Biochemical Systematics and Ecology 48 (2013) 136143Contents lists available at SciVerse ScienceDirectBiochemical Systematics and Ecology

journal homepage: www.elsevier .com/locate/biochemsysecoPhylogenetic analyses and improved resolution of the familyBovidae based on complete mitochondrial genomesChengzhong Yang a, Changkui Xiang b, Wenhua Qi a,c, Shan Xia a,d, Feiyun Tu a,Xiuyue Zhang a, Timothy Moermond a, Bisong Yue a,e,*a Sichuan Key Laboratory of Conservation Biology on Endangered Wildlife, College of Life Sciences, Sichuan University,Chengdu 610064, PR Chinab School of Industry Manufacturing, Chengdu University, Chengdu 610106, PR Chinac School of Life Sciences and Engineering, Chongqing Three Gorges University, Chongqing 404100, PR ChinadDepartment of Science Education, Chengdu Normal University, Chengdu 611130, PR ChinaeKey Laboratory of Bio-resources and Eco-environment (Ministry of Education), College of Life Sciences, Sichuan University,Chengdu 610064, PR Chinaa r t i c l e i n f o

Article history:Received 2 July 2012Accepted 15 December 2012Available online 20 January 2013

Keywords:Naemorhedus goralMitochondrial genomeMolecular phylogenyBovidae* Corresponding author. Key Laboratory of Bio-rChengdu 610064, PR China. Tel.: 86 28 85412488;

E-mail address: bsyue@yahoo.com (B. Yue).

0305-1978/$ see front matter 2012 Elsevier Ltdhttp://dx.doi.org/10.1016/j.bse.2012.12.005a b s t r a c t

Efforts have been made to investigate the phylogeny of the family Bovidae; however, therelationships within this group still remain controversial. To further our understanding ofthe relationships, we sequenced the mitochondrial genome of the Himalayan goral, Nae-morhedus goral, an IUCN Redlist near threatened conservation dependent species. Then weconducted molecular phylogenetic relationships of the Bovidae based on Bayesian andMaximum Likelihood methods. The results indicate that the basal divergence within theBovidae is between the Bovinae and a strongly supported clade of the remaining Bovidaespecies. The two Neotragus species (the suni and pygmy antelope) clustered with theimpala, Aepyceros melampus (Aepycerotinae), and together they formed the most basal ofthe non-Bovinae. All the genera of the Antilopinae clustered together except Neotragus,which suggested that the Antilopinae was a paraphyletic subfamily. The present studyconfirmed a close relationship between the genera Capricornis and Naemorhedus whilesupporting their designation as separate genera and suggested that the Capricornis-Nae-morhedus-Ovibos clade (serows, gorals, and the muskox) should be placed in the Caprinae.Bison, Bos, and Tragelaphus (bison & cattle and kudus and nyalas) were paraphyletic. Thevery close relationship between Bison and Bos suggested that Bos and Bison should beintegrated into a single Bos genus. Saiga and Pantholops (the Chiru or Tibetan Antelope),unique genera which have sometimes been lumped together, were placed in differentgroups: Saiga within the Antilopinae and Pantholops at the base of the Caprinae. Our re-sults also supported a new taxonomy which places the three species of Hemitragus intothree monospecific genera: the genus Hemitragus is restricted to the Himalayan tahr, andtwo new genera are created: Arabitragus for the Arabian tahr and Nilgiritragus for theNilgiri tahr.

2012 Elsevier Ltd. All rights reserved.esources and Eco-environment (Ministry of Education), College of Life Sciences, Sichuan University,fax: 86 28 85414886.

. All rights reserved.

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C. Yang et al. / Biochemical Systematics and Ecology 48 (2013) 136143 1371. Introduction

The phylogenetic status of the conservation dependent gorals, Naemorhedus species, and the related species of Capricornis,the serows, remains uncertain and needs to be clarified (Corbet and Hill, 1992; Groves and Grubb, 1985; Mead, 1989). Due tolimitations of collecting samples from threatened Naemorhedus and Capricornis species, it is difficult to perform molecularphylogenetic analysis to find out their precise classification scheme. Fortunately, more molecular data samples from thesespecies have become available, andwe here contribute a complete genetic analysis fromNaemorhedus goral, the last species ofthis genus to be sampled. The Himalayan goral, N. goral, a small, elusive high mountain goat antelope of the family Bovidae,has a narrow distribution zone in China occurring only along the southern border of Tibet. Owing to excessive hunting, andhabitat loss, its wild populations have been declining for decades. Consequently, the Himalayan goral is protected in Chinaunder the Wild Animal Protection Law as a Category II key species (Li et al., 2000) and listed by IUCN as near threatened. N.goral along with the other three goral species may be considered as conservation dependent species and all four are listed inAppendix I of CITES (Duckworth and MacKinnon, 2008). In order to domesticate, genetically improve, and systematicallyconserve the Himalayan goral, it is necessary to define the genetic characteristics of this species and its relationship to otherspecies within the family Bovidae.

The bovids (oxen, sheep, goats, antelopes and allies) include 137 living and more than 300 fossil species (Savage andRussell, 1983). They are found in Africa, Europe, Asia and North America, with the great majority being in Africa. The Bovi-dae includes more species than any other extant family of largemammals, but their phylogenetic relationships remain largelyunresolved, showing that Simpsons (1945) view that Bovidae is one of the most troublesome groups of mammals to classifystill applies today.

The origin, development, and relationships within the Bovidae are poorly understood and opinions on these topics differwidely. Thus, the classification of the bovids, particularly with respect to the recognition of the subfamilies and tribes, isnoteworthy for its lack of consensus. Numerous versions of bovid taxonomy exist (e.g., Simpson, 1945; Haltenorth, 1963;Gentry,1992; Grubb, 2001), and controversy persists over which versionmost accurately reflects phyletic relationships. Thereis considerable disagreement in the allocation of genera to tribes and subfamilies, from the five subfamilies and 13 tribes ofSimpson (1945) to the 10 subfamilies and 28 tribes of Haltenorth (1963). The most recent version of bovid taxonomy (Grubb,2001) proposes 9 subfamilies and 17 tribes for the extant bovid species.

Intertribal relationships also have resulted in considerable difference of opinion. Although monophyly of the majority ofthe subfamilies and tribes is supported by morphological and molecular data, the evolutionary relationships among most ofthem are still surrounded by controversy. Therefore, the identity of sister taxa among these subfamilies or tribes, and in-terrelationships among genera and species within them remain uncertain. This is reflected in the growing literature, whichencompasses paleontological, morphological, and molecular data, all of which attempt to clarify various aspects of bovidevolution among tribes and subfamilies (e.g., Georgiadis et al., 1990; Gentry, 1992) and within them (e.g., Janecek et al., 1996).

The phylogenetic relationships within Bovidae have been investigated using both mitochondrial and nuclear sequences(Hassanin and Douzery, 1999, 2003; Gatesy and Arctander, 2000a,b). However, these studies did not resolve conclusively thephylogenetic relationships within Bovidae. Phylogenetic reconstruction based on a single gene or a short DNA segment maysometimes compromise phylogenetic accuracy (Hernandez-Fernandez and Vrba, 2005) and may be responsible for dis-crepancies between different studies. Complete mitochondrial genomes have been commonly applied to phylogeneticanalysis at different levels: Hassanin et al. (2012) reconstructed the phylogenetic tree of Cetartiodactyla including theBovidae. However, the phylogenetic position of phylogenetic relationships within Bovidae may be affected when morespecies are sampled (Zwick and Hillis, 2002). Here, after sequencing the complete mitochondrial genome of N. goral, weconducted the phylogenetic analyses within the Bovidae based on complete mitochondrial genomes, which will improve ourunderstanding of evolutionary biology of this animal group.

2. Materials and methods

2.1. DNA sample

Muscle of the wild N. goral was obtained from the Mount Qomolangma (Everest) Nature Reserve in Tibet Province. TotalDNA was extracted following the method of Sambrook and Russell (2001).

2.2. PCR amplification and sequencing

The N. goral mitochondrial genome was amplified in twenty-three overlapping fragments using the long and accuratepolymerase chain reaction (LA-PCR) technique according to the manufacturers instructions (TaKaRa, Beijing, China). A set ofprimers described by Hassanin et al. (2009) were used in the amplification. PCR cycling was carried out on a PTC-100 thermalcycler (BioRad, Hercules, CA, USA). The amplificationwas carried out in 25 ml reaction volumes with 2.5 ml of 10 ExTaq buffer(Mg2 Free), 1.03.0 ml dNTP (2.5 mM each), 2.0 ml MgCl2 (25 mM), 1 ml of each primer (10 uM), 0.2 ml ExTaq polymerase (5 U/ml), and approximately 200 ng total genomic DNA as the template. PCR conditions were an initial pre-denaturation for 5 minat 95 C, followed by 35 cycles of 30 s denaturations at 94 C, 45 s annealing at 5061 C, 13 min extensions at 72 C, anda final extension at 72 C for 10 min. The PCR products were purified using the DNA Agarose Gel Extraction Kit (Omega Bio-

C. Yang et al. / Biochemical Systematics and Ecology 48 (2013) 136143138Tek, Norcross, GA, USA). The purified DNA was then sequenced on an ABI 3730 DNA Analyzer using a BigDye chemistry kit(Applied Biosystems, Inc., Carlsbad, CA, USA), in which the same PCR primers were used.2.3. Sequence analysis

DNA sequences were analyzed using the softwareMEGA 4.0 program (Tamura et al., 2007). The locations of protein-codingand rRNA genes were determined by comparison with corresponding known sequences from three other species: Naemo-rhedus baileyi, Naemorhedus caudatus and Naemorhedus griseus. The tRNA genes were identified with tRNAscan-SE v.1.21(Lowe and Eddy, 1997). Two tRNA genes, which were not found with tRNAscan-SE, were identified by comparison withhomologues from N. caudatus.2.4. Phylogenetic analysis

To further address the phylogenetic relationships within the Bovidae, the 12 heavy-strand encoded protein-coding geneswere aligned according to Nikaido et al. (2001). After removal of gaps and ambiguous sites adjacent to gaps, 10,713 nucle-otides were obtained. Multiple alignments of the 12 of the concatenated protein-coding genes from N. goral and 90 otherspecies (Table 1) were performed using Clustal X (Tompson et al., 1997) with the default setting. Two species of Tragulidae (aTable 1The sequences GenBank accession numbers for the 91 species used for phylogenetic analysis.

Species GenBank no. Species GenBank no.

Addax nasomaculatus JN632591 Naemorhedus baileyi JN632663Aepyceros melampus JN632592 Naemorhedus caudatus NC_013751Alcelaphus buselaphus JN632594 Naemorhedus goral (This study) JX188255Ammotragus lervia NC_009510 Naemorhedus griseus JN632664Antilope cervicapra NC_012098 Nanger dama JN632665Antidorcas marsupialis JN632596 Nanger granti JN632666Bison bison JN632601 Nanger soemmerringii JN632667Bison bonasus NC_014044 Neotragus batesi JN632668Bos grunniens NC_006380 Neotragus moschatus JN632669Bos indicus NC_005971 Oreamnos americanus FJ207535Bos javanicus JN632605 Oreotragus oreotragus JN632675Bos primigenius NC_013996 Oryx beisa JN632676Boselaphus tragocamelus EF536350 Oryx dammah NC_016421Bubalus bubalis NC_006295 Oryx gazella NC_016422Bubalus depressicornis EF536351 Oryx leucoryx JN632679Budorcas taxicolor NC_013069 Ourebia ourebi JN632680Capra caucasica JN632609 Ovis ammon HM236188Capra falconeri FJ207525 Ovis aries NC_001941Capra ibex FJ207526 Ovis canadensis NC_015889Capra nubiana FJ207527 Ovis vignei HM236186Capricornis crispus NC_012096 Ovibos moschatus FJ207536Capricornis sumatraensis FJ207534 Pantholops hodgsonii NC_007441Capricornis swinhoei NC_010640 Pelea capreolus JN632684Cephalophus adersi JN632611 Philantomba maxwellii JN632685Cephalophus callipygus JN632614 Philantomba monticola JN632687Cephalophus dorsalis JN632615 Procapra gutturosa JN632689Cephalophus jentinki JN632616 Procapra przewalskii NC_014875Connochaetes gnou JN632626 Pseudois nayaur FJ207537Connochaetes taurinus JN632627 Pseudois schaeferi NC_016689Damaliscus pygargus FJ207530 Pseudoryx nghetinhensis EF536352Dorcatragus megalotis JN632631 Raphicerus campestris JN632693Eudorcas rufifrons JN632633 Redunca arundinum JN632694Gazella bennettii JN632635 Redunca fulvorufula JN632695Gazella cuvieri JN632636 Rupicapra pyrenaica FJ207538Gazella dorcas JN632637 Rupicapra rupicapra FJ207539Gazella gazella JN632640 Saiga tatarica JN632700Hemitragus jayakari FJ207523 Sylvicapra grimmia JN632701Hemitragus jemlahicus FJ207531 Syncerus caffer EF536353Hippotragus equinus JN632647 Taurotragus derbianus EF536354Hippotragus niger JN632648 Tetracerus quadricornis EF536355Hyemoschus aquaticus (Out Group) JN632650 Tragelaphus angasii JN632702Kobus ellipsiprymnus JN632651 Tragelaphus eurycerus JN632703Kobus leche JN632652 Tragelaphus imberbis EF536356Litocranius walleri JN632653 Tragelaphus oryx JN632704Madoqua kirkii JN632654 Tragulus kanchil (Out Group) JN632709Madoqua saltiana JN632655

C. Yang et al. / Biochemical Systematics and Ecology 48 (2013) 136143 139mouse-deer, Tragulus [javanicus] kanchil, and a chevrotain, Hyemoschus aquaticus) were used as an out group to root the treeof Bovidae (Table 1). Bayesian phylogenetic analyses were conducted using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003).The best fitting model (TIM3I G) of sequence evolution for Bayesian analyses were obtained byModeltest 3.7 (Posada andCrandall, 1998) under the Akaike Information Criterion (AIC). Four independent Markov chains Monte Carlo (MCMC) ransimultaneously for ten million generations, sampling one tree per 200 generations, and the first 25% of the samples werediscarded as the burn-in. At program termination, the average standard deviation of split frequencies was 0.003260. Tracerv1.3 (Rambaut and Drummond, 2005) was used to check chain convergence and parameter mixing. Maximum likelihood(ML) analyses were performed in RAxMLWeb-Servers (Stamatakis et al., 2008) using default parameters with 1000 bootstrapreplicates.

3. Results

3.1. Some characteristics of the mitochondrial genome

The whole sequence of the Himalayan goral, N. goral, mitochondrial genome and its genomic structure was determinedand deposited in GenBankwith the accession number JX188255. The total length of the sequencewas 16,555 bp. As presentedin Table 2, the N. goral was found to consist of 13 protein-coding genes, two rRNA genes (12S rRNA and 16S rRNA), 22 tRNAgenes, and a control region. These components were the same as for other mammalianmitochondrial genomes. Most of thesegenes were encoded on the H strand, except for the ND6 gene and eight tRNA genes (tRNA-Gln, tRNA-Ala, tRNA-Asn, tRNA-Cys, tRNA-Tyr, Trna-Ser, tRNA-Pro, tRNA-Glu), whichwere encoded on the L strand. The base composition of the H strandwas:Table 2Mitochondrial genome organization of the Himalayan goral, Naemorhedus goral.

Genea Position Length (bp) Codon Intergenic nucleotideb Strandc

From To Start Stop

tRNA-Phe 1 68 68 0 H12S rRNA 69 1025 957 0 HtRNA-Val 1026 1092 67 0 H16S rRNA 1093 2659 1657 0 HtRNA-Leu(UUR) 2660 2734 75 0 HND1 2737 3692 956 ATG TA 2 HtRNA-Ile 3693 3761 69 0 HtRNA-Gln 3759 3830 72 3 LtRNA-Met 3833 3901 69 2 HND2 3902 4945 1044 ATA TAG 0 HtRNA-Trp 4944 5010 67 2 HtRNA-Ala 5012 5080 69 1 LtRNA-Asn 5082 5154 73 1 LOL 5155 5189 35 0 LtRNA-Cys 5187 5254 68 3 LtRNA-Tyr 5255 5322 68 0 LCOI 5324 6868 1545 ATG TAA 1 HtRNA-Ser(UCN) 6866 6934 69 3 LtRNA-Asp 6942 7009 68 7 HCOII 7011 7694 684 ATG TAA 1 HtRNA-Lys 7698 7765 68 3 HATP8 7767 7967 201 ATG TAA 1 HATP6 7928 8608 681 ATG TAA 40 HCOIII 8608 9388 781 ATG T 1 HtRNA-Gly 9392 9460 69 3 HND3 9461 9807 347 ATA TA 0 HtRNA-Arg 9808 9876 69 0 HND4L 9877 10173 297 ATG TAA 0 HND4 10167 11544 1378 ATG T 7 HtRNA-His 11545 11613 69 0 HtRNA-Ser(AGY) 11614 11673 60 0 HtRNA-Leu(CUN) 11675 11743 69 1 HND5 11744 13564 1821 ATT TAA 0 HND6 13548 14075 528 ATG TAA 17 LtRNA-Glu 14076 14144 69 0 LCytb 14149 15288 1140 ATG AGA 4 HtRNA-Thr 15292 15361 70 3 HtRNA-Pro 15362 15427 66 0 LD-loop 15428 16555 1128 0 H

a tRNA abbreviations follow the IU-PAC-IUB three-letter code.b Negative numbers indicate that adjacent genes overlap.c H and L indicate genes transcribed on the heavy and light strand, respectively.

C. Yang et al. / Biochemical Systematics and Ecology 48 (2013) 136143140A, 33.6%; G, 13.2%; T, 27.2%; C, 26.0%, which reflected the typical AT-rich pattern commonly seen in the vertebrate mito-chondrial genomes.

3.2. Phylogenetic analysis

Phylogenetic trees of the Bovidae constructed using BI and ML methods showed a similar topology (Fig. 1). The basaldivergence within Bovidae was between the Bovinae and a strongly supported clade of the remaining non-bovinine species(PP 1.00, BS 78) (Fig. 1). Within the Bovinae, the trees indicated that Bison & Bos (bison and cattle), and Tragelaphus (kudusand nyalas) were paraphyletic. The non-Bovinae clade included the Cephalophinae, Antilopinae, Reduncinae, Peleinae,Alcelaphinae, Hippotraginae, Caprinae, Aepycerotinae and Clade A (which consisted of two species ofNeotragusdthe suni andthe pygmy antelope). Clade A clustered with the impala, Aepyceros melampus (Aepycerotinae), both of which formed a mostbasal clade in non-Bovinae (PP 1.00, BS 73) (Fig. 1). Within the Cephalophinae (duikers), all genera clustered together andno polyphyletic phenomenon appeared. The Cephalophinae was associated with the Antilopinae. Within the Antilopinae, allFig. 1. Molecular phylogenetic tree derived from complete DNA sequence of 12 mitochondrial protein-coding genes using Bayesian inference and ML analysis.The numbers beside the nodes are Bayesian posterior probabilities (PP) and bootstrap support (BS). Tragulus (javanicus) kanchil and Hyemoschus aquaticus wereset as out-groups.

C. Yang et al. / Biochemical Systematics and Ecology 48 (2013) 136143 141the genera clustered together except Neotragus (Clade A), which clustered with the Aepycerotinae. The saiga, Saiga tatarica,was placed in the Antilopinae (PP 1.00, BS 79). The subfamily Peleinae, which is composed of only one species, the GreyRhebok (Pelea capreolus), nested within the Reduncinae (reedbucks, Redunca, waterbucks and kobs, Kobus), which indicatedthe Reduncinae is non-monophyletic. The Hippotraginae (the sable and roan antelopes) sistered to the Alcelaphinae(hartebeests and wildebeests), and the Oryx species appeared polyphyletic.

Within the Caprinae (goats and sheep), the consensus tree (1) placed Pantholops hodgsonii (the chiru or Tibetan antelope)at the basal position of the Caprinae with strong support values (PP 1.0, BS 100); (2) confirmed that Capricornis andNaemorhedus (serows and gorals) have a very close relationship (PP 1.00, BS 100); and (3) indicated that Hemitragus (thetahrs) was very likely a polyphyletic genus with the two species showing different affiliations.

4. Discussion

In the past few decades, extensive efforts have been made to investigate the Bovidae phylogeny; however, relationshipswithin this group remain unclear. One possibility is that the DNA sequences from different markers are highly divergent intheir evolutionary rates and that the substitution rate for each marker varies among lineages examined; therefore, phylo-genetic reconstruction based upon a single gene or a short DNA segment is highly likely to produce an incorrect tree topology(Nikaido et al., 1999). The complete mitochondrial genome provides a higher level of support than those based on individualor partial mitochondrial genes (Krzywinski et al., 2006). Mitochondrial DNAs, especially those encoding protein, have beenfrequently utilized as a powerful tool for evolutionary studies of animals (Boore and Brown, 1998). The present studydemonstrated that the phylogenetic analyses based on complete mitochondrial genomes served well to resolve relationshipswithin the Bovidae.

Two main clades have been consistently retrieved within the Bovidae, a basal group comprising the Bovinae and a large,more derived assemblage, which includes all the other subfamilies (Allard et al., 1992; Hassanin and Douzery, 1999). Theresult of this study strongly supported the division of the Bovidae into those two clades with full posterior probability andbootstrap value (PP 1.00, BS 100). This finding appeared solid and rejected the subdivision into Aegodontia and Boodontiapreviously suggested by Schlosser (1904).

Antilopinae is, from a phylogenetic standpoint, probably the least understood subfamily of the Bovidae (Rebholz andHarley, 1999). The taxonomy of this subfamily has presented formidable confusion ever since the early attempts at classi-fication by Sclater and Thomas (1897). The present study revealed that Neotragus (the suni and the pygmy antelope) clusteredwith the Aepycerotinae (the impala), and together they formed themost basal clade of the non-Bovinae, which also suggestedthat the Antilopinae was a paraphyletic subfamily. Paraphyletic phenomena may be among the possible reasons for thedifficult taxonomy of the Antilopinae.

The Cephalophinae (the duikers) is one group whose taxonomic placement is very difficult because its species presenta complex assemblage of primitive characters. It has been placed as a sister group of various other subfamilies: for example,the Bovinae (Gentry, 1992), Reduncinae (reedbucks, Redunca, waterbucks and kobs, Kobus) (Gatesy and Arctander, 2000a),Antilopinae (Matthee and Davis, 2001), Neotragini (Kingdon, 1982a,b), a clade composed of the Caprinae (goats & sheep),Alcelaphinae (hartebeest and wildebeest) and Hippotraginae (the sable and roan antelopes) (Castresana, 2001), a cladecontaining the Reduncinae, Alcelaphinae and Hippotraginae (Gatesy and Arctander, 2000b) and, finally, all the other non-bovine groups (Georgiadis et al., 1990). However, the molecular evidence from the present study revealed a sister relation-ship between the Cephalophinae and the Antilopinae, which was strongly supported by a Bayesian tree with full posteriorprobability (PP 1.00). The relationship was also supported by an ML tree, but the bootstrap value was not high (BS 58).

Pantholops and Saiga were originally considered close relatives and placed in their own tribe within the Caprinae(Simpson, 1945). Over the past century, these problematic genera have bounced back and forth between the Antilopinae andthe Caprinae (Schaller, 1977; Gentry, 1992). The result of our present study strongly supported the hypothesis that Saiga beplaced within the Antilopinae and that Pantholops (the Chiru or Tibetan Antelope) be placed within the Caprinae (Hassaninet al., 2012).

Based on Hassanins classification revision (Hassanin and Douzery, 1999), the long-tailed goral (N. caudatus) was placed inan enlarged tribe, called Caprini sensu lato, within the subfamily Antilopinae (Bovidae). The genera Capricornis and Naemo-rhedus were classified into three and four species, respectively (Corbet and Hill, 1992; Groves and Grubb, 1985; Mead, 1989);however, the phylogenetic relationships of these genera and species have remained under question. The present study con-firmed a close relationship between Capricornis and Naemorhedus (PP 1.00, BS 100) while supporting their designation asseparate genera. And the results suggested that the Capricornis-Naemorhedus-Ovibos clade (serows, gorals, and the muskox)should be placed in the Caprinae, which is consistent with the previous studies (Hernandez-Fernandez and Vrba, 2005;Ropiquet andHassanin, 2005). A previous study (Ropiquet andHassanin, 2004) indicated thatNaemorhedus,Ovibos,Oreamnos(themountain goat), andRupicapra (the chamois) clustered together basedon threemarkers (cytochromeb,12S rRNAandexon4 of the K-casein gene). However, the present study as well as a recent report (Hassanin et al., 2012) based onmtDNA genomesconsistently placedOvibos (themuskox)withNaemorhedus-Capricornis, andOreamnos (themountain goat) with Budorcas (thetakin), and, while both clades fell clearly in the Caprinae, the two clades were distinct and distant from each other (Fig. 1).

Our consensus tree showed a very close relationship between Bison and Bos (the bison and cattle) under the subfamilyBovinae. Due to their close phylogenetic relationship, it has been suggested that Bos and Bison should be integrated intoa single Bos genus (Hernandez-Fernandez and Vrba, 2005). Bubalus bubalis (the water buffalo) was observed more distantly

C. Yang et al. / Biochemical Systematics and Ecology 48 (2013) 136143142related from the Bos-Bison group as previously reported (Hassanin and Douzery, 1999). The close relationship of Bison and Boscompared to a more distant relationship to Bubalus was also supported from morphological, paleontological, and repro-ductive data (Miyamoto et al., 1989). Molecular data of the Bovini suggested two lineages: buffalo (Bubalus [water buffalos &anoas] and Synceros [the African cape buffalo]) versus cattle (Bos, Bison) (Lenstra and Bradley, 1999; Simpson, 1945).

The phylogenetic trees from the present study in general confirmed the monophyly of the Bovinae and the Caprinae;however the Antilopinae appeared to be polyphyletic, which is consistent with a previous study (Hernandez-Fernandez andVrba, 2005).

Most of the previous phylogenetic studies based on DNA indicated a close relationship between Hemitragus (tahrs) withCapra (goats) (Gatesy et al., 1997; Hassanin and Douzery, 1999). Indeed, in the present study, the close relation of Hemitragusjemlahicus (the Himalayan tahr) with Capra confirmed the relationship. However, our result revealed that Hemitragus jayakari(the Arabian tahr) clustered with Ammotragus (the Barbary sheep). Thus, we suggest Hemitragus is polyphyletic. A previousstudy (Ropiquet and Hassanin, 2005) also indicated that Hemitragus jemlahicus was associated with Capra, but that Hemi-tragus jayakariwas allied with Ammotragus lervia (as confirmed in our study), and that Hemitragus hylocrius (the Nilgiri tahr)was a sister-group of Ovis (sheep). Combining with the morphological, cytogenetic and biogeographic information(Lundrigan,1996; Schaller, 1977; Benirschke and Kumamoto,1982, 1980), we support a new taxonomywhich places the threespecies of Hemitragus into three monospecific genera: the genus Hemitragus is then restricted to the Himalayan tahr, and twonew genera are created: Arabitragus for the Arabian tahr and Nilgiritragus for the Nilgiri tahr (Ropiquet and Hassanin, 2005).

In the present study, the trees generated frommtDNA genomes provided reasonable phylogenetic relationships within theBovidae. However, the mitogenomic dataset of the Bovidae still lacks representatives of some key taxa such as the Huntershartebeest (Beatragus hunteri) and the extinct Balearian mouse-goat (Myotragus balearicus), which are crucial for estab-lishing the Bovidae mitogenomic tree and may affect the relationships in a phylogenetic analysis (Lalueza-Fox et al., 2005).Although using mitogenomic datasets to restructure the phylogenetic tree of Bovidae acquired high support values, they canalso be systematically biased, just like the effect of a single gene or a short DNA segment (Nikaido et al., 1999). The familyBovidae is difficult to classify in part because it appears to represent a rapid early radiation into many forms without clearconnections among them. Furthermore, certain morphological traits have evolved several times within the family to createconvergence that obscures true phylogenetic relationships (Gentry, 1992). Therefore, a more complete mitochondrial genomeset, including more key species as well as multiple nuclear markers, is needed to achieve an unambiguous resolution of thephylogeny of the Bovidae.Acknowledgments

This research was funded by National Science and Technology Support Project of China (2012BAC01B06). Wewould like tothank Guo Cai for his help in sample collection.References

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Phylogenetic analyses and improved resolution of the family Bovidae based on complete mitochondrial genomes1. Introduction2. Materials and methods2.1. DNA sample2.2. PCR amplification and sequencing2.3. Sequence analysis2.4. Phylogenetic analysis

3. Results3.1. Some characteristics of the mitochondrial genome3.2. Phylogenetic analysis

4. DiscussionAcknowledgmentsReferences