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 Cientı́fico 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.
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 (hump-
less) 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 (MacHugh 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).
324 The Journal of Heredity 2002:93(5)
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 (59TTCCGACCACTCAGCCAATG-39 and 59CCTAGAGGGCATTCTCACTGGG-39), 2.5 U
Taq DNA polymerase, 200 lM of each
dNTP, 1.5 mM MgCl2, 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-TTCCGACCACTCAGCCAATG-39, 59-TGCTGGTGCTCAAGATGC-39, and 59-GCTCGTGATCTAATGGTAAG-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 – thrtRNA –
pro
tRNA and phetRNA – 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 pxy 2 (px
1 py)/2] was obtained as implemented by
the ARLEQUIN software (Schneider et al.
2000). Analysis of molecular variance
(AMOVA), pairwise FST 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
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
326 The Journal of Heredity 2002:93(5)
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
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 PortugueseEuropean (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
Among continents
Among populations within continent
Within populations
Haplotypes
1 B. indicus
No B. indicus
1 B. indicus
No B. indicus
65.03
1.75
33.22
32.15
2.57
64.68
73.16
8.16
18.68
44.06a
16.87
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).
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 FST 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 FST 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
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
(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
Figure 3. Neighbor-joining tree based on pairwise
FST 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
328 The Journal of Heredity 2002:93(5)
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 protRNA 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 AMOVA; 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 FST
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
FST distances (Figure 3b), the AA haplo-
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 pvalue 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. Since eight of
the nine haplotypes found here have not
been described in European and African
cattle populations, it can be concluded
that these haplotypes might have been
lost, or alternatively, that further studies
are required.
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Received April 17, 2001
Accepted August 8, 2002
Corresponding Editor: James Womack