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Abstract Hog deer （Axis porcinus） is a small mammal and listed in the International Union for Conservation of Nature. However， phylogenetic position of hog deer within Axis genus has remained controversial. In the present study， we first assembled complete mitochondrial genome of Chinese hog deer reared in Chengdu Zoo， Sichuan， by the second-generation sequencing technology. This newly assembled mitochondrial genome of hog deer is 16 376 bp in length and consists of 13 protein-encoding genes， 23 transfer RNA genes and 2 ribosomal RNA genes. Phylogenetic analyses based on complete mitochondrial genome and cytochrome b gene sequences revealed that hog deer is closely clustered together and placed with sister taxon of spotted deer （A. Axis）， which therefore supported monophyletic statue of Axis genus. Furthermore， considerable genetic differentiation， up to 139 mutations of complete mitochondrial genome was revealed between geographical populations of hog deer in France and Southeast Asia. However， only six variable sites （nucleotide diversity of 0.000 07） and four haplotypes （haplotype diversity of 0.533） were totally detected among ten newly sequenced Chinese hog deer. The results provide a better understanding on the phylogeny of hog deer.
Key words Hog deer; Complete mitochondrial genome; Phylogeny; Diversity
Received： July 21， 2020 Accepted： September 14， 2020
Supported by The Chengdu Giant Panda Breeding Research Foundation Project （CPF2017-07）.
Wei WANG （1984-）， male， P. R. China， associate researcher， devoted to research about animal genetic breeding and propagation.
*Corresponding author. E-mail： 240190769@qq.com; E-mail： wws20062127@163.com.
Hog deer （Axis porcinus） is a small animal in the subfamily Cervinae and mainly distributed in South and Southeast Asia， such as Pakistan， India， Myanmar， Thailand and China[1]. Because the wild hog deer have dramatically disappeared as a result of habitat destruction as well as intensive hunting， it has been listed as an endangered species since 2008 in International Union for Conservation of Nature （IUCN）. Although there is historical record that wild hog deer once lived in Gengma and Cangyuan counties of Western Yunnan， wild population has been acknowledged to be almost extinct in China[2-3]. Fortunately， a captive population of about 50 hog deer has been persistently kept in Chengdu Zoo in Sichuan Province during the past two decades， which therefore provides a well conserved gene pool for this species[4]. Deer of the subfamily Cervinae have been phylogeneticcally classified into four genera of Cervus， Axis， Dama and Elaphurus[5]， among which Axis genus contains hog deer and other three species， including spotted deer （A. axis）， calamian deer （A. calamianensis） and bawean deer （A. kuhlii）. Due to the morphological， geographical and behavioural complexity， it has long been controversial about the phylogenic relationships among Axis taxa. According to morphological characters[6] and mitochondrial cytochrome b gene sequences[6-8]， Axis genus was suggested to be paraphyletic with a close affinity of hog deer to timor deer （C. timorensis） and sambar （C. unicolor）. However， the monophyletic status of Axis genus was also supported by complete mitochondrial genome[9]， nuclear gene[10] and D-loop regionsequences[11]. According to geographical distributions， it has also been proposed that hog deer could be divided into two subspecies， including the Southeast subspecies （A. p. annamiticus） from China， Thailand， Laos， Cambodia and Vietnam， and the Indian subspecies （A. p. porcinus） distributed in Pakistan， Nepal， India， Bangladesh and Burma[12]. Furthermore， the subspecies of A. p. annamiticus was also thought to be a new species taxon based on morphological characters[13]. However， the phylogeny among different geographical populations of hog deer should be carefully further evaluated by more molecular evidence.
The mitochondrial DNA sequence has been widely used as a genetic marker in studies of population and evolutionary biology[14]. Direct sequencing of complete mitochondrial genome is largely facilitated with the obviously improved throughput since the advent of second-generation sequencing technology[15-16]. Meanwhile， many algorithms have been also proposed to accurately assemble the mitochondrial genome from short reads[17]. In the present study， we first assembled complete mitochondrial genome of hog deer sampled in Chengdu Zoo by strategy of low-coverage sequencing of genomic DNA. Subsequently， the phylogenetic positions of Chinese hog deer and genetic diversity were investigated.
Material and Methods
Ethics statement
In the present study， blood sample was collected by veterinarian at annual health inspection. The study design and all experimental methods were approved by Animal Care and Use Committee in Chengdu Zoo.
Sampling， DNA extraction and sequencing
All animals sequenced in the present study were also used in our previous study， which was intended to provide genome-wide SNPs by restriction-site-associated DNA sequencing[4]. Briefly， venous whole blood was collected among 11 unrelated hog deer reared in Chengdu Zoo， Chengdu， China. Genomic DNA was extracted and purified from blood tissue samples according to the protocol of the Animal Genomic DNA Kit （Tiangen， Beijing）. Finally， a total of 10 deer were successfully subjected to whole-genome shotgun sequencing according to the required DNA concentration and quality （GE， American）. Sequencing libraries were first prepared with the insert fragment of -270 bp in length according to manufacturer’s instructions （Illumina， San Diego， USA）， in which the whole DNA consisting of both nuclear and mitochondrial genomes was used. The whole-genome sequencing was subsequently conducted using Illumina HiSeqTM 4000 platform （Illumina， San Diego， USA） at BGI-Shenzhen （Shenzhen， China） with production of 150-bp length of paired-end reads.
Assembly of mitochondrial genome
Raw reads in fast q format were first filtered according to the recommended quality criteria[18]， which contains the cutting of adaptor segments and removing of low-quality sequences. The low-quality read belongs to one of the following types： （i） reads containing adaptor sequences， （ii） reads containing unambiguous bases of N more than 10% of the total length， and （iii） reads containing low-quality bases （Q<5） more than 50% of the total length. After getting clean reads， complete mitochondrial genomes were assembled by two steps. First， all clean reads were mapped against the published reference sequence of mitochondrial genome of hog deer （Gen Bank accession number： NC_020681） using Bowtie2 v2.3.2 with the default parameters[19]. Second， the generated SAM file for each sample was individually subjected to assembly of mitochondrial genome using SPAdes tool v3.0.0[20]. Subsequently， annotation of the newly assembled mitochondrial genome of hog deer was performed on MITOS Web Server[21] with default parameters.
Phylogenetic and genetic diversity analyses
To investigate the phylogenetic position of Chinese hog deer， complete mitochondrial genome sequences of 22 species in Cervinae， including a published mitochondrial genome of hog deer collected in France， were retrieved from Gen Bank （Table 1）. All sequences were first aligned by ClustalW method included in the program MEGA7.0[22]. Subsequently， we employed the Neighbor-Joining method and Kimura 2-parameter model for constructing phylogenetic treewith 1000 bootstrap replications[22]， in which two species of tufted deer （Elaphodus cephalophus） and black muntjac （Muntiacus crinifrons） were also included as out groups. In addition to complete mitochondrial genomes， we also constructed Neighbor-Joining tree based on cytochrome b sequences by additionally including five sequences of hog deer which are available in the Gen Bank （AY035874， AY157734， DQ379301， EU878394 and FJ556558）.
For the ten complete mitochondrial genomes of hog deer newly sequenced in the present study， we aligned them to the published reference sequences （accession number： NC_020681） and exported the variable sites. Subsequently， the haplotype diversity and nucleotide diversity were estimated using DnaSPv5[23]. Results and Discussion
Due to the inherent merits of mitochondrial DNA， including matrilineal inheritance， high evolutionary rate and no recombination， it has been widely used for studying genetic diversity， population structure and phylogenetic relationship in both human and animals[14， 24-25]. In the early studies， partial mitochondrial fragments hundreds of base pairs （bp） long， such as D-loop region[26] and cytochrome b gene[27]， are more preferable perhaps because of difficulties in sequencing long sequence with hundreds of thousands of bp in length. However， the high-throughput sequencing technologies largely alleviate sequencing of complete mitochondrial genomes[16]. In the present study， we totally generated 184.7 million raw paired-end reads among ten Chinese hog deer after quality filtering. The sequenced reads are expected to have ～1.5 X coverage of genome with reference to the reported genome size of 3 438 Mb of C. elaphus （Gen Bank SRA project： PRJNA324173）， which therefore would be enough for assembling mitochondrial genome[28].
After all clean reads were mapped against the published mitochondrial genome of hog deer[9]， it was revealed that the mean coverage of reads is up to 132 X and therefore guarantees the confident assembly[29-30]. We successfully assembled complete mitochondrial genome sequences for Chinese hog deer with circular DNA molecule of 16 376 bp in length， which is only one bp longer than this reference sequence. The newly assembled mitochondrial genome has A+T content of 62.59% for overall composition， which also doesn’t show significant difference among protein-encoding， tRNA and rRNA genes （Table 2）. Based on the ab initio annotation[21]， we totally detected 13 protein-encoding genes， 23 transfer RNA genes and 2 ribosomal RNA genes with the predicted boundaries （Table 3）. The sequence features of mitochondrial genome of Chinese hog deer newly sequenced in this study are consistent with the previous reports for the related animals in the subfamily Cervinae[31- 32].
In our recently published paper[4]， we comprehensively detected genome-wide SNPs for the same population of Chinese hog deer reared in Chengdu Zoo， by which， however， the phylogenetic relationship of hog deer was still remained to be unsolved. It was early reported in multiple studies[5， 7， 8， 33] that either partial or complete mitochondrial cytochrome b gene sequences supported a non-monophyletic status of Axis genus， which confidently clustered hog deer into Cervus genus. However， Gilbert and colleagues （2006） argued that all these studies actually employed a same DNA sequence of cytochrome b gene （Accession no.， AY035874） from a hog deer in India[8]， which， more importantly， was doubted to derive from sample contamination or species misidentification. In addition to the independently sequenced cytochrome b gene sequences of hog deer sampled in France[10]， the complete mitochondrial genome sequence also revealed the close affinity between hog deer and spotted deer， and hence suggested the monophyletic status of Axis genus[9]. However， this controversial issue should be carefully re-evaluated by sequencing additional sample. In the present study， we newly obtained the complete mitochondrial genome sequence in Chinese hog deer and then constructed a phylogenetic tree by including other 21 species available within Cervinae （Fig. 1A）. Our results supported the previous reports[9-10] that both hog deer and spotted deer are first clustered together as a monophyletic Axis genus， which further has a close relationship with swamp deer （R. duvaucelii）. From these mitochondrial genome sequences， we further extracted cytochrome b gene sequences and similarly performed phylogenetic analysis （Fig. 1B）， in which five additional sequences of hog deer with different geographical origins were also included. The overall phylogenetic relationship among these species revealed by cytochrome b gene sequences is comparable with that supported by complete genome sequences. However， seven hog deer were obviously separated into two distant phylogenetic positions， i.e.， the two early published sequences were placed into Cervus genus， whereas the remaining five later sequenced hog deer were clustered within Axis genus. This result could be explained by either sequencing error or actual difference among different geographical populations. However， two sequences of Chinese hog deer， one of both was newly sequenced in the present study， were separately placed， which therefore exclude the possible genetic differentiation among geographical populations of hog deer.
To well know the genetic structure and diversity is essential for efficiently conserving gene pool. We recently investigated the genome-wide SNPs in the captive population of hog deer in Chengdu Zoo and revealed sufficient DNA polymorphism[4]. Because mitochondrial DNA is maternally inherited， the matrilineal component of Chinese hog deer was further analyzed by ten complete mitochondrial genome sequences obtained in the present study. Within the complete mitochondrial genome， a total of 139 mutations were first revealed between Chinese hog deer and the previously sequenced hog deer sampled in France （Fig. 2）. Together with phylogenetic relationship provided by cytochrome b gene sequences （Fig. 1B）， we conclude that there are considerable genetic differentiation between geographical populations in France and Southeast Asia （China and Thailand）. However， only six variable sites （nucleotide diversity of 0.000 07） and four haplotypes （haplotype diversity of 0.533） were detected among the ten sequenced Chinese hog deer （Fig. 2）. This result would suggest deficiency in matrilineal component for this captive population of hog deer， which should be paid with more attentions.
Conclusions
In the present study， we successfully reconstructed complete mitochondrial genome of Chinese hog deer using high-throughput sequencing technology. The phylogenetic analyses supported a monophyletic status of Axis genus and also revealed considerable genetic differentiation among different geographical populations. However， the matrilineal components would be deficient in the captive population of hog deer reared in Chengdu Zoo， Sichuan. Acknowledgements
Author Contributions Wang Wei and Yan Huijuan conceived the study. Wang Wei， Yan Huijuan and Yu Jianqiu designed and performed the computational experiments. Wang Wei wrote the manuscript. We are grateful to Professor Chen of Sichuan Agricultural University for helping with the statistical analysis.
Ethics statement
In the present study， blood sample was collected by veterinarian at annual health inspection. The study design and all experimental methods were approved by Animal Care and Use Committee in Chengdu Zoo.
References
[1] GAUBERT P， GREENGRASS EJ， SAN EDL. Axis porcinus. The IUCN red list of threatened species[J]. 2015， DOI： 10.2305/IUCN.UK.2015-4.RLTS， T136223A45220931.en.
[2] WANG S. China red data book of endangered animals： Mammalian[M]. Beijing： Science Press， 1998.
[3] SMITH A， XIE Y. A guide to the mammals of China[M]. Princeton： Princeton University Press， 2008.
[4] WANG W， YAN H， YU J， et al. Discovery of genome-wide SNPs by RAD-seqand the genetic diversity of captive hog deer （Axis porcinus） [J]. PloS one， 2017， 12， e0174299.
[5] PITRA C， FICKEL J， MEIJAARD E， et al. Evolution and phylogeny of old world deer[J]. Molecular Phylogenetics and Evolution， 2004（33）： 880-895.
[6] GEIST V. Deer of the world. Their evolution， behavior and ecology[J]. Journal of Wildlife Management， 2000（64）： 606.
[7] LIU X， WANG Y， LIU Z， et al. Phylogenetic relationships of Cervinae based on sequence of mitochondrial cytochrome b gene[J]. Zoological Research， 2003（24）： 27-33.
[8] LUDT CJ， SCHROEDER W， ROTTMANN O， et al. Mitochondrial DNA phylogeography of red deer （Cervus elaphus） [J]. Molecular Phylogenetics and Evolution， 2004（31）： 1064-83.
[9] HASSANIN A， DELSUC F， ROPIQUET A， et al. Pattern and timing of diversification of Cetartiodactyla （Mammalia， Laurasiatheria）， as revealed by a comprehensive analysis of mitochondrial genomes[J]. Comptes Rendus Biologies， 2012（335）： 32-50.
[10] GILBERT C， ROPIQUET A， HASSANIN A. Mitochondrial and nuclear phylogenies of Cervidae （Mammalia， Ruminantia）： systematics， morphology， and biogeography[J]. Molecular Phylogenetics and Evolution， 2006（40）： 101-117.
[11] RANDI E， MUCCI N， CLARO-HERGUETA F， et al. A mitochondrial DNA control region phylogeny of the Cervinae： speciation in Cervus and implications for conservation[J]. In Animal Conservation Forum Cambridge University Press， 2001（4）： 1-11. [12] BISWAS T， MATHUR VB. A review of the present conservation scenario of hog deer （Axis porcinus） in its native range[J]. Indian Forester， 2000（126）：1068-1084.
[13] GROVES C， GRUBB P. Ungulate taxonomy[J]. Baltimore： The Johns Hopkins University Press， 2011， USA.
[14] GALTIER N， NABHOLZ B， GLéMIN S， et al. Mitochondrial DNA as a marker of molecular diversity： A reappraisal[J]. Molecular Ecology， 2009（18）： 4541-4550.
[15] MARDIS ER. The impact of next-generation sequencing technology on genetics[J]. Trends in Genetics， 2008（24）：133-141.
[16] GREEN RE， MALASPINAS AS， KRAUSE J， et al. A complete Neandertal mitochondrial genome sequence determined by high-throughput sequencing[J]. Cell， 2008（134）： 416-426.
[17] HAHN C， BACHMANN L， CHEVREUX B. Reconstructing mitochondrial genomes directly from genomic next-generation sequencing reads—A baiting and iterative mapping approach[J]. Nucleic Acids Research， 2013， 41， e129-e129.
[18] LI R， FAN W， TIAN G， et al. The sequence and de novo assembly of the giant panda genome[J]. Nature， 2010（463）： 311.
[19] LANGMEAD B， SALZBERG S. Fast gapped-read alignment with Bowtie 2[J]. Nature Methods， 2012（9）： 357-359.
[20] BANKEVICH A， NURK S， ANTIPOV D， et al. SPAdes： a new genome assembly algorithm and its applications to single-cell sequencing[J]. Journal of computational biology， 2012（19）： 455-77.
[21] BERNT M， DONATH A， JüHLING F， et al. MITOS： improved de novo metazoan mitochondrial genome annotation[J]. Molecular phylogenetics and evolution， 2013（69）： 313-9.
[22] SUDHIR K， GLEN S， KOICHIRO T. MEGA7： Molecular evolutionary genetics analysis version 7.0[J]. Molecular Biology and Evolution （submitted）， 2015.
[23] LIBRADO P， ROZAS J. DnaSP v5： A software for comprehensive analysis of DNA polymorphism data[J]. Bioinformatics， 2009（25）： 1451-1452.
[24] MACAULAY V， HILL C， ACHILLI A， et al. Single， rapid coastal settlement of Asia revealed by analysis of complete mitochondrial genomes[J]. Science， 2005（308）： 1034-1036.
[25] LAI SJ， LIU YP， LIU YX， et al. Genetic diversity and origin of Chinese cattle revealed by mtDNA D-loop sequence variation[J]. Molecular phylogenetics and evolution， 2006（38）： 146-154.
[26] SBIS E， TANZARIELLO F， REYES A， et al. Mammalian mitochondrial D-loop region structural analysis： identification of new conserved sequences and their functional and evolutionary implications[J]. Gene， 1997（205）： 125-140. [27] JOHNS GC， AVISE JC. A comparative summary of genetic distances in the vertebrates from the mitochondrial cytochrome b gene[J]. Molecular Biology and Evolution， 1998（15）： 1481-1490.
[28] HAMOY IG， RIBEIRO-DOS-SANTOS AM， ALVAREZ L， et al. A protocol for mtGenome analysis on large sample numbers[J]. Bioinformatics and biology insights， 2014（8）： 127.
[29] SIMS D， SUDBERY I， ILOTT NE， et al. Sequencing depth and coverage： key considerations in genomic analyses[J]. Nature Reviews Genetics， 2014（15）： 121-132.
[30] NUNEZ JC， OLEKSIAK MF. A cost-effective approach to sequence hundreds of complete mitochondrial genomes[J]. PloS one， 2016， 11， e0160958.
[31] LIU YH， LIU XX， ZHANG MH. The complete mitochondrial genome of Sika deer Cervus Nippon hortulorum （Artiodactyla： Cervidae） and phylogenetic studies[J]. Mitochondrial DNA Part A， 2016（27）： 2967-2968.
[32] LI Y， BA H， YANG F. Complete mitochondrial genome of Cervus elaphus songaricus （Cetartiodactyla： Cervinae） and a phylogenetic analysis with related species[J]. Mitochondrial DNA Part A， 2016（27）： 620-621.
[33] GUHA S， GOYAL SP， KASHYAP VK. Molecular phylogeny of musk deer： A genomic view with mitochondrial 16S rRNA and cytochrome b gene[J]. Molecular phylogenetics and evolution， 2007（42）： 585-597.
Editor： Yingzhi GUANG Proofreader： Xinxiu ZHU
Key words Hog deer; Complete mitochondrial genome; Phylogeny; Diversity
Received： July 21， 2020 Accepted： September 14， 2020
Supported by The Chengdu Giant Panda Breeding Research Foundation Project （CPF2017-07）.
Wei WANG （1984-）， male， P. R. China， associate researcher， devoted to research about animal genetic breeding and propagation.
*Corresponding author. E-mail： 240190769@qq.com; E-mail： wws20062127@163.com.
Hog deer （Axis porcinus） is a small animal in the subfamily Cervinae and mainly distributed in South and Southeast Asia， such as Pakistan， India， Myanmar， Thailand and China[1]. Because the wild hog deer have dramatically disappeared as a result of habitat destruction as well as intensive hunting， it has been listed as an endangered species since 2008 in International Union for Conservation of Nature （IUCN）. Although there is historical record that wild hog deer once lived in Gengma and Cangyuan counties of Western Yunnan， wild population has been acknowledged to be almost extinct in China[2-3]. Fortunately， a captive population of about 50 hog deer has been persistently kept in Chengdu Zoo in Sichuan Province during the past two decades， which therefore provides a well conserved gene pool for this species[4]. Deer of the subfamily Cervinae have been phylogeneticcally classified into four genera of Cervus， Axis， Dama and Elaphurus[5]， among which Axis genus contains hog deer and other three species， including spotted deer （A. axis）， calamian deer （A. calamianensis） and bawean deer （A. kuhlii）. Due to the morphological， geographical and behavioural complexity， it has long been controversial about the phylogenic relationships among Axis taxa. According to morphological characters[6] and mitochondrial cytochrome b gene sequences[6-8]， Axis genus was suggested to be paraphyletic with a close affinity of hog deer to timor deer （C. timorensis） and sambar （C. unicolor）. However， the monophyletic status of Axis genus was also supported by complete mitochondrial genome[9]， nuclear gene[10] and D-loop regionsequences[11]. According to geographical distributions， it has also been proposed that hog deer could be divided into two subspecies， including the Southeast subspecies （A. p. annamiticus） from China， Thailand， Laos， Cambodia and Vietnam， and the Indian subspecies （A. p. porcinus） distributed in Pakistan， Nepal， India， Bangladesh and Burma[12]. Furthermore， the subspecies of A. p. annamiticus was also thought to be a new species taxon based on morphological characters[13]. However， the phylogeny among different geographical populations of hog deer should be carefully further evaluated by more molecular evidence.
The mitochondrial DNA sequence has been widely used as a genetic marker in studies of population and evolutionary biology[14]. Direct sequencing of complete mitochondrial genome is largely facilitated with the obviously improved throughput since the advent of second-generation sequencing technology[15-16]. Meanwhile， many algorithms have been also proposed to accurately assemble the mitochondrial genome from short reads[17]. In the present study， we first assembled complete mitochondrial genome of hog deer sampled in Chengdu Zoo by strategy of low-coverage sequencing of genomic DNA. Subsequently， the phylogenetic positions of Chinese hog deer and genetic diversity were investigated.
Material and Methods
Ethics statement
In the present study， blood sample was collected by veterinarian at annual health inspection. The study design and all experimental methods were approved by Animal Care and Use Committee in Chengdu Zoo.
Sampling， DNA extraction and sequencing
All animals sequenced in the present study were also used in our previous study， which was intended to provide genome-wide SNPs by restriction-site-associated DNA sequencing[4]. Briefly， venous whole blood was collected among 11 unrelated hog deer reared in Chengdu Zoo， Chengdu， China. Genomic DNA was extracted and purified from blood tissue samples according to the protocol of the Animal Genomic DNA Kit （Tiangen， Beijing）. Finally， a total of 10 deer were successfully subjected to whole-genome shotgun sequencing according to the required DNA concentration and quality （GE， American）. Sequencing libraries were first prepared with the insert fragment of -270 bp in length according to manufacturer’s instructions （Illumina， San Diego， USA）， in which the whole DNA consisting of both nuclear and mitochondrial genomes was used. The whole-genome sequencing was subsequently conducted using Illumina HiSeqTM 4000 platform （Illumina， San Diego， USA） at BGI-Shenzhen （Shenzhen， China） with production of 150-bp length of paired-end reads.
Assembly of mitochondrial genome
Raw reads in fast q format were first filtered according to the recommended quality criteria[18]， which contains the cutting of adaptor segments and removing of low-quality sequences. The low-quality read belongs to one of the following types： （i） reads containing adaptor sequences， （ii） reads containing unambiguous bases of N more than 10% of the total length， and （iii） reads containing low-quality bases （Q<5） more than 50% of the total length. After getting clean reads， complete mitochondrial genomes were assembled by two steps. First， all clean reads were mapped against the published reference sequence of mitochondrial genome of hog deer （Gen Bank accession number： NC_020681） using Bowtie2 v2.3.2 with the default parameters[19]. Second， the generated SAM file for each sample was individually subjected to assembly of mitochondrial genome using SPAdes tool v3.0.0[20]. Subsequently， annotation of the newly assembled mitochondrial genome of hog deer was performed on MITOS Web Server[21] with default parameters.
Phylogenetic and genetic diversity analyses
To investigate the phylogenetic position of Chinese hog deer， complete mitochondrial genome sequences of 22 species in Cervinae， including a published mitochondrial genome of hog deer collected in France， were retrieved from Gen Bank （Table 1）. All sequences were first aligned by ClustalW method included in the program MEGA7.0[22]. Subsequently， we employed the Neighbor-Joining method and Kimura 2-parameter model for constructing phylogenetic treewith 1000 bootstrap replications[22]， in which two species of tufted deer （Elaphodus cephalophus） and black muntjac （Muntiacus crinifrons） were also included as out groups. In addition to complete mitochondrial genomes， we also constructed Neighbor-Joining tree based on cytochrome b sequences by additionally including five sequences of hog deer which are available in the Gen Bank （AY035874， AY157734， DQ379301， EU878394 and FJ556558）.
For the ten complete mitochondrial genomes of hog deer newly sequenced in the present study， we aligned them to the published reference sequences （accession number： NC_020681） and exported the variable sites. Subsequently， the haplotype diversity and nucleotide diversity were estimated using DnaSPv5[23]. Results and Discussion
Due to the inherent merits of mitochondrial DNA， including matrilineal inheritance， high evolutionary rate and no recombination， it has been widely used for studying genetic diversity， population structure and phylogenetic relationship in both human and animals[14， 24-25]. In the early studies， partial mitochondrial fragments hundreds of base pairs （bp） long， such as D-loop region[26] and cytochrome b gene[27]， are more preferable perhaps because of difficulties in sequencing long sequence with hundreds of thousands of bp in length. However， the high-throughput sequencing technologies largely alleviate sequencing of complete mitochondrial genomes[16]. In the present study， we totally generated 184.7 million raw paired-end reads among ten Chinese hog deer after quality filtering. The sequenced reads are expected to have ～1.5 X coverage of genome with reference to the reported genome size of 3 438 Mb of C. elaphus （Gen Bank SRA project： PRJNA324173）， which therefore would be enough for assembling mitochondrial genome[28].
After all clean reads were mapped against the published mitochondrial genome of hog deer[9]， it was revealed that the mean coverage of reads is up to 132 X and therefore guarantees the confident assembly[29-30]. We successfully assembled complete mitochondrial genome sequences for Chinese hog deer with circular DNA molecule of 16 376 bp in length， which is only one bp longer than this reference sequence. The newly assembled mitochondrial genome has A+T content of 62.59% for overall composition， which also doesn’t show significant difference among protein-encoding， tRNA and rRNA genes （Table 2）. Based on the ab initio annotation[21]， we totally detected 13 protein-encoding genes， 23 transfer RNA genes and 2 ribosomal RNA genes with the predicted boundaries （Table 3）. The sequence features of mitochondrial genome of Chinese hog deer newly sequenced in this study are consistent with the previous reports for the related animals in the subfamily Cervinae[31- 32].
In our recently published paper[4]， we comprehensively detected genome-wide SNPs for the same population of Chinese hog deer reared in Chengdu Zoo， by which， however， the phylogenetic relationship of hog deer was still remained to be unsolved. It was early reported in multiple studies[5， 7， 8， 33] that either partial or complete mitochondrial cytochrome b gene sequences supported a non-monophyletic status of Axis genus， which confidently clustered hog deer into Cervus genus. However， Gilbert and colleagues （2006） argued that all these studies actually employed a same DNA sequence of cytochrome b gene （Accession no.， AY035874） from a hog deer in India[8]， which， more importantly， was doubted to derive from sample contamination or species misidentification. In addition to the independently sequenced cytochrome b gene sequences of hog deer sampled in France[10]， the complete mitochondrial genome sequence also revealed the close affinity between hog deer and spotted deer， and hence suggested the monophyletic status of Axis genus[9]. However， this controversial issue should be carefully re-evaluated by sequencing additional sample. In the present study， we newly obtained the complete mitochondrial genome sequence in Chinese hog deer and then constructed a phylogenetic tree by including other 21 species available within Cervinae （Fig. 1A）. Our results supported the previous reports[9-10] that both hog deer and spotted deer are first clustered together as a monophyletic Axis genus， which further has a close relationship with swamp deer （R. duvaucelii）. From these mitochondrial genome sequences， we further extracted cytochrome b gene sequences and similarly performed phylogenetic analysis （Fig. 1B）， in which five additional sequences of hog deer with different geographical origins were also included. The overall phylogenetic relationship among these species revealed by cytochrome b gene sequences is comparable with that supported by complete genome sequences. However， seven hog deer were obviously separated into two distant phylogenetic positions， i.e.， the two early published sequences were placed into Cervus genus， whereas the remaining five later sequenced hog deer were clustered within Axis genus. This result could be explained by either sequencing error or actual difference among different geographical populations. However， two sequences of Chinese hog deer， one of both was newly sequenced in the present study， were separately placed， which therefore exclude the possible genetic differentiation among geographical populations of hog deer.
To well know the genetic structure and diversity is essential for efficiently conserving gene pool. We recently investigated the genome-wide SNPs in the captive population of hog deer in Chengdu Zoo and revealed sufficient DNA polymorphism[4]. Because mitochondrial DNA is maternally inherited， the matrilineal component of Chinese hog deer was further analyzed by ten complete mitochondrial genome sequences obtained in the present study. Within the complete mitochondrial genome， a total of 139 mutations were first revealed between Chinese hog deer and the previously sequenced hog deer sampled in France （Fig. 2）. Together with phylogenetic relationship provided by cytochrome b gene sequences （Fig. 1B）， we conclude that there are considerable genetic differentiation between geographical populations in France and Southeast Asia （China and Thailand）. However， only six variable sites （nucleotide diversity of 0.000 07） and four haplotypes （haplotype diversity of 0.533） were detected among the ten sequenced Chinese hog deer （Fig. 2）. This result would suggest deficiency in matrilineal component for this captive population of hog deer， which should be paid with more attentions.
Conclusions
In the present study， we successfully reconstructed complete mitochondrial genome of Chinese hog deer using high-throughput sequencing technology. The phylogenetic analyses supported a monophyletic status of Axis genus and also revealed considerable genetic differentiation among different geographical populations. However， the matrilineal components would be deficient in the captive population of hog deer reared in Chengdu Zoo， Sichuan. Acknowledgements
Author Contributions Wang Wei and Yan Huijuan conceived the study. Wang Wei， Yan Huijuan and Yu Jianqiu designed and performed the computational experiments. Wang Wei wrote the manuscript. We are grateful to Professor Chen of Sichuan Agricultural University for helping with the statistical analysis.
Ethics statement
In the present study， blood sample was collected by veterinarian at annual health inspection. The study design and all experimental methods were approved by Animal Care and Use Committee in Chengdu Zoo.
References
[1] GAUBERT P， GREENGRASS EJ， SAN EDL. Axis porcinus. The IUCN red list of threatened species[J]. 2015， DOI： 10.2305/IUCN.UK.2015-4.RLTS， T136223A45220931.en.
[2] WANG S. China red data book of endangered animals： Mammalian[M]. Beijing： Science Press， 1998.
[3] SMITH A， XIE Y. A guide to the mammals of China[M]. Princeton： Princeton University Press， 2008.
[4] WANG W， YAN H， YU J， et al. Discovery of genome-wide SNPs by RAD-seqand the genetic diversity of captive hog deer （Axis porcinus） [J]. PloS one， 2017， 12， e0174299.
[5] PITRA C， FICKEL J， MEIJAARD E， et al. Evolution and phylogeny of old world deer[J]. Molecular Phylogenetics and Evolution， 2004（33）： 880-895.
[6] GEIST V. Deer of the world. Their evolution， behavior and ecology[J]. Journal of Wildlife Management， 2000（64）： 606.
[7] LIU X， WANG Y， LIU Z， et al. Phylogenetic relationships of Cervinae based on sequence of mitochondrial cytochrome b gene[J]. Zoological Research， 2003（24）： 27-33.
[8] LUDT CJ， SCHROEDER W， ROTTMANN O， et al. Mitochondrial DNA phylogeography of red deer （Cervus elaphus） [J]. Molecular Phylogenetics and Evolution， 2004（31）： 1064-83.
[9] HASSANIN A， DELSUC F， ROPIQUET A， et al. Pattern and timing of diversification of Cetartiodactyla （Mammalia， Laurasiatheria）， as revealed by a comprehensive analysis of mitochondrial genomes[J]. Comptes Rendus Biologies， 2012（335）： 32-50.
[10] GILBERT C， ROPIQUET A， HASSANIN A. Mitochondrial and nuclear phylogenies of Cervidae （Mammalia， Ruminantia）： systematics， morphology， and biogeography[J]. Molecular Phylogenetics and Evolution， 2006（40）： 101-117.
[11] RANDI E， MUCCI N， CLARO-HERGUETA F， et al. A mitochondrial DNA control region phylogeny of the Cervinae： speciation in Cervus and implications for conservation[J]. In Animal Conservation Forum Cambridge University Press， 2001（4）： 1-11. [12] BISWAS T， MATHUR VB. A review of the present conservation scenario of hog deer （Axis porcinus） in its native range[J]. Indian Forester， 2000（126）：1068-1084.
[13] GROVES C， GRUBB P. Ungulate taxonomy[J]. Baltimore： The Johns Hopkins University Press， 2011， USA.
[14] GALTIER N， NABHOLZ B， GLéMIN S， et al. Mitochondrial DNA as a marker of molecular diversity： A reappraisal[J]. Molecular Ecology， 2009（18）： 4541-4550.
[15] MARDIS ER. The impact of next-generation sequencing technology on genetics[J]. Trends in Genetics， 2008（24）：133-141.
[16] GREEN RE， MALASPINAS AS， KRAUSE J， et al. A complete Neandertal mitochondrial genome sequence determined by high-throughput sequencing[J]. Cell， 2008（134）： 416-426.
[17] HAHN C， BACHMANN L， CHEVREUX B. Reconstructing mitochondrial genomes directly from genomic next-generation sequencing reads—A baiting and iterative mapping approach[J]. Nucleic Acids Research， 2013， 41， e129-e129.
[18] LI R， FAN W， TIAN G， et al. The sequence and de novo assembly of the giant panda genome[J]. Nature， 2010（463）： 311.
[19] LANGMEAD B， SALZBERG S. Fast gapped-read alignment with Bowtie 2[J]. Nature Methods， 2012（9）： 357-359.
[20] BANKEVICH A， NURK S， ANTIPOV D， et al. SPAdes： a new genome assembly algorithm and its applications to single-cell sequencing[J]. Journal of computational biology， 2012（19）： 455-77.
[21] BERNT M， DONATH A， JüHLING F， et al. MITOS： improved de novo metazoan mitochondrial genome annotation[J]. Molecular phylogenetics and evolution， 2013（69）： 313-9.
[22] SUDHIR K， GLEN S， KOICHIRO T. MEGA7： Molecular evolutionary genetics analysis version 7.0[J]. Molecular Biology and Evolution （submitted）， 2015.
[23] LIBRADO P， ROZAS J. DnaSP v5： A software for comprehensive analysis of DNA polymorphism data[J]. Bioinformatics， 2009（25）： 1451-1452.
[24] MACAULAY V， HILL C， ACHILLI A， et al. Single， rapid coastal settlement of Asia revealed by analysis of complete mitochondrial genomes[J]. Science， 2005（308）： 1034-1036.
[25] LAI SJ， LIU YP， LIU YX， et al. Genetic diversity and origin of Chinese cattle revealed by mtDNA D-loop sequence variation[J]. Molecular phylogenetics and evolution， 2006（38）： 146-154.
[26] SBIS E， TANZARIELLO F， REYES A， et al. Mammalian mitochondrial D-loop region structural analysis： identification of new conserved sequences and their functional and evolutionary implications[J]. Gene， 1997（205）： 125-140. [27] JOHNS GC， AVISE JC. A comparative summary of genetic distances in the vertebrates from the mitochondrial cytochrome b gene[J]. Molecular Biology and Evolution， 1998（15）： 1481-1490.
[28] HAMOY IG， RIBEIRO-DOS-SANTOS AM， ALVAREZ L， et al. A protocol for mtGenome analysis on large sample numbers[J]. Bioinformatics and biology insights， 2014（8）： 127.
[29] SIMS D， SUDBERY I， ILOTT NE， et al. Sequencing depth and coverage： key considerations in genomic analyses[J]. Nature Reviews Genetics， 2014（15）： 121-132.
[30] NUNEZ JC， OLEKSIAK MF. A cost-effective approach to sequence hundreds of complete mitochondrial genomes[J]. PloS one， 2016， 11， e0160958.
[31] LIU YH， LIU XX， ZHANG MH. The complete mitochondrial genome of Sika deer Cervus Nippon hortulorum （Artiodactyla： Cervidae） and phylogenetic studies[J]. Mitochondrial DNA Part A， 2016（27）： 2967-2968.
[32] LI Y， BA H， YANG F. Complete mitochondrial genome of Cervus elaphus songaricus （Cetartiodactyla： Cervinae） and a phylogenetic analysis with related species[J]. Mitochondrial DNA Part A， 2016（27）： 620-621.
[33] GUHA S， GOYAL SP， KASHYAP VK. Molecular phylogeny of musk deer： A genomic view with mitochondrial 16S rRNA and cytochrome b gene[J]. Molecular phylogenetics and evolution， 2007（42）： 585-597.
Editor： Yingzhi GUANG Proofreader： Xinxiu ZHU