African-Derived Mitochondria in South American Native Cattle Breeds (Bos taurus): Evidence of a New Taurine Mitochondrial Lineage

African-Derived Mitochondria in South American Native Cattle Breeds (Bos taurus): Evidence of a New Taurine Mitochondrial Lineage M. M. Miretti, H. A. Pereira, Jr., M. A. Poli, E. P. B. Contel, and J. A. Ferro This article reports the nucleotide diversity within the control region of 42 mitochondrial chromosomes belonging to five South American native cattle breeds (Bos taurus). Analysis of these data in conjunction with B. taurus and B. indicus sequences from Africa, Europe, the Near East, India, and Japan allowed the recognition of eight new mitochondrial haplotypes and their relative positions in a phylogenetic network. The structure of genetic variation among different hypothetical groupings was tested through the molecular variance decomposition, which was best explained by haplotype group components. Haplotypes surveyed were classified as European-related and African-related. Unexpectedly, two haplotypes within the African cluster were more divergent from the African consensus than the latter from the European consensus. A neighbor-joining tree shows the position of two haplotypes compared to European/African mitochondrial lineage splitting. This different and putatively ancestral mitochondrial lineage (AA) is supported by the calibration of sequence divergence based on the Bos Bison separation. The European/African mitochondria divergence might be subsequent (67,100 years before present) to that between AA and Africans (84,700 years before present), also preceding domestication times. These genetic data could reflect the haplotype distribution of Iberian cattle five centuries ago. From the Departmento de Tecnologia, FCAV, UNESP, Via de Acesso Prof. P. D. Castellane km 5, 14884-900 Jaboticabal, SP, Brazil (Miretti, Ferro, and Pereira); Departmento de Genética, FMRP, USP, Av. Bandeirantes 3900, 14049-900, Ribeirão Preto, SP, Brazil (Miretti and Contel); and Instituto de Genética, CNIA-INTA, Castelar, Argentina (Poli). The authors would like to thank F. Holgado, Andre Carvalho, and Dr. Junqueira for allowing blood collection of an Argentinean Creole, Caracu, and Mocho Nacional population, respectively; Dr. F. Meirelles for providing Nellore DNA; Dr. M. Naves for valuable comments; Dr. D. Bradley for providing the Portuguese cattle mtdna sequences, Dr. J. Sereno, CPAP/ EMBRAPA, and Dr. M. Lara, Pantaneiro and Curraleiro blood samples. This work was funded by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Fundação de Apoio ao Ensino, Pesquisa e Assitência do HC da FMRP, USP (FAEPA). Address correspondence to M. M. Miretti at the address above, or e-mail: jesus@ fcav.unesp.br. Ó 2002 The American Genetic Association 93:323 330 The comprehension of phenotypic differences and genetic variations among extant cattle world populations provided clues about their origin, evolutionary history, and in particular their relationship with early human civilizations. Wild aurochs (Bos primigenius), spread over the whole Near East and Europe in the post-pleistocene period and domesticated by Neolithic civilizations in Western Asia, have been considered the common ancestors of all modern cattle breeds (Epstein and Mason 1984; Payne 1991). The analysis of European B. primigenius DNA samples from tissues dated 12,000 years before present (YBP) showed that B. primigenius sequences were phylogenetically closer to extant B. taurus than to B. indicus (Bailey et al. 1996). The extent of divergence between zebuine and taurine cattle, based on variations within the control region of mitochondrial DNA (mtdna) between Bos and Bison, was estimated as 740,000 YBP (Loftus et al. 1994), well before domestication times. This was interpreted as evidence for a predomestic and separate origin of zebuine (humped) and taurine (humpless) cattle population ancestors. Taurine mitochondrial diversity can be grouped within two major mitochondrial haplotype clusters. There is a single highly frequent haplotype in each group representing European and African taurine cattle breeds, namely the African consensus (Afcons) and the European consensus (Eucons), respectively (Bradley et al. 1996). Both mitochondrial types differ in three substitutions within a 240 bp DNA fragment of the control region and are linked to a number of variants through one or a few mutation steps. European extant cattle, including the Iberian peninsula breeds, are presumably descendants of the Near East B. primigenius populations; nevertheless, mitochondrial haplotypes of African origin have also been found in Portuguese cattle breeds (Cymbron et al. 1999). Consequently Iberian cattle appear to have received influence from eastern Sahara B. taurus descents before the spreading of B. indicus in Africa (Mac- Hugh et al. 1997), which is consistent with the demographic history of the Iberian peninsula. Given that all the 323

American native cattle have their roots in the Iberian cattle (B. taurus), one would expect to find mitochondria of European as well as of African origin among them. However, current genetic and historical data do not permit us to discern whether those animals first introduced in South America were exclusively European taurus or if African taurus were already present. In any case, mtdna information from South American native breeds could tell us if the Eucons and the Afcons have a similar distribution pattern in America. During the first two decades of the colonization of America, Europeans introduced a relatively small number of animals (Ne;200) (Rabasa 1993; Rouse 1977). Cattle spread throughout South America 50 years later (Salazar and Cardozo 1986), eventually suffering population contraction and expansion periods (Giberti 1974; Hernández 1881). Given this scenario, one would expect to observe a reduced number of mitochondrial haplotypes shared by South American native cattle breeds. With the aim to uncover the source of mitochondrial origin and the nature of mtdna diversity, we examined the nucleotide variation within the mitochondrial control region in Argentinean and Brazilian native cattle breeds as representatives of the South American descendants of the Iberian cattle. We also investigated the contribution of European and African cattle breeds to the formation of the native cattle herds in these two countries. Materials and Methods Animals and DNA Extraction Genomic DNA was extracted from blood or semen samples from 42 animals according to Sambrook et al. (1995) and Zadworny and Kuhnlein (1990) and was used for polymerase chain reaction (PCR) amplification and sequencing. These 42 animals represent the Argentinean Creole (AC, n 5 12) and four Brazilian native cattle breeds: Caracu (CC, n 5 8), Curraleiro (CU, n 5 6), Mocho Nacional (MN, n 5 6), and Pantaneiro (PP, n 5 10). Unrelated AC males were carefully selected based on Breed Association records and pedigree information to represent different AC strains distributed throughout Argentina. Animals from the Brazilian breeds were chosen according to herd records, avoiding relatives except in PP, where no reproductive records are available due to the management nature of this breed (Mazza et al. 1994). mtdna Amplification and Sequencing A 1.39 kb DNA fragment covering the whole mitochondrial control region was amplified by PCR using 200 ng of bovine genomic DNA, 10 pmol each primer (59- TTCCGACCACTCAGCCAATG-39 and 59- CCTAGAGGGCATTCTCACTGGG-39), 2.5 U Taq DNA polymerase, 200 lm of each dntp, 1.5 mm MgCl 2, and 13 reaction buffer. Amplification reactions were performed in a PE9600 cycler (Perkin-Elmer Cetus, Norwalk, CT) starting with a 3 min denaturation step at 948C, followed by 30 cycles of 1 min at 948C, 45 s at 608C, 1 min at 728C, and a 4 min final extension period at 728C. A total of 20 50 ng of purified PCR product was used as template for sequencing reactions with the following primers: 59-TTCCGACCACT- CAGCCAATG-39, 59-TGCTGGTGCTCAA- GATGC-39, and 59-GCTCGTGATCTAATG- GTAAG-39. A consensus sequence of approximately 800 bp for each animal resulted from contig assembling of sequence reads in both strands. Point mutations were confirmed through chromatogram inspection and by at least two sequence reads covering these positions. PCR and sequencing primers were designed based on the alignment of all Bos mtdna sequences recovered from the GenBank. However, as no B. indicus coding sequence bordering the control region was available, we obtained the nucleotide sequence of CYTB thr trna pro trna and phe trna 12rRNA genes on the upstream and downstream side of the control region, respectively, from a B. indicus animal (Nellore). Nucleotide positions at the mtdna control region were numbered according to the B. taurus mtdna reference sequence (Anderson et al. 1982) (accession no. NC001567). Sequence Analysis mtdna nucleotide sequences obtained in this work (GenBank accession nos. AF517787 AF517828) were aligned to B. taurus, B. indicus, and Bison bison sequences recovered from the GenBank (accession nos. AB003793 AB003801, AF016060 AF016063, AF016066 AF016097, AF022916 AF022924, AF034438 AF034446, AF336383 AF336748, AF083353 AF083354, AF162484 AF162485, BBU12946 BBU12959, L27714 L27715, L27720 L27723, L27728 L27733, L27736 L27737, U51806 U51842, U87633 U87650, U87893, U92230 U92244) using CLUSTALX software (Thompson et al. 1997) totaling 545 Bos mtdna sequences. Positions in the alignment showing gaps were excluded from the analysis. Interhaplotypic distances were estimated using the substitution model of Tamura and Nei (1993), allowing rate heterogeneity among sites. The value for the rate heterogeneity parameter a of the gamma distribution was estimated from the 240 bp (16023 16262 of the reference sequence) fragment of the control region mtdna sequences from B. taurus and B. indicus using the PUZZLE program (Strimmer and von Haeseler 1996) according to recommendations of Meyer et al. (1999). The corrected average population pairwise sequence difference [p 5 p xy 2 (p x 1 p y )/2] was obtained as implemented by the ARLEQUIN software (Schneider et al. 2000). Analysis of molecular variance (AMOVA), pairwise F ST distance estimates, and the construction of a minimum spanning network (MSN) were also performed using ARLEQUIN. Additional phylogenies were constructed using the NEIGHBOR program incorporated in the PHYLIP package (Felsenstein 1993). Results Variation in mtdna Nucleotide Sequence The nucleotide sequence variation within the first 620 bp of the control region has been examined in 42 animals representing five native cattle breeds from Argentina and Brazil (Figure 1). Sequence alignments revealed the presence of nine haplotypes, eight of them not previously reported. Only haplotype EA1 matched perfectly with the reference sequence (Eucons), which was one of the two most frequently observed in this study (38.09%). The other highly represented haplotype (AA1; 33.33%) shared an identical sequence state with the African consensus (Afcons; Figure 1) at the three differentiating positions (16050, 16113, 16255) that split B. taurus mtdna sequences into two main clusters, the European taurine and the African taurine mitochondria, as described by Bradley et al. (1996). Among the seven remaining haplotypes, four are variations of the European consensus (EA2, EA3, EA4, EA5) and three are closer to the African consensus (AA2, AA3, AA4). The haplotype distribution varied according to the breeds as shown in Figure 1. For instance, Africanrelated haplotypes were absent in CU animals and were found in only one AC sample, the haplotype AA2. AA2 is four 324 The Journal of Heredity 2002:93(5)

mutational events away from the haplotype AA1 frequently observed in most of the Brazilian breeds studied in this work, but differs in two substitutions from the African consensus. The remaining AC animals only bore mitochondria from European origin, as did all the CU animals. We did not detect the presence of any B. indicus haplotype in the sample assayed; all the mtdna sequences clustered within the taurine branch in an neighbor-joining tree (Figure 2). We have also sequenced the corresponding mtdna fragment of one B. indicus individual from an imported Indian Nellore strain as PCR and sequencing control (Nell; Figure 1). Genetic Structure The genetic structure of the population was investigated through the AMOVA as implemented by the ARLEQUIN software (Schneider et al. 2000). It performs the analysis of variance (ANOVA) of the gene frequencies taking into account compositional differences among haplotypes. The hierarchical analysis decomposes the total variance into covariance components due to interindividual and interpopulational differences. Accordingly, a particular genetic structure can be tested defining alternative groups of populations. A global AMOVA was performed, considering an mtdna control region fragment of 240 bp of our 42 sequences added to those recovered from the GenBank, namely 209 European, 103 African, 48 Anatolian, 37 Middle East taurine breeds, 33 Japanese Black cattle (B. taurus), and 25 from Indian B. indicus breeds. More importantly, a further 49 mtdna sequences from six Portuguese cattle breeds (Cymbron et al. 1999) were incorporated into the analysis separately. Results indicate that 65.03% of the variance was accounted for by the eight continental subdivisions, 1.75% due to an among-breeds, within-continents component and 33.22% within breeds (Table 1, first column). In order to closely examine the partition of the variance components within the B. taurus mitochondrial lineages, a new round of analyses were carried out excluding the Indian breeds. Consequently the percentage of variation accounted for among continents dropped to 32.15%, increasing the amount of variance due to differences within breeds (64.68%) (Table 1, second column). A portion of the intrabreed variation observed after dropping the B. indicus sequences could result in part from Figure 1. mtdna sequence variations observed in 42 individuals from one Argentinean and four Brazilian native cattle breeds. All mitochondrial control region sequences obtained in this work, added to the one representing the African consensus (Afcons; Bradley et al. 1996), were aligned to the reference sequence of Anderson et al. (1982) (Eucons). Nucleotide positions were also numbered according to Eucons. Identity with the reference sequence is denoted by a dash, variations in the base letter involved in the substitution and deletions by a colon (:). The first two letters of the sequence identification, on the left-hand side, define the breed name (AC, Argentinean Creole; CC, Caracu; CU, Curraleiro; MN, Mocho Nacional; PP, Pantaneiro). Haplotype identification is given on the right side. Nell is the complete control region nucleotide sequence from a B. indicus representative animal of the Nellore breed. Miretti et al African-Derived Mitochondria 325

whereas 17 and 11 mtdna sequences pertained to the African-related group. AMOVA of this haplotype clustering significantly reduced the within-breed variation from 64.68% to 39.08% (Table 1) but raised the variance contribution of the among-groups component to 44.06%. Figure 2. Phylogenetic relationships (unrooted neighbor-joining tree) for B. taurus lineages inferred from interhaplotypic distances estimated from mtdna control region sequences (16023 16262). The shaded areas circumscribe haplotypes belonging to the two major taurine lineages (African taurine and European taurine) and the Near Eastern cluster. Note the divergent branch conducting to the AA1 AA4 haplotypes. The AA3 haplotype is assumed to be AA1 when only the 240 bp fragment is considered (Figure 1). B. indicus is represented by the Nell sequence in Figure 1. sequence differences between Africanand European-related haplotypes within South American and Portuguese breeds. To investigate whether this subdivision actually influenced the variance distribution, and to what extent, haplotypes from Argentinean, Brazilian, and Portuguese breeds sharing the characteristic African consensus sequence state at the three nucleotide positions T16050, C16113, and C16255 (TCC) were pooled apart from those closely related to the European consensus sequence (CTT). To perform this new analysis, all sequences were aligned and classified accordingly. The clustering was inspected by the neighbor-joining tree (not shown) and by the haplotype-sharing option of the ARLEQUIN software. Mitotypes from 23 and 38 samples from the South American and Portuguese breeds, respectively, presented European-related haplotypes, Divergence Times Between Cattle Populations and Haplotypes The average corrected pairwise sequence difference between populations was estimated under the Tamura and Nei (1993) model of rate heterogeneity. The a value obtained from these data was 0.30 for the 240 bp fragment. It is assumed, based on paleontological evidence, that cattle and bison diverged at least 1 million years before present, which allows the calibration of mtdna sequence diversity. Differences of approximately 10% in the mutation rate between Bison B. taurus (Bb-Bt) and Bison B. indicus (Bb-Bi) lineages were observed. For instance, it reached 8.92% representing 89,230 years when applying the time depth estimation procedure described in Bradley et al. (1996). Hence the estimation of the age of the most recent common ancestor (MRCA) was carried out, averaging the rate of change between branches according to the relative rate method of Kumar and Hedges (1998) and Ingman et al. (2000). We then applied two other methods in order to check for consistency. First, the method used by Bradley et al. (1996) resulted in estimates similar to those previously obtained by Loftus et al. (1994) for the MRCA age for Bi-Bt (770,000 years, p 5 0.1555), and for Eu-Af divergence as well (61,500 years). Second, following the procedure of Vigilant et al. (1991), 88 substitutions (86 Ts, 2 Tv) were found within a 240 bp region in the alignment of 544 Bos mtdna control region sequences. As four Tv were observed between cattle and bison, the nucleotide differences accumulated within this sequence stretch, since their divergence 1 million years ago was 35.83%. This corresponds to a substitution rate of 1 Ts in 5,814 years, considering both mitochondrial lineages (1 Ts/ 11,628 years, one lineage rate) or to a B. taurus:b. indicus divergence time of 500,000 years. Finally, the age of the Bi-Bt MRCA based on the whole mitochondrial chromosome nucleotide sequence excluding the control region was 580,000 YBP (Miretti M et al., unpublished results). Once the calibration of the D-loop clock proved to be consistent regarding 326 The Journal of Heredity 2002:93(5)

the Bi-Bt separation, we proceeded with further time divergence estimations between B. taurus subdivisions. Thus, if Argentinean and Brazilian native cattle breeds (AB) are considered as one population, the estimated divergence time from the extant European, African, and Portuguese cattle is 32,900 YBP, 28,200 YBP, and 19,000 YBP, respectively, given a mutation rate l 5 1.0097 3 10 7. However, the reliability of these estimates can be questioned as AB, as well as P, is in fact an admixture of highly divergent haplotype derivatives from distant mitochondrial resources. Nevertheless, more confident information can be obtained from gene-tree analysis instead of a population-based analysis. Therefore we implemented haplotype clustering of B. taurus mtdna sequences in Near Eastern, African, and European taurus-derived haplotypes depending on the sequence state at the five positions as defined earlier (C16057 A16189 C16255, T16050 C16113 C16255, C16050 T16113 T16255, respectively). The AB haplotypes were thus separated into African derived (AA) and European derived (EA), a criterion that was also applied to Portuguese mtdna sequences which were thus subdivided into Portuguese-African (PAf)- and Portuguese- European (PEu)-related haplotypes. From the mean number of pairwise differences (p), comparable divergence times were found between EA-PEu and EA-Eu (2,200 and 2,400 years), and EA-Af and EA-PAf (58,700 and 67,300 years), respectively. Meanwhile, AA-EA presented higher divergence dates (157,000 years), even older than the Eu-Af separation and of similar magnitude to AA-Eu and AA-PEu (170,000 and 168,000 years). Anatolian and Middle Eastern cattle were indistinguishable under a population-based analysis (population average corrected p 5 0.00003 5 175 years), but their intrapopulation nucleotide diversity are among the widest (population average corrected p 5 0.01472 and 0.018444, corresponding to 74,500 and 93,300 years, respectively). Anatolian and Middle Eastern high population diversity might result from either a considerably larger effective population size or longer genetic history, which is in line with the argument of Troy et al. (2001) in that sequences of the center of origin are expected to retain more ancestral variation and show higher haplotypic and nucleotide diversity. Even though this reasoning certainly can not be extended to explain why AB showed the Table 1. Analysis of molecular variance of cattle mtdna sequences Populations highest within-population diversity (population average corrected p 5 0.019525 corresponding to 98,700 years). Portuguese cattle also present substantial within-population diversity (population average corrected p 5 0.01361 corresponding to 68,800 years). Phylogenetic Tree Construction The neighbor-joining tree based on interhaplotypic distances (Figure 2) shows the phylogenetic relationship among B. taurus, B. indicus, and Bison mtdna control region sequences. Within the B. taurus lineage, three clades of sequences can be differentiated corresponding to the African, European, and Near Eastern taurine cattle. Haplotypes observed in Argentinean and Brazilian native cattle are placed within European (EA2, EA3, EA4, EA5) and African (AA1, AA2, AA3, AA4) sequence clusters. Note the extent of divergence in the lineage conducting to one of the most frequently observed haplotypes identified in this survey (AA1). Pairwise F ST values have been adopted to demonstrate the level of genetic distinction between populations (Bradley et al. 1996; Mannen et al. 1998; Roca et al. 2001) and can be used to estimate genetic distance over short time periods (Slatkin 1995). Figure 3 shows phylogenetic reconstruction based on population and haplotype group pairwise F ST values for the 240 bp fragment. All comparisons were significantly different (P, 10 5 ) except differentiation between the An and ME populations, and the PEu and Eu haplotype groups. Both dendrograms clearly show the distinction between the two major clades, B. indicus and B. taurus, as reported by Loftus et al. (1994). However, the branching pattern within B. taurus is markedly different between Haplotypes 1 B. indicus No B. indicus 1 B. indicus No B. indicus Among continents 65.03 32.15 73.16 44.06 a Among populations within continent 1.75 2.57 8.16 16.87 Within populations 33.22 64.68 18.68 39.08 AMOVA testing of two different genetic structures: Populations considers a geographical distribution of haplotypes (American, European, Portuguese, African, Anatolian, Middle Eastern, Japanese, and Indian breeds). Haplotypes data were classified into haplotype groups according to their sequence state at nucleotide positions diagnostic of taurine lineages. The African-related haplotype group includes all those haplotypes sharing T16050 C16113 C16255, whereas the European-related haplotype group includes haplotypes sharing C16050 T16113 T16255. To the Near Eastern-related haplotype group belong haplotypes sharing the following sequence state: 16057C, 16189A, and 16255C. 1 B. indicus and No B. indicus indicate the inclusion and exclusion of B. indicus sequences in the analysis. a Among African-related (AA, PAf, Af), European-related (EA, PEu, Eu), and Near Eastern-related haplotype groups (NE) and Japanese Black cattle (Jp1, Jp2). these two approaches, essentially contrasting distinctive features of population trees and gene trees. In the populationbased analysis, European (Eu) and Japanese Black (Jp) and Portuguese (P), Anatolian (An), and Middle Eastern (ME) cattle populations constitute two subgroups sharing clusters identified as European taurine cattle, corroborating the findings of Cymbron et al. (1999) and Mannen et al. (1998). Instead, African taurine breeds (Af) and Argentinean Brazilian native cattle (AB) are represented as distinct terminal branches of the neighbor-joining tree (Figure 3a). In the haplotype group analysis (gene tree), EA and PEu haplotypes were grouped within the European taurine haplotype cluster, constituted by most of the European sequences and a subdivision of the Japanese Black population (Jp2). On the other side, PAf haplotypes grouped together with the African taurine sequences (Af). Unexpectedly the AA subset of haplotypes observed in Brazilian native cattle constituted a distinct terminal branch excluded from both clusters (Figure 3b). Inspection of sequence alignment led to the observation of further sequencestate differences in four positions when comparing these AA haplotypes to Af and PAf sequences. Notably, the Africanrelated Argentinean Brazilian haplotypes, except AA2, shared the nucleotides CCTA at positions 16053, 16122, 16139, and 16196 (Figure 1), but this was not observed in mtdna sequences from Africa and Portugal. Conversely TTCG was found in Af and PAf, and in the European consensus as well. Consequently these haplotypes (AA1, AA3, AA4) differ from other African-derived mtdna sequences in at least these four positions, disregarding the other three Miretti et al African-Derived Mitochondria 327

Figure 3. Neighbor-joining tree based on pairwise F ST distances. (a) Population-based analysis. Note that the Argentinean Brazilian (AB) and African (Af) populations are separately grouped from the European (Eu), Japanese Black (Jp), Portuguese (P), Anatolian (An), and Middle Eastern (ME) cattle mtdna haplotypes, both within the taurine cluster. (b) Haplotype group analysis. Haplotype groups were classified into African-related, European-related, and Near Eastern-related according to their sequence state at positions 16050, 16057, 16113, 16185, and 16255. AA and PAf are African-derived haplotypes observed in South American and Portuguese cattle breeds, respectively; Jp1 and Jp2, Japanese Black; EA and PEu, European-derived haplotypes observed in South American and Portuguese breeds, respectively; Bi, B. indicus. substitutions that separate them from the European consensus. To focus on the relationship among mtdna haplotypes, a MSN was constructed based on substitutions observed in the 240 bp fragment of 520 B. taurus mtdna sequences (Figure 4). Two predominant and putatively separate ancestral mitochondrial types, defined as the European consensus (Eucons) and the African taurine consensus (Afcons) by Bradley et al. (1996), can be seen. Each sequence forms the center of radiation of a number of variants linked to it by mostly one or only a few substitutions. The most frequently observed African-related Argentinean Brazilian haplotype, AA1, is connected to the African consensus through four substitutions (large dark circle). Also note that the distance, in terms of substitution number, linking AA1 and Afcons is greater than the separation between Afcons and Eucons. Despite the fact that hypothetical relationships of the primary B. taurus haplotypes proposed by Troy et al. (2001) include unsampled intermediate nodes, our MSN displayed other equally parsimonious paths connecting the Eucons and Afcons. In these alternatives, which differ in the way that substitutions separating both clusters are ordered, haplotypes representing all the intermediate nodes were clearly identified, providing consistency to our MSN. Discussion We have assessed the nucleotide diversity in the control region of the mtdna in Argentinean Creole and four Brazilian native cattle breeds. This is the first report on mtdna concerning South American native cattle breeds, which allowed analysis of these data in conjunction with those from European, Anatolian, Middle Eastern, African, Japanese, and Indian cattle. All nine haplotypes described here are of B. taurus origin. The absence of B. indicus mitochondria was unexpected given that there is evidence, based on Y chromosome dimorphism (Britto 1998), of zebuine introgression into some Brazilian native breeds (Lara et al. 1997; Mazza et al. 1994). This result points toward a male-mediated introgression of zebu genes. Conversely Giovambattista et al. (2000) and Sinópoli (1993) observed only submetacentric Y chromosomes in different Argentinean Creole populations. We amplified and sequenced the mtdna control region of a B. indicus animal as a positive control, and primers were also designed to avoid any known polymorphic site. For instance, we found two substitution sites within the pro trna gene at positions 15745 (B. taurus C, B. indicus T) and 15755 (B. taurus A, B. indicus G), a region commonly used to place amplification primers. Regarding B. taurus mtdna haplotypes, we showed that information is lost when data analysis is only performed considering haplotypes as they occur in breeds (population-based phylogeny and AMO- VA; Figure 3 and Table 1). Under this assumption, estimates of nucleotide diversity and divergence times based only on the corrected MSPD between geographical subdivisions (populations) lead to a partial, and possibly wrong, interpretation of results due to the admixture of mtdna from both African and European taurine mitochondrial lineages within South American and Portuguese native cattle breeds. This reduces the divergence time between populations, inflates the variance component within breeds (Table 1), and distorts the branching pattern of dendrograms constructed with population pairwise F ST genetic distance (Figure 3). Indeed, the shorter separation time between the Argentinean Brazilian native cattle and Portuguese breeds (AB-P, 19,000 YBP) in comparison with its divergence from European (32,900 YBP) and African (28,200 YBP) breeds can be explained by the mitochondrial African component observed within the South American and Portuguese breeds. We then classified mtdna sequences into Near Eastern, African, and European taurine mitochondrial haplotype clusters (Figures 1 and 2), and subsequently subdivided them according to their geographic origin. Divergence estimates based on haplotype group comparisons were more reliable than population-based analysis. Regarding the European mitochondrial lineage of the taurine cluster, we estimate a divergence time of 2,400 YBP between European-related South American haplotypes and European haplotypes (EA-Eu). The distribution of the EA haplotypes within the breeds studied seems to represent the haplotype distribution described for European cattle, with Eucons as predominant and some closely related haplotypes. These haplotypes could have been introduced into America with either Portuguese or Spanish cattle, in spite of the fact that, disregarding EA1, they were absent in the Portuguese cattle breeds already studied (Cymbron et al. 1999) and that no mtdna information is available from Spanish cattle presumably introduced into the Americas. A different picture was found concerning the African-related Argentinean Brazilian haplotypes (AA). In the neighborjoining tree constructed with pairwise F ST distances (Figure 3b), the AA haplo- 328 The Journal of Heredity 2002:93(5)

type groups do not share any cluster with either the African (Af) or the African-related haplotypes of Portuguese cattle (PAf). Instead, it occupies an ancestral position regarding the separation of B. taurus mitochondrial lineages. In addition, divergence time estimations support the ancestrality of the AA haplotype group, which might have separated from all the other African-derived haplotypes 84,700 YBP, and 170,000 YBP from the European-derived haplotypes. Considering haplotypes separately, AA1, AA3, and AA4 were classified as Africanrelated due to their nucleotide composition TCC at positions 16050, 16113, and 16255. However, variations at four other positions 16053, 16122, 16139, and 16196 distinguished them from the two major taurine mitochondrial haplotype lineages: the European and the African taurine (Figures 1, 2, and 4). Notably Eucons and Afcons are more closely related to each other than Afcons to AA1. In this sense European and African mitochondrial lineages diverged 67,100 YBP, whereas African and AA showed older separation times (84,700 YBP). Pairwise haplotype group divergence between AA-PAf (80,100 years) is also consistent with ancestral separation of the mitochondrial lineage that gave rise to AA1, AA3, and AA4 haplotypes. Two other Pleistocene B. primigenius haplotypes are also distantly related to Eucons and Afcons (Bailey et al. 1996), but in this case substitutions involve other positions. Finally, age estimates should be cautiously interpreted, as they explicitly depend on the mutation rate considered (1.0097 3 10 7 ). We take time divergence in years as a relative measure of divergence between populations and haplotype groups, but the corresponding p- value for each age estimate can be obtained given the mutation rate, thus allowing comparison with other studies. The separation of African and European mitochondrial lineages should have occurred prior to the African and European cattle population expansion events 9,000 YBP and 5,000 YBP, respectively (Bailey et al. 1996). The profound population growth, which is apparent in the MSN topology, could be a consequence of the domestication process. Based on these observations, Bradley et al. (1996) suggested the existence of two independent domestication events involving two different strains of taurine progenitors. In this sense, if European and African mitochondrial types are indeed relicts Figure 4. Minimum spanning network (MSN) of taurine mtdna haplotypes. The MSN was constructed based on substitutions within the control region (240 bp) of mtdna haplotypes. Haplotypes having CTT at positions 16050, 16113, and 16255 are placed within the European consensus circle (Eucons), whereas those presenting TCC at these positions were classified as African consensus (Afcons) sequences. Haplotypes can be viewed as internal or terminal nodes, represented by circles interconnected by lines that correspond to a mutational step. The area of the circles is proportional to the haplotype frequency, except that the Eucons circle size should be twice as big, and short cross-hatched lines indicate further mutational events separating two haplotypes. Not all the alternative paths are shown and not all the haplotypes are represented. of temporally and spatially separate domestic origins (Bradley et al. 1996), it is tempting to interpret our genetic data as evidence of a further taurine subdivision that could have diverged from the taurine branch even before the European-African mitochondria split. This supposedly former taurine mitochondrial lineage, represented by AA1, AA3, and AA4 haplotypes found in South American cattle, was absent in African and Portuguese cattle surveys, even in the comprehensive cattle mtdna diversity study recently published (Troy et al. 2001), which precludes any inference about its geographical origin. Then, and for further analysis, it might be more appropriate to consider these haplotypes as part of another major mitochondrial lineage within B. taurus. It is also feasible that the high proportion of AA1 in South American native cattle, while absent in others, may not reflect European or African taurine history, but the evolutionary process during its prosperous American life. The AA1 haplotype was frequent in most of the Brazilian native breeds, which could be a sign of their introduction by Portuguese conquest through the Brazilian coastal line. But no consistency can be found in the Portuguese cattle study (Cymbron et al. 1999), since, although they reported haplotypes of African origin, the most closely related to AA1 differs in five substitutions. The African-related haplotype found in Argentinean Creole, AA2, belongs to a mitochondrial lineage different from those observed in Brazilian breeds and was also absent in Portuguese cattle. This could represent a hint for further investigations of native cattle breeds of other South American countries. Even though mtdna analysis should be extended to other African and European breeds, specifically Spanish cattle, before any conclusion can be drawn, our results might be mirroring a picture of the mitochondrial lineage distribution among cattle breeds in the Iberian peninsula five centuries ago. 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