MOLECULAR

PHYLOGENETICS
AND
EVOLUTION
Molecular Phylogenetics and Evolution 33 (2004) 896–907
www.elsevier.com/locate/ympev

Molecular phylogeny of the tribe Bovini (Bovidae, Bovinae)

and the taxonomic status of the Kouprey, Bos sauveli Urbain 1937
Alexandre Hassanin*, Anne Ropiquet
FRE 2695 – Origine, Structure et Evolution de la Biodiversité, Département Systématique et Evolution, Muséum National dÕHistoire
Naturelle, Case postale No. 51, 55, rue Buffon, 75005 Paris, France
Service de Systématique Moléculaire, Muséum National dÕHistoire Naturelle, 43, rue Cuvier, 75005 Paris, France

Received 24 May 2004; revised 22 July 2004

Available online 29 September 2004

Abstract

The kouprey is a very rare bovid species of the Indochinese peninsula, and no living specimen has been described for a long time,
suggesting that it is possibly extinct. Its systematic position within the tribe Bovini remains confused since the analyses of morpho-
logical characters have led to several conflicting hypotheses. Some authors have also suggested that it could be a hybrid species pro-
duced by the crossing of the banteng with gaur, zebu, or water buffalo. Here we performed a molecular phylogeny of the tribe Bovini
to determine the taxonomic status of the kouprey. DNA was extracted from the holotype specimen preserved in the MNHN col-
lections. Phylogenetic analyses were carried out on a matrix including all the taxonomic diversity described in the tribe Bovini, and
2065 nucleotide characters, representing three different markers, i.e., the promotor of the lactoferrin and two mitochondrial genes
(cytochrome b and subunit II of the cytochrome c oxidase). The results show that the kouprey belongs to the subtribe Bovina, and
that three different clades can be evidenced into this group: the first includes the domestic ox, zebu, and European bison; the second
incorporates the yak and American bison; and the third contains the kouprey, banteng and gaur. All hypotheses involving hybrid-
ization for the origin of the kouprey can be rejected, confirming that it is a real wild species. Molecular datings and biogeographic
inferences suggest that the kouprey diverged from banteng and gaur during the Plio-Pleistocene of Asia. In addition, several molec-
ular signatures were detected in the cytochrome b gene, permitting a molecular identification of the kouprey. We propose a conser-
vation project based on a molecular taxonomy approach for tracking the kouprey in Indochina in order to determine whether some
populations still survive in the wild.

2004 Elsevier Inc. All rights reserved.

Keywords: Phylogeny; DNA; Kouprey; Bos sauveli; Bovidae; Bovinae; Bovini; Hybridization; Asia; Indochina; Biogeography

1. Introduction kouprey is confined to a small area of the Indochinese

The kouprey (Bos sauveli) is one of the most seriously
threatened large mammals in the world (MacKinnon
and Stuart, 1988). The first specimen to be scientifically
described (Urbain, 1937) was a young male calf sent to
the Vincennes Zoo near Paris, which has been collected
in the village of Tchep in the northern region of Cambo-
dia by Dr. R. Sauvel, after whom it was named. The
*
Corresponding author. Fax: +33 1 40 79 30 63.
E-mail address: hassanin@mnhn.fr (A. Hassanin).
peninsula comprising the northern plains of Cambodia,
the Dongrak Mountains of eastern Thailand, the south-
ernmost provinces of Laos, and the western edge of
Vietnam, along the Cambodia border (MacKinnon
and Stuart, 1988). No living specimen has been recently
observed, suggesting that the species may be extinct.
The kouprey is lighter in build than other wild cattle
found in the Indochinese region, i.e., banteng (Bos java-
nicus) and gaur (Bos frontalis). Adult females have a
characteristic grey colour, which gives the animal its lo-
cal name (grey ox), and readily distinguishes them from

1055-7903/$ - see front matter
2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.ympev.2004.08.009
 A. Hassanin, A. Ropiquet / Molecular Phylogenetics and Evolution 33 (2004) 896–907 897

the reddish-brown females of banteng and the dark,

blackish-brown females of gaur. The male loses its grey
flanks with age, becoming increasingly black. Both sexes
have a very long tail and notched nostrils. Adult males
have a pronounced dewlap, and have wide-spaced taper-
ing horns with characteristic fraying near the tips. The
horns of female kouprey are gracefully lyriform,
explaining why they have been sometimes confused with
horns of Pseudonovibos spiralis (Dioli, 1995; Hoffman,
1986), a putative new species described by Peter and Fei-
ler (1994a,b). However, DNA analyses have shown that
the horns used to formally describe P. spiralis were bo-
gus and were in fact derived from the horns of domestic
cattle (Bos taurus) (Hassanin et al., 2001; Thomas et al.,
2001). As a consequence, the horns described in Hoff-
man (1986) as belonging to a female kouprey must
now be regarded as simple horns from B. taurus.
Within the family Bovidae, all authors agree to con-
sider the kouprey as being a member of the tribe Bovini
(Coolidge, 1940; Bohlken, 1961; Geraads, 1992; Groves,
1981). According to the classifications of Simpson
(1945), Grubb (1993) and McKenna and Bell (1997),
thirteen species may be ranged into the tribe Bovini,
including B. taurus, which groups two major breeds of
domestic cattle, i.e., no hump cattle (B. t. taurus) and
humped zebu (B. t. indicus). The natural distribution
of Bovini incorporates North America with the Ameri-
can bison (Bison bison), Europe with the European bison Fig. 1. Morphological hypotheses for the systematic position of the
or wisent (Bison bonasus), Africa with the African buf- kouprey within the tribe Bovini. Because of important conflicts
falo (Syncerus caffer), but most of the diversity is found between the classifications proposed in the literature, we used common
in Asia with nine species: B. sauveli (kouprey), B. fronta- names rather than scientific names. For convenience, the following
lis (gaur), Bos grunniens (yak), B. javanicus (banteng), classification was however used in the main text (Grubb, 1993;
McKenna and Bell, 1997; Simpson, 1945): African buffalo (Syncerus
Bubalus bubalis (water buffalo), Bubalus depressicornis caffer), Water buffalo (Bubalus bubalis), Anoa (Bubalus depressicornis),
(anoa), Bubalus mephistopheles (short-horned water buf- American bison (Bison bison), European bison (Bison bonasus), Yak
falo), Bubalus mindorensis (tamaraw), and Bubalus (Bos grunniens), Aurochs and its domesticates (Bos taurus), Gaur (Bos
quarlesi (mountain anoa). frontalis), Banteng (Bos javanicus), and Kouprey (Bos sauveli).
Phylogenetic relationships between species of the
tribe Bovini remain poorly understood. In particular, closely related to the banteng (Fig. 1). But two years
the taxonomic status of the kouprey is a real enigma later, he concluded that the three skulls of kouprey pre-
and many different hypotheses have been proposed in served in the MNHN collections of Paris are castrated
the literature (Fig. 1). The kouprey was originally de- domestic cattle (Bohlken, 1963). All the hypotheses of
scribed as Bos (Bibos) sauveli (Urbain, 1937), the subge- hybridization were considered as unfounded by Pfeffer
nus Bibos indicating close affinities with other wild cattle and Kim-San (1967), who suggested strong affinities
of Indochina, i.e., banteng and gaur. On the basis of a with banteng and gaur, as originally proposed by Urb-
full description of an adult bull, Coolidge (1940) created ain (1937). More recent morphological investigations
a new genus for it, Novibos, on the grounds of primitive have led to different conclusions (Fig. 1): Groves
characters by comparison with more advanced wild cat- (1981) proposed an association with the aurochs (B. t.
tle and bison (Fig. 1). Edmond-Blanc (1947) was the first primigenius), i.e., the wild ancestor of domestic cattle,
author to consider the kouprey as being a hybrid pro- while the cladistic analyses performed by Geraads
duced by the crossing of banteng with one of the three (1992) suggested a sister-group relationship with the
following species inhabiting Indochina: gaur, zebu, or clade composed of banteng, gaur, and aurochs with its
water buffalo. The hypothesis of crossbreeding was fol- domesticates.
lowed by Bohlken (1958), who concluded that the kou- Molecular studies based on restriction-endonuclease
prey was obtained by hybridization of the banteng with site mapping of nuclear-ribosomal DNA (Wall et al.,
zebu. Three years later, Bohlken (1961) recognised 1992) or sequences of the mitochondrial cytochrome b
however that the kouprey belongs to a distinct species gene (Hassanin and Douzery, 1999a) have suggested a
898 A. Hassanin, A. Ropiquet / Molecular Phylogenetics and Evolution 33 (2004) 896–907

clear distinction within the tribe Bovini, with on the one
hand, all species of Bos (cattle) and Bison (bisons), and
on the other hand, all species of Bubalus (Asian buffa-
loes) and Syncerus (African buffaloes). By using a taxo-
nomic sample including the saola or Vu Quang ox
(Pseudoryx nghetinhensis), which is the latest described
bovid species (Dung et al., 1993), and by analysing mito-
chondrial and nuclear markers, Hassanin and Douzery
(1999b) have defined three subtribes into the tribe Bovi-
ni: (i) the subtribe Bovina, which incorporates all species
ranged into the two genera Bos and Bison; (ii) the sub-
tribe Bubalina, which contains all species of Bubalus
and Syncerus; and (iii) the subtribe Pseudoryina, only
represented by P. nghetinhensis. The relationships within
the subtribe Bubalina have been clarified by sequencing
the complete cytochrome b gene for all living species of
Bubalus (Tanaka et al., 1996). By contrast, the relation-
ships within the subtribe Bovina are still confused, but
two results emerged consitently from mitochondrial se-
quences: (1) the American bison is associated with the
yak rather than with the European bison; and (2) ban-
teng and gaur are grouped together (Janecek et al.,
1996; Verkaar et al., 2004).
In the present study, we performed a molecular phy-
logeny of the tribe Bovini for determining the systematic
status of the kouprey. The latter has been included in the
analyses by extracting DNA from a bone sample of the
holotype specimen preserved in the MNHN collections.
Three different markers, representing a total of 2065
characters, were analysed for a taxonomic sample incor-
porating all the diversity described into the tribe Bovini.
They include one nuclear fragment, i.e., the promotor of
the lactoferrin (Lf), and two mitochondrial genes, i.e.,
the cytochrome b (Cyb) and the subunit II of the cyto-
chrome c oxidase (CO2). Bayesian and Maximum Parsi-
mony (MP) methods were used for tree reconstruction,
and the three markers were analysed separately and also
combined to answer the following questions: (1) Is the
kouprey a hybrid or a real species? (2) What is the tax-
onomic status of the kouprey? (3) What are the phyloge-
netic relationships between genera and species of the
tribe Bovini? By estimating molecular divergences times,
and by inferring ancestral biogeographic areas, we also
proposed an evolutionary scenario for explaining how
bovines diversified during the Neogene.
2. Materials and methods
2.1. Taxonomic sample
The taxonomic sample used for this study contains 20
taxa (Table 1). The ingroup is the tribe Bovini, repre-
sented by seven species of the subtribe Bovina, three spe-
cies of the subtribe Bubalina, and the saola (P.
nghetinhensis), the unique member of the subtribe

Table 1
Taxonomic sample
Taxon Species–common name Accession numbers
Cyb CO2 Lf
Cervidae Muntiacus reevesi–Chinese muntjak AF527537 AF527537 AY122037
Bovidae, Antilopinae
Aepycerotini Aepyceros melampus–Impala AF036289 AY689194a AF091659
Alcelaphini Damaliscus pygargus–Blesbok AF036287 AY689195a AF091653
Caprini sensu lato Ovis aries–Sheep AF034730 AF010406 AF091651
Bovidae, Bovinae
Boselaphini Boselaphus tragocamelus–Nilgai AJ222679 U62566 AF091642
Tetracerus quadricornis–Four-horned antelope AF036274 AY689196a AF091643
Bovini, Bovina Bison bison–American bison AF036273 AY689197a AF091639
Bison bonasus–European bison AY689186a AY689198a Pitra et al., 1997
Bos frontalis–Gaur AY689187a AY689199a AY689191a
Bos grunniens–Yak AF091631 AY689200a AF091638
Bos javanicus–Banteng AY689188a AY689201a AY689192a
Bos sauveli–Kouprey AY689189a AY689202a AF281090
Bos taurus taurus–Humpless domestic cattle V00654 V00654 AF281088
Bos taurus indicus–Zebu AY689190a AY689203a AY689193a
Bovini, Bubalina Bubalus bubalis–Water buffalo D88634 AY689204a AF281089
Bubalus depressicornis–Lowland anoa AF091632 U18822 AF091641
Syncerus caffer–African buffalo AF036275 U18825 AF09164
Bovini, Pseudoryina Pseudoryx nghetinhensis–Saola AF091635 AY689205a AF091660
Tragelaphini Tragelaphus imberbis–Lesser kudu AF036279 U18815 AF091649
Tragelaphus strepsiceros–Greater kudu AF036280 AY689206a AF091648
a
Sequences produced during this study.
 A. Hassanin, A. Ropiquet / Molecular Phylogenetics and Evolution 33 (2004) 896–907
Pseudoryina. The outgroup is composed of eight species
belonging to four different taxa of the suborder Rumi-
nantia: the family Cervidae with Muntiacus; the subfam-
ily Antilopinae sensu Kingdon (1982, 1997) with
Aepyceros, Damaliscus, and Ovis; and the two other
tribes of the subfamily Bovinae: the tribe Tragelaphini,
which is represented by Tragelaphus imberbis and Tra-
gelaphus strepsiceros; and the tribe Boselaphini, which
contains Boselaphus and Tetracerus.
2.2. DNA extraction and sequencing
Total DNAs were extracted from blood for B. bison,
B. grunniens, B. javanicus, B. bubalis, and T. strepsiceros,
from cells for Damaliscus pygargus, from skin for Bos
frontalis and P. nghetinhensis, and from hair for B. t.
indicus. The protocol is described in Winnepenninckx
et al. (1993). DNA was also extracted from bone sample
of MNHN specimens using the procedure detailed in
Hassanin et al. (1998b) for B. sauveli (Laboratoire
dÕAnatomie Comparée, MNHN CG 1940–51, holotype
skull of the kouprey), Aepyceros melampus (Zoothèque,
MNHN CG 1996-645), B. bonasus, and Tetracerus
quadricornis (Zoothèque, MNHN CG 1986-464).
The entire cytochrome b gene was amplified by the
polymerase chain reaction (PCR) with the primers given
in Hassanin et al. (1998b) and Hassanin and Douzery
(1999a). The subunit II of the cytochrome c oxidase gene
(positions 7374-7955 of the B. taurus sequence – Acces-
sion No. NC_001567) was amplified with the four fol-
lowing primers: U1: 50 -G TGA AAA TCC YGT ACA
YCT CAT-30 , U314: 50 -ACH ATA GGR CAY CAR
TGA TA-30 , L315 (reverse): 50 -A RTC TGT RTA
YTC RTA GCT TCA-30 , and L582 (reverse): 50 -CC
RCA RAT YTC TGA RCA TTG-30 . The promotor seg-
ment of the lactoferrin-encoding gene, i.e., positions
322–647 of the B. taurus sequence (Accession No.
L19985), was generated using the primers given in Hass-
anin and Douzery (1999b). The standard PCR condi-
tions were as follows: 3 min at 94 C; 35 cycles of
denaturation/annealing/extension with 1 min at 94 C
for denaturation, 1 min at 55 C for annealing, and
1 min at 72 C for extension; and 7 min at 72 C.
The PCR products were purified using the Montage
PCR Centrifugal Filter Devices (Millipore). Both strands
of all amplicons were directly sequenced using the
CEQ2000 Dye terminator cycle Sequencing Quick start
kit, and the sequencing reactions were run on a Beckman
CEQ2000 automatic capillary sequencer. The sequences
have been deposited in the EMBL/GenBank/DDBJ dat-
abases under the Accession Nos. specified in Table 1.
2.3. Phylogenetic analyses
DNA alignments were performed with Se-Al v2.0a11
(Sequence Alignment Editor Version 2.0 alpha 11; An-
899
drew Rambaut, software available at http://evolve.
zoo.ox.ac.uk/). The alignment of the complete Cyb gene
includes 1140 nucleotides (nt), the one of the CO2 gene
consists of 582 nt, and the one of the Lf promotor in-
cludes 339 nt with four unambiguous indels (insertion–
deletion) coded as additional characters at the end of
the matrix.
The three markers were analysed separately and also
combined to benefit from the maximum number of
molecular characters. Bayesian and Maximum Parsi-
mony (MP) methods were used for the tree reconstruc-
tion analyses.
Bayesian analyses were performed with Mr. Bayes
3.0b4 (Huelsenbeck and Ronquist, 2001). The Bayesian
approach evaluates the posterior probability (PPB) of a
tree given the character matrix, i.e., the probability that
the tree is correct. The posterior probability is obtained
after combining the prior probabilities of a tree and of
the data with the likelihood of the data given that tree.
The Bayesian approach combines the advantages of
defining an explicit probability model of character evo-
lution and of obtaining a rapid approximation of poster-
ior probabilities of trees through the use of Markov
chains Monte Carlo (MCMC). The likelihood model
chosen is the General Time Reversible model (GTR,
Yang, 1994) with among-site substitution rate heteroge-
neity described by a gamma distribution with eight cat-
egories and a fraction of sites constrained to be
invariable (GTR + I + C8). Different models were used
for each of the seven partitions corresponding to the
Lf promotor and the three codon-positions for Cyb
and CO2. All analyses were conducted with five inde-
pendent Markov chains run for 1,000,000 Metropolis-
coupled MCMC generations, with tree sampling every
100 generations and burn-in after 3000 trees.
The MP analyses were conducted on PAUP 3.1.1
(Swofford, 1993) with differential weighting of the char-
acter-state transformations as detailed in Hassanin et al.
(1998a,b): for each substitution type (i.e., A–G, C–T,
A–C, A–T, C–G, G–T, and indels), the amount of
homoplasy was measured through the consistency index
excluding uninformative characters (CIex), and the sat-
uration was assessed graphically by plotting the pairwise
number of observed differences against the correspond-
ing pairwise number of inferred substitutions calculated
by PAUP (the slope of the linear regression [S] was used
to evaluate the level of saturation). The CIex and S val-
ues were calculated by distinguishing each of the three
codon-positions separately for Cyb and CO2. The MP
tree was found by heuristic search using 1000 replicates
of random stepwise addition. Support for individual
branches was assessed by Bootstrap Percentages (BP)
(Felsenstein, 1985) computed after 1000 replicates of
the closest stepwise addition option.
The divergence times were estimated under BASEML
in the PAML 3.14b software (Yang, 2003). We used the
900 A. Hassanin, A. Ropiquet / Molecular Phylogenetics and Evolution 33 (2004) 896–907

local clock model for combined analysis of multiple-
partition data, which allows the branch group rates
to vary in different ways among the data partitions
(Yang and Yoder, 2003). We assigned independent
rates for several groups of branches on the tree corre-
sponding to the following clades: Antilopinae, Bovinae,
Boselaphini, Tragelaphini, Bovini, Bubalina, and Bovi-
na. The combined analysis was performed using a
GTR model with a gamma distribution with eight cat-
egories for each of the seven following partitions: three
codon positions separately for Cyb and CO2, and the
Lf promotor. The first appearance of bovids in the fos-
sil record was used as a calibration point for molecular
datings (Vrba and Schaller, 2000): the bovid divergence
was fixed at 18.5 Mya with an interval between 18 and
20 Mya. Divergence times were estimated on each of
the 27 different topologies compatible with the consen-
sus tree (Fig. 3) deduced from Bayesian and MP anal-
yses (Fig. 2). For each node of interest, 27 dates were
thus calculated, and the values presented in Fig. 3
are averages calculated by using the youngest and old-
est dates.
2.4. Biogeography analyses
Biogeography analyses were conducted from DIVA
1.1 (Ronquist, 1996). DIVA is a program for recon-
structing ancestral distributions in a phylogeny using
dispersal-vicariance analysis (DIVA). This method,
in which ancestral distributions are inferred, is based
on a three-dimensional cost matrix derived from a
simple biogeographic model (Ronquist, 1997).
The biogeographic inferences were done using four
distinct geographical areas (i.e., Africa, America, Asia,
and Europe), and they were performed on the 27 dif-
ferent topologies compatible with the consensus tree
(Fig. 3) deduced from Bayesian and MP analyses
(Fig. 2).

Fig. 2. Molecular phylogeny of the tribe Bovini. The trees were obtained from the combined analysis of the three markers, i.e., cytochrome b (Cyb),
subunit II of the cytochrome c oxidase (CO2), and the promotor of the lactoferrin (Lf). Two methods of tree reconstruction were used for the
phylogenetic analyses: the Bayesian approach (A), and the Maximum Parsimony (MP) method using a differential weighting of the character-state
transformations (B). The values indicated on the branches of the Bayesian tree correspond to posterior probabilities (PPB), whereas the values on the
phylogramm of 579997 steps correspond to Bootstrap Percentages (BP). The white values in the black spots are the PPB or BP obtained with the
combination of the three markers. The other values are the PPB or BP obtained with each of the three markers independently; from left to right: Cyb,
CO2, and Lf. Dash indicates that the node was not retrieved with the marker, but no alternative hypothesis was supported by PPB greater than 0.75
or BP greater than 60. Asterisk indicates that an alternative hypothesis was supported by a PPB greater than 0.75 or a BP greater than 60.
 A. Hassanin, A. Ropiquet / Molecular Phylogenetics and Evolution 33 (2004) 896–907 901

Fig. 3. Biogeographic inferences and molecular datings. The presented tree is a strict consensus of the phylogenetic analyses performed with Bayesian
and MP methods on the combined matrix including all the three molecular markers (see Fig. 2). Biogeographic inferences and molecular datings were
conducted on the 27 different topologies compatible with this consensus tree. Biogeographic analyses were done with DIVA using four distinct
geographical areas, i.e., Africa (Af), America (Am), Asia (As), and Europe (Eu). For the outgroup taxa, we have considered an Asian origin for the
family Cervidae, and an African origin for the subfamily Antilopinae (Kingdon, 1982, 1997). The inferred ancestral areas are shown on the branches,
and the values in brackets indicate the frequency of their occurrence when several possible areas were found in the 27 analyses. The molecular datings
were estimated with PAML for each of the 27 different topologies. The divergence times indicated on the nodes were calculated by averaging out the
youngest and oldest ages obtained from the 27 analyses. For each node, the interval of age estimations is represented by the width of the grey bar.

3. Results and discussion
3.1. Inter-generic relationships within the subfamily
Bovinae
The three different markers (Cyb, CO2, Lf) were
analysed separately and also combined, representing a
total of 2065 sites (including four sites with indels in
the Lf). In Figs. 2A and B are presented the phyloge-
netic trees performed with Bayesian and MP methods,
respectively. They are very similar except a few topo-
logical differences, which are not strongly supported.
The subfamilies Antilopinae and Bovinae were found
monophyletic (PPB/BP = 1/96 and 1/92, respectively),
and this bovid dichotomy was independently retrieved
with each of the three markers (see values in Fig. 2).
This division of the family Bovidae into two subfami-
lies is in agreement with previous molecular studies
(Gatesy et al., 1997; Gatesy and Arctander, 2000;
Hassanin and Douzery, 1999a,b; Hassanin and Douz-
ery, 2003; Matthee and Davis, 2001), and was initially
proposed by Kingdon (1982, 1997) on the basis of dif-
ferences concerning horn type, thermoregulation, and
glands.
902 A. Hassanin, A. Ropiquet / Molecular Phylogenetics and Evolution 33 (2004) 896–907
The analyses combining the three markers suggest
that all of the three tribes currently described into the
subfamily Bovinae are monophyletic: (1) the tribe Bos-
elaphini, which includes two monospecific genera of In-
dia: Boselaphus (nilgai) and Tetracerus (four-horned
antelope) (PPB = 1; BP = 100); (2) the tribe Tragela-
phini, which is endemic of Africa, and is represented
here by T. imberbis (lesser kudu) and T. strepsiceros
(greater kudu) (PPB = 1; BP = 100); and (3) the tribe
Bovini, which groups together all species ranged into
the genera Bos, Bison, Bubalus, Syncerus and Pseudoryx
(PPB = 1; BP = 87). Three subtribes can be defined into
the tribe Bovini: (1) the subtribe Bovina, which incorpo-
rates all species of Bos and Bison found in Eurasia and
America (PPB = 1; BP = 100); (2) the subtribe Bubalina,
which associates the Asian genus Bubalus with the Afri-
can genus Syncerus (PPB = 1; BP = 100); and (3) the
subtribe Pseudoryina, which consists in only P. nghe-
tinhensis (saola) confined to the Indochinese peninsula.
Independent analyses of all the three markers agree with
the monophyly of Boselaphini, Tragelaphini, Bovina,
and Bubalina (PPB = 0.89-1; BP = 63-100). By contrast,
separate analyses of the mitochondrial markers do not
support the monophyly of Bovini, but other alternatives
are not robust: Pseudoryx and Tragelaphini are sister-
group with CO2 (PPB = 0.53; BP = 51), whereas Buba-
lina are basal to all other Bovinae with Cyb
(PPB = 0.61; BP = 34). All Bovini are however grouped
together with the Lf nuclear marker (PPB = 1; BP = 91),
and four signatures are diagnostic of this grouping,
including one deletion of seven nucleotides (ATA-
TWGC in position 136), and three punctual mutations
(positions 101:G fi A, 280: A fi T, and 284:R fi C).
The lack of signal in the mitochondrial genes for sup-
porting the monophyly of Bovini may be explained by
two different processes: (1) saturation of nucleotide sub-
stitutions, as the mitochondrial genome evolves at much
higher rates than the nuclear one (e.g., Burger et al.,
2003; Li, 1997), and (2) the rapid diversification of the
tribe Bovini into three subtribes (Bovina, Bubalina,
and Pseudoryina) soon after its separation from Bosela-
phini and Tragelaphini, as molecular datings (Fig. 3)
indicate that Bovini diversified only two millions years
ago after the emergence of Bovinae (13.68 ± 1.85 and
15.78 ± 1.36 Mya for the most recent common ancestors
of Bovini and Bovinae, respectively).
Interrelationships among the three tribes of Bovinae
are confused. According to morphologists, Boselaphini
may be grouped with Bovini (Gentry, 1992; Groves,
1981), or with Tragelaphini (Kingdon, 1982). However,
all the three possible solutions have been supported by
different molecular datasets: Boselaphini + Tragelaphini
(Allard et al., 1992; Gatesy et al., 1997; Hassanin and
Douzery, 1999b), Bovini + Boselaphini (Gatesy and
Arctander, 2000; Janecek et al., 1996), Bovini + Tragel-
aphini (Hassanin and Douzery, 2003). Two alternative
hypotheses emerged from the present analyses: (1) a sis-
ter-group relationship between Bovini and Tragelaphini,
or (2) an association of Boselaphini with Tragelaphini.
The first hypothesis is found with CO2 alone
(PPB = 0.63; BP = 31), and with the MP analysis com-
bining all the three markers (BP = 61). The second
hypothesis is supported by independent analyses of
Cyb (PPB = 0.48; BP = 25) and Lf genes (PPB = 0.71;
BP = 62), and by the Bayesian analysis combining all
the three markers (PPB = 0.59). These topological con-
flicts for inter-tribal relationships reflect the explosive
diversification of bovid tribes during the Middle Mio-
cene, as previously suggested by paleontological and
molecular data (Gentry, 1994; Hassanin and Douzery,
1999a).
The monophyly of the subtribe Bovina is robust
(PPB = 1; BP = 100) and members of this group share
a large amount of molecular signatures: six in the Cyb
gene (positions 574: A, 636: C, 823: T, 884: C, 1108:
G, and 1128: A), one in the CO2 gene (position 342:
G), and four in the Lf promotor (positions 249: T,
287: T, 316: C, and 335: A). This result is not surprising
given that numerous morphological studies already evi-
denced strong affinities between species ranged into Bos
and Bison (Fig. 1). Four clades of Bovina emerged ro-
bustly from our molecular analyses: (1) the grouping
of the two major breeds of domestic cattle, i.e., no hump
taurine (B. t. taurus) and humped zebu (B. t. indicus)
(PPB = 1; BP = 100), and (2) their association with the
wisent (B. bonasus) (PPB = 0.92; BP = 70); (3) the clus-
tering of the kouprey (B. sauveli) with the two other
Indochinese wild cattle species, i.e., banteng (B. javani-
cus) and gaur (B. frontalis) (PPB = 1; BP = 90); and (4)
the sister-group relationship between the yak (B. grunn-
iens) and American bison (B. bison) (PPB = 1; BP = 99).
These relationships suggest that Bos and Bison are para-
phyletic, and imply therefore that the genus Bison
should be regarded as a synonym of Bos. The polyphyly
of Bison was already proposed on the basis of mitochon-
drial markers (Janecek et al., 1996; Verkaar et al., 2004).
However, the two species of Bison can produce com-
pletely fertile hybrid offsprings and have similar AFLP
patterns (Buntjer et al., 2002) as well as Y chromosomal
sequences (Verkaar et al., 2004). Two main explanations
have been provided for interpreting this conflict between
mitochondrial and nuclear genomes (Janecek et al.,
1996; Verkaar et al., 2004): (1) lineage sorting, which im-
plies that two distinct mitochondrial lineages coexisted
in the bison-yak branch until the recent divergence of
Amercian bison and wisent; or (2) interspecific hybrid-
ization, which involves that the wisent emerged by intro-
gression of bison bulls in another ancestral species,
probably related to the extinct aurochs (B. t. primige-
nius) given that mitochondrial sequences associate B.
bonasus with B. taurus, and that the humpless domestic
cattle (B. t. taurus) and humped zebu (B.t. indicus) were
 A. Hassanin, A. Ropiquet / Molecular Phylogenetics and Evolution 33 (2004) 896–907
domesticated from two separate aurochsen populations
during the Holocene (Bradley et al., 1996).
3.2. Biogeographic evolution of the Bovinae during the
Neogene
The African origin of Antilopinae is upheld by the
fact that most tribes of this subfamily have unquestion-
able African origins, i.e., Aepycerotini, Alcelaphini,
Cephalophini, Hippotragini, Neotragini, Oreotragini,
and Reduncini (Gentry and Heizmann, 1996; Kingdon,
1982; Vrba, 1985). The origin of Bovinae is more enig-
matic since fossils of Boselaphini have been described
in the Middle Miocene of Africa, Asia, and Europe
(Vrba, 1985). Our molecular datings confirm that extant
members of the subfamily Bovinae share a common
ancestor in the Middle Miocene (at 15.78 ± 1.36 Mya),
but biogeographic inferences suggest three possible
ancestral areas for their emergence (Fig. 3): Africa (fre-
quency of this hypothesis: 6%), Asia (38%), or an en-
larged area including Africa and Asia (56%).
According to Kingdon (1982), the two subfamilies of
Bovidae developed separately in two different continents
at the beginning of the Neogene, with Antilopinae in
Africa and Bovinae in Eurasia. An Asian or Eurasian
origin for extant Bovinae seems effectively more likely
since it is supported by two major arguments. (1) Even
if low sea level stands may have facilitated inter-conti-
nental faunal exchanges between Africa, Europe, and
India during the Middle Miocene (Van der Made,
1999), Bovinae, which are adapted to moister habitats,
were not expected to disperse in Africa because a rela-
tively arid ecological barrier is thought to have existed
between Africa and Eurasia during this epoch (Thomas,
1979). By contrast, Antilopinae, which are smaller ani-
mals adapated to drier habitats, were expected to dis-
perse more easily in this continent. (2) The fossil
record of the early Middle Miocene reveals that bovid
remains of Africa are in fact related to gazelles and cap-
rines, i.e., Antilopinae, while most fossils from Eurasia
have been linked to boselaphines, i.e., Bovinae (Gentry,
1994; Gentry and Heizmann, 1996).
Most of the evolution of the tribe Bovini occurred in
the Old World: the earliest fossil for this tribe is known
from the Miocene of Africa, dated around 6–7 Mya, but
remains of Bovini come from levels slightly younger in
the Indian subcontinent, perhaps around 6 Mya (Gen-
try, 1990). Paradoxically, all palaeontologists consider
that Bovini came from Eurasia originally, and entered
in Africa in the Late Miocene between 5–7 Mya (Gentry,
1990; Geraads, 1992; Vrba, 1985). Our biogeography
inferences argue also in favour of this view since 82%
of the analyses suggest an Asian origin whereas only
18% suggest an enlarged ancestral area including both
Africa and Asia. Our molecular estimations reveal that
a long hiatus exists in the fossil record of Bovini since
903
their common ancestor is dated at 13.68 ± 1.85 Mya,
while the most ancient remains are dated at 6–7 Mya.
Our interpretation is that there is a strong taphonomic
bias for Bovini. Whether an organism is preserved
greatly depends on the local environment in which it
died. Animals from humid forests are rarely preserved
because they decay rapidly in these regions. Bovini are
large grazers in moist habitats, having an optimum pre-
ferred habitat of riverine forest and grassland (Kingdon,
1982). The fossilization of Bovini is therefore less ex-
pected than bovids occupying drier and more open ter-
rains. This taphonomic bias is also corroborated by
the fact that most Miocene Bovini are African species
related to the only one extant bovine genus endemic to
sub-Saharan Africa, i.e., Syncerus (Gentry, 1990), which
today inhabits a wide range of habitats, including
savannah.
Our molecular datings indicate that three groups
emerged at around 8–9 Mya (Fig. 3): Boselaphini in In-
dia at 8.09 ± 1.55 Mya, Bubalina in Africa and Asia at
9.12 ± 1.64 Mya, and Tragelaphini in Africa at
8.87 ± 1.45 Mya. During this period of time, significant
increases in altitudes of the Himalayan mountains and
Tibetan plateau were assumed to strongly affect the cli-
mates of Asia, with enhanced aridity in the Asian inte-
rior and onset of the Indian and East Asian monsoons
(Zhisheng et al., 2001). At this time, the North of the
African desert zone was displaced northwards to be over
Mediterranean, and central and eastern North Africa
became seasonnaly humid (Griffin, 2002). Consequently,
these climatic changes may have promoted the dispersal
of Bubalina and Tragelaphini from Asia to Africa. The
hypothesis involving an Asian origin for Bubalina and
Tragelaphini is supported by their extant geographical
distribution and by their fossil record. Extant Bubalina
are found in Asia with five species of Bubalus, as well
as in Africa with S. caffer, but, as pointed out previ-
ously, their center of origin is undoubtedly in Asia. By
contrast, Tragelaphini are now an exclusively African
group, and their fossil record is restricted to the sub-
Saharan regions of Africa, and is not anterior to the
Late Miocene (Kingdon, 1997; Vrba, 1985). Because of
their ecology, and a strong East African bias in their ex-
tant distribution, Kingdon (1997) however suggested
that they derived from an Asian immigrant, which were
relatively large and adapted to strong seasonal habitats.
According to that, it seems likely that Tragelaphini en-
tered in Africa in company of some Bubalina species,
since both groups appeared suddenly in East Africa at
around 6–7 Mya (Gentry, 1990; Vrba, 1985).
3.3. Taxonomic status of the Kouprey
In agreement with all studies based on morphological
characters (Fig. 1), the present molecular analyses
strongly support the placement of the kouprey into the
904 A. Hassanin, A. Ropiquet / Molecular Phylogenetics and Evolution 33 (2004) 896–907

subtribe Bovina (PPB = 1; BP = 100). Within Bovina,
the kouprey is clearly enclosed with the two other South
East Asian Bos species, i.e., banteng and gaur (PPB = 1;
BP = 90). Although in contradiction with the conclu-
sions of Coolidge (1940) and Groves (1981), this result
is in perfect accord with the view of Pfeffer and Kim-
San (1967). In addition, it is consistent with the former
designation of these three species as belonging to the
subgenus Bibos (Urbain, 1937). Molecular estimations
of divergence times indicate that banteng, gaur, and
kouprey shared a common ancestor at 2.6 ± 0.5 Mya,
and that these three lineages separated in a very short
period of time, i.e., between 2.6 and 2.3 ± 0.5 Mya.
These molecular estimations fit perfectly with the palea-
ontological record, which indicates firstly that remains
related to the extant banteng and gaur are found in
the Pleistocene of Asia, and secondly that these two lin-
eages cannot be traced back any earlier (Badam and
Grigson, 1990; Hooijer, 1958). After 2.6 Mya, the Indian
and East Asian summer mosoon becomes more variable
and at times weaker, whereas the East Asian winter mo-
soon intensifies. This period is also marked by the begin-
ning of the major Northern Hemisphere glaciation,
which appears to have influenced, and was probably
influenced by the modifications of the Asian mosoons
(Zhisheng et al., 2001). This suggests therefore that the
rapid diversification of Asian wild cattle was a conse-
quence of important climatic changes.
The kouprey has been interpreted as being a hybrid
generated by the crossing of the banteng (B. javanicus)
with one of the three following species: B. frontalis, B.
taurus, or B. bubalis (Bohlken, 1958; Edmond-Blanc,
1947). These hypotheses are plausible since data from
hybridization indicate that in captivity the banteng is
able to hybridize with the gaur, but also with domestic
cattle and yak (Van Gelder, 1977). With this respect, it
is relevant to note that several cases of hybridization be-
tween banteng and domestic zebu have been reported in
the literature (Nijman et al., 2003). If the kouprey is a
hybrid of banteng or gaur with another species, its
mtDNA genome is expected to be identical or very sim-
ilar to the one of its maternal parental species. For test-
ing this possibility, phylogenetic analyses were carried
out by integrating all the complete and partial sequences
of CO2 and Cyb deposited in the EMBL/GenBank/
DDBJ databases (Fig. 4). The results from Bayesian
analyses show that all specimens of B. frontalis are
grouped together in a monophyletic assemblage distinct
from the one composed of all specimens of B. javanicus,
and that the kouprey is never found related to any pop-
ulation of banteng or gaur. Consequently, these molec-
ular analyses do not favour any hypothesis of

Fig. 4. Phylogenetic analyses including all the diversity for banteng and gaur sequences. The phylogenetic trees were carried out with the Bayesian
approach, and the values on the branches correspond to posterior probabilities greater than 0.5. The analyses were done by including all sequences of
banteng (Bos javanicus) and gaur (Bos frontalis) available in the EMBL/GenBank/DDBJ databases. Banteng and gaur sequences produced during
this study are indicated by black circle. The tree in (A) was found by analysing the sequences of the subunit II of the cytochrome c oxidase (CO2),
whereas the tree in (B) was obtained with the cytochrome b gene sequences (Cyb).
 A. Hassanin, A. Ropiquet / Molecular Phylogenetics and Evolution 33 (2004) 896–907
hybridization, and involve that the kouprey is a real spe-
cies of wild cattle distinct from B. frontalis and B.
javanicus.
3.4. Morphological and molecular identification of the
Kouprey
Different species of Bovini are found in Indochina,
i.e., B. frontalis, B. javanicus, B. sauveli, Bubalus bubalus,
and P. nghetinhensis. Morphologically, the three species
of wild cattle (genus Bos) are clearly distinct from the
water buffalo (Bubalus) and saola (Pseudoryx), but their
distinction is more difficult. As in most gregarious herbi-
vores evolving in seasonal environments, they develop-
ped an important sexual dimorphism (Geist, 1987),
which is expressed differently in each species. The three
species of the subgenus Bibos may be therefore diagnosed
as follows (Coolidge, 1940; Lekagul and McNeely, 1977;
MacKinnon and Stuart, 1988; Pfeffer and Kim-San,
1967): (1) both sexes of banteng exhibit a characteristic
white rump patch as well as a white band around the
muzzle; females are usually brown or reddish brown,
whereas the males are blackish brown or blue-black,
and their horns are more upright and considerably smal-
ler than in the males; (2) the general coloration of gaur is
dark reddish brown to almost blackish brown; males are
approximately one-fourth larger and heavier than fe-
males, and have a large hump over the shoulders; (3) in
the kouprey, the young are brownish, the adult females
are a characteristic grey colour, and the old males are a
very rich, dark brown. The kouprey is a graceful animal
when compared to banteng and gaur. In addition, males
have a very pronounced dewlap, which appears lesser
developed or absent in the banteng and gaur. The horns
are widely spread and recurved forward in the bulls,
while they are typically lyred-shaped in the females.
The close scrutiny of Cyb sequences reveals that this
mitochondrial gene may be particularly useful for diag-
nosing the kouprey. Indeed, several nucleotide signa-
tures are found in this marker for diagnosing the
subgenus Bibos, as well as all the three species of this
taxon: the subgenus Bibos can be recognized by the pres-
ence of a T nucleotide in position 975; our sequence of
B. frontalis exhibits a characteristic T in position 717,
as well as the other complete Cyb gene of gaur deposited
in the databases (Accession No. AY079128); our se-
quence of B. javanicus is diagnosed by a G nucleotide
in positions 84, 123, and 531, and these three signatures
are also found in all other available sequences of ban-
teng (see Accession Nos. in Fig. 4); the Cyb gene of
the kouprey is diagnosed by seven automorphic substi-
tutions, including five transitions (positions 631:
A fi G, 686: T fi C, 919:C fi T, 1043:T fi C, and
1057: C fi T) and two transversions (positions 360:
A fi T, 361: C fi A). DNA was extracted from another
specimen of kouprey preserved in the Museum of Bour-
905
ges (France). By sequencing two fragments of the Cyb
gene, corresponding to positions 163–405 and 894–
1140, we retrieved all signatures, excepting the transition
T fi C in position 1043 (unpublished data). This indi-
cates that most of these signatures can be used at the
species level for diagnosing other specimens of B. sauv-
eli. Since some specimens were already misattributed
to B. sauveli by the past (Hoffman, 1986), molecular tax-
onomy appears as the best way for validating the aut-
enthicity of the museum specimens of this very rare
species.
According to the IUCN (2003), the kouprey is classi-
fied as critically endangered, the banteng as endangered,
and the gaur as vulnerable. Despite the presence of key
morphological characters for distinguishing the three
species of the subgenus Bibos found in sympatry in
Indochina, it remains difficult to know whether the kou-
prey still survives in the wild because this animal is rare
and is generally shy towards humans (MacKinnon and
Stuart, 1988). The molecular signatures found in the
Cyb gene may be used for tracking the kouprey in Indo-
china. We proposed a conservation project that should
be carried out in three steps: (1) collecting fecal samples
in different areas of the forests of Cambodia, Thailand,
Laos and Vietnam; (2) extracting DNA from these sam-
ples, and (3) sequencing a short fragment of the Cyb
gene (200–300 nt) for determining to what species the
feces materials belong. These molecular investigations
would allow us to know whether the kouprey still sur-
vives in some Indochinese regions or not.
Acknowledgments
We are grateful to Jacques Cuisin, Jean-Marc Pons,
Francis Renoult, Daniel Robineau, and Michel Tranier
for the bone samples from MNHN specimens, to Jean-
Luc Berthier, Gerard Dousseau, Jean-Francois Marja-
rie, Claire Rejaud, and Jacques Rigoulet for blood or
hair samples from American bison, gaur, and yak, to
Jean-Marie Carenton for blood from banteng, to Phi-
lippe Chardonnet for tissue from greater kudu, to Gau-
thier Dobigny for hairs from zebu, to Françoise
Hergueta-Claro for cells from blesbok, to Bui Xuan
Nguyen for blood from water buffalo, to Herbert Tho-
mas for skin samples from saola, and to Simon Tillier
for bone from European bison. We also acknowledge
the reviewers for their helpful comments on the first ver-
sion of the manuscript.
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