Clare D. Marsden

Bottlenecks and selective sweeps during domestication have increased deleterious genetic variation in dogs

Significance Dogs have an integral role in human society, and recent evidence suggests they have a unique bond that elicits a beneficial hormonal response in both dogs and human handlers. Here, we show this relationship has a dark side. Small population size during domestication and strong artificial selection for breed-defining traits has unintentionally increased the numbers of deleterious genetic variants. Our findings question the overly typological practice of breeding individuals that best fit breed standards, a Victorian legacy. This practice does not allow selection to remove potentially deleterious variation associated with genes responsible for breed-specific traits. Population bottlenecks, inbreeding, and artificial selection can all, in principle, influence levels of deleterious genetic variation. However, the relative importance of each of these effects on genome-wide patterns of deleterious variation remains controversial. Domestic and wild canids offer a powerful system to address the role of these factors in influencing deleterious variation because their history is dominated by known bottlenecks and intense artificial selection. Here, we assess genome-wide patterns of deleterious variation in 90 whole-genome sequences from breed dogs, village dogs, and gray wolves. We find that the ratio of amino acid changing heterozygosity to silent heterozygosity is higher in dogs than in wolves and, on average, dogs have 2–3% higher genetic load than gray wolves. Multiple lines of evidence indicate this pattern is driven by less efficient natural selection due to bottlenecks associated with domestication and breed formation, rather than recent inbreeding. Further, we find regions of the genome implicated in selective sweeps are enriched for amino acid changing variants and Mendelian disease genes. To our knowledge, these results provide the first quantitative estimates of the increased burden of deleterious variants directly associated with domestication and have important implications for selective breeding programs and the conservation of rare and endangered species. Specifically, they highlight the costs associated with selective breeding and question the practice favoring the breeding of individuals that best fit breed standards. Our results also suggest that maintaining a large population size, rather than just avoiding inbreeding, is a critical factor for preventing the accumulation of deleterious variants.

Spatiotemporal dynamics of gene flow and hybrid fitness between the M and S forms of the malaria mosquito, Anopheles gambiae

Significance Populations of the African malaria vector, Anopheles gambiae, are structured into M and S forms. All current work assumes the two rarely hybridize. Here we show this assumption is false. We demonstrate (i) significant exchange of genes between the two forms, even though (ii) hybrids have reduced fitness and (iii) the gene exchange process is spatially and temporally dynamic. For malaria, it is important to determine if genes for traits like insecticide resistance are shared between forms. For evolutionary biologists, this work confirms that this mosquito is a good model for studying if and how species may evolve in cases where there is ongoing gene flow. The M and S forms of Anopheles gambiae have been the focus of intense study by malaria researchers and evolutionary biologists interested in ecological speciation. Divergence occurs at three discrete islands in genomes that are otherwise nearly identical. An “islands of speciation” model proposes that diverged regions contain genes that are maintained by selection in the face of gene flow. An alternative “incidental island” model maintains that gene flow between M and S is effectively zero and that divergence islands are unrelated to speciation. A “divergence island SNP” assay was used to explore the spatial and temporal distributions of hybrid genotypes. Results revealed that hybrid individuals occur at frequencies ranging between 5% and 97% in every population examined. A temporal analysis revealed that assortative mating is unstable and periodically breaks down, resulting in extensive hybridization. Results suggest that hybrids suffer a fitness disadvantage, but at least some hybrid genotypes are viable. Stable introgression of the 2L speciation island occurred at one site following a hybridization event.

Asymmetric introgression between the M and S forms of the malaria vector, Anopheles gambiae, maintains divergence despite extensive hybridization.

The suggestion that genetic divergence can arise and/or be maintained in the face of gene flow has been contentious since first proposed. This controversy and a rarity of good examples have limited our understanding of this process. Partially reproductively isolated taxa have been highlighted as offering unique opportunities for identifying the mechanisms underlying divergence with gene flow. The African malaria vector, Anopheles gambiae s.s., is widely regarded as consisting of two sympatric forms, thought by many to represent incipient species, the M and S molecular forms. However, there has been much debate about the extent of reproductive isolation between M and S, with one view positing that divergence may have arisen and is being maintained in the presence of gene flow, and the other proposing a more advanced speciation process with little realized gene flow because of low hybrid fitness. These hypotheses have been difficult to address because hybrids are typically rare (<1%). Here, we assess samples from an area of high hybridization and demonstrate that hybrids are fit and responsible for extensive introgression. Nonetheless, we show that strong divergent selection at a subset of loci combined with highly asymmetric introgression has enabled M and S to remain genetically differentiated despite extensive gene flow. We propose that the extent of reproductive isolation between M and S varies across West Africa resulting in a ‘geographic mosaic of reproductive isolation’; a finding which adds further complexity to our understanding of divergence in this taxon and which has considerable implications for transgenic control strategies.

Spatial and temporal patterns of neutral and adaptive genetic variation in the endangered African wild dog (Lycaon pictus)

Deciphering patterns of genetic variation within a species is essential for understanding population structure, local adaptation and differences in diversity between populations. Whilst neutrally evolving genetic markers can be used to elucidate demographic processes and genetic structure, they are not subject to selection and therefore are not informative about patterns of adaptive variation. As such, assessments of pertinent adaptive loci, such as the immunity genes of the major histocompatibility complex (MHC), are increasingly being incorporated into genetic studies. In this study, we combined neutral (microsatellite, mtDNA) and adaptive (MHC class II DLA‐DRB1 locus) markers to elucidate the factors influencing patterns of genetic variation in the African wild dog (Lycaon pictus); an endangered canid that has suffered extensive declines in distribution and abundance. Our genetic analyses found all extant wild dog populations to be relatively small (Ne < 30). Furthermore, through coalescent modelling, we detected a genetic signature of a recent and substantial demographic decline, which correlates with human expansion, but contrasts with findings in some other African mammals. We found strong structuring of wild dog populations, indicating the negative influence of extensive habitat fragmentation and loss of gene flow between habitat patches. Across populations, we found that the spatial and temporal structure of microsatellite diversity and MHC diversity were correlated and strongly influenced by demographic stability and population size, indicating the effects of genetic drift in these small populations. Despite this correlation, we detected signatures of selection at the MHC, implying that selection has not been completely overwhelmed by genetic drift.

Morphological Differentiation May Mediate Mate-Choice between Incipient Species of Anopheles gambiae s.s.

The M and S molecular forms of Anopheles gambiae s.s. have been considered incipient species for more than ten years, yet the mechanism underlying assortative mating of these incipient species has remained elusive. The discovery of the importance of harmonic convergence of wing beat frequency in mosquito mating and its relation to wing size have laid the foundation for exploring phenotypic divergence in wing size of wild populations of the two forms. In this study, wings from field collected mosquitoes were measured for wing length and wing width from two parts of the sympatric distribution, which differ with respect to the strength of assortative mating. In Mali, where assortative mating is strong, as evidenced by low rates of hybridization, mean wing lengths and wing widths were significantly larger than those from Guinea-Bissau. In addition, mean wing widths in Mali were significantly different between molecular forms. In Guinea-Bissau, assortative mating appears comparatively reduced and wing lengths and widths did not differ significantly between molecular forms. The data presented in this study support the hypothesis that wing beat frequency may mediate assortative mating in the incipient species of A. gambiae and represent the first documentation of a morphological difference between the M and S molecular forms.

Diversity, Differentiation and Linkage Disequilibrium: Prospects for Association Mapping in the Malaria Vector, Anopheles arabiensis

Association mapping is a widely applied method for elucidating the genetic basis of phenotypic traits. However, factors such as linkage disequilibrium and levels of genetic diversity influence the power and resolution of this approach. Moreover, the presence of population subdivision among samples can result in spurious associations if not accounted for. As such, it is useful to have a detailed understanding of these factors before conducting association mapping experiments. Here we conducted whole-genome sequencing on 24 specimens of the malaria mosquito vector, Anopheles arabiensis, to further understanding of patterns of genetic diversity, population subdivision and linkage disequilibrium in this species. We found high levels of genetic diversity within the An. arabiensis genome, with ~800,000 high-confidence, single- nucleotide polymorphisms detected. However, levels of nucleotide diversity varied significantly both within and between chromosomes. We observed lower diversity on the X chromosome, within some inversions, and near centromeres. Population structure was absent at the local scale (Kilombero Valley, Tanzania) but detected between distant populations (Cameroon vs. Tanzania) where differentiation was largely restricted to certain autosomal chromosomal inversions such as 2Rb. Overall, linkage disequilibrium within An. arabiensis decayed very rapidly (within 200 bp) across all chromosomes. However, elevated linkage disequilibrium was observed within some inversions, suggesting that recombination is reduced in those regions. The overall low levels of linkage disequilibrium suggests that association studies in this taxon will be very challenging for all but variants of large effect, and will require large sample sizes.

Chromosome inversions, genomic differentiation and speciation in the African malaria mosquito Anopheles gambiae.

The African malaria vector, Anopheles gambiae, is characterized by multiple polymorphic chromosomal inversions and has become widely studied as a system for exploring models of speciation. Near complete reproductive isolation between different inversion types, known as chromosomal forms, has led to the suggestion that A. gambiae is in early stages of speciation, with divergence evolving in the face of considerable gene flow. We compared the standard chromosomal arrangement (Savanna form) with genomes homozygous for j, b, c, and u inversions (Bamako form) in order to identify regions of genomic divergence with respect to inversion polymorphism. We found levels of divergence between the two sub-taxa within some of these inversions (2Rj and 2Rb), but at a level lower than expected and confined near the inversion breakpoints, consistent with a gene flux model. Unexpectedly, we found that the majority of diverged regions were located on the X chromosome, which contained half of all significantly diverged regions, with much of this divergence located within exons. This is surprising given that the Bamako and Savanna chromosomal forms are both within the S molecular form that is defined by a locus near centromere of X chromosome. Two X-linked genes (a heat shock protein and P450 encoding genes) involved in reproductive isolation between the M and S molecular forms of A. gambiae were also significantly diverged between the two chromosomal forms. These results suggest that genes mediating reproductive isolation are likely located on the X chromosome, as is thought to be the case for the M and S molecular forms. We conclude that genes located on the sex chromosome may be the major force driving speciation between these chromosomal forms of A. gambiae.

An analysis of two island groups as potential sites for trials of transgenic mosquitoes for malaria control

Considerable technological advances have been made towards the generation of genetically modified mosquitoes for vector control. In contrast, less progress has been made towards field evaluations of transformed mosquitoes which are critical for evaluating the success of, and hazards associated with, genetic modification. Oceanic islands have been highlighted as potentially the best locations for such trials. However, population genetic studies are necessary to verify isolation. Here, we used a panel of genetic markers to assess for evidence of genetic isolation of two oceanic island populations of the African malaria vector, Anopheles gambiae s.s. We found no evidence of isolation between the Bijagós archipelago and mainland Guinea‐Bissau, despite separation by distances beyond the known dispersal capabilities of this taxon. Conversely, the Comoros Islands appear to be genetically isolated from the East African mainland, and thus represent a location worthy of further investigation for field trials. Based on assessments of gene flow within and between the Comoros islands, the island of Grande Comore was found to be genetically isolated from adjacent islands and also exhibited local population structure, indicating that it may be the most suitable site for trials with existing genetic modification technologies.

A new multiplex SNP genotyping assay for detecting hybridization and introgression between the M and S molecular forms of Anopheles gambiae.

The M and S forms of Anopheles gambiae have been the subject of intense study, but are morphologically indistinguishable and can only be identified using molecular techniques. PCR‐based assays to distinguish the two forms have been designed and applied widely. However, the application of these assays towards identifying hybrids between the two forms, and backcrossed hybrids in particular, has been problematic as the currently available diagnostic assays are based on single locus and/or are located within a multicopy gene. Here, we present an alternative genotyping method for detecting hybridization and introgression between M and S molecular forms based on a multilocus panel of single‐nucleotide polymorphisms (SNPs) fixed between the M and S forms. The panel of SNPs employed is located in so‐called islands of divergence leading us to describe this method as the ‘Divergence Island SNP’ (DIS) assay. We show this multilocus SNP genotyping approach can robustly and accurately detect F1 hybrids as well as backcrossed individuals.

Inferring the ancestry of African wild dogs that returned to the Serengeti-Mara

An endangered population of African wild dogs (Lycaon pictus) disappeared from the Serengeti-Mara area in 1991. The reasons for the extinction are not well understood, but disease was implicated in the disappearance. In 2001, wild dogs naturally re-established themselves in the region. We conducted genetic profiling on samples collected prior and subsequent to this event, as well as samples from three geographically close populations, to determine the potential source of colonisers. Contrary to expectations, we found no evidence of re-colonisation from these nearby wild dog populations. Rather, our analyses suggest that the re-established animals are primarily derived from the same genetic population as the pre-extinction animals, indicating that wild dogs are likely to have persisted in the Serengeti-Mara after 1991. We also detected some migrants that could be derived from genetically distinct populations outside the recovery area. Overall, we did not detect a decline in genetic diversity at either neutral microsatellites or major histocompatibility complex loci, indicating that the supposed disappearance of wild dogs in the Serengeti-Mara did not substantially impact genetic variation of the population.