Discussion
We observed a continent-wide allele-frequency cline with decreasing frequencies in a northerly direction for both the DE and SAE loci; two groups of microsatellites that are largely independent of one another (interlocus distance <29 Mb at only two out of 72 DE-SAE locus pairs). We could attribute these clines to selection (DE loci: P= 0.083, SAE loci: P = 0.043, all loci: P = 0.013). Further, we observed an LD cline at the two most closely linked locus pairs (P = 0.014, other three physically linked locus pairs:P = 0.99), with decreasing interlocus associations between male-deleterious-trait-associated alleles in a northerly direction. Most of the interlocus associations at the five physically linked locus pairs were positive (P = 0.030). Continent-wide positive LD in combination with an LD cline (combined P = 0.020) is indicative of genome-wide selection resulting in increased frequencies of deleterious-allele haplotypes (relative to linkage equilibrium). Relatively high DE allele frequencies and near-significant positive LD in East Africa indicate that the male-deleterious alleles are active across the whole latitudinal range. Apparently, there is continent-wide and genome-wide selection for male-deleterious traits with selection strength decreasing from south to north. The only exceptions are the populations in HiP and Nairobi NP, where male-deleterious alleles appear to be under negative selection. According to Figure 2, these two populations seem to have lost many (if not most in Nairobi NP) of their male-deleterious alleles. The selection pressures in East and southern Africa must be high to prevent destruction of the allele-frequency clines and haplotypes by LD decay. These results provide strong support for earlier reported observations of genome-wide selection in KNP and HiP (van Hooft et al., 2018; van Hooft et al., 2019; van Hooft et al., 2014).
Common explanations for allele-frequency clines due to selection assume the advance of a favourable mutation (in our case male-deleterious alleles), an environmental gradient, or a combination of the two (Charlesworth & Charlesworth, 2010; de Jong, Collins, Beldade, Brakefield, & Zwaan, 2013). The advance of a favourable mutation assumes South Africa as the place of origin of the male-deleterious alleles. However, we consider it unlikely that all male-deleterious alleles originated in a relatively small area. The presence of a linear environmental gradient seems an unlikely explanation considering the wide range of habitats and climates in the sampled region that overall are non-linearly distributed. To our knowledge, there is no linear disease gradient in this region either. Further, the advance of a favourable mutation, an environmental gradient or a disease gradient would not explain the high frequencies of the male-deleterious alleles, particularly those also deleterious to females (linked to the DE alleles), which normally are under negative selection (Henn, Botigue, Bustamante, Clark, & Gravel, 2015). It is also implausible that the allele-frequency clines are a result of the selective agent being present only in the most southern part of the range (e.g. South Africa) considering the occurrence of positive LD in East Africa. LD decay is expected to be very fast in the absence of selection (6-31% per generation at the locus pairs analysed in East Africa, considering the chromosomal distances involved).
Instead of the aforementioned explanations, we argue that the continent-wide allele-frequency clines are caused by the same sex-ratio meiotic gene-drive system that earlier has been hypothesized to be the ultimate selective agent in KNP and HiP, although the male-deleterious alleles and gene-drive system have been shown to interact with diseases, such as BTB, and other environmental factors, such as droughts (van Hooft et al., 2018; van Hooft et al., 2019; van Hooft et al., 2014). We hypothesize that there is a similar continent-wide frequency cline of gene-drive alleles, also with decreasing frequencies in a northerly direction, considering that in KNP DE and SAE allele-frequency clines co-occurred with an allele-frequency cline of a Y-chromosomal haplotype linked to a Y-suppressor gene (Y haplotype 557) (van Hooft et al., 2014). According to this hypothesis, the gene-drive system originated in southern Africa and subsequently spread north, thereby forming a selection gradient. Relatively low frequencies of Y haplotype 557 in other southern African populations (except HiP) support this hypothesis (frequency in KNP: 0.24, 95% CI: 0.19, 0.30; average frequency in four other populations: 0.07, 95% CI: 0.02, 0.18 (Smitz et al., 2014; van Hooft et al., 2010)). The male-deleterious alleles (which occur on the autosomes) probably originated at various localities throughout Africa, considering that mutations leading to such alleles can in principle occur in any population, after which they formed an allele-frequency cline in response to the aforementioned selection gradient (formed by the gene-drive system, which occurs on the sex chromosomes).
When the gene-drive system is incomplete, selection may become negative, resulting in relatively low DE and SAE allele frequencies. This has earlier has been argued for HiP and may also apply for Nairobi NP (van Hooft et al., 2019). In both cases, incompleteness of the gene drive system may be due to strong genetic drift in these small and isolated populations (HiP: ≤ 75 individuals between 1895 and 1930, Nairobi NP: current census size ~150; these small sizes probably contributed to the relatively large pairwise F STvalues with other populations, Figure S1) (Heller, Okello, & Siegismund, 2010; van Hooft et al., 2019).
Our explanation for the allele-frequency clines implies that frequencies of male-deleterious alleles and haplotypes on the autosomes are maintained at specific equilibrium values in each population depending on the local frequencies of the gene-drive alleles on the sex chromosomes. This would require some form of balancing selection (which does not preclude periods of positive or negative selection such as observed in KNP and HiP, respectively (van Hooft et al., 2018; van Hooft et al., 2019; van Hooft et al., 2014)). We previously hypothesized that epigenetic suppression of male-deleterious alleles results in balancing selection of Y-chromosomal genes in the gene-drive system (van Hooft et al., 2018). Taking cognizance of an association between epigenetic suppression and low pre-birth rainfall, we argued that males with many active male-deleterious alleles should tend to produce offspring with suppressed male-deleterious alleles and vice versa (males with few active alleles producing offspring with many active alleles). We further hypothesize here that the same process also results in balancing selection of male-deleterious alleles and haplotypes.
Positive LD at linked loci indicates increased frequencies of deleterious-allele haplotypes relative to linkage equilibrium. We detected long-distance LD at two pairs of loci, both ~28 Mb apart; these pairs occur on chromosomes 1 and 3 in cattle. Long-distance LD appears to occur genome-wide considering that significant LD has also been observed in KNP for theINFNG -GLYCAM1 locus pair, which has an interlocus distance of 18 Mb and occurs on chromosome 5 in cattle (D ’ = 0.28, i.e. LD is at 28% of its maximum possible value) (Ihara et al., 2004; Lane-deGraaf et al., 2015). This is a relatively large distance, especially considering that high haploid diversity indicates a large effective population size for the KNP buffalo (34 mitochondrial D-loop haplotypes with H (gene diversity) = 0.94, 15 Y-chromosomal haplotypes with H = 0.74; census size ~37,000 in 2010) (Greyling, 2007; van Hooft et al., 2018; van Hooft et al., 2007). According to population genetic theory, a large effective population size limits long-distance LD because of increased LD decay with chromosomal distance (Slatkin, 2008). LD in buffalo populations extends across much larger chromosomal distances than in other natural mammal populations that we are aware of for which LD decay has been estimated. In chimpanzees (Pan troglodytes ) and bonobos (Pan paniscus ) LD extends to a distance of ~0.15 Mb, in Arizona wild mice (Mus musculus domesticus ) to a distance of ~0.2 Mb, in Iberian wild boar (Sus scrofa ) to a distance of ~0.5 Mb, and in gray wolf (Canus lupus ) and coyote (Canus latrans ) to a distance of ~5 Mb, even in small or bottlenecked populations (except for the wolf population of Isle Royale, which consisted of just 10-30 individuals) (De Manuel et al., 2016; Gray et al., 2009; Herrero-Medrano et al., 2013; Laurie et al., 2007; Munoz et al., 2019). Further, the half-length of LD (the distance at which LD is 50% of its maximal value) in two isolated Canadian populations of bighorn sheep (Ovis canadensis ) is only 17%-28% of that in KNP buffalo, despite their small size of less than 200 individuals each (4.6-7.5 Mb vs. 26.4 Mb, Text S4) (Miller, Poissant, Kijas, Coltman, & Int Sheep Genomics, 2011; Miller, Poissant, Malenfant, Hogg, & Coltman, 2015).
LD between distant loci in large outbreeding buffalo populations despite fast LD decay due to recombination (~20-31% per generation), indicates strong selection pressures. However, simulation studies indicate that multilocus selection may lower the minimum selection pressure necessary for a given level of LD and may slow down LD decay in a multilocus cline (although even then selection is probably still relatively strong) (Baird, 1995; Hastings, 1984). Further, allele frequency differences between male and female gametes due to sex-specific selection, as hypothesized in KNP based on genetic data from male and female calves (van Hooft et al., 2018), may have resulted in admixture LD between distant loci, especially when selection is strong (Úbeda et al., 2011).
A high number of high-frequency deleterious alleles of seemingly large effect (van Hooft et al., 2018; van Hooft et al., 2014), with many co-occurring in haplotypes (relative to what one would expect if no linkage existed among these alleles), indicates a high genetic load. However, most populations of African buffalo seem to be stable after their recovery from the rinderpest pandemic of 1889-1895, which decimated buffalo populations across the whole of Africa (Bengis, Kock, & Fischer, 2002; van Hooft et al., 2000). At face value, this seems to support the view, advocated by some population geneticists, that genetic load plays a smaller role than one might expect in ecology (Agrawal & Whitlock, 2012).
We suggest the following five reasons, among others that surely exist, as non-mutually exclusive explanations why buffalo populations are still stable.
  1. Male-deleterious alleles are epigenetically suppressed in a large fraction of animals (van Hooft et al., 2018; van Hooft et al., 2019).
  2. Net-deleterious effects on female health are diminished or even prevented by male-deleterious alleles or haplotypes with negative phenotypic effects in males but positive phenotypic effects in females (i.e., associated with SAE microsatellite alleles) (van Hooft et al., 2014).
  3. Negative phenotypic effects only become evident in stressful periods, such as those caused by droughts and disease outbreaks. For example, in 2001 average body condition was lower in southern KNP, a region characterized by relatively high frequencies of DE and SAE alleles, than in northern KNP, but only significantly so at the end of the dry season (Caron, Cross, & Du Toit, 2003). It may even be that male-deleterious alleles are mainly expressed during times of high environmental stress considering that most genetic samples used in the studies on KNP and HiP were collected during dry seasons (van Hooft et al., 2018; van Hooft et al., 2019; van Hooft et al., 2014), and then mainly in animals born during stressful periods because these cohorts do not experience epigenetic suppression (van Hooft et al., 2018; van Hooft et al., 2019).
  4. Selection is mostly soft, where selective death due to male-deleterious alleles would otherwise be replaced by nonselective death due to environmental and ecological conditions, e.g. droughts, diseases and intraspecific resource competition (Agrawal & Whitlock, 2012).
  5. Interspecific competition is limited, thereby minimizing the ecological effects of genetic load (Agrawal & Whitlock, 2012). A recent review has failed to find conclusive evidence for interspecific competition between African buffalo and other large mammals (Prins, 2016), which may be due to ecological separation (Traill, 2004).
Our analyses and results reveal a continent-wide and genome-wide distribution of high-frequency male-deleterious alleles in the African buffalo, with many co-occurring in haplotypes (relative to what one would expect if no linkage existed among these alleles). Since most populations appear to be stable, this indicates that, under specific circumstances, natural populations of mammals can withstand a high genetic load. Nevertheless, we expect that a high genetic load makes many buffalo populations vulnerable to environmental stresses such as droughts and disease outbreaks. Since buffalo play an important role in the maintenance and transmission of a variety of economically important livestock diseases (Michel & Bengis, 2012), which may well have been augmented by a high genetic load, particularly in southern Africa, our results have relevance to livestock husbandry in areas were cattle graze in close proximity to buffalo herds.