* Centre for Cellular and Molecular Biology, Hyderabad, India
Central Institute of Research on Goat, Mathura, India
Department of Biochemistry, University of Oxford, Oxford, U.K.
Correspondence: E-mail: thangs{at}ccmb.res.in.
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Abstract |
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Key Words: goats mtDNA India phylogeography livestock
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Introduction |
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Mitochondrial DNA (mtDNA) contains highly informative polymorphic sites and its simple maternal inheritance without recombination makes it useful for population studies in many organisms (Brown et al. 1986; Vigilant et al. 1991; Loftus et al. 1994; Bradley et al. 1996; Manceau et al. 1999; Luikart et al. 2001). Luikart et al. (2001) carried out a worldwide survey of domestic goat mtDNA diversity and identified three major mtDNA lineages. Lineage A was the most common in all continents. Lineage B was found in the Indian subcontinent, Mongolia, and Southeast Asia. Lineage C was observed in a few samples from Mongolia, Switzerland, and Slovenia. These three lineages were judged to have diverged over 200,000 years ago; this ancient divergence time and the different geographical localizations of the lineages suggested the likelihood of either multiple domestication events or introgression of additional lineages after the original domestication. The predominance of a single lineage in goats was in contradiction to the dual origin of livestock species reported in cattle, sheep, and pigs (MacHugh and Bradley 2001).
This initial survey provides a context for more detailed regional studies. India is a vast subcontinent with about 123 million goats comprising 20 recognized breeds and nondescript (Local) goats, which together make up approximately 20% of the world's goat population (http://fao.org). Indian goat breeds exhibit enormous variations in fecundity; production of meat, milk, and fibre; draughtability; disease resistance; and heat tolerance. However, a previous analysis of Indian goats was confined to a single study by Luikart et al. (2001) using a limited number of samples (14 individuals from five breeds). There is, therefore, a need for an extensive study of Indian goat breeds to understand their origin, divergence, and past migration patterns. Hence, we have undertaken the present investigation of 363 goats belonging to 10 different breeds from different geographic regions (fig. 1). We find evidence for population structure and additional lineages in Indian goats and cannot reconcile the genetic diversity found within the major lineage with domestication starting about 10,000 years ago from a single mtDNA ancestor. Thus, we propose a more complex origin for domestic goats.
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Materials and Methods |
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Sequencing of Amplicons
PCR products were sequenced directly on both strands by adopting the strategy described elsewhere (Thangaraj et al. 2003) using 50 ng (2.0 µl) of PCR product and 4 pM (1.0 µl) of primer, 4 µl of BigDye Terminator ready reaction kit (Perkin Elmer), and 3.0 µl of double distilled water to adjust the volume to 10.0 µl. Cycle sequencing was carried out in a GeneAmp9600 thermal cycler (Perkin Elmer) employing 30 cycles at 96°C for 10 s, 50°C for 5 s, and 60° for 4 min. Extended products were purified by alcohol precipitation followed by washing with 70% alcohol. Purified samples were dissolved in 10 µl of 50% Hi-Di formamide and analyzed in an ABI3700 automated DNA Analyzer (Perkin Elmer).
Data Analysis
Four hundred fifty-seven base pairs from the mtDNA HVRI region of 363 individuals of 10 Indian goat breeds and 14 sequences of six wild goat species (downloaded from NCBI; accession numbers AJ317864 to AJ31787), were aligned in the ClustalX package (Thompson, Higgins, and Gibson 1994). The GENEDOC package was used for formatting the sequences to make them compatible with the desired software. Reduced median networks (Bandelt, Forster, and Rohl 1999) were drawn using the program Network 3.1.1.1 (www.fluxus-engineering.com) with the parameters set to a weight of 2 and threshold 1; estimates ± standard error (SE) were obtained using the same program. Haplotype diversity and its SE, Fu's Fs statistics (Fu 1997), mismatch analysis (Schneider et al. 1999), AMOVA (analysis of molecular variance), mean number pairwise differences, and population pairwise
ST values were computed using ARLEQUIN version 2.001 (http://anthropologie.unige/arlequin) (Excoffier, Smouse, and Quattiro 1992).
ST values were calculated using 100 bootstraps. These
ST values were used to reconstruct an NJ/UPGMA tree in comparison with wild goat breeds using the PHYLIP package version 3.6 (Felsenstein 1993). A Maximum-likelihood tree was reconstructed with Kimura two- parameter model in PAUP* software version 4.0 (Swofford 1993). The substantial heterogeneity in substitution rates among nucleotide sites was estimated under Kimura two-parameter with gamma correction, which has been observed for other mammals (Vigilant et al. 1991; Bradley et al. 1996; Excoffier and Schnedier 1999; Luikart et al. 2001). The Tamura and Nei distances within goats and between goat and sheep were calculated in MEGA version 2.1 (Kumar et al. 2001) using a value for the alpha parameter of 0.29 (Luikart et al. 2001). Pairwise
ST values between breeds were displayed by multidimensional scaling (MDS) using SPSS 11.0; the stress value was 0.12. A Mantel test was carried out to estimate the correlation between geographical distances and genetic distances between breeds (Adams 1999).
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Results |
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The data were used to construct a reduced median network (fig. 2). The network shows considerable diversity, but one high-frequency haplotype, H1, (present in 27 individuals, 7.4% of the total) stands out at the center of a star-shaped phylogeny. It has 21 one-step neighboring haplotypes, of which four contained five or more individuals, so that the total number of individuals at this distance was 51 (14% of the total). Haplotypes two, three, four, and five steps away are represented by 58, 84, 73, and 12 individuals, respectively (16%, 23.1%, 20.1%, and 3.3%). We compared these haplotypes with the A, B, and C lineages described previously (Luikart et al. 2001). Luikart and colleagues reported the presence of lineage A and B in the Indian subcontinent but not lineage C. The H1 haplotype and its neighbors fell within the lineage A, as would be expected from their high frequency in both data sets. A few samples of Indian goats clustered with lineages B and C, demonstrating that C is present in India at low frequency; its previously reported absence was probably the result of the small sample size examined. We also observed additional lineages, D and E as distant from haplotype H1 as lineage B (16 steps, compared with 13 steps for lineage B [fig. 2]), showing that considerable additional diversity exists within Indian domestic goats. These two lineages are found only in the Barbari breed (fig. 1).
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This estimate includes all of the diversity within Indian goat mtDNAs. Several distinct lineages are present (fig. 2), and the times associated with individual lineages, particularly the major one, are also of interest. We therefore estimated the TMRCA for the major lineage using , the average number of mutational steps from the ancestral sequence, assuming that the ancestor was H1. Dates of 42,000 ± 7,000 to 59,000 ± 10,000 years were obtained.
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Discussion |
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Indian goat mtDNA sequences exhibited considerable diversity, and the TMRCA calibrated against the fossil record was 103,000 to 143,000 or 201,000 to 280,000 years. This figure, as noted by Luikart et al. (2001) for their worldwide data set, is considerably older than the date of domestication of goats, approximately 10,000 years ago. Their figure of 201,380 to 281,932 years was derived from changes at the third position in codons of the mtDNA cytochrome b gene. They used different DNA samples and thus their estimate was partly independent, although the same fossil record calibration point was used. The two estimates are in good agreement.
The TMRCA for the most frequent lineage was estimated to lie between 35,000 and 69,000 years, using the same mutation rate and taking into account the uncertainty in the timing of the sheep-goat divergence and the standard (sampling) error of . These periods, despite the uncertainty, are considerably older than the time of domestication. Four explanations are possible: (1) the TMRCA estimate could be too old by a factor of 3.5 or more because of errors in the calculation, (2) domestication could have started earlier than revealed by the archaeological record or (3) involved multiple related lineages and thus incorporated preexisting variation that dates back more than 35,000 years, or, alternatively, (4) mtDNA could have been subject to diversifying selection, so calculations based on the assumption of neutrality are invalid. We note that our TMRCA does not conflict with the expansion time of lineage A of Luikart et al. (2001) because these authors simply assumed, and did not calculate, their expansion time of 10,000 years.
Do errors in the calculation provide a plausible explanation? Four sources of error are possible: diversity within goats, the number of mutational changes between sheep and goats, sheep-goat divergence time, and selection.
Our estimate of 10.0/457 bp mean pairwise differences is in good agreement with the 10.9/481 bp of Luikart et al. (for lineage A) and is thus unlikely to be greatly in error.
The adjustment for multiple hits used by Vigilant et al. (1991) led to a TMRCA of 166,000 to 249,000 years for human mtDNA and 80,000 to 480,000 years by Tamura and Nei (1993) based on a 4 to 6 Myr chimpanzee-human divergence time. A recent estimate for the human mtDNA TMRCA based on diversity within the entire sequence and a 5-Myr divergence time was 171,500 ± 50,000 years (Ingman et al. 2000), so these methods of adjustment for recurrent mutation in the control region lead to reliable time estimates.
Sheep-goat divergence time is established from the fossil record (Savage and Russell 1983, Carroll 1988) and would have to be approximately 1.4 Myr to lead to a TMRCA that overlaps with the 10,000-year domestication time. It seems unlikely that the fossil dating could be so much in error. Similarly, although domestication may have begun a few hundred or even a few thousand years before the first archaeological records, a time of 35,000 years ago in the Paleolithic period is not credible.
Human selection for increased mtDNA diversity could, in principle, have led to higher than expected diversity during the 10,000 years since domestication, but there is no plausible way in which the likely selection for phenotypic characteristics of interest to farmers could have led to such an increase in mtDNA variation. We, thus, conclude that domestication probably involved multiple maternally related goats carrying considerable preexisting mtDNA diversity.
All the domestic goat lineages examined fall into a single monophyletic group that is distinct from all available wild goat sequences (fig. 4). The lineages contributing to domestic goats were therefore derived from an unknown population that may now be rare or extinct. Further investigations of wild goats and archaeological specimens are therefore needed to investigate these ancestors.
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Conclusions |
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The Pashmina goats appear to have had a different demographic history from the other breeds. These goats are adapted to living in a cold environment at high altitude in the Himalayas where the human population density is low. Perhaps early on their numbers reached a limit set by the environment, and cannot expand further. Whether the expansion seen in the other breeds has affected only mtDNA lineages or the entire genome could be assessed by analyzing autosomal and Y-chromosomal markers. If all loci show the same pattern, it is likely that the population has increased in size, but if other loci show different patterns, there could have been selection on the mtDNA.
We find that goats have had a more complex history of domestication than indicated by previous studies. We suggest that the diversity within the major A lineage dates back more than 35,000 years, indicating that domestication involved a considerable number of females 10,000 years ago and that diverse lineages in addition to B and C have been incorporated at low frequency.
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Acknowledgements |
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Footnotes |
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Sequence data from this article have been deposited with GenBank under accession numbers AY155674 to AY156039.
David Goldstein, Associate Editor
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adams, L. 1999. Mantel nonparametric test calculator. Version 2.00. http://www.sci.qut.edu.au/NRS/mantel.html.
Bandelt, H., P. Forster, and A. Rohl. 1999. Median joining networks for inferring intraspecific phylogenies. Mol. Biol. Evol. 16:37-48.[Abstract]
Bradley, D. G., D. E. MacHugh, P. Cunningham, and R. T. Loftus. 1996. Mitochondrial diversity and the origin of the African and European cattle. Proc. Natl. Acad. Sci. USA 93:5131-5135.
Brown, G. G., G. Gadaleta, G. Pepe, and C. Saccone. 1986. Structral conservation and variation in the D loop containing region of vertebrate mitochondrial DNA. J. Mol. Biol. 192:503-511.[ISI][Medline]
Carroll, R. L. 1988. Vertebrate palaentology and evolution. Freeman, New York.
Cavalli-Sforza, L. L., P. Menozzi, and A. Piazza. 1994. The history and geography of human genes. Princeton University Press, Princeton.
Excoffier, L., and S. Schneider. 1999. Why hunter-gatherer populations do not show signs of Pleistocene demographic expansions. Proc. Natl. Acad. Sci. USA 96:10597-10602.
Excoffier, L., P. E. Smouse, and J. M. Quattiro. 1992. Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 131:479-491.
Felsenstein, J. 1993. PHYLIP (phylogeny inference package). Versions 3.5c. Distributed by the author, Department of genetics, University of Washington, Seattle.
Fu, X-Y. 1997. Statistical tests of neutrality of mutations against population growth, hitchhiking and background selection. Genetics 147:915-925.
Ingman, M., H. Kaessmann, S. Pääbo, and U. Gyllensten. 2000. Mitochondrial genome variation and the origin of modern humans. Nature 408:708-713.[CrossRef][ISI][Medline]
Kumar, S., K. Tamura, I. B. Jakobsen, and M. Nei. 2001. MEGA2: molecular evolutionary genetics analysis. Version 2.1. Bioinformatics 17:1244-1245.
Legge, T. 1996. The beginning of caprine domestication in Southwest Asia. Pp. 236262 in D. R. Harris, ed. The Origins and Spread of Agriculture and Pastoralism in Eurasia. Smithsonian Institute, Washington DC.
Loftus, R. T., D. E. MacHugh, D. G. Bradley, P. M. Sharp, and P. Cunningham. 1994. Evidence for two independent domestications of cattle. Proc. Natl. Acad. Sci. USA 91:2757-2761.[Abstract]
Luikart, G., L. Giellly, L. Excoffier, J. D. Vigne, J. Bouuvet, and P. Taberlet. 2001. Multiple maternal origins and weak phylogeographic structure in domestic goats. Proc. Natl. Acad. Sci. USA 98:5927-5932.
MacHugh, D. E., and D. G. Bradley. 2001. Livestock genetic origins: goats buck the trend. Proc. Natl. Acad. Sci. USA 98:5382-5384.
Manceau, V., P. Boursot, L. Despres, and P. Taberlrt. 1999. Systematics of genus Capra as inferred by mitochondrial DNA sequence data. Mol. Phylogenet. Evol. 13:504-510.[CrossRef][ISI][Medline]
Mannen, H., Y. Nagata, and S. Tsuji. 2001. Mitochondrial DNA reveal that domestic goat (Capra hircus) are genetically affected by two subspecies of bezoar (Capra aegagurus). J. Biochem. Genet. 39:145-154.[CrossRef]
Meadow, A. 1996. Animal domestication in the Middle East: a revised view from the eastern margin. Pp. 295320 in G. Possehl, ed. Harappan civilisation. Oxford and IBH, New Delhi.
Misra, V. N. 2001. Prehistoric human colonization of India. J. Biosci. 26:491-531.[ISI][Medline]
Porter, V. 1996. Goats of the world. Farming Press, Ipswich, UK.
Pringle, H. 1998. Neolithic agriculture: reading the signs of ancient animal domestication. Science 282:1448.
Rogers, A. R., and H. Harpending. 1992. Population growth curves in the distribution of pairwise genetic differences. Mol. Biol. Evol. 9:552-569.[Abstract]
Savage, D. E., and D. E. Russell. 1983. Mammalian palaeofaunas of the world. Addison-Wesley, Reading, Mass.
Schneider, S., J. M. Kueffer, D. Roessli, and L. Excoffier. 1999. Arlequin 2.001: A software for population genetic data analysis. Genetics and Biometry Laboratory, University of Geneva, Switzerland.
Seielstad, M. T., E. Minch, and L. L. Cavalli-Sforza. 1998. Genetic evidence for a higher female migration rate in humans. Nat. Genet. 20:278-280.[CrossRef][ISI][Medline]
Swofford, D. L. 1993. PUAP*: phylogenetic analysis using parsimony (*and other methods). Version 4.0. Sinauer Associates, Sunderland, Mass.
Tamura, K., and M. Nei. 1993. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol. Biol. Evol. 10:512-526.[Abstract]
Thangaraj, K., M. B. Joshi, A. G. Reddy, N. J. Gupta, B. Chakraborthy, and L. Singh. 2002. CAG repeat expansion in androgen receptor gene is not associated with male infertility in Indian populations. J. Androl. 23:815-818.
Thangaraj, K., L. Singh, A. G. Reddy, V. R. Rao, S. C. Sehgal, P. A. Underhill, M. Pierson, I. G. Frame, and E. Hagelberg. 2003. Genetic affinities of the Andaman Islanders, a vanishing human population. Curr. Biol. 13:86-93.[ISI][Medline]
Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalities and weight matrix choice. Nucleic Acids Res. 22:4673-4680.[Abstract]
Vigilant, L. M., M. Stoneking, H. Harpending, K. Hawkes, and A. C. Wilson. 1991. African populations and the evolution of mitochondrial DNA. Science 253:1503-1507.[ISI][Medline]
Zeder, M. A., and B. Hesse. 2000. The initial domestication of goats (Capra hircus) in the Zagros Mountain 10,000 years ago. Science 287:2254-2257.
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