Phenotypic differences, spatial distribution and diversity at the Cytb and BMP4 genes in springbok (Antidorcas marsupialis)

Mammalian Biology 77 (2012) 391–396

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Original Investigation

Phenotypic differences, spatial distribution and diversity at the Cytb and BMP4 genes in springbok (Antidorcas marsupialis) E. Van Aswegen a , C. Labuschagne b , J.P. Grobler a,∗ a b

Department of Genetics, University of the Free State, PO Box 339, Bloemfontein 9300, South Africa Inqaba Biotechnical Industries, PO Box 14356, Hatfield 0028, South Africa

a r t i c l e

i n f o

Article history: Received 30 September 2011 Accepted 29 November 2011 Keywords: Antidorcas marsupialis Cytb BMP4 tandem repeat Phenotype

a b s t r a c t The springbok (Antidorcas marsupialis) is a significant contributor to the economically important game ranching sector in Southern Africa. Phenotypic variation between springbok from the Karoo and Kalahari regions has been reported by several sources, with springbok from the Kalahari regarded as the larger form. There is no consensus on whether the two variants are determined by heredity, environment or a combination of the two. We studied variation in 80 individuals from four springbok populations using both a gene widely used for population studies (Cytb) and a gene that effects growth (BMP4). Results from Cytb haplotypes and BMP4 diploid gene sequences reveal moderate differentiation among springbok sampled from different regions. We also found a CA tandem repeat motive with high variability at the 3 end of the BMP4 gene region sequenced (the third exon). There is some support for a hypothesis that nominally short and long fragments at this BMP4 repeat are associated with different populations, which may indicate either neutral genetic differentiation between spatially isolated forms, or a relationship between phenotype and BMP4 genotype. We also present new primer sequences to amplify both a partial fragment of the BMP4 gene region and the complete BMP4 tandem repeat motive in springbok. © 2011 Deutsche Gesellschaft für Säugetierkunde. Published by Elsevier GmbH. All rights reserved.

Introduction Springbok occurs in the arid areas of Southern Africa (Fig. 1) where grasslands or savannas with short-growing grasses occur (Chain et al. 2004). The springbok is a significant contributor to the economically important game ranching sector and is widely utilized in the commercial wildlife ranching and hunting industry in South Africa (SA). It is the game species most extensively cropped in South Africa for the local and export venison market (Hoffman and Wiklund 2006; Hoffman et al. 2007). Phenotypic variation in springbok is well known with several color variants that have been reported (Kruger et al. 1979; Hetem et al. 2009). Normal coloration consists of cinnamon-brown dorsal parts with white ventral areas, separated by a reddish-brown band (Estes 1991). Color aberrations that occur at low frequencies have been exploited by commercial game farmers, leading to a proliferation of black, white and copper colored springbok populations. Size differences between springbok from different regions of Southern Africa have also been reported. The size differences correspond broadly to two geographic regions, the Karoo and Kalahari. Estes (1991) reported that male springbok from the Karoo region

∗ Corresponding author. Tel.: +27 514013844; fax: +27 865187317. E-mail address: [email protected] (J.P. Grobler).

have an average shoulder height of 73 cm and weigh 30.6 kg, compared to 77–87 cm/41 kg for males from the Kalahari region. There is currently no consensus on whether the size variants are determined by heredity, environment or a combination of both. Large body size has been used as a criterion for active selection by farmers resulting in translocation of Kalahari type animals to the Karoo region in South Africa, in an attempt to improve average body size in populations. Anecdotal evidence from some farmers suggested that gains are short-lived and the average size of individuals revert to pre-augmented standards within 2–3 generations; whereas others claim that augmentation followed by selection resulted in sustained improvements in the average body size of herds. Early genetic and taxonomic studies of springbok focused on the status of sub-species in A. marsupialis. Three subspecies of springbok were formerly recognized, with A. m. marsupialis (Zimmermann, 1780) at the southern edge of the distribution range in South Africa; A. m. hofmeyri (Thomas, 1926) in the northern parts of South Africa, as well as in Botswana and Namibia; and with A.m. angolensis (Blaine, 1922) in Angola (Bigalke 1970; Ansell 1972). Robinson (1975) questioned the continued recognition of the three subspecies, based on karyological evidence, allozymes and skull morphology. Subsequently, Peters and Brink (1992) suggested that there may be significant size differences among springbok, corresponding to the ranges of A. m. marsupialis and A. m. hofmeyri. In later genetic studies of springbok, the focus shifted to population

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Fig. 1. The distribution of springbok in Southern Africa (shaded in light grey), with sampling localities ().

genetics (Bigalke et al. 1993) and a possible link between inbreeding and fluctuating asymmetry in this species (Grobler et al. 1999). Studies in conservation genetics routinely aim to use neutral markers, to ensure that the effects of processes such as drift and inbreeding are not masked by the signature of selection. For example, Cytb has often been used to elucidate taxonomic disputes in the Cetartiodactyla (Hassanin and Douzery 1999; Rodríguez et al. 2009) and also the Bovidae, to which the springbok resort (Matthee and Robinson 1999). However, with possible genetic differentiation in springbok potentially expressed as a detectable size difference, the use of a marker with possible adaptive significance relating to bone growth could add valuable insight into the evolutionary biology of this species, considering that the confusion on subspecies status was at least partially based on size differences. One candidate gene region for such analysis is the bone morphogenetic protein (BMP) genes. Among the 20 genes in this group, BMP4 has been widely studied and postulated to play a role in skeletal development (Hogan 1996), bone density (Mangino et al. 1999) and fracture healing (Nakase et al., 1994). In domestic animals, an association between a tandem repeat motive within BMP4 and phenotypic variation in cattle was suggested by Zhong et al. (2010). Since a potential link between BMP4 and bone parameters exists, it was decided to use diversity at this gene as an added measure of differentiation between the two phenotypic forms in springbok. In this paper, we describe a project where diversity at a neutral gene and a presumably more adaptive gene was correlated to geographic origin and phenotypic differences in springbok. Specifically, we aim to describe differences in Cytb and BMP4 variability in springbok from four populations with diverse founding and management histories and investigate the possible link to the phenotype.

Material and methods Sampling Twenty springbok were sampled from each of four farms [with abbreviation used in brackets] (Fig. 1). Springbok described as pure Karoo type were collected from the farm Renostervlak in

the Northern Cape (NC) Province of SA [Karoo]. Pure Kalahari type springbok were sampled from the farm Süs in the Mariental district of Namibia [Kalahari]. Two mixed populations were also sampled: (i) Wonderboom farm (NC, SA), which hosts a population based on former Karoo-type springbok, with intensive directional selection for increased body size and with augmentation using Kalahari type springbok [Selected]. (ii) Jakkalsfontein farm (NC, SA), which hosts a mixed population of both indigenous Karoo- and introduced Kalahari type springbok, but without the intense selection practiced at the Wonderboom locality [Mixed]. Sampling kits (with ethanol-filled tubes) and information pamphlets were distributed to the four farmers, who then collected samples when springbok were hunted or culled on their properties. Muscle tissue samples (approximately 2 cm3 ) were stored in 10 ml collection tubes with 90% ethanol and sent to the Department of Genetics at the University of the Free State.

Genetic analysis Genomic DNA was isolated using the Roche Diagnostics High Pure Template Preparation kit (Roche Diagnostics, Germany) following manufacturers specifications. The Cytb mtDNA gene region was amplified using the universal primers L14724 and H15149, as described by Irwin et al. (1991) and modified by Nagata et al. (1995) (L14724: 5 -GATATGAAAAACCATCGTTG-3 ; H15149: 5 -CTCAGCCTGATATTTGTCCTCA-3 ). Primers to amplify the BMP4 gene region were designed using a selection of mammalian BMP4 gene sequences available on GenBank, based on conserved areas across aligned sequences. The sequences of the designed primers were: BMP4-NEW-F: 5 -CCTCTTTAACCTCAGCAGCATCC-3 ; BMP4NEW-R: 5 -GCTATAAGGAAGCRGTCTGTGTAG-3 . Identical reaction mixtures were used for both gene regions. Mixtures contained 12.5 ␮l Econotaq plus green master mix (Lucigen, Middleton, WI), 1 ␮l of each primer (from a 10 ␮M solution), ±80 ng genomic DNA and 9.5 ␮l ddH2 O, for a final volume of 25 ␮l. Thermal cycling conditions for Cytb consisted of an initial denaturation step of 95 ◦ C for 5 min, followed by 45 cycles each of 95 ◦ C for 30 s, 50 ◦ C for 30 s and 72 ◦ C for 1 min; with a final extension step of 72 ◦ C for 10 min and a hold at 4 ◦ C. Thermal cycling conditions for BMP4

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Table 1 Haplotypes (designated based on GenBank numbers), haplotype frequencies, shared haplotypes and indices of population diversity for Cytb in four populations of springbok. Population Karoo Haplotype JN254621 JN254622 JN254623 JN930833 JN254620 JN254618 JN254619 JN254625 JN254624 JN254626 Proportion unique haplotypes (%) Nucleotide diversity (%)

0.750 0.200 0.050 – – – – – – – 25.0 0.133 (±0.128)

were the same except for an increase in annealing temperature to 59 ◦ C and a lengthened extension step of 2 min at 72 ◦ C during each cycle. Thermal cycling was carried out on an ABI 9700 Thermocycler (Perkin-Elmer Applied Biosystems, Foster City, CA). Amplification products were purified using ExoSaP before cycle sequencing. Sequencing reactions were performed using the ABI Big Dye Terminator Cycle Sequencing kit version 3.1 (Applied Biosystems, Foster City, CA) according to manufacturer’s specification. Products of sequencing reactions were purified using the Zymo Research Sequencing Clean-up kit (Zymo Research, Orange, CA) according to manufacturer’s specification and analyzed on the ABI 3500XL Genetic analyzer (Applied Biosystems, Foster City, CA). Initial screening of BMP4 gene sequences generated revealed a di-allelic tandem repeat motive (with 15–19 repeats of a CA motive) at the 3 -end of the region sequenced and we thus designed an additional primer to specifically amplify this region to enable fragment analyses without the need to sequence the entire gene. This primer, designated BMP4-Fam-F-mid (5 -FAM-GCTGACCACCTCAACTC-3 ), was used in conjunction with primer BMP4-NEW-R described above. The reaction mixtures for fragment analysis contained 12.5 ␮l Econotaq Plus green master mix (Lucigen, Middleton, WI), 1ul of each primer (from 10 ␮M stock solutions), 1 ␮l of the product of previous BMP4 PCR reactions as template, and 9.5 ␮l ddH2 O for a final volume of 25 ␮l. Thermal cycling conditions consisted of an initial denaturation step of 95 ◦ C for 5 min, followed by 35 cycles each of 95 ◦ C for 30 s, 59 ◦ C for 30 s and 72 ◦ C for 2 min; and 72 ◦ C for 10 min. Prior to analyses on the ABI 3130XL Genetic analyzer (Applied Biosystems, Foster City, CA), 1 ␮l of the PCR product was diluted with 9 ␮l water and 1 ␮l of the subsequent dilution mixed with 9.7 ␮l Hi-di Formamide (Applied Biosystems, Foster City, CA) and 0.3 ␮l size standard LIZ500 (Applied Biosystems, Foster City, CA). All primers were synthesised and purified by Inqaba Biotechnical Industries Pty., Ltd., South Africa Statistical analysis Sequences of the Cytb and BMP4 gene regions were aligned and inspected with Mega5 (Tamura et al. 2011), and annotated used Artemis ver. 13.0 (Rutherford et al. 2000) before submission to GenBank. Subsequent statistical analysis was done using three approaches: (i) Cytb gene sequences were analyzed as haplotypes using Mega5 and ARLEQUIN 3.1 (Excoffier et al. 2005). Genetic diversity within populations was quantified as nucleotide diversity and haplotype diversity in each population. A medianjoining network of springbok haplotypes was constructed using NETWORK, version 4.5 (Bandelt et al. 1999). (ii) The BMP4 gene region, excluding the tandem repeat motive, was analyzed as a diploid gene region, with polymorphisms at specific sites treated as

Kalahari

Mixed

0.563 – – 0.188 0.125 0.063 0.063 – – –

0.824 – – – – – – 0.176 – –

433.7 0.199 (±0.169)

17.6 0.153 (±0.141)

Selected 0.889 – – – – – – – 0.056 0.056 11.1 0.055 (±0.076)

Fig. 2. Median-joining network between Cytb haplotypes of springbok. Nodes represent haplotypes, with the size of each node representing the number of individuals that share that haplotype. Cross-bars reflect the number of mutational events between specific haplotype-pairs. Colors used to indicate population(s) of origin of haplotypes: Karoo , Mixed 䊉, Selected 䊉 and Kalahari 䊉; with  denoting a median vector.

heterozygotes at different loci. (iii) After separate amplification of the di-allele tandem repeat region, microsatellite fragments were scored using GeneMarker 1.6 (Softgenetics© ) software. MSToolkit (Park 2001) was used to organize microsatellite data, determine allelic and genotypic frequencies and calculate basic measures of genetic diversity (Unbiased heterozygosity – Hz, Nei 1987 and mean number of alleles per locus – A). Differentiation among pairs of populations was calculated using uncorrected P-distance for Cytb haplotypes and diploid BMP4 sequences, and Fst (Wright 1965) for tandem repeats, using ARLEQUIN. A sequential Bonferroni correction was used to account for Type I errors during these multiple pairwise comparisons (Rice 1989). Results A 405 basepair (bp) length of the Cytb mtDNA gene was successfully amplified in 71 springbok. Ten different haplotypes were recorded in these individuals (GenBank accession nos. JN254618–JN254626 and JN390833). Haplotype frequencies and nucleotide diversity are presented in Table 1. The same haplotype (JN254621) occurred in 54 individuals, representing the most common allele in all populations (75.0% of Karoo; 82.4% of Mixed; 88.9% of Selected; 56.2% of Kalahari). Low numbers of unique haplotypes were however found in all populations (Karoo = 2, Kalahari = 4, Mixed = 1, and Selected = 2). Nucleotide diversity ranged from a low of 0.055% in the Selected group to 0.199 in the Kalahari population (Table 1). A median-joining network showing the relationship among haplotypes is presented in Fig. 2. As expected from haplotype frequencies, most individuals from all populations were

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Table 2 Population differentiation among 4 populations of springbok, based on (i) haplotypes of the Cytb mtDNA gene, (ii) genotypes of the diploid BMP4 gene and (iii) frequencies of the BMP4 tandem repeat region. Population pairs

(i) Differentiation (P-distance) from Cytb haplotypes

(ii) Differentiation (P-distance) from BMP4 diploid sequences

(iii) Fst from the BMP4 tandem repeat area only

Karoo–Kalahari Karoo–Mixed Kalahari–Mixed Karoo–Selected Kalahari–Selected Mixed–Selected

0.092 (p = 0.006) 0.117 (p = 0.013) 0.099 (p = 0.013) 0.073 (p = 0.084) 0.067 (p = 0.008) 0.098 (p = 0.102)

0.035 (p = 0.042) 0.115 (p = 0.009) 0.111 (p = 0.001) 0.030 (p = 0.080) 0.041 (p = 0.018) 0.099 (p = 0.001)

0.097 (p = 0.001) 0.061 (p = 0.061) 0.073 (p = 0.005) 0.008 (p = 0.266) 0.024 (p = 0.119) 0.017 (p = 0.134)

Table 3 Polymorphic loci (nucleotide position), nature of polymorphisms and frequency of polymorphisms at the diploid BMP4 gene in four springbok populations. Locus

69 82 198 199 389 518

Nature of polimorphism

Population

A/G C/T A/T A/G A/G C/T

Unbiased heterozygosity

contained in a single node, with some lower frequency haplotypes occurring in all populations with none of these low-frequency haplotypes occurring in more than one population. P-distance between all population pairs are presented in Table 2(i). Differentiation between the Karoo and Kalahari populations was potentially significant with Fst = 0.092 and P = 0.006. Approximately 800 bp of the BMP4 gene was successfully amplified in all individuals studied. Based on BLAST results, this region shows 98% identity with the third exon of the BMP4 gene in cattle (GenBank accession no. NM 001045877). Polymorphic positions were identified within the gene as well as a 3 di-allelic tandem repeat motive (CA), as described above. The BMP4 data were thus analyzed using two approaches: a 768 bp region (excluding the tandem repeat motive) was analyzed as a diploid gene sequence in all individuals, with variation at the tandem repeatmotive analyzed separately by fragment analysis. For the 768 bp diploid gene sequence, seven specific genotypes were found, with GenBank accession numbers JN254611–JN254617. Heterozygotes were observed at six positions (designated loci) (Table 3). Nonoverlapping heterozygous loci were scored in the Karoo and Kalahari populations, with a mix of Karoo, Kalahari and unique

Karoo

Kalahari

Mixed

Selected

– – – 0.142 0.142 –

– 0.056 0.056 – – –

– – 0.056 – – 0.322

0.214 0.059 0.059 – 0.059 –

0.037

0.014

0.049

0.051

alleles in the Selected population and a combination of Kalahari and unique alleles in the Mixed population. P-distance between these populations was 0.035 with P = 0.042 (Table 2(ii)). Heterozygosity values ranged from 0.014–0.037% in the Karoo and Kalahari populations to 0.049–0.051 in the Mixed and Selected populations (Table 3). The BMP4 tandem repeat area proved highly polymorphic in all populations (Table 4(i)), with 5–8 alleles scored per population (Hz = 0.758–0.844). Differentiation between the Karoo and Kalahari population was potentially significant, with Fst = 0.097 and P = 0.001 Table 2(iii). In line with suggestions that intragenic tandem repeats generate functional variability (Verstrepen et al. 2005), we screened the frequencies of all alleles in the Karoo and Kalahari populations, in an attempt to find possible trends among nominally “short” and “long” repeat motives. Results show that the Kalahari population contains mostly fragments of 290 bp or shorter (60% of alleles), whereas the Karoo contains mostly fragments of 292 bp or longer (76.3%). This pattern holds true for six out of seven alleles at the locus. A subsequent classification of fragments of 286–290 bp as “short” and 292–300 bp as “long” led to notable trends among populations: a total of 23.7% of alleles

Table 4 Diversity at the BMP4 tandem repeat region in four springbok populations. (a) Allele frequencies and diversity at the BMP4 tandem repeat region; and (b) percentage of genotypes with nominally long and short alleles. Population Karoo

Kalahari

Mixed

Selected

(i) Alleles and allele frequencies 286 288 290 292 294 296 298 300 Unbiased heterozygosity Average number of alleles:

– 0.079 0.158 0.368 – 0.290 – 0.105 0.758 5

– 0.375 0.225 0.100 0.150 0.125 – 0.025 0.785 6

0.075 0.050 0.300 0.300 0.175 0.025 0.050 0.025 0.797 8

0.083 0.167 0.194 0.250 0.056 0.194 0.056 – 0.844 7

(ii) Frequencies of genotypic combinations Genotypes with short alleles only Genotypes with long alleles only Genotypes with long and short alleles combined

0.053 0.579 0.368

0.450 0.250 0.300

0.150 0.300 0.550

0.167 0.278 0.555

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scored in the Karoo population were short, whereas a higher proportion of 44.4–60% of alleles scored in the Kalahari and Selected populations classified as short. Conversely, long alleles were the most common type scored in the Karoo population (76.3%), with a lower proportion of long fragments in the Kalahari and Selected populations (40–55.6%). Values for the Mixed population were intermediate to the Karoo and Kalahari values. The numbers of specific genotypes were also investigated to identify trends in the occurrence of nominally long and short alleles (Table 4(ii)). Only 5.3% of Karoo animals possessed genotypes with two short fragments (in either homozygous or heterozygous form). By comparison 16.7% of Selected and 45.0% of Kalahari animals contained only short tandem repeats. Frequencies of long alleles revealed a similar and more robust trend: 57.9% of Karoo animals possessed two nominally long alleles, whereas 25% of Kalahari type animals had genotypes with two long tandem repeats. Values for the Mixed populations (25–30%) were intermediate to Karoo and Kalahari (Table 4(ii)). The Mixed population also contained a high proportion of combined long and short alleles (55.0–55.5%).

Discussion Under an assumption of completely neutral genetic drift between the Karoo and Kalahari types, similar patterns of differentiation would be expected for different genes. Results from Cytb haplotypes and BMP4 diploid gene sequences in springbok revealed similar patterns of polymorphism. These patterns conform to the geographic origins of animals and thus do not provide any evidence of a link between genotype and phenotype. For the mtDNA Cytb gene region, the same haplotype was shared by 76% of individuals, comprising individuals from all populations sampled (56–89% of individuals from each population). Nevertheless, there were unique haplotypes in all populations, with such population-specific haplotypes accounting for 25% and 44% of haplotypes in the Karoo and Kalahari populations respectively. The Mixed and Selected populations contained low numbers of unique haplotypes not found in the Karoo or Kalahari populations, which may have been present in the source populations from which the former two populations were founded. As for Cytb, results from the diploid BMP4 gene sequences revealed polymorphism that showed a link to geographic origin and partly confirm population history in the Mixed and Selected populations. Two distinct heterozygous loci were identified in each of the geographically separate Karoo and Kalahari populations, with no shared polymorphism. Genotypes found in the Selected population included a mix of Karoo and Kalahari specific alleles, likely caused by mixing of Karoo and Kalahari type springbok during the management history of this population. The presence of unique alleles in the Selected population may be attributed to the legacy of resident springbok on the property, present before the start of selection and translocations. Genotypic frequencies in the Mixed population were less informative, with a combination of Kalahari and unique alleles, but no nominal Karoo alleles. Differentiation among population pairs were investigated with the hypothesis that under a model of linkage between genotype and phenotype, the distance between Karoo and Kalahari (the extreme phenotypes) should be greater than the distance between Kalahari and Selected (potentially with convergent phenotypes). Similarly distances between Karoo and Selected (with slightly dissimilar phenotypes) should be greater compared to Karoo–Kalahari (with more similar phenotypes). Under a model with no link between genotype and phenotype, distance between composite populations and pure populations should reflect admixture but not the patterns described above. For Cytb haplotypes, the P-distance between

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Karoo and Kalahari was 0.092, whereas the distances between Kalahari–Selected and Karoo–Selected is lower and very similar (0.067 and 0.073). With no major difference between the latter two values, the P-distance values most likely reflect neutral admixture. For the BMP4 diploid gene, P-distance between Karoo–Kalahari as well as Kalahari–Selected and Karoo–Selected are all very similar (0.035, 0.041 and 0.030). In fact, the value for Karoo–Selected (0.030) indicates slightly more identity between these populations compared to P-distance for the Kalahari–Selected pair (0.041). Overall, these patterns support the results from Cytb haplotypes in suggesting only neutral drift. In contrast to the results from Cytb and BMP4 gene sequences, the distribution of specific allele sizes for the BMP4 tandem repeat region provided good support for a hypothesis of interaction between fragment length and phenotype-based management history, in addition to confirming geographic origin. Only 23.7% of alleles scored in the Karoo population were classified as short, compared to 60% of alleles scored in the Kalahari population. The intensively managed Selected population had an intermediate number of short alleles (44.4%), with a comparable proportion in the Mixed population (42.5%). In terms of genotypes, only 5.3% of Karoo animals possessed genotypes consisting of two short fragments (in either homozygous or heterozygous combinations), whereas 45.0% of Kalahari animals contained combinations of short alleles alone. Values for artificially managed populations were intermediate, with 16.7% combinations of short alleles in the Selected population and 15% in Mixed population. In all cases, trends from long alleles were similar (though opposite). A conservative interpretation of the results from the BMP4 repeat region would be that that the observed polymorphism is also neutral, as with the gene sequences analyzed. In this scenario, the spatially separated Karoo and Kalahari populations contain predominantly shorter (Karoo) or longer (Kalahari) alleles of the tandem repeat, representing neutral divergence due to drift or similarly random evolutionary processes. The Selected and Mixed populations have a histories of artificial management that include augmentation from extra-limital populations and the signature of these events are evident from the mixture of pure “Karoo” and “Kalahari” genotypes observed in these populations. It was noted that levels of diversity in the two artificially managed populations were higher compared to levels in the populations that were conserved as pure regional types, which provide further evidence of admixture occurring during the management history of the former two populations. A second possible interpretation of our results may be that the tandem repeat polymorphism observed at the BMP4 gene is indeed related to the body-size differences. Variation in the numbers of tandem repeats present in coding sequences, regulatory regions and introns have been shown to influence gene expression and create quantitative alterations in phenotypes (Verstrepen et al. 2005; Vinces et al. 2009). The patterns of diversity at the BMP4 gene may thus be more directly linked to body size difference, as proposed by Zhong and colleagues (2010) for cattle. In this paper, we present new data on patterns of genetic diversity at the Cytb and BMP4 gene regions in springbok. Our data show slight differences between geographically isolated Karoo and Kalahari springbok that were detectable from sequences of both genes, and from fragment lengths at the BMP4 tandem repeat region. These results can be explained by neutral drift between populations, but there is also support for a more direct link between BMP4 tandem repeat allele length and body-size differences in springbok. We suggest that future research should include a larger number of pure and admixed populations. Larger sample sizes could also provide insight into the origin of the unique alleles found in admixed population, but not in the Karoo or Kalahari springbok. Furthermore, body size in Karoo and Kalahari populations and selection

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gains in mixed populations should be quantified and correlated to the occurrence of nominally short and long alleles of the BMP4 tandem repeat. Such analysis should also include phenotypic variation within admixed populations, in contrast to the treatment of populations as homogeneous phenotypic units during the current study. Acknowledgments The authors wish to thank Dr. N.E van Aswegen and Dr. J. Brink for valuable discussions on genes associated with limb development, Mr. D van Aswegen for the collection of the samples during culling of springbok on the various farms in SA and the four landowners for permission to sample their populations. Samples from Namibia were collected under permit no. 1507/2010. Funding was provided by the National Research Foundation of South Africa (NRF) and the University of the Free State. We would like to thank two anonymous reviewers for valuable comments on the first version of this paper. References Ansell, W.F.H., 1972. Order Artiodactyla. In: Meester, J., Ä Setzer, H.W. (Eds.), The Mammals of Africa: An Identification Manual, 15. Smithsonian Institution Press, Washington, DC, pp. 1–84. ˝ Bandelt, H.-J., Forster, P., Rohl, A., 1999. Median-joining networks for inferring intraspecific phylogenies. Mol. Biol. Evol. 16, 37–48. Bigalke, R.C., 1970. Observations on springbok populations. Zool. Africana 5, 59–70. Bigalke, R.C., Hartl, G.B., Berry, M.P.S., Van Hensbergen, H.J., 1993. Population genetics of the springbok Antidorcas marsupialis – a preliminary study. Acta Theriol. 38, 103–111. Blaine, G., 1922. Notes on the zebras and some antelopes of Angola. Proc. Zool. Soc. Lond. 1922, 333–336. Chain III, W.J., Krausman, P.R., Germain, H.L., 2004. Mammalian species: Antidorcas marsupialis. Am. Soc. Mammal., 753. Estes, R.D., 1991. The Behavior Guide to African Mammals: Including Hoofed Mammals, Carnivores Primates. Russel Friedman Books, Halfway House. Excoffier, L., Laval, G., Schneider, S., 2005. Arlequin, Version 3.1: An Integrated Software Package for Population Genetics Data Analysis Computational and Molecular Population Genetics Laboratory (CMPG). Institute of Zoology, University of Berne, Switzerland. Grobler, J.P., Taylor, P., Pretorius, D.M., Anderson, P.C., 1999. Fluctuating asymmetry and allozyme variation in an isolated springbok (Antidorcas marsupialis) population from the Chelmsford Nature Reserve. Acta Theriol. 44 (2), 183–193. Hassanin, A., Douzery, E.J.P., 1999. The tribal radiation of the family Bovidae (Artiodactyla) and the evolution of the mitochondrial Cytochrome b gene. Mol. Phylogenet. Evol. 13, 227–243. Hetem, R.S., de Witt, B.A., Fick, L.G., Fuller, A., Kerley, G.I.H., Meyer, L.C.R., Mitchell, D., Maloney, S.K., 2009. Body temperature, thermoregulatory behaviour and pelt characteristics of three colour morphs of springbok (Antidorcas marsupialis). Comp. Biochem. Physiol. A 152, 379–388.

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