Genetic evidence from mitochondrial DNA corroborates the origin of Tibetan chickens

Abstract

Chicken is the most common poultry species and is important to human societies. Tibetan chicken (Gallus gallus domesticus) is a breed endemic to China that is distributed mainly on the Qinghai-Tibet Plateau. However, its origin has not been well characterized. In the present study, we sequenced partial mitochondrial DNA (mtDNA) control region of 239 and 283 samples from Tibetan and Sichuan indigenous chickens, respectively. Incorporating 1091 published sequences, we constructed the matrilineal genealogy of Tibetan chickens to further document their domestication history. We found that the genetic structure of the mtDNA haplotypes of Tibetan chickens are dominated by seven major haplogroups (A-G). In addition, phylogenetic and network analyses showed that Tibetan chickens are not distinguishable from the indigenous chickens in surrounding areas. Furthermore, some clades of Tibetan chickens may have originated from game fowls. In summary, our results collectively indicated that Tibetan chickens may have diverged from indigenous chickens in the adjacent regions and hybridized with various chickens.

About the Authors:

Long Zhang

Contributed equally to this work with: Long Zhang, Pu Zhang, Qingqing Li, Uma Gaur, Yiping Liu

Affiliation: Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, China

Pu Zhang

Contributed equally to this work with: Long Zhang, Pu Zhang, Qingqing Li, Uma Gaur, Yiping Liu

Affiliation: Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, China

Qingqing Li

Contributed equally to this work with: Long Zhang, Pu Zhang, Qingqing Li, Uma Gaur, Yiping Liu

Current address: Kunming Xianghao Technology Co. Ltd., Kunming, China

Affiliation: Life Science College, Southwest Forestry University, Kunming, China

Uma Gaur

Contributed equally to this work with: Long Zhang, Pu Zhang, Qingqing Li, Uma Gaur, Yiping Liu

Affiliation: Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, China

Yiping Liu

Contributed equally to this work with: Long Zhang, Pu Zhang, Qingqing Li, Uma Gaur, Yiping Liu

Affiliation: Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, China

Qing Zhu

Affiliation: Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, China

Xiaoling Zhao

Affiliation: Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, China

Yan Wang

Affiliation: Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, China

Huadong Yin

Affiliation: Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, China

Yaodong Hu

Affiliation: Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, China

Aiping Liu

Affiliation: College of Food Science, Sichuan Agricultural University, Ya'an, China

Diyan Li

* E-mail: [email protected]

Affiliation: Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, China

Introduction

As one of the most extensively distributed domesticated animals, chicken not only provides humans with a stable source of protein, but also serves important roles in numerous aspects of human society [1–3]. Tibetan chicken is an indigenous breed at high altitudes and is well known for its adaptability to survive in hypoxic conditions [4]. For example, the hatchability of Tibetan chicken eggs incubated in high attitude is greater than 70%, whereas hatchability is less than 40% for low altitude breeds [5, 6]. The hypoxia adaptation is achieved in Tibetan chickens via an increased red blood cell (RBC) count and blood oxygen affinity, together with a decreased mean cell volume and reduced susceptibility to hypocapnia [7]. To date, numerous genetic association studies demonstrated the hypoxia adaptability of Tibetan chickens living at high altitudes [8–11], yet the origin and evolution of this breed endemic to China is unknown.

Mitochondrial DNA (mtDNA) is inherited maternally and is often employed in population genetic analyses due to its high copy number, haploid nature and absence of/rare recombination events [12, 13]. The relative high mutation rate makes it suitable in studying domestic animals with recent timescale [14], numerous studies have focused on reconstructing the matrilineal history of domestic chickens by studying the control region of mtDNA. In initial researches, Fumihito et al [15, 16] have suggested a monophyletic origin of domestic chickens; these authors propose that the domestic chickens’ primary wild ancestor is a group of red jungle fowls found in the forests of Southeast Asia and India, which was spread to other parts of the world when people domesticated chickens, resulting in many chicken breeds. In contrast, with the increasing sample size, other authors have provided evidence of numerous and independent domestication events. Liu et al [17] suggested the involvement of multiple matrilines in domestication events in southern China, South Asia and Southeast Asia. Meanwhile, Kanginakudru et al [18] found evidences of domestication in Indian birds from G. g. spadiceus, G. g. gallus and G. g. murghi, demonstrating multiple domestication pathways of Indian and other domestic chickens. Recent studies involving the analysis of chicken mtDNA sequences from ancient sequences indicated some potential time, region, and pattern of chicken domestication in several regions [19–22], thus suggesting the key role of control region sequences in determining the history of chickens domestications.

In the present study, we used mtDNA partial control region sequences to investigate (i) the genetic diversity of Tibetan and indigenous fowls; (ii) the evolutionary status of Tibetan chickens as well as the surrounding indigenous populations; and (iii) the potential origin of Tibetan chickens. Our findings will increase the understanding about the matrilineal origin of Tibetan chickens in China.

Materials and methods

Sampling and DNA extraction

In total, 284 and 239 blood samples were collected from 5 low-altitude Sichuan chicken populations (Leshan, Chengdu, Dazhou, Yaan and Panzhihua) and 7 Tibetan chicken populations from Qinghai-Tibet Plateau localities (Diqing, Linzhi, Lhasa, Shannan, Haiyan, Aba and Ganzi), respectively (Fig 1). Genomic DNA was extracted from blood samples using phenol/chloroform following standard procedures [23]. The partial control region (approximately 500 bp) was amplified using the following previously published primers [2]: L16750: 5’-AGGACTACGGCTTGAAAAGC-3’ and H522: 5’-ATGTGCCTGACCGAGGAACCAG-3’. PCR amplification was performed in a 25-μL volume containing 1.25 μL of template genomic DNA, 12.5 μL of 2× Taq PCR Master Mix, 1.25 μL of each primer (10 pmol/μL), and 8.75 μL of ddH₂O. The PCR conditions were as follows: 95°C for 3 min; followed by 33 cycles of 94°C for 35 s, 58.4°C for 35 s and 72°C for 1 min; and a final extension at 72°C for 10 min. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Institute of Animal Genetic and Breeding, Sichuan Agricultural University, Chengdu, China, under permit No. YCS-B20100804. Published control region sequences of chickens from localities in Xinjiang, Qinghai, Sichuan, Yunnan, Tibet and Red jungle fowls were downloaded from the database of National Center for Biotechnology Information (NCBI). The quality of sequences were checked according to the workflow described previously [24, 25]. Sequences with potential artificial recombination, surplus of mutations or phantom mutations were not used for further analysis. Taken together, we analyzed 1613 sequences in this study, including 1091 retrieved from NCBI and 522 sequences from our samples.

[Figure omitted. See PDF.]

Fig 1. Map of sampling localities and distributions of the domestic haplogroups in each group.

Note: Codes 1–7 represent the sampling localities of Tibetan chicken populations from Diqing (27°49′ N, 99°42′ E), Linzhi (3100 m; 29°39′ N, 94°21′ E), Lhasa (3650 m; 29°38′ N, 91°06′ E), Shannan (3700 m; 29°13 N, 91°44′ E), Haiyan (3260 m; 36°53′ N, 100°59′ E), Aba (3300 m; 32°54′ N, 101°42′ E) and Ganzi (3390 m; (31°37′ N, 99°59′ E), respectively. Codes 8–12 represent the sampling localities of Sichuan lowland chicken populations from Leshan (400 m; 29°33′ N, 103°45′ E), Chengdu (540 m; 30°34′ N, 104°03′ E), Dazhou (800 m; 30°12′ N, 107°27′ E), Ya’an (900 m; 30°01′ N, 103°02′ E) and Panzhihua (1400 m; 26°35′ N, 101°43′ E), respectively. Ti: Tibetan chickens; SC: Sichuan indigenous chickens; YN: Yunnan indigenous chickens; XJ: Xinjiang indigenous chickens; QH: Qinghai indigenous chickens; RJFs: red jungle fowls.

https://doi.org/10.1371/journal.pone.0172945.g001

Sequence analysis

The nucleotide diversity and haplotypes of all sequences were estimated with DnaSP 5.0 [26]. Network 5.0 software [27] was used to draw maximum parsimony median-joining (MP) network plots. All sequences were aligned with CLUSTAL W [28] prior to phylogenetic analysis using the Bayesian Evolutionary Analysis by Sampling Trees (BEAST) 1.7 [29]. The GTR substitution model with the invariant sites and gamma distribution (GTR+I+G) was selected as the best-fitting model using the jModelTest 2.1.1 [30]. The length of MCMC was set to 30,000,000 and sampled every 1,000 generations with 10% burn-in. The maximum clade credibility tree was created using TreeAnnotator [29]. DomeTree and MitotoolPy were used to identify mutations and standard haplogroups in each sequences [31].

Results

Genetic diversity of Tibetan and lowland fowls

In this study we obtained partial control region sequences of 522 individuals (S1 dataset), together with those published control region sequences from adjacent geographic regions, we generated a dataset containing 1613 sequences, including 276 Tibetan chickens, 461 Sichuan indigenous chickens, 743 Yunnan indigenous chickens, 14 indigenous chickens from Qinghai, 39 indigenous chickens from Xinjiang and 80 Red jungle fowls (RJFs). The overall information of Tibetan chickens as well as chickens from different populations were summarized in Table 1, within the Tibetan chickens from different populations, the number of polymorphic sites in each population varies from 17 in Linzhi to 27 in Haiyan, and the unique sequences from Aba, Diqing, Ganzi, Haiyan, Lhasa, Linzhi, Shannan and the unknown was 11, 9, 11, 15, 15, 5, 8 and 9, respectively. The highest haplotype diversity was found in Lhasa, while the average number of nucleotide differences of RJFs was greater than others. For those Tibetan chicken sequences retrieved from NCBI, the value of average number of nucleotide differences is higher than other Tibetan chicken populations, it means that those sequences are potentially obtained from different regions. Tajima's D test was not significant in all populations, indicating that neither balancing selection nor purifying selection occurred in all chicken populations. Taken together, a total of 42 haplotypes coming from 47 polymorphic sites were identified in Tibetan chickens, and the overall haplotype diversity, nucleotide diversity and average nucleotide differences were 0.925, 0.016 and 7.685, respectively. All of the genetic diversity parameters in Tibetan chickens did not have much differences from other lowland chickens of different localities, suggesting that the genetic diversity of Tibetan chickens is similar to the indigenous chickens of adjacent regions.

[Figure omitted. See PDF.]

Table 1. Genetic diversity indices of Tibetan and lowland fowls.

https://doi.org/10.1371/journal.pone.0172945.t001

Phylogenetic relationships among Tibetan and indigenous chickens

To study the relationships among different populations, a phylogenetic tree of haplotypes was constructed by Bayesian method. Simultaneously, MitotoolPy was used to classify all the haplotypes as well as the sequences to different clades by diagnostic mutational motifs. As illustrated in Fig 2, the phylogenetic relationships generated by BEAST software is in agreement with the previous studies [2, 17]. Except for the paraphyletic haplogroup D, all haplogroups with more than one individual were supported by a robust posterior possibility value. Notably, like the neighbor-joining tree built of control region sequences, the RJFHap 6 which belongs to haplogroup C by diagnostic mutational motifs, was classified into clade D in our phylogenetic tree. As shown in Table 2 and Fig 1, clade A, B and G harbored haplotypes from different populations in this study, while no sequence from Xinjiang and Qinghai indigenous chickens was found in clade F and clade G. Clade D contained haplotype from Tibet Plateau, Xinjiang and RJFs, Clade H contained only indigenous chickens that were restricted to Yunnan, and only a few haplotypes represented a small number of RJFs belonging to clade W, X, Y and Z. As for Clade I, the rare south Asia haplotypes, was not found in those sequences. Those results clearly indicated that hybridization among Chinese chickens is extremely common.

[Figure omitted. See PDF.]

Table 2. Geographical distribution of the major clades of different populations.

https://doi.org/10.1371/journal.pone.0172945.t002

[Figure omitted. See PDF.]

Fig 2. Phylogenetic tree of control region haplotypes constructed by Bayesian method.

Values in parentheses are Bayesian posterior probability of haplogroups (if haplogroups with more than one individual) identified by Liu et al [17] and Miao et al [2]. Only Bayesian posterior probability of the clade higher than 0.5 are indicated above branches. Red lines: red jungle fowls (RJFs); dark green lines: Yunnan indigenous chickens (YN); pink lines: Sichuan indigenous chickens (SC); blue lines: Qinghai indigenous chickens (QH); yellow lines: Xinjiang indigenous chickens (XJ) and light green lines: Tibetan chickens (Ti).

https://doi.org/10.1371/journal.pone.0172945.g002

Median-joining network profiles of Tibetan chickens in comparison to other populations

To investigate the relationships of haplotypes, we constructed network plot for Tibetan chickens with and without the chickens of adjacent regions (Fig 3A and 3B). The median-joining network revealed a complicated pattern among Tibetan chickens and other fowls (Fig 3A). Nevertheless, the majority of the haplogroups exhibited a star-like distribution profile, indicating a recent exponential population growth. In clade A, B, F and G, the core haplotypes occurred in Tibetan chickens, RJFs and indigenous chickens in adjacent provinces. As for clade C and E, sequences of RJFs were identified on the periphery of the plot; whereas, Tibetan chickens shared main haplotypes with other indigenous chickens. Importantly, only Tibetan chickens formed the core haplotype in clade D and this core haplotype differed at least by two mutation distance from other haplotypes. Fig 3B presented the network within Tibetan chickens sequenced in this study (the sequences of Tibetan chickens downloaded from NCBI may come from more than one population, and therefore, were not included in Fig 3B), and it was also consistent with a star like pattern. Seven clades (A-G) were found in Lhasa and the remaining Tibetan chickens clustered from three clades in Aba to six clades in Ganzi. It is noteworthy that, there were high frequency of samples from Diqing and Ganzi in clade F and G. However, those private haplotypes identified in clade D were, without expectation, almost distributed in all populations of Tibetan chickens (Fig 3B).

[Figure omitted. See PDF.]

Fig 3. Maximum parsimony median-joining network of Tibetan chickens with (A) and without the chickens of adjacent regions (B).

The domestic clades (A-H) are labeled. Node sizes are proportional to haplotype frequencies. The lines linking the nodes are proportional to the mutation steps. Black nodes indicate inferred steps not identified in the sampled populations. Colors within the circles represent chickens from different localities.

https://doi.org/10.1371/journal.pone.0172945.g003

Discussion

In contrast to slowly mutating genes, which provide data about ancient history, the control region of mitochondrial DNA exhibits high mutation rates and provides information about recent evolutionary history. To reconstruct the recent past, we used this marker to address the issue of the domestication event of Tibetan chickens. By extent the sample size, we, found for the first time, haplotypes not only belong to A, E, F and G [2, 17], but also dispersed in clade B, C and D. These results suggested that they are originated from different distinct maternal lineages.

Although Tibetan chickens have adapted to the extreme environment through specific genetic mechanisms [32, 33], our phylogenetic tree and network analysis indicated that they are not distinguishable from the indigenous chickens in surrounding areas. In addition, the frequencies and distribution of haplotypes of Tibetan chickens were similar to indigenous fowls in the adjacent regions. Furthermore, a total of seven haplogroups were found in Tibetan chickens which is higher than the indigenous population in each adjacent regions. Collectively, those evidences indicated that Tibetan chickens are most likely diverged from indigenous chickens in the surrounding areas of China rather than directly from red jungle fowls. In the history, two important trade routes namely Tangbo Ancient Road and Tea-horse Ancient Road linked the Qinghai-Tibet Plateau and the surrounding regions of China [34, 35]. Thus, as an easily carried species, chicken is probably introduced to Tibet Plateau from the northwest and southwest of China. However, the Qinghai-Tibet Plateau samples possess a great number of unique haplotypes (Fig 3A). There were a lot of haplotypes of Tibetan chickens presented in clade D and only a few shared with Xinjiang indigenous chickens. Our previous findings indicated that the distribution of clade D is associated with the dispersal of cockfighting cultures [17, 36], and indeed, Tibetan chickens appear more aggressive than other common chickens. Therefore, we propose that game fowls may have been introduced to Tibetan chickens. Nevertheless, it is hard to make conclusion about the origin of those haplotypes. Because almost all of these haplotypes shared an undiscovered mutation linkage at 355 and 391 sites [2] (S1 Table) and only a few samples (39 individuals) in Xinjiang have been sequenced. More importantly, we noticed that these private haplotypes are widely distributed in Tibetan chicken populations, suggesting that potential maternal lineages of game fowls have been introduced to Qinghai-Tibet Plateau for a long time.

As aforementioned, the 49 samples in Lhasa were assigned to seven domestic clades, because Lhasa is the economic center of Tibetan region, highest haplotype diversity and haplogroups distribution of fowls in this place may be caused by numerous trade activities. In contrast, some Tibetan populations possess specific haplotypes, for example, the results presented in Fig 3 indicated that the majority of samples of Diqing and Ganzi were classified into clade F and G. On the account of haplogroups F and G were mainly distributed in Yunnan and shared their core haplotypes with Tibetan chickens. We speculate that the origin of Tibetan chickens in Diqing and Ganzi can be traced back to Yunnan. However, we found that only a few individuals in Sichuan were distributed in Clade F and G. It is unlikely that the Sichuan act as a passageway for the gene flow from Diqing Tibetan chickens to Ganzi. Thus, we propose that the Tibetan chickens in Diqing are directly originated from Yunnan while a large number of individuals in Ganzi are introduced from the Diqing by breeding activities. In addition, sequences of clade F and G were absent in many places of Tibetan highland, indicating that and hybridization between Tibetan chickens and Yunnan indigenous fowls happened recently.

In summary, we sequenced and compared Tibetan chickens with different chicken populations in this study. The results indicated that the Tibetan chickens exhibit multiple origins, and they may originate from indigenous fowls in Yunnan, Sichuan, Xinjiang and/or game fowls. Additionally, breeding activities and hybridization may exist within or among different populations and the maternal lineages of Tibetan chickens is very complex. The obtained mtDNA phylogeny extends our understanding of the matrilineal history of chickens and more genetics or genomics studies need to be conducted in order to reveal the clear gene flow between or among different populations in the future.

Supporting information

[Figure omitted. See PDF.]

S1 Dataset. Partial mtDNA control region sequences of 284 Sichuan indigenous chickens and 239 Tibetan chickens.

https://doi.org/10.1371/journal.pone.0172945.s001

(TXT)

S1 Table. Summary of haplogroups and detail variants of each sequence used in this study.

https://doi.org/10.1371/journal.pone.0172945.s002

(XLSX)

Acknowledgments

This work was supported by the Program of Sichuan Agricultural University (02920400), the National Natural Science Foundation of China (NSFC31402063), the Sichuan Provincial Department of Science and Technology Program (2015JQO023), the Key Discipline Construction Project of Biology in Yunnan province (50097505), the Doctoral Scientific Research Foundation of Southwest Forestry University (111441) and the project of Thirteen Five-Year Province Crops and Livestock and Poultry Breeding Research (2016NYZ0043). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We also thank Binlong Chen for help during sample collection.

Author Contributions

1. Conceptualization: LZ QL DL.

2. Data curation: LZ YL QZ XZ YW.

3. Formal analysis: LZ PZ YL XZ YW YH DL.

4. Funding acquisition: QL YL DL.

5. Investigation: LZ PZ XZ HY.

6. Methodology: LZ PZ YL YW.

7. Project administration: QL YL DL.

8. Resources: QL XZ AL.

9. Supervision: YL QZ DL.

10. Validation: YL QZ DL.

11. Visualization: LZ PZ UG DL.

12. Writing – original draft: LZ PZ.

13. Writing – review & editing: LZ PZ YL UG DL.

Citation: Zhang L, Zhang P, Li Q, Gaur U, Liu Y, Zhu Q, et al. (2017) Genetic evidence from mitochondrial DNA corroborates the origin of Tibetan chickens. PLoS ONE 12(2): e0172945. https://doi.org/10.1371/journal.pone.0172945

References

1. Wu Z, Kaiser P. Antigen presenting cells in a non-mammalian model system, the chicken. Immunobiology. 2011;216(11):1177–83. pmid:21719145

2. Miao Y, Peng M, Wu G, Ouyang Y, Yang Z, Yu N, et al. Chicken domestication: an updated perspective based on mitochondrial genomes. Heredity. 2013;110(3):277–82. pmid:23211792

3. Hillier LW, Miller W, Birney E, Warren W, Hardison RC, Ponting CP, et al. Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution. Nature. 2004;432(7018):695–716. pmid:15592404

4. Liu C, Zhang L, Song M, Bao H, Zhao C, Li N. Highly efficient dissociation of oxygen from hemoglobin in Tibetan chicken embryos compared with lowland chicken embryos incubated in hypoxia. Poultry science. 2009;88(12):2689–94. pmid:19903969

5. Zhang H, Wu C, Chamba Y. Hatchability of miniature laying chicken and its hybrids at high altitude. Scientia Agricultura Sinica. 2006;39(7):1507–10.

6. Zhang H, Wang X, Chamba Y, Ling Y, Wu C. Influences of hypoxia on hatching performance in chickens with different genetic adaptation to high altitude. Poultry science. 2008;87(10):2112–6. pmid:18809874

7. Zhang H, Wu C, Chamba Y, Ling Y. Blood characteristics for high altitude adaptation in Tibetan chickens. Poultry science. 2007;86(7):1384–9. pmid:17575186

8. Zhao X, Wu N, Zhu Q, Gaur U, Gu T, Li D. High-altitude adaptation of Tibetan chicken from MT-COI and ATP-6 perspective. Mitochondrial DNA Part A. 2016;27(5):3280–8.

9. Sun J, Zhong H, Chen S-Y, Yao Y-G, Liu Y-P. Association between MT-CO3 haplotypes and high-altitude adaptation in Tibetan chicken. Gene. 2013;529(1):131–7. pmid:23850731

10. Bao H, Zhao C, Li J, Wu C. Association of MT-ND5 gene variation with mitochondrial respiratory control ratio and NADH dehydrogenase activity in Tibet chicken embryos. Animal genetics. 2007;38(5):514–6. pmid:17614984

11. Bao H, Zhao C, Zhang L, Li J, Wu C. Single-nucleotide polymorphisms of mitochondrially coded subunit genes of cytochrome c oxidase in five chicken breeds: Full-Length Research Paper. DNA Sequence. 2008;19(5):461–4.

12. Wani CE, Yousif IA, Ibrahim ME, Musa HH. Molecular Characterization of Sudanese and Southern Sudanese Chicken Breeds Using mtDNA D-Loop. Genetics research international. 2014;2014.

13. Moore WS. Inferring phylogenies from mtDNA variation: mitochondrial-gene trees versus nuclear-gene trees. Evolution. 1995:718–26.

14. Wang GD, Xie HB, Peng MS, Irwin D, Zhang YP. Domestication genomics: evidence from animals. Annu Rev Anim Biosci. 2014;2(1):65–84.

15. Fumihito A, Miyake T, Sumi S, Takada M, Ohno S, Kondo N. One subspecies of the red junglefowl (Gallus gallus gallus) suffices as the matriarchic ancestor of all domestic breeds. Proceedings of the National Academy of Sciences. 1994;91(26):12505–9.

16. Fumihito A, Miyake T, Takada M, Shingu R, Endo T, Gojobori T, et al. Monophyletic origin and unique dispersal patterns of domestic fowls. Proceedings of the National Academy of Sciences. 1996;93(13):6792–5.

17. Liu YP, Wu GS, Yao YG, Miao YW, Luikart G, Baig M, et al. Multiple maternal origins of chickens: out of the Asian jungles. Molecular phylogenetics and evolution. 2006;38(1):12–9. pmid:16275023

18. Kanginakudru S, Metta M, Jakati R, Nagaraju J. Genetic evidence from Indian red jungle fowl corroborates multiple domestication of modern day chicken. BMC evolutionary biology. 2008;8(1):1.

19. Storey AA, Ramírez JM, Quiroz D, Burley DV, Addison DJ, Walter R, et al. Radiocarbon and DNA evidence for a pre-Columbian introduction of Polynesian chickens to Chile. Proceedings of the National Academy of Sciences. 2007;104(25):10335–9.

20. Xiang H, Gao J, Yu B, Zhou H, Cai D, Zhang Y, et al. Early Holocene chicken domestication in northern China. Proceedings of the National Academy of Sciences. 2014;111(49):17564–9.

21. Thomson VA, Lebrasseur O, Austin JJ, Hunt TL, Burney DA, Denham T, et al. Using ancient DNA to study the origins and dispersal of ancestral Polynesian chickens across the Pacific. Proceedings of the National Academy of Sciences. 2014;111(13):4826–31.

22. Flink LG, Allen R, Barnett R, Malmström H, Peters J, Eriksson J, et al. Establishing the validity of domestication genes using DNA from ancient chickens. Proceedings of the National Academy of Sciences. 2014;111(17):6184–9.

23. Sambrook J, Russell DW. Molecular cloning: a laboratory manual 3rd edition. Coldspring-Harbour Laboratory Press, UK. 2001.

24. Shi NN, Fan L, Yao YG, Peng MS, Zhang YP. Mitochondrial genomes of domestic animals need scrutiny. Molecular ecology. 2014;23(22):5393–7. pmid:25294192

25. Peng MS, Shi NN, Yao YG, Zhang YP. Caveats about interpretation of ancient chicken mtDNAs from northern China. Proceedings of the National Academy of Sciences. 2015;112(16):E1970–E1.

26. Librado P, Rozas J. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics. 2009;25(11):1451–2. pmid:19346325

27. Bandelt HJ, Forster P, Röhl A. Median-joining networks for inferring intraspecific phylogenies. Molecular biology and evolution. 1999;16(1):37–48. pmid:10331250

28. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic acids research. 1994;22(22):4673–80. pmid:7984417

29. Drummond AJ, Suchard MA, Xie D, Rambaut A. Bayesian phylogenetics with BEAUti and the BEAST 1.7. Molecular biology and evolution. 2012;29(8):1969–73. pmid:22367748

30. Darriba D, Taboada GL, Doallo R, Posada D. jModelTest 2: more models, new heuristics and parallel computing. Nature methods. 2012;9(8):772-.

31. Peng MS, Fan L, Shi NN, Ning T, Yao YG, Murphy RW, et al. DomeTree: a canonical toolkit for mitochondrial DNA analyses in domesticated animals. Molecular ecology resources. 2015;15(5):1238–42. pmid:25655564

32. Zhang Q, Gou W, Wang X, Zhang Y, Ma J, Zhang H, et al. Genome Resequencing Identifies Unique Adaptations of Tibetan Chickens to Hypoxia and High-Dose Ultraviolet Radiation in High-Altitude Environments. Genome biology and evolution. 2016;8(3):765–76. pmid:26907498

33. Wang MS, Li Y, Peng M-S, Zhong L, Wang ZJ, Li Q-Y, et al. Genomic analyses reveal potential independent adaptation to high altitude in Tibetan chickens. Molecular biology and evolution. 2015:msv071.

34. Sigley G. The Ancient Tea Horse Road and the politics of cultural heritage in Southwest China: regional identity in the context of a rising China. Cultural heritage politics in china: Springer; 2013. p. 235–46.

35. Yang S, Zhang H, Mao H, Yan D, Lu S, Lian L, et al. The local origin of the Tibetan pig and additional insights into the origin of Asian pigs. PloS one. 2011;6(12):e28215. pmid:22163285

36. Liu YP, Zhu Q, Yao YG. Genetic relationship of Chinese and Japanese gamecocks revealed by mtDNA sequence variation. Biochemical genetics. 2006;44(1–2):18–28.