The Muntiacus vuquangensis had 92.1% similarities with Muntiacus crinifrons, 94.3% Muntiacus muntjak, 92.5% with Muntiacus reevesi, 94.3% Muntiacus rooseveltorum, 94.3% with Muntiacus truongsonensis. The Muntiacus crinifrons had 91.9% similarities with Muntiacus muntjak, 92.1% with Muntiacus reevesi, 92.5% Muntiacus rooseveltorum, 91.9% with Muntiacus truongsonensis. The Muntiacus muntjak had 92.3% similarities with Muntiacus reevesi, 97.0% Muntiacus rooseveltorum, 100.0% with Muntiacus truongsonensis. The Muntiacus reevesi had 93.2% similarities with Muntiacus rooseveltorum, 92.3% with Muntiacus truongsonensis. And the Muntiacus rooseveltorum had 97.0% similarities with Muntiacus truongsonensis (as shown in Table 2).

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 1st+2nd+3rd+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.

Figure 1
Molecular phylogenetic tree of Muntiacus species (16s rRNA gene) by maximum likelihood method.
Figure 2
Molecular phylogenetic tree of Muntiacus species (Cty-b gene) by maximum likelihood method.

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 1st+2nd+3rd+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.

4. 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, 1999BOORE, J.L., 1999. Animal mitochondrial genomes. Nucleic Acids Research, vol. 27, no. 8, pp. 1767-1780.PMid:10101183.). The phylogenetic analyses of genus Muntiacus have long been studied (James et al., 2008JAMES, J., RAMAKRISHNAN, U. and DATTA, A., 2008. Molecular evidence for the occurrence of the leaf deer Muntiacus putaoensis in Arunachal Pradesh, north-east India. Conservation Genetics, vol. 9, no. 4, pp. 927-931.; Timmins and Duckworth, 2016TIMMINS, R.J., and DUCKWORTH, J.W., 2016. Muntiacus putaoensis. In: INTERNATIONAL UNION FOR CONSERVATION OF NATURE AND NATURAL RESOURCES – IUCN, ed. The IUCN Red List of Threatened Species 2016: e.T136479A22159478. Gland.). The complete mitochondrial genome of Muntiacus putaoensis was similar to other species of muntjac deer (Wu and Fang, 2005WU, H.L. and FANG, S.G., 2005. Mitochondrial DNA genetic diversity of black muntjac (Muntiacus crinifrons), an endangered species endemic to China. Biochemical Genetics, vol. 43, no. 7-8, pp. 407-416. PMid:16187164.; Shi et al., 2003SHI, Y.F., XIANGNIAN, S., JIAN, L.I., XIAOMEI, Z. and HAIJUN, Z., 2003. Sequence and organization of the complete mitochondrial genome of the Indian muntjac (Muntiacus muntjak). Dong Wu Xue Bao, vol. 49, pp. 629-636.; Zhang et al., 2004ZHANG, X.M., SHAN, X.N., SHI, Y.F., ZHANG, H.J., LI, J. and ZHENG, A.L., 2004. Sequence and organization of Muntiacus reevesi mitochondrial genome. Yi chuan=. Hereditas, vol. 26, no. 6, pp. 849-853. PMid:15640115.; Hassanin et al., 2012HASSANIN, A., DELSUC, F., ROPIQUET, A., HAMMER, C., VAN VUUREN, B.J., MATTHEE, C., RUIZ-GARCIA, M., CATZEFLIS, F., ARESKOUG, V., NGUYEN, T.T. and COULOUX, A., 2012. Pattern and timing of diversification of Cetartiodactyla (Mammalia, Laurasiatheria), as revealed by a comprehensive analysis of mitochondrial genomes. Comptes Rendus Biologies, vol. 335, no. 1, pp. 32-50. PMid:22226162.). 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)JAMES, J., RAMAKRISHNAN, U. and DATTA, A., 2008. Molecular evidence for the occurrence of the leaf deer Muntiacus putaoensis in Arunachal Pradesh, north-east India. Conservation Genetics, vol. 9, no. 4, pp. 927-931. 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)AMATO, G., EGAN, M.G. and RABINOWITZ, A., 1999. A new species of muntjac, Muntiacus putaoensis (Artiodactyla: Cervidae) from northern Myanmar. Animal Conservation, vol. 2, no. 1, pp. 1-7. 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)LI, G.G., ZHANG, M.X., SWA, K., MAUNG, K.W. and QUAN, R.C., 2017. Complete mitochondrial genome of the leaf muntjac (Muntiacus putaoensis) and phylogenetics of the genus Muntiacus. Zoological Research, vol. 38, no. 5, pp. 310-316. PMid:29181905.. 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)LE, M., NGUYEN, T.V., DUONG, H.T., NGUYEN, H.M., DINH, L.D., DO, T., NGUYEN, H.D. and AMATO, G., 2014. Discovery of the Roosevelt’s Barking Deer (Muntiacus rooseveltorum) in Vietnam. Conservation Genetics, vol. 15, no. 4, pp. 993-999.. James et al. (2008)JAMES, J., RAMAKRISHNAN, U. and DATTA, A., 2008. Molecular evidence for the occurrence of the leaf deer Muntiacus putaoensis in Arunachal Pradesh, north-east India. Conservation Genetics, vol. 9, no. 4, pp. 927-931. 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)LI, G.G., ZHANG, M.X., SWA, K., MAUNG, K.W. and QUAN, R.C., 2017. Complete mitochondrial genome of the leaf muntjac (Muntiacus putaoensis) and phylogenetics of the genus Muntiacus. Zoological Research, vol. 38, no. 5, pp. 310-316. PMid:29181905., which indicates that Muntiacus truongsonensis was closely related to Muntiacus putaoensis based on cytochrome b genome sequences.

Acknowledgements

We are thankful to the Department of Microbiology, University of Haripur, especially to Dr. Irshad ul Haq for data collection and data analysis. The project was funded by Pakistan Agricultural Research Council (PARC) Islamabad Pakistan, under Agricultural Linkages Programme (ALP) with project number AS 156. The authors would like to extend their sincere appreciation to the Researchers Supporting Project Number (RSP-2021/180), King Saud University, Riyadh, Saudi Arabia.

References

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Publication Dates

  • Publication in this collection
    27 Aug 2021
  • Date of issue
    2023

History

  • Received
    30 Jan 2021
  • Accepted
    02 Mar 2021
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