Molecular Insights into the Evolution of the Family Bovidae: A Nuclear DNA Perspective

Conrad A. Matthee and Scott K. Davis

Department of Zoology, University of Stellenbosch, Stellenbosch, South Africa
Department of Animal Science, Texas A&M University


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 
The evolutionary history of the family Bovidae remains controversial despite past comprehensive morphological and genetic investigations. In an effort to resolve some of the systematic uncertainties within the group, a combined molecular phylogeny was constructed based on four independent nuclear DNA markers (2,573 characters) and three mitochondrial DNA genes (1,690 characters) for 34 bovid taxa representing all seven of the currently recognized bovid subfamilies. The nuclear DNA fragments were analyzed separately and in combination after partition homogeneity tests were performed. There was no significant rate heterogeneity among lineages, and retention index values indicated the general absence of homoplasy in the nuclear DNA data. The conservative nuclear DNA data were remarkably effective in resolving associations among bovid subfamilies, which had a rapid radiation dating back to approximately 23 MYA. All analyses supported the monophyly of the Bovinae (cow, nilgai, and kudu clade) as a sister lineage to the remaining bovid subfamilies, and the data convincingly suggest that the subfamilies Alcelaphinae (hartebeest, tsessebe, and wildebeest group) and Hippotraginae (roan, sable, and gemsbok clade) share a close evolutionary relationship and together form a sister clade to the more primitive Caprinae (represented by sheep, goat, and muskox). The problematic Reduncinae (waterbuck, reedbuck) seem to be the earliest-diverging group of the Caprinae/Alcelaphinae/Hippotraginae clade, whereas the Antilopinae (gazelle and dwarf antelope clade) were always polyphyletic. The sequence data suggest that the initial diversification of the Bovidae took place in Eurasia and that lineages such as the Cephalophinae and other enigmatic taxa (impala, suni, and klipspringer) most likely originated, more or less contemporaneously, in Africa.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 
Evolution within the family Bovidae (cow, sheep, and antelope) is characterized by global immigrations, adaptive radiations, and mass extinctions which, in concert, gave rise to the 49 extant genera and more than 140 species known today (Wells 1957Citation ; Vrba 1985Citation ; Kingdon 1989Citation ; Nowak 1999Citation ). The majority of these species are endemic to the African continent, and the complex evolution of the group was molded by a wide variety of mechanisms and natural events, including temperature adaptation, feeding ecology, vegetation physiognomy, rifting, and climatic fluctuations (Kingdon 1989Citation ). The oldest bovid fossils are attributable to the subfamily Bovinae and are known from France and sub-Saharan Africa, where the group evidently first appeared approximately 23 MYA (Vrba 1985Citation ; Kingdon 1989Citation ).

Following Nowak (1999)Citation , with slight modifications from the morphological (Gentry 1992Citation ) and current genetic literature (Gatesy et al. 1997Citation ; Hassanin and Douzery 1999aCitation ; Matthee and Robinson 1999Citation ), at least 7 monophyletic subfamilies and more than 12 tribes can be identified. The subfamilies include the earliest-diverging Bovinae (cow, eland, and nilgai group) and the more recent Cephalophinae (duikers), Antilopinae (gazelles, saiga, springbok, steenbok, dik-dik, and oribi group), Caprinae (sheep and goat group), Hippotraginae (roan and gemsbok group), Reduncinae (waterbuck, lechwe, and gray rhebok group), and Alcelaphinae (hartebeest and wildebeest group). Although monophyly of the majority of the subfamilies is supported by morphological data (Vrba 1985Citation ; Kingdon 1989Citation ; Gentry 1992Citation ), fossil evidence (Gentry 1992Citation ), allozymes (Georgiadis, Kat, and Oketch 1990Citation ), serum immunology (Lowenstein 1986Citation ), cytogenetic data (Modi, Gallagher, and Womack 1996Citation ; Robinson et al. 1998Citation ), and DNA sequence analyses (Allard et al. 1992Citation ; Gatesy et al. 1992, 1997Citation ; Geraads 1992Citation ; Groves and Shields 1996Citation ; Janecek et al. 1996Citation ; Essop, Harley, and Baumgarten 1997Citation ; Hassanin and Douzery 1999a, 1999bCitation ; Matthee and Robinson 1999Citation ; Rebholz and Harley 1999Citation ), the evolutionary relationships among most bovid subfamilies remain uncertain.

The systematic description of the group is further clouded by problematic taxa such as the impala (Aepyceros), the suni (Neotragus), and the klipspringer (Oreotragus). In the past, the impala was grouped with the Alcelaphinae (Vrba 1984Citation ; Lowenstein 1986Citation ), with the Antilopinae (Kingdon 1989Citation ), with the Reduncinae (Murray 1984Citation ), or as a distinct subfamily, Aepycerotinae (Ansell 1971Citation ; Vrba 1979Citation ; Gentry 1992Citation ). Although most conventional classification systems place the suni and klipspringer within the dwarf antelope tribe Neotragini (subfamily Antilopinae; see Nowak 1999Citation ), there is no molecular (Hassanin and Douzery 1999a, 1999bCitation ; Matthee and Robinson 1999Citation ) or strong morphological evidence (Oboussier 1979Citation ; Gentry 1992Citation ) to support these findings. A recent cytochrome b study (Matthee and Robinson 1999Citation ) suggested that the klipspringer was a sister taxon to Cephalophinae, a finding initially proposed by Oboussier (1979)Citation based on brain morphology. Additionally, the molecular cytochrome b data suggested that the suni was a sister taxon to the impala. It is important to realize that statistical support for the above-mentioned associations is weak, and no robust conclusions could thus be reached (Hassanin and Douzery 1999aCitation ; Matthee and Robinson 1999Citation ; Rebholz and Harley 1999Citation ).

Based on cladistic and phenetic analyses of 112 skeletal characters taken from the horn-core, skull, tooth, and postcranium, Gentry (1992)Citation concluded that bovids cluster around four foci in ascending evolutionary sequence: Bovinae; Antelopinae and some dwarf antelopes; Caprinae; and a group of African antelopes containing the impala, Alcelaphinae, Hippotraginae, and Reduncinae. This conclusion, however, was based on the fact that the early derived bovids (i.e., Bovinae) share more primitive similarities than divergently advanced ones with each other and hence associate more closely. Furthermore, the morphological data demonstrated a low consistency index value clearly indicative of a large degree of parallelism in bovid history (Gentry 1992Citation ).

The incomplete nature of the bovid Miocene fossil record (Vrba 1985Citation ), together with morphological parallelisms (Gentry 1992Citation ) and rapid cladogenesis (Vrba 1985Citation ; Georgiadis, Kat, and Oketch 1990Citation ; Allard et al. 1992Citation ; Gatesy et al. 1997Citation ), led many authors to conclude that the subfamilial relationships within the Bovidae could be resolved only through comprehensive species sampling and by using data derived from multiple sources (Allard et al. 1992Citation ; Gentry 1992Citation ; Gatesy et al. 1997Citation ; Matthee and Robinson 1999Citation ). The retrieval of phylogenetic information from mtDNA data has widely been thought to be confounded by the rapid radiation within the Bovidae (Allard et al. 1992Citation ; Gatesy et al. 1997Citation ; Matthee and Robinson 1999Citation ; Rebholz and Harley 1999Citation ). In the mtDNA molecular analyses, the unresolved nodes persist even when complex weighting schemes are used in an attempt to reduce homoplasy/noise (Gatesy et al. 1997Citation ; Hassanin and Douzery 1999a, 1999bCitation ). Rapid radiations become more problematic as evolutionary time increases (Matthee and Robinson 1997Citation ) and, given the Miocene origin of the Bovidae, might explain why few well-supported associations among bovid subfamilies were retrieved by earlier mtDNA studies.

Nuclear DNA sequence data can offer a powerful alternative to mtDNA data (Robinson et al. 1997Citation ; Stanhope et al. 1998Citation ; Gatesy et al. 1999Citation ; von Dornum and Ruvolo 1999Citation ), and the extension of data from additional sources might provide more robust phylogenies for problematic taxa. The nuclear genome of mammals is approximately 166,000 times as large as the mitochondrial genome and also provides sets of markers that potentially segregate independently. Despite this rich source of data, phylogenetic studies based on nuclear DNA genes remain limited, and the majority of past investigations have been at the higher systematic level (i.e., among orders and families of vertebrates with origins more than 30 MYA; Robinson et al. 1997Citation ; Stanhope et al. 1998Citation ; Gatesy et al. 1999Citation ). A recent artiodactyl investigation by Matthee et al. (2001)Citation provided a suite of eight nuclear genetic markers that showed potential to resolve systematic uncertainties at both the higher and the lower taxonomic levels in vertebrates (e.g., among subfamilies of vertebrates with origins generally dating back less than 20 Myr). In the present investigation, we were particularly interested in the utility of these nuclear DNA intron sequences in addressing systematic questions at the lower taxonomic level (see above). In this study, we extended our initial investigation of the Artiodactyla by sequencing 4 of the 8 nuclear DNA fragments for an additional 22 bovid taxa.

In an attempt to resolve the phylogeny of the family Bovidae, a complete mitochondrial DNA data set originating from the three mtDNA genes (Gatesy et al. 1997Citation ; Matthee and Robinson 1999Citation ) was also included in combined analyses. The evolutionary rates and selective constraints operating on different DNA genes vary and could therefore potentially have two benefits for this study. First, highly supported nodes retrieved from the analyses of independent and combined nuclear and mitochondrial data can be considered robust (Hillis 1995Citation ). Second, because of different evolutionary rates among nuclear and mtDNA genes, these data should provide phylogenetic signal at different levels of the tree (Halanych and Robinson 1999Citation ; B. Jansen van Vuuren and T. J. Robinson, personal communication). For example, the rapid evolutionary radiations of the Bovidae near the Miocene-Pliocene boundary (the basal and internal radiations of subfamilies; Vrba 1985Citation ) might be better recovered by the nuclear DNA sequences, while phylogenetic signal from the mid-Pleistocene radiations (more terminal nodes/radiation within subfamilies; Vrba 1985Citation ) might be more prominent in the faster-evolving mitochondrial DNA data.

The aims of this investigation were therefore threefold: (1) to investigate the value of conservative nuclear DNA intron sequences in recovering a robust phylogeny for a reasonably closely related group of vertebrates characterized by a rapid radiation, (2) to provide a more robust phylogeny for the bovid subfamilies, and (3) to obtain a nuclear DNA perspective on the puzzling phylogenetic position of the impala, suni, and klipspringer.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 
Specimens Used
Sequence data from 34 bovid taxa were used for this investigation. At least two representatives of all seven currently recognized bovid subfamilies were included (table 1 ). Based on the availability of sequence data, the giraffe (Giraffa camelopardalis) and the reindeer (Rangifer tarandus) were used as outgroups. Unfortunately no ribosomal mitochondrial data were available for the reindeer, and in these instances, the sambar deer (Cervus unicolor) was used as the second outgroup.


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Table 1 Taxonomic Designations of the 36 Species Used in the Present Study

 
Data Collection
Total genomic DNA was extracted from fibroblast cells or DMSO/NaCl-preserved ear tissue following standard phenol/chloroform/iso-amyl alcohol DNA procedures (Amos and Hoelzel 1991Citation ). Conserved primers located in the protein-coding region of the nuclear DNA fragments were used to amplify four regions located on different chromosomes of the cow; these were B-Spectrin nonerythrocytic 1 (SPTBN1), Protein-Kinase C 1 (PRKC1), Kappa-casein (Kap-cas), and Thyrotropin (Thy; Matthee et al. 2001)Citation . The primers were originally selected from a suite of gene mapping markers currently used to detect target inserts in bovine and ovine bacterial artificial chromosome (BAC) libraries, and although these primers are located within conserved coding regions, most of the sequence data were obtained from faster-evolving intron regions. BLASTN searches were performed against published mouse, cow, or human sequences to confirm the authenticity of the coding regions present in the fragments, and there were no indications that any of the regions sequenced were located in isochores with different GC contents (see Bernardi 2000)Citation .

To obtain comparable taxon sampling between the mtDNA and nuclear DNA data sets, we sequenced an additional 12 mtDNA gene fragments from seven taxa. The universal cytochrome b primers (L14724 and H15915; see Matthee and Robinson 1999Citation for details) were used to obtain complete gene sequences for the gemsbok (Gazella oryx) and the gerenuk (Litocranius walleri) and the published ribosomal primers (see Gatesy et al. [1997Citation ] for details) were used to generate the missing 12S rRNA and 16S rRNA sequences for the gerenuk (L. walleri), the Cape grysbok (Raphicerus melanotis), Sharp's grysbok (Raphicerus sharpei), the suni (Neotragus moschatus), the gray duiker (Sylvicapra grimmia), and the klipspringer (Oreotragus oreotragus).

PCR procedures followed standard methodology, and the reaction mixes contained approximately 100–200 ng total genomic DNA. Variable MgCl2 concentrations (1.0–2.5 mM) were used to achieve optimal primer annealing and to reduce nonspecific amplification. Before purification with QIAquick (Qiagen Ltd.), a 5-µl sample of each PCR reaction mix was screened on 1% agarose gels, together with a size standard ({lambda} cut with HindIII). The purified PCR products were cycle-sequenced using BigDye terminator chemistry (Perkin Elmer, Applied Biosystems), and all of the sequenced products were purified using Centrisep spin columns (Princeton separations) and analyzed on a 377 ABI automated sequencer.

Sequence data were obtained for all genes in all specimens with the exception of the Kap-cas fragment in oribi. Several attempts, including the development of additional primers, failed to amplify the kappa-casein fragment for this taxon. The oribi was therefore excluded from the Kap-cas analyses. The sequence data were verified by sequencing both strands. Heterozygous changes in the nuclear DNA data occurred at low frequencies (less than 0.1% cases) and were confirmed on the chromatograms. They mostly involved transitional changes, and these changes were designated as one of the two possible states based on the frequencies in the other individuals.

Sequence Analyses
All sequences were aligned manually, and the exon/intron boundaries for the nuclear genes were defined using BLASTN searches in GenBank. The aligned database has been deposited in EMBL (accession number DS42701), and all of the individual sequences can be obtained from GenBank (accession numbers AF210154AF210240 and AF249973AF249984). Small areas of ambiguous alignment in the 16S rRNA region (39 characters) were excluded before the phylogenetic analyses were performed. All alignment gaps were treated as missing characters, and unique synapomorphic nuclear insertions or deletions were indicated on the final trees. These unique indels were considered only when they had clearly defined alignment borders. Each gene was tested for rate heterogeneity among lineages using Tajima's (1993)Citation relative-rate test. The deer was used as reference taxon each time, and the chi-square probability values were corrected for multiple testing by applying the Bonferroni correction.

In order to test for congruence among genes, the different nuclear DNA segments were analyzed separately using maximum-parsimony and neighbor-joining methods in PAUP, version 4.0b2a, written by David L. Swofford. Due to computational time constraints, no maximum-likelihood analyses were performed for the individual genes. The independent nuclear analyses (Miyamoto and Fitch 1995Citation ) were followed by combined approaches (de Queiroz 1993Citation ), and in this instance, maximum-likelihood analyses were included. Before the data were combined, partition homogeneity tests were performed to identify possible conflicting phylogenetic signals in our data (see also Liu and Miyamoto 1999Citation ). First, the four nuclear DNA segments were tested prior to the generation of a nuclear DNA gene tree. Second, the four nuclear DNA data sets were tested together with the three mtDNA data sets (i.e., seven data partitions) before analyses of the total molecular evidence. In the latter instance, the maximum number of trees saved during each replicate was restricted to 1,000 due to time constraints.

Parsimony analyses utilized 100 replicates of a random taxon addition sequence followed by the heuristic search option with TBR branch swapping. Previous cytochrome b analyses showed that saturation was present at third-codon-position transitions in the cytochrome b gene (Hassanin and Douzery 1999a, 1999bCitation ; Matthee and Robinson 1999Citation ; Rebholz and Harley 1999Citation ), and based on these results, there is seemingly good evidence to exclude these transitions from the analyses. With the exception of this weighting scheme, no differential weighting was used in the present study. However, because removing characters in order to reduce homoplasy does not fundamentally increase general congruence (Vidal and Lecointre 1998Citation ), third-position changes were also included to test for congruence among weighting schemes. Distance and maximum-likelihood analyses were based on the HKY85 (Hasegawa, Kishino, and Yano 1985Citation ) correction, which adjusts for the differences in transition : transversion ratio and also takes into account unequal nucleotide frequencies. Gamma shape parameters were estimated under maximum likelihood for each analysis using the neighbor-joining topology as a starting point. These parameters were included in all of our distance analyses so as to correct for among-sites rate variation.

Nodal support for the parsimony and neighbor-joining analyses was assessed by 1,000 bootstrap iterations, while 100 replicates were performed for maximum-likelihood. For the maximum-likelihood bootstrap analyses, the neighbor-joining tree was used each time as a starting point, and, due to time constraints, the settings for maximum-likelihood did not include among-sites rate variation. Additionally, decay index values (Bremer 1994Citation ) for the nodes were calculated using AutoDecay, version 4.0 (Eriksson 1998Citation ), with default settings. Finally, alternative hypotheses were compared using the Kishino and Hasegawa (1989)Citation test in PAUP, version 4.0b2a, under maximum likelihood.


    Results and Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 
Characteristics of the Nuclear DNA Data
The aligned nuclear DNA data comprised a total of 2,572 bp, of which 1,870 bp were derived from intron regions and the remaining 702 bp were coding (table 2 ). The number of variable characters for each gene ranged from 26.3% for Kap-cas to 32.6% for SPTBN1. Although 25% more taxa were used than in Matthee et al. (2001)Citation , the number of parsimony-informative characters was only half that found previously when the evolutionarily more diverse taxa were included. This finding clearly corroborates the slow substitution rate for nuclear DNA data and, as similarly suggested by the high retention index (RI) values (RI > 0.69 for all nuclear fragments), clearly insinuates that very little saturation is present.


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Table 2 Breakdown of the Statistics Derived from each Gene Used in this Study

 
There were differences in base composition when the gene fragments for the Bovidae were compared with each other. Although both Kap-cas and PRKC1 were characterized by an adenine-plus-thymine bias, the former gene had low numbers of guanine (15%) while the latter had low numbers of cytosine (11.5%; table 2 ). Importantly, 45% of the Kap-cas gene fragment was coding, while only 13% of PRKC1 was represented by exon sequence (table 2 ). It seems reasonable to argue that these results reflect the differences in selection pressures between intron and exon sequences (see also Groth and Barrowclough 1999Citation ).

Sequence Divergence
Based on sequence divergence values, all nuclear DNA segments accumulated changes fairly slowly. The average sequence diversity for the members of the family Bovidae when all nuclear genes were combined was 4.23% (±1.24%), and the divergence values ranged from 0.74% between the lechwe (Kobus lechwe) and the waterbuck (K. ellipsiprymnus) to 6.56% between the Cape grysbok (R. campestris) and the lesser kudu (Tragelaphus imberbis). When the same taxa were compared for the cytochrome b mtDNA gene, the average sequence divergence among taxa was 13.72% (±0.99%); this value roughly translates to a 3.2-fold higher substitution rate in cytochrome b than in the nuclear DNA sequences.

When the nuclear genes were analyzed independently, the Kap-cas segment had the lowest sequence diversity (3.65% ± 1.33%), while the highest was found for the SPTBN1 region (5.03% ± 1.70%). The Thy and PRKC1 genes were intermediate, with average diversities of 3.80% (±1.22%) and 3.93% (±1.63%), respectively. These values clearly reflect different selection constraints on the different genes included in our study. The difference in evolutionary rates among genes is probably best illustrated by the comparison between Thy and SPTBN1, where both gene segments had similar numbers of coding (25%) and noncoding (75%) characters and one (SPTBN1) evolved at least 25% faster than the other (Thy).

Saturation Analyses
From the RI values, it is evident that the nuclear DNA data show little homoplasy (RI = 0.73), while most of the mtDNA characters are homoplastic (RI = 0.45; table 2 ). The exclusion of the presumably randomized signal present at third codon positions of cytochrome b (see above) resulted in higher statistical support (bootstrap and decay index values) for the subfamilial clusterings and lower statistical support for the closely related taxa (e.g., within the Alcelaphinae). This is not unexpected given the rate at which phylogenetic signal accumulates coupled with the effects of the randomization of phylogenetic signal over time (Matthee and Robinson 1997, 1999Citation ). Given the focus of the present investigation (subfamilial), we chose to present the bootstrap topology derived from the combined data, where third-codon transitions of cytochrome b were excluded (see below).

Rate Heterogeneity Analyses
Among-sites rate variation was limited in the nuclear DNA data (ranging from 0.765 to 0.938), and, as expected from previous reports, {propto} values for the protein-coding cytochrome b gene were much lower (0.480), indicating more among-sites rate heterogeneity for this rapidly evolving protein marker. The proportion of variable sites ranged from 0.08 for the Kap-cas gene to 0.34 for SPTBN1; the mtDNA data had a higher proportion of variable sites (table 2 ).

Previous mtDNA studies of bovids which relied on cytochrome b (Matthee and Robinson 1999Citation ) and 12S rRNA gene sequences (Allard et al. 1992Citation ) showed either limited or nonexistent rate heterogeneity among bovid taxa. The nuclear DNA evidence suggested a similar trend. After adjusting P values for multiple testing (2,196 pairwise comparisons in the present nuclear DNA study), there was no significant rate heterogeneity present among the bovid taxa examined.

Independent Gene Analyses
Separate analyses of each gene revealed a high number of most-parsimonious trees (ranging from 29 to 1,950; table 2 ). This is primarily the result of terminal polytomies within the monophyletic subfamilies Bovinae, Caprinae, and Alcelaphinae. When the genes were analyzed independently, bootstrapping showed a lack of support for the deeper nodes (defining subfamilies), probably due to the low number of synapomorphies present (65–141 for 36 taxa; table 2 ). However, all four gene trees consistently supported the distinction between the Bovinae and the other bovid subfamilies, and both equally weighted parsimony and neighbor-joining analyses recovered most subfamilies within the Bovidae with high bootstrap support (fig. 1 ). The monophyly of both the Reduncinae and the Cephalophinae was supported by of all four genes, and at least three of four genes supported the monophyly of the remaining five subfamilies as suggested by previous studies (Bovinae, Antilopinae, Hippotraginae, Caprinae, and Alcelaphinae; fig. 1 ). This result is striking given the relatively short nuclear DNA fragments (526–854 bp) that were analyzed independently. Both PRKC1 and Kap-cas had <70 parsimony-informative characters (table 2 ) for the 36 taxa included, and both fragments recovered at least 10 nodes with bootstrap support >70 when analyzed using parsimony and neighbor-joining analyses (fig. 1 ). In sharp contrast, when a collateral comparison was made among the 16S rRNA gene (which has 70 parsimony-informative characters for the same number of taxa; table 2 ) and these two nuclear DNA fragments, just four nodes are recovered with bootstrap support >70 (these nodes were those supporting the monophyly of Oryx, Kobus, Redunca, and Raphicerus; data not shown).



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Fig. 1.—Nuclear DNA phylogenetic trees showing the respective gene trees for the family Bovidae. Bootstrap values are presented at nodes, and the numbers above the diagonal branches represent the parsimony analyses, while those below the branches represent neighbor-joining support. Unique indel events are indicated by the triangles, and subfamily designations are indicated as follows: {block} = Bovinae; # = Hippotraginae; = Alcelaphinae; {square} = Caprinae; * = Reduncinae; O = Antilopinae; {diamond} = Cephalophinae; ? = uncertain status

 
Partition Homogeneity Tests
The partition homogeneity test indicated no significant differences in the phylogenetic signal between the mitochondrial and the nuclear DNA segments used in this study (P = 0.14). In contrast, support for combination of the independent nuclear DNA fragments was marginal when 100 repartitions were performed (P = 0.07). The conflicts between the independent gene data sets were often due to differences in the phylogenetic placement of the suni (Neotragus) and the klipspringer (Oreotragus; fig. 1 ). Most of the controversial associations (the position of the suni, the impala, and the klipspringer) were not well supported by bootstrapping (fig. 1 ) and, following the suggestions made by Remsen and DeSalle (1998)Citation and Liu and Miyamoto (1999)Citation , the nuclear DNA data were therefore combined into a single data set.

Combined Analyses
Far greater resolution was obtained when the nuclear DNA data set was analyzed without partitioning. The 762 variable and 349 parsimony-informative characters of the combined nuclear analyses (table 2 ) supported several previous molecular evolutionary suggestions and also provided new insights into the evolutionary relationships among bovid subfamilies (see below). Tree topologies obtained from maximum-likelihood, parsimony, and neighbor-joining analyses of the complete nuclear DNA data set were largely congruent with that obtained when the mitochondrial and nuclear DNA data were combined (fig. 2 Go ). Although the mtDNA certainly provided additional phylogenetic information, the additional resolution was mostly confined to the tips of the tree (see below). The additional informative characters not only caused an increase in the number of nodes recovered, but also caused an increase in bootstrap support (fig. 1 cf. fig. 2 ). Because the choice of outgroup might cause alterations in the ingroup topology (Milinkovitch and Lyons-Weiler 1998Citation ), we also analyzed the nuclear DNA data using midpoint rooting. Apart from slight alterations in the bootstrap support, the main findings of our study remained unaltered.



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Fig. 2.—Analysis of the combined data (four nuclear genes and three mtDNA genes). Bootstrap values are presented at nodes, with numbers above the diagonal branches representing parsimony values; those below the branches indicate neighbor-joining and maximum likelihood support. Unique indel events are indicated by the triangles, and subfamily designations are indicated as follows: {block} = Bovinae; # = Hippotraginae; = Alcelaphinae; {square} = Caprinae; * = Reduncinae; O = Antilopinae; {diamond} = Cephalophinae; ? = uncertain status

 
Resolution Within Bovid Subfamilies
There were noticeable differences in phylogenetic resolution among genera within bovid subfamilies. For example, the analyses of the combined nuclear DNA data were convincing in resolving the associations within the Reduncinae (bootstrap support for the nodes ranged between 72% and 99%) but failed to provide robust support for the evolutionary relationships between genera within the Alcelaphinae (nodes were unresolved or had bootstrap support lower than 56%). This is probably a reflection of the evolutionary rate of the genes used in this study, the taxa sampled, and different patterns of speciation within subfamilies. For example, the well-resolved Reduncinae clade either originated in Africa or immigrated from Eurasia during the late Miocene approximately 10–12 MYA (Vrba 1985Citation ; Kingdon 1989Citation ). Additionally, the three Reduncinae genera (Kobus, Redunca, and Pelea) are well separated in geological time (Skinner and Smithers 1990Citation ) and showed marked morphological and geographic differences (Kingdon 1989Citation ). In sharp contrast, the African Alcelaphinae, represented by Damaliscus, Alcelaphus, Connochaetus, and Beatragus, are characterized by a more rapid radiation; this occurred approximately 5–6 MYA (Vrba 1979Citation ) and did not allow for the accumulation of high numbers of synapomorphic nuclear DNA characters. In contrast, the rapidly evolving mtDNA cytochrome b gene (which evolves at roughly 3.2 times the rate of the nuclear DNA data used here) retrieved a well-supported phylogeny for the Alcelaphinae (Matthee and Robinson 1999Citation ). It therefore comes as no surprise that the combined total DNA analyses contained herein recovered three additional nodes within subfamilies (those defining Raphicerus; Ovis plus Capra, and Beatragus plus Damaliscus; see fig. 3 ).



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Fig. 3.—Combined maximum-likelihood topology for all genes and all taxa included in this study. Branch lengths are drawn proportionally, and the numbers above or to the right of the diagonals represent the bootstrap support for each node. Decay index values (where present) are shown in bold below the branches

 
This study provides strong genetic evidence for the monophyly of the Bovinae, the Hippotraginae, the Alcelaphinae, the Caprinae, the Reduncinae, and the Cephalophinae; it also suggests that the Antilopinae is polyphyletic (figs. 2 and 3 ), thereby supporting earlier investigations (Hassanin and Douzery 1999aCitation ; Matthee and Robinson 1999Citation ). While the high bootstrap values (>99%), 23 decay steps, and 2 unique indel events all point to the monophyletic status of six Antilopinae genera included in this study (Gazella, Antidorcas, Litocranius, Ourebia, Madoqua, and Raphicerus); importantly, however, the klipspringer (Oreotragus) and the suni (Neotragus) never formed part of this assemblage (figs. 2 and 3 ). When all the data were combined, the maximum-likelihood scores of constrained topologies which enforce the monophyly of the "conventional" Antilopinae (which include the suni and the klipspringer within the subfamily Antilopinae) were significantly worse than the maximum-likelihood score of the tree presented in figure 3 . When both the suni and the klipspringer were constrained to form part of the Antilopinae, the comparison between the alternative hypotheses resulted in t = 5.87 (P < 0.0001). When only the suni was enforced within the Antilopinae, the value was t = 4.79 (P < 0.0001). Finally, when only the klipspringer was enforced within the Antilopinae, the value was t = 4.40 (P < 0.0001). Although the nonmonophyly of the Antilopinae was underscored by previous molecular (Gatesy et al. 1997Citation ; Hassanin and Douzery 1999aCitation ; Matthee and Robinson 1999Citation ; Rebholz and Harley 1999Citation ) and morphological investigations (Gentry 1992Citation ), no statistical support could be found from the nuclear DNA data included in this study to support the associations between the suni and the impala (Hassanin and Douzery 1999aCitation ; Matthee and Robinson 1999Citation ) and the klipspringer and the Cephalophinae (Oboussier 1979Citation ; Hassanin and Douzery 1999aCitation ; Matthee and Robinson 1999Citation ). Moreover, widespread support for these evolutionary relationships was clearly weak and inconsistent in the mitochondrial DNA studies of Hassanin and Douzery (1999a) and Matthee and Robinson (1999)Citation . This suggests that the mtDNA placement of the suni as a sister taxon to the impala and that of the klipspringer as a ancient cephalophid are artifacts probably caused by typical mtDNA biases (Felsenstein 1978Citation ; Swofford et al. 1996Citation ; Huelsenbeck 1997Citation ; Hassanin and Douzery 1999aCitation ; Matthee and Robinson 1999Citation ).

Apart from the questionable placement of the suni and the klipspringer, the nuclear DNA data also failed to resolve the phylogenetic position of the impala, giving credence to previous suggestions that the impala should be recognized as a distinct evolutionary lineage, the Aepycerotonidae (Ansell 1971Citation ; Vrba 1979Citation ; Gentry 1992Citation ). Moreover, the average nuclear DNA sequence divergence values separating the three problematical taxa from the remaining bovid subfamilies are larger than or of similar magnitude to the values separating the currently recognized subfamilies (suni = 4.78% ± 0.51%; klipspringer = 3.84% ± 0.62%; impala = 3.99% ± 0.57%). Coupled with the absence of nuclear DNA sequence saturation and the apparent lack of significant rate heterogeneity among lineages, it seems reasonable to argue that the suni, the klipspringer, and the impala are unique species not particularly closely related to any of the recognized bovid tribes/subfamilies. All three are probably older, independent lineages that originated in Africa during the middle Miocene (approximately 15 MYA; Vrba 1985Citation ; Kingdon 1989Citation ).

Resolution Among Bovid Subfamilies
The combined nuclear DNA data proved to be informative in recovering evolutionary relationships among some bovid subfamilies (fig. 2 ). The Bovidae are characterized by a basal division which separates the Bovinae (cow, nilgai, kudu clade) from the remaining bovid taxa. This finding is supported by four unique indels (fig. 2 ) and is consistent across independent gene analyses and analytical methods. The bootstrap support for the basal split in the Bovidae tree was 100% in all the combined analyses (fig. 2 ), and more than 21 decay steps support each of the two clades as being monophyletic (fig. 3 ). Given morphological (Kingdon 1989Citation ), cytogenetic (Buckland and Evens 1978Citation ; Robinson et al. 1998Citation ), and other molecular evidence (Beintema et al. 1986Citation ; Lowenstein 1986Citation ; Gatesy et al. 1997Citation ; Matthee and Robinson 1999Citation ), this finding appears solid and clearly rejects the dental Aegodontia/Boodontia split previously suggested by Schlosser (1904)Citation .

The central result of the nuclear DNA data was the resolution we obtained within the clade comprising the Alcelaphinae, the Hippotraginae, and the Caprinae (fig. 2 ). The combined DNA analyses strongly suggest a close evolutionary link between the African Alcelaphinae and Hippotraginae (one unique indel event; bootstrap support higher than 99% in all instances; and 10 decay steps), and this clade forms a sister assemblage to the more morphologically conservative Caprinae, whose origin might be Eurasian (Vrba 1985Citation ). Furthermore, the maximum-likelihood score of the tree presented in figure 2 is significantly better than the best alternative hypothesis, which does not support the monophyly of this group (t = 2.76; P = 0.0057). This proposal enjoys varying paleontological support. Gentry (1990)Citation noted that the Alcelaphinae and Hippotraginae share 26 out of 112 selected morphological characteristics, leading him to conclude that Hippotragini are more closely associated with the Alcelaphini than any other bovid tribe. However, he is also of the opinion that the link between the Caprinae and the African antelope is weak. The caprids are characterized by a combination of symplesiomorphic morphological characters (Gentry 1992Citation ), and in spite of previous mtDNA studies, the evolutionary placement of the Caprinae had remained enigmatic (Gatesy et al. 1997Citation ; Hassanin and Douzery 1999aCitation ; Matthee and Robinson 1999Citation ).

The Caprinae notwithstanding, the tribe Reduncini (herein referred to as the subfamily Reduncinae) is the most difficult group to place among the bovid tribes on morphological grounds (Gentry 1992Citation ). They differ morphologically from the Hippotragini (herein referred to as the Hippotraginae) and share many plesiomorphic morphological features with several members of the Bovinae. Although there is some molecular support from both nuclear (see PRKC1 in fig. 1 in the present study and Hassanin and Douzery [1999bCitation ]) and mtDNA cytochrome b sequences (Hassanin and Douzery 1999aCitation ) to suggest that the Reduncinae form part of an assemblage comprising the Caprinae, the Hippotraginae, and the Alcelaphinae, a definitive statement on the group's phylogenetic position is precluded by inconsistencies in topologies, weak bootstrap support, and low decay index values.

The phylogenetic placement of the Antilopinae and the Cephalophinae, together with the problematic suni, impala, and klipspringer (see above), was not a consistent finding in our investigation. Considerable instability occurred among these taxa when the nuclear genes were analyzed separately, and this phenomenon was reflected by the polytomy found in the combined analyses (fig. 2 ). As with earlier palaeontological, morphological, and molecular analyses (Vrba 1985Citation ; Lowenstein 1986Citation ; Kingdon 1989Citation ; Georgiadis, Kat, and Oketch 1990Citation ; Gentry 1992Citation ; Modi, Gallagher, and Womack 1996Citation ; Gatesy et al. 1997Citation ; Robinson et al. 1998Citation ; Hassanin and Douzery 1999aCitation ; Matthee and Robinson 1999Citation ), the polytomy of the parsimony tree (fig. 2 ) and the short internal branches of the maximum-likelihood tree (fig. 3 ) suggest a rapid radiation within the Bovidae, possibly during the Miocene (Vrba 1985Citation ).

Bovidae Evolution
The outcome of this study, in conjunction with conclusions from previous molecular (Gatesy et al. 1997Citation ; Matthee and Robinson 1999Citation ; Matthee et al. 2001Citation ), morphological, and paleontological studies (Vrba 1985Citation ; Kingdon 1989Citation ; Gentry 1992Citation ), have significantly improved our understanding of bovid evolution. It has been suggested that the antelope lineages became separated from the main Asian boselaphine/bovine lineage near the Oligocene/Miocene boundary approximately 23 MYA (Kingdon 1989Citation ). This early radiation could have resulted from climatic adaptations and/or continental separation of populations (Kingdon 1989Citation ).

Based on the fossil evidence, it is not clear whether the Antilopinae originated in Eurasia or sub-Saharan Africa (Vrba 1985Citation ). Petrified remains reveal that the Antilopinae and the Caprinae were present in Africa near 14 MYA, with Vrba (1985)Citation suggesting that these two evolutionary lineages probably originated in Eurasia at a much earlier date (also see Kingdon 1989Citation ; Gentry 1992Citation ). Wells (1957)Citation stated that fossil remains of the Reduncinae are unrecorded in the African Pliocene record, and the only remains predating 10 MYA (which are vaguely related to this group) are found in India. More recently, Vrba (1985)Citation speculated that the group either originated in Africa during the late Miocene or immigrated from Eurasia. If the molecular association between the Reduncinae and the Caprinae holds, the data at hand would support a Eurasian origin for both the Reduncinae and the Caprinae. The ancestral stock of these lineages probably immigrated into Africa, where they developed into the highly specialized grassland antelopes now represented by the contemporary Reduncinae, Alcelaphinae, and Hippotraginae (Vrba 1985Citation ).

It is noteworthy that the average sequence divergence of 3.68% (±0.32%) between the African tragelaphines (kudu, eland) and the other bovine tribes (buffalo, cow, and nilgai) approximates the 4.14% (±0.54%) average sequence divergence value separating the four mainly African lineages (impala, suni, klipspringer, Cephalophinae) and the Reduncinae/Caprinae/Hippotraginae/Alcelaphinae clade. If the molecular data hold, it seems reasonable that the early diversification of the Bovidae was followed by an African immigration of diverse members belonging to both of the major bovid lineages (Bovinae and a larger "mainly African clade"). Kingdon (1989)Citation has speculated that the larger Eurasian boselaphines adapted to moister habitats in Eurasia, and the ancestral antelope (represented by the smaller eotragine/neotragines) adapted to drier habitats, making their radiation into Africa possible. It follows that the immigration of the Bovidae into Africa was accompanied by rapid secondary adaptations to open habitats, causing numerous speciation events. The rapid speciation events are clearly corroborated by the short internal branches found in this study (fig. 3 ) and, once again, the similarity in sequence divergence distances among most of the subfamilial evolutionary lineages. From these values, it would seem that the African endemics (impala, suni, klipspringer, duikers) originated in Africa at more or less the same time as the first Eurasian immigrations occurred, and these taxa thus represent most likely ancient survivors of the initial bovid radiation in Africa.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 
We thank D. Gallagher Jr., E. Harley, and T. J. Robinson for material/DNA. Comments on earlier drafts of this manuscript were provided by M. Cunningham, E. Douzery, B. Jansen van Vuuren, J. Gatesy, S. Matthee, and T. J. Robinson. This work was conducted while C.A.M. was a postdoctoral fellow at Texas A&M University. Financial support from the National Research Foundation (South Africa) and the Skye Foundation and Charitable Trust is gratefully acknowledged.


    Footnotes
 
Steve Palumbi, Reviewing Editor

1 Keywords: Bovidae systematics nuclear DNA evolution phylogeny Back

2 Address for correspondence and reprints: Conrad A. Matthee, Department of Zoology, University of Stellenbosch, Private Bag X1, Matieland, 7602, South Africa. E-mail: cam{at}maties.sun.ac.za Back


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 Introduction
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Accepted for publication March 14, 2001.