Chromosome arm‐specific patterns of polymorphism associated with chromosomal inversions in the major African malaria vector,Anopheles funestus

Chromosomal inversions facilitate local adaptation of beneficial mutations and modulate genetic polymorphism, but the extent of their effects within the genome is still insufficiently understood. The genome of Anopheles funestus, a malaria mosquito endemic to sub‐Saharan Africa, contains an impressive number of paracentric polymorphic inversions, which are unevenly distributed among chromosomes and provide an excellent framework for investigating the genomic impacts of chromosomal rearrangements. Here, we present results of a fine‐scale analysis of genetic variation within the genome of two weakly differentiated populations of Anopheles funestus inhabiting contrasting moisture conditions in Cameroon. Using population genomic analyses, we found that genetic divergence between the two populations is centred on regions of the genome corresponding to three inversions, which are characterized by high values of F_ST, absolute sequence divergence and fixed differences. Importantly, in contrast to the 2L chromosome arm, which is collinear, nucleotide diversity is significantly reduced along the entire length of three autosome arms bearing multiple overlapping chromosomal rearrangements. These findings support the idea that interactions between reduced recombination and natural selection within inversions contribute to sculpt nucleotide polymorphism across chromosomes in An. funestus.     

Correlated patterns of genetic diversity and differentiation across an avian family

Comparative studies of closely related taxa can provide insights into the evolutionary forces that shape genome evolution and the prevalence of convergent molecular evolution. We investigated patterns of genetic diversity and differentiation in stonechats (genus Saxicola), a widely distributed avian species complex with phenotypic variation in plumage, morphology and migratory behaviour, to ask whether similar genomic regions have become differentiated in independent, but closely related, taxa. We used whole‐genome pooled sequencing of 262 individuals from five taxa and found that levels of genetic diversity and divergence are strongly correlated among different stonechat taxa. We then asked whether these patterns remain correlated at deeper evolutionary scales and found that homologous genomic regions have become differentiated in stonechats and the closely related Ficedula flycatchers. Such correlation across a range of evolutionary divergence and among phylogenetically independent comparisons suggests that similar processes may be driving the differentiation of these independently evolving lineages, which in turn may be the result of intrinsic properties of particular genomic regions (e.g. areas of low recombination). Consequently, studies employing genome scans to search for areas important for reproductive isolation or adaptation should account for corresponding regions of differentiation, as these regions may not necessarily represent speciation islands or evidence of local adaptation.     

Evolution of putative barrier loci at an intermediate stage of speciation with gene flow in campions (Silene)

Understanding the origin of new species is a central goal in evolutionary biology. Diverging lineages often evolve highly heterogeneous patterns of genetic differentiation; however, the underlying mechanisms are not well understood. We investigated evolutionary processes governing genetic differentiation between the hybridizing campions Silene dioica (L.) Clairv. and S. latifolia Poiret. Demographic modelling indicated that the two species diverged with gene flow. The best‐supported scenario with heterogeneity in both migration rate and effective population size suggested that a small proportion of the loci evolved without gene flow. Differentiation (F _ST) and sequence divergence (d _XY) were correlated and both tended to peak in the middle of most linkage groups, consistent with reduced gene flow at highly differentiated loci. Highly differentiated loci further exhibited signatures of selection. In between‐species population pairs, isolation by distance was stronger for genomic regions with low between‐species differentiation than for highly differentiated regions that may contain barrier loci. Moreover, differentiation landscapes within and between species were only weakly correlated, suggesting that linked selection due to shared recombination and gene density landscapes is not the dominant determinant of genetic differentiation in these lineages. Instead, our results suggest that divergent selection shaped the genomic landscape of differentiation between the two Silene species, consistent with predictions for speciation in the face of gene flow.     

Genomic islands of speciation in Anopheles gambiae

The African malaria mosquito, Anopheles gambiae sensu stricto (A. gambiae), provides a unique opportunity to study the evolution of reproductive isolation because it is divided into two sympatric, partially isolated subtaxa known as M form and S form. With the annotated genome of this species now available, high-throughput techniques can be applied to locate and characterize the genomic regions contributing to reproductive isolation. In order to quantify patterns of differentiation within A. gambiae, we hybridized population samples of genomic DNA from each form to Affymetrix GeneChip microarrays. We found that three regions, together encompassing less than 2.8 Mb, are the only locations where the M and S forms are significantly differentiated. Two of these regions are adjacent to centromeres, on Chromosomes 2L and X, and contain 50 and 12 predicted genes, respectively. Sequenced loci in these regions contain fixed differences between forms and no shared polymorphisms, while no fixed differences were found at nearby control loci. The third region, on Chromosome 2R, contains only five predicted genes; fixed differences in this region were also verified by direct sequencing. These “speciation islands” remain differentiated despite considerable gene flow, and are therefore expected to contain the genes responsible for reproductive isolation. Much effort has recently been applied to locating the genes and genetic changes responsible for reproductive isolation between species. Though much can be inferred about speciation by studying taxa that have diverged for millions of years, studying differentiation between taxa that are in the early stages of isolation will lead to a clearer view of the number and size of regions involved in the genetics of speciation. Despite appreciable levels of gene flow between the M and S forms of A. gambiae, we were able to isolate three small regions of differentiation where genes responsible for ecological and behavioral isolation are likely to be located. We expect reproductive isolation to be due to changes at a small number of loci, as these regions together contain only 67 predicted genes. Concentrating future mapping experiments on these regions should reveal the genes responsible for reproductive isolation between forms.     

Genomic Islands of Speciation in Anopheles gambiae

The African malaria mosquito, Anopheles gambiae sensu stricto (A. gambiae), provides a unique opportunity to study the evolution of reproductive isolation because it is divided into two sympatric, partially isolated subtaxa known as M form and S form. With the annotated genome of this species now available, high-throughput techniques can be applied to locate and characterize the genomic regions contributing to reproductive isolation. In order to quantify patterns of differentiation within A. gambiae, we hybridized population samples of genomic DNA from each form to Affymetrix GeneChip microarrays. We found that three regions, together encompassing less than 2.8 Mb, are the only locations where the M and S forms are significantly differentiated. Two of these regions are adjacent to centromeres, on Chromosomes 2L and X, and contain 50 and 12 predicted genes, respectively. Sequenced loci in these regions contain fixed differences between forms and no shared polymorphisms, while no fixed differences were found at nearby control loci. The third region, on Chromosome 2R, contains only five predicted genes; fixed differences in this region were also verified by direct sequencing. These “speciation islands” remain differentiated despite considerable gene flow, and are therefore expected to contain the genes responsible for reproductive isolation. Much effort has recently been applied to locating the genes and genetic changes responsible for reproductive isolation between species. Though much can be inferred about speciation by studying taxa that have diverged for millions of years, studying differentiation between taxa that are in the early stages of isolation will lead to a clearer view of the number and size of regions involved in the genetics of speciation. Despite appreciable levels of gene flow between the M and S forms of A. gambiae, we were able to isolate three small regions of differentiation where genes responsible for ecological and behavioral isolation are likely to be located. We expect reproductive isolation to be due to changes at a small number of loci, as these regions together contain only 67 predicted genes. Concentrating future mapping experiments on these regions should reveal the genes responsible for reproductive isolation between forms.     

Complex genome evolution in Anopheles coluzzii associated with increased insecticide usage in Mali

In certain cases, a species may have access to important genetic variation present in a related species via adaptive introgression. These novel alleles may interact with their new genetic background, resulting in unexpected phenotypes. In this study, we describe a selective sweep on standing variation on the X chromosome in the mosquito Anopheles coluzzii, a principal malaria vector in West Africa. This event may have been influenced by the recent adaptive introgression of the insecticide resistance gene known as kdr from the sister species Anopheles gambiae. Individuals carrying both kdr and a nearly fixed X‐linked haplotype, encompassing at least four genes including the P450 gene CYP9K1 and the cuticular protein CPR125, have rapidly increased in relative frequency. In parallel, a reproductively isolated insecticide‐susceptible A. gambiae population (Bamako form) has been driven to local extinction, likely due to strong selection from increased insecticide‐treated bed net usage.     

Prominent intraspecific genetic divergence within Anopheles gambiae sibling species triggered by habitat discontinuities across a riverine landscape

The Anopheles gambiae complex of mosquitoes includes malaria vectors at different stages of speciation, whose study enables a better understanding of how adaptation to divergent environmental conditions leads to evolution of reproductive isolation. We investigated the population genetic structure of closely related sympatric taxa that have recently been proposed as separate species (An. coluzzii and An. gambiae), sampled from diverse habitats along the Gambia river in West Africa. We characterized putatively neutral microsatellite loci as well as chromosomal inversion polymorphisms known to be associated with ecological adaptation. The results revealed strong ecologically associated population subdivisions within both species. Microsatellite loci on chromosome‐3L revealed clear differentiation between coastal and inland populations, which in An. coluzzii is reinforced by a unusual inversion polymorphism pattern, supporting the hypothesis of genetic divergence driven by adaptation to the coastal habitat. A strong reduction of gene flow was observed between An. gambiae populations west and east of an extensively rice‐cultivated region apparently colonized exclusively by An. coluzzii. Notably, this ‘intraspecific’ differentiation is higher than that observed between the two species and involves also the centromeric region of chromosome‐X which has previously been considered a marker of speciation within this complex, possibly suggesting that the two populations may be at an advanced stage of differentiation triggered by human‐made habitat fragmentation. These results confirm ongoing ecological speciation within these most important Afro‐tropical malaria vectors and raise new questions on the possible effect of this process in malaria transmission.     

Nuclear DNA recapitulates the cryptic mitochondrial lineages of Lumbricus rubellus and suggests the existence of cryptic species in an ecotoxological soil sentinel

Mitochondrial DNA analysis has revealed two distinct phylogenetic lineages within the ecotoxological sentinel earthworm model Lumbricus rubellus Hoffmeister, 1843. The existence of these lineages could complicate ecotoxicological studies that use the species as a sentinel for soil contamination testing, as they may respond differently to contamination; however, as mitochondrial haplotypes are not always expected to segregate in the same way as chromosomal DNA in natural populations, we further investigated this issue by using nuclear DNA markers (microsatellites) to measure genetic diversity, differentiation, and gene flow in sympatric populations of the two L. rubellus lineages at two sites in South Wales. Our results show that sympatric populations of the two lineages are more genetically differentiated than geographically distant populations of the same lineage, and Bayesian clustering analysis revealed no evidence of gene flow between the lineages at either site. Additionally, DNA sequencing of these microsatellite loci uncovered substantial differentiation between lineages at homologous flanking regions. Overall our findings indicate a high degree of nuclear genetic differentiation between the two lineages of L. rubellus, implying reproductive isolation at the two study sites and therefore the potential existence of cryptic species. The existence of two cryptic taxa has major implications for the application of L. rubellus as an ecotoxicological sentinel. It may therefore be necessary to consider the lineages as separate taxa during future ecotoxicological studies. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 780–795.     

Arm-specific dynamics of chromosome evolution in malaria mosquitoes

Background  

The malaria mosquito species of subgenus Cellia have rich inversion polymorphisms that correlate with environmental variables. Polymorphic inversions tend to cluster on the chromosomal arms 2R and 2L but not on X, 3R and 3L in Anopheles gambiae and homologous arms in other species. However, it is unknown whether polymorphic inversions on homologous chromosomal arms of distantly related species from subgenus Cellia nonrandomly share similar sets of genes. It is also unclear if the evolutionary breakage of inversion-poor chromosomal arms is under constraints.     

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.     

Chromosomal plasticity and evolutionary potential in the malaria vector <Emphasis Type="Italic">Anopheles gambiae</Emphasis> sensu stricto: insights from three decades of rare paracentric inversions

Background  

In the Anopheles gambiae complex, paracentric chromosomal inversions are non-randomly distributed along the complement: 18/31 (58%) of common polymorphic inversions are on chromosome arm 2R, which represents only ~30% of the complement. Moreover, in An. gambiae sensu stricto, 6/7 common polymorphic inversions occur on 2R. Most of these inversions are considered markers of ecological adaptation that increase the fitness of the carriers of alternative karyotypes in contrasting habitats. However, little is known about the evolutionary forces responsible for their origin and subsequent establishment in field populations.     

Multiple chromosomal inversions contribute to adaptive divergence of a dune sunflower ecotype

Both models and case studies suggest that chromosomal inversions can facilitate adaptation and speciation in the presence of gene flow by suppressing recombination between locally adapted alleles. Until recently, however, it has been laborious and time‐consuming to identify and genotype inversions in natural populations. Here we apply RAD sequencing data and newly developed population genomic approaches to identify putative inversions that differentiate a sand dune ecotype of the prairie sunflower (Helianthus petiolaris) from populations found on the adjacent sand sheet. We detected seven large genomic regions that exhibit a different population structure than the rest of the genome and that vary in frequency between dune and nondune populations. These regions also show high linkage disequilibrium and high heterozygosity between, but not within, arrangements, consistent with the behaviour of large inversions, an inference subsequently validated in part by comparative genetic mapping. Genome–environment association analyses show that key environmental variables, including vegetation cover and soil nitrogen, are significantly associated with inversions. The inversions colocate with previously described “islands of differentiation,” and appear to play an important role in adaptive divergence and incipient speciation within H. petiolaris.     

Ecological genetic studies in the chromosomal form Mopti ofAnopheles gambiae s.str. in Mali,West Africa

Among the sibling species of the AfrotropicalAnopheles gambiae complex, the nominal taxon (An. gambiae s.str.) is the major malaria vector. Its bionomics suggest a man-dependent speciation process which involves, in West Africa, various incipient species chromosomally recognized by different combinations of 2R paracentric inversions. One of the most recent evolutionary steps of such a speciation process appears to be the chromosomal form Mopti, which is associated with dry season irrigation in arid zones, and is characterized by a remarkable ecological flexibility related to three 2R alternative arrangements, namelybc, u and +, whose expected karyotypes are found in Hardy-Weinberg equilibrium. The study of this chromosomal polymorphism in samples from a 16-locality transect in Mali shows wide variations and highly significant correlation with both temporal and spatial climatic differences. Mosquitoes homokaryotypic for 2Rbc are the actual dry season and arid areas breeders. The regular rise of 2Rbc frequency, up to fixation, during each dry season, corresponds to the South-North clinal increase of the same arrangement along the transect, from about 30% in the humid savanna to near fixation in the South-Saharan zone. This coherent ecological genetics case provides full support to the hypothesis of the adaptive nature of paracentric inversions. Moreover, the very peculiar system of combinations of contiguous 2R inversions, utilized by Mopti as well as by other chromosomal forms ofAn. gambiae, suggests a process of polygenic reorganization based on linkage disequilibria and involving the inversions as driving selection units.     

Linkage disequilibrium network analysis (LDna) gives a global view of chromosomal inversions,local adaptation and geographic structure

Recent advances in sequencing allow population‐genomic data to be generated for virtually any species. However, approaches to analyse such data lag behind the ability to generate it, particularly in nonmodel species. Linkage disequilibrium (LD, the nonrandom association of alleles from different loci) is a highly sensitive indicator of many evolutionary phenomena including chromosomal inversions, local adaptation and geographical structure. Here, we present linkage disequilibrium network analysis (LDna), which accesses information on LD shared between multiple loci genomewide. In LD networks, vertices represent loci, and connections between vertices represent the LD between them. We analysed such networks in two test cases: a new restriction‐site‐associated DNA sequence (RAD‐seq) data set for Anopheles baimaii, a Southeast Asian malaria vector; and a well‐characterized single nucleotide polymorphism (SNP) data set from 21 three‐spined stickleback individuals. In each case, we readily identified five distinct LD network clusters (single‐outlier clusters, SOCs), each comprising many loci connected by high LD. In A. baimaii, further population‐genetic analyses supported the inference that each SOC corresponds to a large inversion, consistent with previous cytological studies. For sticklebacks, we inferred that each SOC was associated with a distinct evolutionary phenomenon: two chromosomal inversions, local adaptation, population‐demographic history and geographic structure. LDna is thus a useful exploratory tool, able to give a global overview of LD associated with diverse evolutionary phenomena and identify loci potentially involved. LDna does not require a linkage map or reference genome, so it is applicable to any population‐genomic data set, making it especially valuable for nonmodel species.     

Genomic islands of speciation or genomic islands and speciation?

Populations of the malaria mosquito, Anopheles gambiae, are comprised of at least two reproductively isolated, sympatric populations. In this issue, White et al. (2010) use extensive sampling, high‐density tiling microarrays, and an updated reference genome to clarify and expand our knowledge of genomic differentiation between these populations. It is now clear that DNA near the centromeres of all three chromosomes are in near‐perfect disequilibrium with each other. This is in stark contrast to the remaining 97% of the assembled genome, where fixed differences between populations have not been found, and many polymorphisms are shared. This pattern, coupled with direct evidence of hybridization in nature, supports models of “mosaic” speciation, where ongoing hybridization homogenizes variation in most of the genome while loci under strong selection remain in disequilibrium with each other. However, unambiguously demonstrating that selection maintains the association of these pericentric “speciation islands” in the face of gene flow is difficult. Low recombination at all three loci complicates the issue, and increases the probability that selection unrelated to the speciation process alters patterns of variation in these loci. Here, we discuss these different scenarios in light of this new data.     

Lack of gene flow: Narrow and dispersed differentiation islands in a triplet of Leptidea butterfly species

Genome scans in recently separated species can inform on molecular mechanisms and evolutionary processes driving divergence. Large‐scale polymorphism data from multiple species pairs are also key to investigate the repeatability of divergence—whether radiations tend to show parallel responses to similar selection pressures and/or underlying molecular forces. Here, we used whole‐genome resequencing data from six wood white (Leptidea sp.) butterfly populations, representing three closely related species with karyomorph variation, to infer the species' demographic history and characterize patterns of genomic diversity and differentiation. The analyses supported previously established species relationships, and there was no evidence for postdivergence gene flow. We identified significant intraspecific genetic structure, in particular between karyomorph extremes in the wood white (L. sinapis)—a species with a remarkable chromosome number cline across the distribution range. The genomic landscapes of differentiation were erratic, and outlier regions were narrow and dispersed. Highly differentiated (F_ST) regions generally had low genetic diversity (θ_π), but increased absolute divergence (D_XY) and excess of rare frequency variants (low Tajima's D). A minority of differentiation peaks were shared across species and population comparisons. However, highly differentiated regions contained genes with overrepresented functions related to metabolism, response to stimulus and cellular processes, indicating recurrent directional selection on a specific set of traits in all comparisons. In contrast to the majority of genome scans in recently diverged lineages, our data suggest that divergence landscapes in Leptidea have been shaped by directional selection and genetic drift rather than stable recombination landscapes and/or introgression.     

Polymorphic chromosomal inversions in Anopheles moucheti,a major malaria vector in Central Africa

Anopheles moucheti Evans (Diptera: Culicidae) is a major vector of malaria in forested areas of Central Africa. However, few genetic tools are available for this species. The present study represents the first attempt to characterize chromosomes in An. moucheti females collected in Cameroon. Ovarian nurse cells contained polytene chromosomes, which were suitable for standard cytogenetic applications. The presence of three polymorphic chromosomal inversions in An. moucheti was revealed. Two of these inversions were located on the 2R chromosome arm. The homology between the 2R chromosome arms of An. moucheti and Anopheles gambiae Giles was established by fluorescent in situ hybridization of six An. gambiae genic sequences. Mapping of the probes on chromosomes of An. moucheti detected substantial gene order reshuffling between the two species. The presence of polytene chromosomes and polymorphic inversions in An. moucheti provides a new basis for further population genetic, taxonomic and ecological studies of this neglected malaria vector.     

The genomic bases of morphological divergence and reproductive isolation driven by ecological speciation in Senecio (Asteraceae)

Ecological speciation, driven by adaptation to contrasting environments, provides an attractive opportunity to study the formation of distinct species, and the role of selection and genomic divergence in this process. Here, we focus on a particularly clear‐cut case of ecological speciation to reveal the genomic bases of reproductive isolation and morphological differences between closely related Senecio species, whose recent divergence within the last ~200 000 years was likely driven by the uplift of Mt. Etna (Sicily). These species form a hybrid zone, yet remain morphologically and ecologically distinct, despite active gene exchange. Here, we report a high‐density genetic map of the Senecio genome and map hybrid breakdown to one large and several small quantitative trait loci (QTL). Loci under diversifying selection cluster in three 5 cM regions which are characterized by a significant increase in relative (F_ST), but not absolute (d_XY), interspecific differentiation. They also correspond to some of the regions of greatest marker density, possibly corresponding to ‘cold‐spots’ of recombination, such as centromeres or chromosomal inversions. Morphological QTL for leaf and floral traits overlap these clusters. We also detected three genomic regions with significant transmission ratio distortion (TRD), possibly indicating accumulation of intrinsic genetic incompatibilities between these recently diverged species. One of the TRD regions overlapped with a cluster of high species differentiation, and another overlaps the large QTL for hybrid breakdown, indicating that divergence of these species may have occurred due to a complex interplay of ecological divergence and accumulation of intrinsic genetic incompatibilities.