http://informahealthcare.com/mdn ISSN: 1940-1736 (print), 1940-1744 (electronic) Mitochondrial DNA, Early Online: 1–8 ! 2014 Informa UK Ltd. DOI: 10.3109/19401736.2014.880887
ORIGINAL ARTICLE
DNA barcoding revises a misidentification on musk deer Chengzhong Yang1,2, Zhen Xiao1, Yuan Zou3, Xiuyue Zhang3, Bo Yang4, Yinghong Hao5, Timothy Moermond3, and Bisong Yue2
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1
Chongqing Key Laboratory of Animal Biology, College of Life Sciences, Chongqing Normal University, Chongqing, P.R. China, 2Key Laboratory of Bio-resources and Eco-environment, Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, P.R. China, 3Sichuan key Laboratory of Conservation Biology on Endangered Wildlife, College of Life Sciences, Sichuan University, Chengdu, P.R. China, 4China Conservation and Research Center for the Giant Panda, Wolong, Sichuan, P.R. China, and 5Shanxi Pangquangou National Nature Reserve, Jiaocheng, P.R. China Abstract
Keywords
As an endangered animal group in China, musk deer (genus Moschus) have attracted the attention of deer biologists and wildlife conservationists. Clarifying the taxonomic status and distribution of musk deer species is important to determine the conservation status for each species and establish appropriate conservation strategies. There remains some uncertainty about the species determination of the musk deer in the Guandi Forest District of Shanxi Province, China. The musk deer in Shanxi would appear to represent an extension of the geographical distribution of either the Forest Musk Deer from the southwest or the Siberian Musk Deer from the northeast, or possibly both. The musk deer population in Shanxi Province provides an interesting and significant case to test the value of applying molecular methods to make a genetic species identification. In order to clarify the species status of the Shanxi musk deer, we sequenced 627 bp of the COI gene and &723 bp of the D-loop gene in 12 musk deer samples collected from the Guandi Forest District, and the two reference samples collected from Sichuan. Genetic analyses from the data suggest that all of the samples from the Guandi Forest District are M. berezovskii rather than M. moschiferus. It is most likely that the most previous studies had wrong species identification. And it is the first time we use DNA barcoding to prove that Shanxi is a new distribution of M. berezovskii.
Barcoding, COI, D-loop, Moschus berezovskii, Moschus moschiferus
Introduction Musk deer (Moschus spp.) are solitary ruminants distributed throughout the forest and mountainous parts of Asia, from just north of the Arctic Circle southward to the northern edge of Mongolia and to Korea, and further south through China and the Himalayan region to Northern India, Myanmar, and Northern Vietnam (Flerov, 1952; Green, 1986). China is one of the important range countries for musk deer, with all the recognized species of musk deer occurring in China (Yang et al., 2003). Due to excessive hunting and habitat loss, the wild populations of all the musk deer species have been declining for decades (Meng et al., 2003a). Consequently, all species of musk deer are on the World Conservation Union IUCN Red List of Threatened Species (IUCN, 2004), as well as in the Appendices of CITES (the Convention on International Trade in Endangered Species of Wild Fauna and Flora) (Zhang, 1998). In China, all the musk deer species are now listed as Category I key species under the Wild Animal Protection Law (Meng et al., 2003b). Despite the great interest in musk deer, the definitive designation of Moschus species and subspecies remains a matter of question. For example, relatively isolated population of musk
Correspondence: Bisong Yue, Key Laboratory of Bio-resources and Ecoenvironment, Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610064, P.R. China. Tel: +86 28 85412488. Fax: +86 28 85414886. E-mail: [email protected]
History Received 29 September 2013 Revised 31 December 2013 Accepted 5 January 2014 Published online 3 February 2014
deer in Anhui Province has been variously regarded as a subspecies of M. moschiferus or as a subspecies of M. berezovskii, and, most recently, elevated to full species status as the Anhui Musk Deer, M. anhuiensis (Smith & Xie, 2009). Likewise, the Himalayan Musk Deer, M. leucogster, and the Black Musk Deer, M. fuscus, have both been previously designated as subspecies of the Alpine Musk Deer, M. chrysogaster. These various designations of different musk deer populations remain under question and are not based on genetics. The number of species of the genus Moschus in China is still under question (Sheng, 1998), and a variety of classification systems has been proposed (Cai & Feng, 1981; Groves, 1975; Groves et al., 1995; Groves & Feng, 1986; Grubb, 1982; Sheng, 1998; Wang et al., 1993; Yang & Feng, 1998). Species identification based on morphological characters is a traditional basis for classification; however, species identification is liable to be influenced by subjective diagnosis of morphological characters and geographic variation. When the species identification based on morphological characters is inadequate to resolve the problem, molecular methods may provide an answer. The musk deer population in Shanxi Province provides an interesting and significant case to test the value of applying molecular methods to make a genetic species identification. There are records of Siberian Musk Deer there (Groves et al., 1995; Hao et al., 1991, 1990, 1993, 1994; Liao & Li, 2008; Wang & Zhao, 2011; Yang et al., 2003). Smith & Xie (2008) show a number of records of both species in Shanxi. If this is true, these Shanxi
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records of the Siberian Musk Deer would represent a somewhat isolated population existing southwest of the populations in northeast China, and the records of the Forest Musk Deer would represent a somewhat isolated population existing northeast of the main center of the Forest Musk Deer populations in south central China (Smith & Xie, 2008). It is even more interesting, or, rather, surprising that Smith & Xie (2009) show only M. moschiferus records in Shanxi (i.e. in the Chinese edition published 1 year later than the English edition). The records of M. berezovskii shown in Shanxi on their 2008 map were removed from the map in 2009 (Smith & Xie, 2008, 2009). The question is whether the musk deer in Shanxi are Siberian Musk Deer, or Forest Musk Deer, or both? The musk deer in Shanxi Province in the Guandi Forest District, which includes the Pangquangou Nature Reserve, have been identified as Siberian Musk Deer, and a number of ecological and behavioral studies have been conducted on these purported Siberian Musk Deer (Hao et al., 1991, 1990, 1993, 1994; Liao & Li, 2008; Wang & Zhao, 2011). However, some scholars doubt the validity of the musk deer diagnosis there (e.g. Zuomo Liu, personal communication). They postulate that the musk deer in the Guandi Forest District are Forest Musk Deer (M. berezovskii) rather than Siberian Musk Deer (M. moschiferus), but no evidence has been provided. This study attempts the ‘‘re-identification’’ of the musk deer of the Guandi Forest District using molecular genetic methods to provide a more definitive assessment of the species of these interesting region which lies in between the adjacent ranges of the Siberian Musk Deer and the Forest Musk Deer. For species identification, a certain fragment of the mitochondrial gene COI, coding for a subunit of the enzyme cytochrome oxidase, has become widely known and used as ‘‘the DNA barcode’’ for the identification of most animal species (Hebert et al., 2003a,b). What makes this sequence particularly suitable for such tasks is the fact that it varies noticeably between species and very little between the individuals of a given species (Gross, 2012). The COI gene enables accurate animal species identification only in the case where adequate reference sequence data exist (Dawnay et al., 2007). Forensic wildlife cases require accurate species identification based on similarity between mitochondrial DNA of unknown samples and reference data (Dalton & Kotze, 2011). In the present study, the mitochondrial COI gene was used to identify the species of musk deer samples collected from the Guandi Forest District. And then, in order to further assess the accuracy
of the species identification based on COI, D-loop gene was also used.
Materials and methods Specimens and DNA extraction Twelve specimens of musk deer were obtained from the Guandi Forest District, Shanxi Province, China from 1995 to 2011. Other two reference samples of Forest Musk Deer (M. berezovskii) were collected from Miyaluo, Sichuan Province, China. The collection information is listed in Table 1. Total DNA was extracted following the method of Sambrook & Russell (2001). Polymerase chain reaction amplification and sequencing The purified DNA was used as a template to amplify a 627 bp fragment of COI gene and &723 bp fragment D-loop gene. The primers for amplifying segment of COI were: HCO2198-50 TAA ACT TCA GGG TGA CCA AAA AAT CA 30 and LCOI490-50 GGT CAA CAA ATC ATA AAG ATA TTG G 30 (Dalton & Kotze, 2011; Folmer et al., 1994). The primers for amplifying segment of D-loop gene were designed based on the Moschus sequences from GenBank: lsdloopF-50 CCT CCC TAA GAC TCA AGG AAG AA 30 ; lsdloopR-50 TCG TGC CTA CCA TTA TGG TGA TG 30 . PCR cycles were 5 min at 95 C followed by 35 cycles of 30 s at 95 C, 50 s at 54 C, and 1 min at 72 C, with a final extension at 72 C for 10 min. The polymerase chain reaction products were electrophoresed on a 1.0% agarose gel and purified with the DNA Agarose Gel Extraction Kit (Omega Bio-Tek, Norcross City, GA). The purified fragments were sequenced directly using an ABI 3730XL automatic sequencer (Applied Biosystems, Foster City, CA). Data analysis To determine whether the sequences were of mitochondrial origin or represented paralogous sequences in the nucleus, the following methods were used: All DNA sequence electropherograms were examined for distinguishing features of nuclear copies, including double peaks resulting from potential coamplification of mtDNA and nuclear DNA sequences, unexpected insertions/deletions, frameshifts or stop codons, and mismatches in overlapping sequence for a given taxon from different amplification products. Features consistent with mitochondrial origin that we observed in our sequences are (1) presence of a conserved reading frame in
Table 1. The informations of the specimens – the unknown samples and the reference samples in this study. GenBank no. Specimen No. Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 Sample 11 Sample 12 FMD 1 FMD 2
Sex
Sample type
Origin
Date sampled
COI
D-loop
Female Male Female Male Female Male Male Male Female Female Female Male Male Female
Skin Skin Skin Skin Skin Skin Skin Skin Skin Skin Skin Skin Blood Muscle
Guandi Forest District, Shanxi Guandi Forest District, Shanxi Guandi Forest District, Shanxi Guandi Forest District, Shanxi Guandi Forest District, Shanxi Guandi Forest District, Shanxi Guandi Forest District, Shanxi Guandi Forest District, Shanxi Guandi Forest District, Shanxi Guandi Forest District, Shanxi Guandi Forest District, Shanxi Guandi Forest District, Shanxi Miyaluo, Sichuan Miyaluo, Sichuan
Sept. 5, 2011 Mar. 16, 2011 Aug. 28, 2011 Apr. 13, 1995 Dec. 5, 2010 Dec. 5, 2010 Jun. 3, 1997 Dec. 4, 2005 Jul. 7, 2007 May 8, 2004 Dec. 24, 2010 Dec. 23, 2009 Oct. 10, 2006 Aug. 20, 2009
JX627384 JX627385 JX627386 JX627387 JX627388 JX627389 JX627390 JX627391 JX627392 JX627393 JX627394 JX627395 JX627396 JX627397
KJ000676 KJ000677 KJ000678 KJ000679 KJ000680 KJ000681 KJ000682 KJ000683 KJ000684 KJ000685 KJ000686 KJ000687 KJ000688 KJ000689
FMD means Forest Musk Deer (M. berezovskii).
DNA barcoding of musk deer
DOI: 10.3109/19401736.2014.880887
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protein-coding genes among all taxa with decreasing rates of variability at third, first, and second codon positions, respectively, and (2) absence of extra stop codons, frameshifts, or unusual amino acid substitutions. Further, we found no evidence of sequence changes yielding losses of known secondary structure for tRNA and rRNA genes that would indicate translocation to the nucleus and loss of function. Where possible, we have compared our sequences with homologs from GenBank for conspecific individuals. In all such cases, we found nearly identical matching. A total of 627 bp of the COI gene and &723 bp of the D-loop gene of the 14 musk deer samples were included in the analyses. Measuring genetic differentiation was based on Kimura two parameter (K2P) distance (Nei & Kumar, 2000). Phylogenetic analysis All of the reference sequences included in phylogenetic analyses are listed in Table S1 (Supporting Information, Table S1). After being aligned and deleted gaps, the sequences data sets for treeconstruction were obtained. The unrooted neighbor-joining (NJ) tree based on COI and D-loop were established in MEGA 4 (Tamura et al., 2007). Additionally, in the GenBank database, the length and number of D-loop sequences for each musk deer species are not identical (from 229 to 923 bp). For making the
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most of the sequence information from GenBank, we constructed two D-loop trees in the present study: one was based on 229 bp and the other was 446 bp.
Results Genetic variations of the COI gene A 627-bp fragment of the mitochondrial COI gene was successfully sequenced for all the specimens, and was deposited in GenBank with the accession numbers JX627384 to JX627397 (Table 1). The 12 unknown samples present four haplotypes (Hap1 to Hap4). Samples 1, 2, 3, 5, 6, 7, 8, 9 and 11 all share Hap1; the remaining three samples (4, 10 and 12) each has a different haplotypes (Hap2, Hap3 and Hap4, respectively). The genetic distance within the four haplotypes ranges from 0.2% to 1.1%. M. berezovskii is the species whose genetic distance to the 12 unknown samples is the shortest (0.2–1.6%). The genetic distance within species, genus, family and order are 0–1.6%, 2.5–7.0%, 2.4–19.4%, and 14.5–21.2%, respectively (Figure 1). There are 70 variable sites within the musk deer COI sequences. Among them, the variable sites between the four haplotypes (Hap1–Hap4) and M. berezovskii sequences are the least (Figure 2). By comparing among the alignment of musk deer sequences, it would be found that the four haplotypes were the most similar to that of M. berezovskii
Figure 1. The distribution of pairwise K2P genetic distance. (a) within species; (b) within genus; (c) within family; (d) within order.
Figure 2. Variable sites analyses on regions of alignment of musk deer COI gene sequences. Matched sites are represented by dots.
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Figure 3. Neighbor-joining (NJ) analysis of Kimura 2-parameter (K2P) distance of COI sequences. The first number behind the species name is the Voucher ID, and the second is the GenBank accession number.
DNA barcoding of musk deer
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DOI: 10.3109/19401736.2014.880887
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Figure 4. Variable sites analyses on regions of alignment of musk deer D-loop gene sequences. Matched sites are represented by dots; insertions and deletions were compensated by introducing alignment gaps (dash).
sequences. As showed in the NJ tree (Figure 3), all COI sequences of the 12 unknown samples cluster in Moschidae. Within Moschidae, M. berezovskii and all the unknown samples cluster together, showing a closer relationship with each other than either has with M. chrysogaster or M. moschiferus. Genetic variations of the D-loop gene A &723 bp length of the D-loop gene was also sequenced from all specimens, and was deposited in GenBank with the accession numbers KJ000676 to KJ000689 (Table 1). The 12 unknown samples display six haplotypes (H1–H6). Samples 1, 10, 11 and 12 share the H1; samples 2, 4 and 9 share the H2; samples 5 and 7 share the H3; and the remaining three samples (3, 6 and 8) each has a different haplotypes (H4, H5 and H6, respectively). Similar to the COI gene, the results of comparing variable sites of the D-loop sequences also showed that the six (H1–H6) haplotypes were the most similar to that of M. berezovskii sequences (Figure 4). Though the length and number of D-loop sequences for each species is different, the NJ trees of Moschidae based on the length of 229-bp and 446-bp D-loop sequences showed a similar topology (Figures 5 and 6). As showed in these two figures, all of the 12 unknown
samples nest within M. berezovskii. Within the Moschidae, all members cluster together with each musk deer species (Figures 5 and 6).
Discussion In the present study, the genetic distance based on COI among the 12 unknown samples is in the range of intraspecific distance (Figure 1), which indicated that the 12 unknown samples belong to the same species. The analysis of the genetic distance between the 12 unknown samples and the two reference species further indicated that the unknown samples are most likely conspecific with M. berezovskii. The result is also consistent with the phylogenetic relationships of the COI-tree (Figure 3) and D-loop trees (Figures 5 and 6) as well as the variable sites analyses (Figures 2 and 4). All lines of evidence suggest that all of the 12 unknown samples should be ascribed to M. berezovskii rather than to M. moschiferus. Stoeckle & Hebert (2008) indicated that mitochondrial sequence divergences are strongly linked to the process of speciation. In the present study, all of the conspecific pairs were observed to have K2P COI divergence smaller than 1.6%, while the interspecific pairs had divergences greater than 2.4%. Indeed, 98% of sister species pairs of vertebrates showed a divergence
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Figure 5. Neighbor-joining (NJ) analysis of Kimura 2-parameter (K2P) distance of 294-bp of the D-loop sequences. The species name is followed by its GenBank accession number.
greater than 2% (Johns & Avise, 1998). Therefore, some congeneric pairs with low divergences in the present study (approximate to 2%) may represent recent histories of reproductive isolation (Hebert et al., 2003a). Many previous studies conducted in Guandi Forest District about musk deer were ostensibly about M. moschiferus (Hao et al., 1991, 1990, 1993, 1994; Liao & Li, 2008; Wang & Zhao, 2011; Yang et al., 2003). The samples used in the present study were collected from the year 1995 to 2011, which covered the time of some previous study there (Hao et al., 1994; Liao & Li, 2008; Wang & Zhao, 2011; Yang et al., 2003). However, all of the samples are M. berezovskii, and not even one M. moschiferus was found. It is most likely that the previous studies had wrong species identification. Nevertheless, first, we must emphasize that it is very difficult for us to sample the threatened musk deer species from the Guandi Forest District. Due to this issue, it is not yet possible to make a definitive conclusion as to whether the M. moschiferus once occurred or still occurs in the Guandi Forest District. Lack of evidence does not constitute evidence of a lack of M. moschiferus. Even so, our study did serve to show the ability of genetic species identification to provide definitive identification to the species level of the samples
examined, and prove that Shanxi is a new distribution of M. berezovskii. In addition to the small sample size of the present study, if both species were present in the Guandi Forest District in the past, other processes could confound genetic studies. For example, introgressive hybridization and incomplete lineage sorting are two important evolutionary processes that often confound phylogenetic inference (Leache´ & McGuire, 2006; McGuire et al., 2007). Introgressive hybridization can cause discrepancy of mtDNA and nDNA in a hybrid zone, and incomplete lineage sorting can result in a confused allele distribution as two taxa diverge. In the present study, only mtDNA sequences were used to identify species of musk deer collected from the Guandi Forest District. Although our present data indicated that all of the samples from the Guandi Forest District are M. berezovskii, due to limited sampling and lack of nuclear markers, this conclusion awaits further corroboration by more genetic evidence and additional samples from the area. In spite of these caveats, however, there remains the most possibility raised by this study that only one musk deer species occurs within Shanxi, and that species is M. berezovskii. And it is the first time we use DNA barcoding to prove that Shanxi is a new distribution of M. berezovskii.
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DOI: 10.3109/19401736.2014.880887
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Figure 6. Neighbor-joining (NJ) analysis of Kimura 2-parameter (K2P) distance of 446-bp of the D-loop sequences. The species name is followed by its GenBank accession number.
Acknowledgements We are grateful to Professor Yuanjun Zhao, editors and two anonymous referees for their encouragement, support, comments, and suggestions.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article. This research was funded by National Science and Technology Support Project of China (2012BAC01B06).
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Green MJB. (1986). The distribution, status and conservation of the Himalayan Musk Deer (Moschus leucogaster). Biol Conserv 35: 347–75. Gross M. (2012). Barcoding biodiversity. Curr Biol 22:R73–6. Groves CP. (1975). The taxonomy of Moschus (Mammalia, Artiodactyla) with particular reference to the Indian Region. J Bombay Nat Hist Soc 72:662–76. Groves CP, Feng ZJ. (1986). The status of musk deer from Anhui Province, China. Acta Theriol Sinica 6:101–6. Groves CP, Wang YP, Grubb P. (1995). Taxonomy of musk deer, genus Moschus (Moschidae, Mammalia). Acta Theriol Sinica 15:181–97. Grubb P. (1982). The systematics of Sino-Himalayan Musk-deer (Moschus), with particular reference to the species described by Hodgson B H. Saugetierk Mitt 30:127–35. Hao Y, An W, Yang X, Guo Y, Han B, Meng X. (1993). Natural resources of Moschus moschiferus in Shanxi Pangquangou Nature Reserve. Sichuan J Zool 12:34–5. Hao Y, An W, Zhang Z, Hao Y, Meng X. (1994). Food habits study of Moschus moschiferus. Chin J Zool 29:46–50. Hao Y, Wu J, Wang J. (1990). Some ecological information of Moschus moschiferus in the Pangquangou Nature Reserve. Sichuan J Zool 9: 44–6. Hao Y, Wu J, Wang J, Wang J. (1991). Ecological features of Moschus moschiferus in the Pangquangou Nature Reserve. Chin J Ecol 10: 16–18. Hebert PDN, Cywinska A, Ball SL, deWaard JR. (2003b). Biological identifications through DNA barcodes. Proc R Soc London Ser, B: Biol Sci 270:313–21. Hebert PDN, Ratnasingham S, deWaard J R. (2003a). Barcoding animal life: Cytochrome c oxidase subunit 1 divergences among closely related species. Proc R Soc London B 270:96–9.
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IUCN. (2004). IUCN Red List of Threatened Species. IUCN Species Survival Commission, The World Conservation Union. Available at: www.iucnredlist.org. (Accessed 18 August 2010). Johns GC, Avise JC. (1998). A comparative summary of genetic distances in the vertebrates from the mitochondrial cytochrome b gene. Mol Biol Evol 15:1481–90. Leache´ AD, McGuire JA. (2006). Phylogenetic relationships of horned lizards (Phrynosoma) based on nuclear and mitochondrial data: Evidence for a misleading mitochondrial gene tree. Mol Phylogenet Evol 39:628–44. Liao X, Li Q. (2008). The geographical distribution reports of Moschus moschiferus L. in Shanxi. J Shanxi Agri Sci 36:55–6. McGuire JA, Linkem CW, Koo MS, Hutchison DW, Lappin KA, Orange DL, Lemos-Espinal J, et al. (2007). Mitochondrial introgression and incomplete lineage sorting through space and time: Phylogenetics of crotaphytid lizards. Evolution 61:2879–97. Meng X, Yang Q, Feng Z, Xia L, Jiang Y Wang P. (2003b). Timing and synchrony of parturition in Alpine Musk Deer (Moschus sifanicus). Folia Zool 52:39–50. Meng X, Yang Q, Xia L, Feng Z, Jiang Y, Wang P. (2003a). The temporal estrous patterns of female Alpine Musk Deer in captivity. Appl Anim Behav Sci 82:75–85. Nei M, Kumar S. (2000). Molecular evolution and phylogenetics. New York, NY: Oxford University Press. Sambrook J, Russell DW. (2001). Molecular cloning: A laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Press.
Sheng H. (1998). Genus Moschus in China. In: Wang S, editor. China Red data book of endangered animals. Beijing: Science Press. p 231–45. Smith AT, Xie Y, editors. (2008). A guide to the mammals of China. Princeton, NJ: Princeton University Press. Smith AT, Xie Y, editors. (2009). A guide to the mammals of China. Changsha: Hunan Education Press. Stoeckle MY, Hebert PDN. (2008). Barcode of life. Sci Am 299:82–8. Tamura K, Dudley J, Nei M, Kumar S. (2007). MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–9. Wang G, Zhao Y. (2011). Survey of wildlife resources in Pangquangou. J Econ Anim 15:134–7. Wang Y, Ma S, Li C. (1993). The taxonomy, distribution, and status of forest musk deer in China. In: Ohtaishi N, Sheng HL, editors. Deer of China: Biology and management. Tokyo: Elsevier. p 22–30. Yang Q, Feng Z. (1998). Status and conservation of musk deer at the southeast of the Qinghai-Xizang (Tibet) Plateau. In: Hu J, Wu Y, editors. Resources and conservation of vertebrates. Chengdu: Sichuan Sciences and Technology Publishing House. p 80–7. Yang Q, Meng X, Xia L, Feng Z. (2003). Conservation status and causes of decline of musk deer (Moschus spp.) in China. Biol Conserv 109: 333–42. Zhang E. (1998). Musk deer. In: Wemmer C, editor. Deer, status survey and conservation action plan. Gland, Switzerland: IUCN/SSC Deer Specialist Group, IUCN, p 72–7.
Supplementary material available online Supplementary Table S1
ORIGINAL ARTICLE
DNA barcoding revises a misidentification on musk deer Chengzhong Yang1,2, Zhen Xiao1, Yuan Zou3, Xiuyue Zhang3, Bo Yang4, Yinghong Hao5, Timothy Moermond3, and Bisong Yue2
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1
Chongqing Key Laboratory of Animal Biology, College of Life Sciences, Chongqing Normal University, Chongqing, P.R. China, 2Key Laboratory of Bio-resources and Eco-environment, Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, P.R. China, 3Sichuan key Laboratory of Conservation Biology on Endangered Wildlife, College of Life Sciences, Sichuan University, Chengdu, P.R. China, 4China Conservation and Research Center for the Giant Panda, Wolong, Sichuan, P.R. China, and 5Shanxi Pangquangou National Nature Reserve, Jiaocheng, P.R. China Abstract
Keywords
As an endangered animal group in China, musk deer (genus Moschus) have attracted the attention of deer biologists and wildlife conservationists. Clarifying the taxonomic status and distribution of musk deer species is important to determine the conservation status for each species and establish appropriate conservation strategies. There remains some uncertainty about the species determination of the musk deer in the Guandi Forest District of Shanxi Province, China. The musk deer in Shanxi would appear to represent an extension of the geographical distribution of either the Forest Musk Deer from the southwest or the Siberian Musk Deer from the northeast, or possibly both. The musk deer population in Shanxi Province provides an interesting and significant case to test the value of applying molecular methods to make a genetic species identification. In order to clarify the species status of the Shanxi musk deer, we sequenced 627 bp of the COI gene and &723 bp of the D-loop gene in 12 musk deer samples collected from the Guandi Forest District, and the two reference samples collected from Sichuan. Genetic analyses from the data suggest that all of the samples from the Guandi Forest District are M. berezovskii rather than M. moschiferus. It is most likely that the most previous studies had wrong species identification. And it is the first time we use DNA barcoding to prove that Shanxi is a new distribution of M. berezovskii.
Barcoding, COI, D-loop, Moschus berezovskii, Moschus moschiferus
Introduction Musk deer (Moschus spp.) are solitary ruminants distributed throughout the forest and mountainous parts of Asia, from just north of the Arctic Circle southward to the northern edge of Mongolia and to Korea, and further south through China and the Himalayan region to Northern India, Myanmar, and Northern Vietnam (Flerov, 1952; Green, 1986). China is one of the important range countries for musk deer, with all the recognized species of musk deer occurring in China (Yang et al., 2003). Due to excessive hunting and habitat loss, the wild populations of all the musk deer species have been declining for decades (Meng et al., 2003a). Consequently, all species of musk deer are on the World Conservation Union IUCN Red List of Threatened Species (IUCN, 2004), as well as in the Appendices of CITES (the Convention on International Trade in Endangered Species of Wild Fauna and Flora) (Zhang, 1998). In China, all the musk deer species are now listed as Category I key species under the Wild Animal Protection Law (Meng et al., 2003b). Despite the great interest in musk deer, the definitive designation of Moschus species and subspecies remains a matter of question. For example, relatively isolated population of musk
Correspondence: Bisong Yue, Key Laboratory of Bio-resources and Ecoenvironment, Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610064, P.R. China. Tel: +86 28 85412488. Fax: +86 28 85414886. E-mail: [email protected]
History Received 29 September 2013 Revised 31 December 2013 Accepted 5 January 2014 Published online 3 February 2014
deer in Anhui Province has been variously regarded as a subspecies of M. moschiferus or as a subspecies of M. berezovskii, and, most recently, elevated to full species status as the Anhui Musk Deer, M. anhuiensis (Smith & Xie, 2009). Likewise, the Himalayan Musk Deer, M. leucogster, and the Black Musk Deer, M. fuscus, have both been previously designated as subspecies of the Alpine Musk Deer, M. chrysogaster. These various designations of different musk deer populations remain under question and are not based on genetics. The number of species of the genus Moschus in China is still under question (Sheng, 1998), and a variety of classification systems has been proposed (Cai & Feng, 1981; Groves, 1975; Groves et al., 1995; Groves & Feng, 1986; Grubb, 1982; Sheng, 1998; Wang et al., 1993; Yang & Feng, 1998). Species identification based on morphological characters is a traditional basis for classification; however, species identification is liable to be influenced by subjective diagnosis of morphological characters and geographic variation. When the species identification based on morphological characters is inadequate to resolve the problem, molecular methods may provide an answer. The musk deer population in Shanxi Province provides an interesting and significant case to test the value of applying molecular methods to make a genetic species identification. There are records of Siberian Musk Deer there (Groves et al., 1995; Hao et al., 1991, 1990, 1993, 1994; Liao & Li, 2008; Wang & Zhao, 2011; Yang et al., 2003). Smith & Xie (2008) show a number of records of both species in Shanxi. If this is true, these Shanxi
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records of the Siberian Musk Deer would represent a somewhat isolated population existing southwest of the populations in northeast China, and the records of the Forest Musk Deer would represent a somewhat isolated population existing northeast of the main center of the Forest Musk Deer populations in south central China (Smith & Xie, 2008). It is even more interesting, or, rather, surprising that Smith & Xie (2009) show only M. moschiferus records in Shanxi (i.e. in the Chinese edition published 1 year later than the English edition). The records of M. berezovskii shown in Shanxi on their 2008 map were removed from the map in 2009 (Smith & Xie, 2008, 2009). The question is whether the musk deer in Shanxi are Siberian Musk Deer, or Forest Musk Deer, or both? The musk deer in Shanxi Province in the Guandi Forest District, which includes the Pangquangou Nature Reserve, have been identified as Siberian Musk Deer, and a number of ecological and behavioral studies have been conducted on these purported Siberian Musk Deer (Hao et al., 1991, 1990, 1993, 1994; Liao & Li, 2008; Wang & Zhao, 2011). However, some scholars doubt the validity of the musk deer diagnosis there (e.g. Zuomo Liu, personal communication). They postulate that the musk deer in the Guandi Forest District are Forest Musk Deer (M. berezovskii) rather than Siberian Musk Deer (M. moschiferus), but no evidence has been provided. This study attempts the ‘‘re-identification’’ of the musk deer of the Guandi Forest District using molecular genetic methods to provide a more definitive assessment of the species of these interesting region which lies in between the adjacent ranges of the Siberian Musk Deer and the Forest Musk Deer. For species identification, a certain fragment of the mitochondrial gene COI, coding for a subunit of the enzyme cytochrome oxidase, has become widely known and used as ‘‘the DNA barcode’’ for the identification of most animal species (Hebert et al., 2003a,b). What makes this sequence particularly suitable for such tasks is the fact that it varies noticeably between species and very little between the individuals of a given species (Gross, 2012). The COI gene enables accurate animal species identification only in the case where adequate reference sequence data exist (Dawnay et al., 2007). Forensic wildlife cases require accurate species identification based on similarity between mitochondrial DNA of unknown samples and reference data (Dalton & Kotze, 2011). In the present study, the mitochondrial COI gene was used to identify the species of musk deer samples collected from the Guandi Forest District. And then, in order to further assess the accuracy
of the species identification based on COI, D-loop gene was also used.
Materials and methods Specimens and DNA extraction Twelve specimens of musk deer were obtained from the Guandi Forest District, Shanxi Province, China from 1995 to 2011. Other two reference samples of Forest Musk Deer (M. berezovskii) were collected from Miyaluo, Sichuan Province, China. The collection information is listed in Table 1. Total DNA was extracted following the method of Sambrook & Russell (2001). Polymerase chain reaction amplification and sequencing The purified DNA was used as a template to amplify a 627 bp fragment of COI gene and &723 bp fragment D-loop gene. The primers for amplifying segment of COI were: HCO2198-50 TAA ACT TCA GGG TGA CCA AAA AAT CA 30 and LCOI490-50 GGT CAA CAA ATC ATA AAG ATA TTG G 30 (Dalton & Kotze, 2011; Folmer et al., 1994). The primers for amplifying segment of D-loop gene were designed based on the Moschus sequences from GenBank: lsdloopF-50 CCT CCC TAA GAC TCA AGG AAG AA 30 ; lsdloopR-50 TCG TGC CTA CCA TTA TGG TGA TG 30 . PCR cycles were 5 min at 95 C followed by 35 cycles of 30 s at 95 C, 50 s at 54 C, and 1 min at 72 C, with a final extension at 72 C for 10 min. The polymerase chain reaction products were electrophoresed on a 1.0% agarose gel and purified with the DNA Agarose Gel Extraction Kit (Omega Bio-Tek, Norcross City, GA). The purified fragments were sequenced directly using an ABI 3730XL automatic sequencer (Applied Biosystems, Foster City, CA). Data analysis To determine whether the sequences were of mitochondrial origin or represented paralogous sequences in the nucleus, the following methods were used: All DNA sequence electropherograms were examined for distinguishing features of nuclear copies, including double peaks resulting from potential coamplification of mtDNA and nuclear DNA sequences, unexpected insertions/deletions, frameshifts or stop codons, and mismatches in overlapping sequence for a given taxon from different amplification products. Features consistent with mitochondrial origin that we observed in our sequences are (1) presence of a conserved reading frame in
Table 1. The informations of the specimens – the unknown samples and the reference samples in this study. GenBank no. Specimen No. Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 Sample 11 Sample 12 FMD 1 FMD 2
Sex
Sample type
Origin
Date sampled
COI
D-loop
Female Male Female Male Female Male Male Male Female Female Female Male Male Female
Skin Skin Skin Skin Skin Skin Skin Skin Skin Skin Skin Skin Blood Muscle
Guandi Forest District, Shanxi Guandi Forest District, Shanxi Guandi Forest District, Shanxi Guandi Forest District, Shanxi Guandi Forest District, Shanxi Guandi Forest District, Shanxi Guandi Forest District, Shanxi Guandi Forest District, Shanxi Guandi Forest District, Shanxi Guandi Forest District, Shanxi Guandi Forest District, Shanxi Guandi Forest District, Shanxi Miyaluo, Sichuan Miyaluo, Sichuan
Sept. 5, 2011 Mar. 16, 2011 Aug. 28, 2011 Apr. 13, 1995 Dec. 5, 2010 Dec. 5, 2010 Jun. 3, 1997 Dec. 4, 2005 Jul. 7, 2007 May 8, 2004 Dec. 24, 2010 Dec. 23, 2009 Oct. 10, 2006 Aug. 20, 2009
JX627384 JX627385 JX627386 JX627387 JX627388 JX627389 JX627390 JX627391 JX627392 JX627393 JX627394 JX627395 JX627396 JX627397
KJ000676 KJ000677 KJ000678 KJ000679 KJ000680 KJ000681 KJ000682 KJ000683 KJ000684 KJ000685 KJ000686 KJ000687 KJ000688 KJ000689
FMD means Forest Musk Deer (M. berezovskii).
DNA barcoding of musk deer
DOI: 10.3109/19401736.2014.880887
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protein-coding genes among all taxa with decreasing rates of variability at third, first, and second codon positions, respectively, and (2) absence of extra stop codons, frameshifts, or unusual amino acid substitutions. Further, we found no evidence of sequence changes yielding losses of known secondary structure for tRNA and rRNA genes that would indicate translocation to the nucleus and loss of function. Where possible, we have compared our sequences with homologs from GenBank for conspecific individuals. In all such cases, we found nearly identical matching. A total of 627 bp of the COI gene and &723 bp of the D-loop gene of the 14 musk deer samples were included in the analyses. Measuring genetic differentiation was based on Kimura two parameter (K2P) distance (Nei & Kumar, 2000). Phylogenetic analysis All of the reference sequences included in phylogenetic analyses are listed in Table S1 (Supporting Information, Table S1). After being aligned and deleted gaps, the sequences data sets for treeconstruction were obtained. The unrooted neighbor-joining (NJ) tree based on COI and D-loop were established in MEGA 4 (Tamura et al., 2007). Additionally, in the GenBank database, the length and number of D-loop sequences for each musk deer species are not identical (from 229 to 923 bp). For making the
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most of the sequence information from GenBank, we constructed two D-loop trees in the present study: one was based on 229 bp and the other was 446 bp.
Results Genetic variations of the COI gene A 627-bp fragment of the mitochondrial COI gene was successfully sequenced for all the specimens, and was deposited in GenBank with the accession numbers JX627384 to JX627397 (Table 1). The 12 unknown samples present four haplotypes (Hap1 to Hap4). Samples 1, 2, 3, 5, 6, 7, 8, 9 and 11 all share Hap1; the remaining three samples (4, 10 and 12) each has a different haplotypes (Hap2, Hap3 and Hap4, respectively). The genetic distance within the four haplotypes ranges from 0.2% to 1.1%. M. berezovskii is the species whose genetic distance to the 12 unknown samples is the shortest (0.2–1.6%). The genetic distance within species, genus, family and order are 0–1.6%, 2.5–7.0%, 2.4–19.4%, and 14.5–21.2%, respectively (Figure 1). There are 70 variable sites within the musk deer COI sequences. Among them, the variable sites between the four haplotypes (Hap1–Hap4) and M. berezovskii sequences are the least (Figure 2). By comparing among the alignment of musk deer sequences, it would be found that the four haplotypes were the most similar to that of M. berezovskii
Figure 1. The distribution of pairwise K2P genetic distance. (a) within species; (b) within genus; (c) within family; (d) within order.
Figure 2. Variable sites analyses on regions of alignment of musk deer COI gene sequences. Matched sites are represented by dots.
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Figure 3. Neighbor-joining (NJ) analysis of Kimura 2-parameter (K2P) distance of COI sequences. The first number behind the species name is the Voucher ID, and the second is the GenBank accession number.
DNA barcoding of musk deer
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DOI: 10.3109/19401736.2014.880887
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Figure 4. Variable sites analyses on regions of alignment of musk deer D-loop gene sequences. Matched sites are represented by dots; insertions and deletions were compensated by introducing alignment gaps (dash).
sequences. As showed in the NJ tree (Figure 3), all COI sequences of the 12 unknown samples cluster in Moschidae. Within Moschidae, M. berezovskii and all the unknown samples cluster together, showing a closer relationship with each other than either has with M. chrysogaster or M. moschiferus. Genetic variations of the D-loop gene A &723 bp length of the D-loop gene was also sequenced from all specimens, and was deposited in GenBank with the accession numbers KJ000676 to KJ000689 (Table 1). The 12 unknown samples display six haplotypes (H1–H6). Samples 1, 10, 11 and 12 share the H1; samples 2, 4 and 9 share the H2; samples 5 and 7 share the H3; and the remaining three samples (3, 6 and 8) each has a different haplotypes (H4, H5 and H6, respectively). Similar to the COI gene, the results of comparing variable sites of the D-loop sequences also showed that the six (H1–H6) haplotypes were the most similar to that of M. berezovskii sequences (Figure 4). Though the length and number of D-loop sequences for each species is different, the NJ trees of Moschidae based on the length of 229-bp and 446-bp D-loop sequences showed a similar topology (Figures 5 and 6). As showed in these two figures, all of the 12 unknown
samples nest within M. berezovskii. Within the Moschidae, all members cluster together with each musk deer species (Figures 5 and 6).
Discussion In the present study, the genetic distance based on COI among the 12 unknown samples is in the range of intraspecific distance (Figure 1), which indicated that the 12 unknown samples belong to the same species. The analysis of the genetic distance between the 12 unknown samples and the two reference species further indicated that the unknown samples are most likely conspecific with M. berezovskii. The result is also consistent with the phylogenetic relationships of the COI-tree (Figure 3) and D-loop trees (Figures 5 and 6) as well as the variable sites analyses (Figures 2 and 4). All lines of evidence suggest that all of the 12 unknown samples should be ascribed to M. berezovskii rather than to M. moschiferus. Stoeckle & Hebert (2008) indicated that mitochondrial sequence divergences are strongly linked to the process of speciation. In the present study, all of the conspecific pairs were observed to have K2P COI divergence smaller than 1.6%, while the interspecific pairs had divergences greater than 2.4%. Indeed, 98% of sister species pairs of vertebrates showed a divergence
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Figure 5. Neighbor-joining (NJ) analysis of Kimura 2-parameter (K2P) distance of 294-bp of the D-loop sequences. The species name is followed by its GenBank accession number.
greater than 2% (Johns & Avise, 1998). Therefore, some congeneric pairs with low divergences in the present study (approximate to 2%) may represent recent histories of reproductive isolation (Hebert et al., 2003a). Many previous studies conducted in Guandi Forest District about musk deer were ostensibly about M. moschiferus (Hao et al., 1991, 1990, 1993, 1994; Liao & Li, 2008; Wang & Zhao, 2011; Yang et al., 2003). The samples used in the present study were collected from the year 1995 to 2011, which covered the time of some previous study there (Hao et al., 1994; Liao & Li, 2008; Wang & Zhao, 2011; Yang et al., 2003). However, all of the samples are M. berezovskii, and not even one M. moschiferus was found. It is most likely that the previous studies had wrong species identification. Nevertheless, first, we must emphasize that it is very difficult for us to sample the threatened musk deer species from the Guandi Forest District. Due to this issue, it is not yet possible to make a definitive conclusion as to whether the M. moschiferus once occurred or still occurs in the Guandi Forest District. Lack of evidence does not constitute evidence of a lack of M. moschiferus. Even so, our study did serve to show the ability of genetic species identification to provide definitive identification to the species level of the samples
examined, and prove that Shanxi is a new distribution of M. berezovskii. In addition to the small sample size of the present study, if both species were present in the Guandi Forest District in the past, other processes could confound genetic studies. For example, introgressive hybridization and incomplete lineage sorting are two important evolutionary processes that often confound phylogenetic inference (Leache´ & McGuire, 2006; McGuire et al., 2007). Introgressive hybridization can cause discrepancy of mtDNA and nDNA in a hybrid zone, and incomplete lineage sorting can result in a confused allele distribution as two taxa diverge. In the present study, only mtDNA sequences were used to identify species of musk deer collected from the Guandi Forest District. Although our present data indicated that all of the samples from the Guandi Forest District are M. berezovskii, due to limited sampling and lack of nuclear markers, this conclusion awaits further corroboration by more genetic evidence and additional samples from the area. In spite of these caveats, however, there remains the most possibility raised by this study that only one musk deer species occurs within Shanxi, and that species is M. berezovskii. And it is the first time we use DNA barcoding to prove that Shanxi is a new distribution of M. berezovskii.
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Figure 6. Neighbor-joining (NJ) analysis of Kimura 2-parameter (K2P) distance of 446-bp of the D-loop sequences. The species name is followed by its GenBank accession number.
Acknowledgements We are grateful to Professor Yuanjun Zhao, editors and two anonymous referees for their encouragement, support, comments, and suggestions.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article. This research was funded by National Science and Technology Support Project of China (2012BAC01B06).
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Supplementary material available online Supplementary Table S1
