Introgressive hybridization is now recognized as a widespread phenomenon, but its role in evolution remains contested. Here we use newly available reference genome assemblies to investigate phylogenetic relationships and introgression in a medically important group of Afrotropical mosquito sibling species. We have identified the correct species branching order to resolve a contentious phylogeny, and show that lineages leading to the principal vectors of human malaria were among the first to split. Pervasive autosomal introgression between these malaria vectors means that only a small fraction of the genome, mainly on the X chromosome, has not crossed species boundaries. Our results suggest that traits enhancing vectorial capacity may be gained through interspecific gene flow, including between non-sister species.

Chromosomal inversion polymorphisms play an important role in adaptation to environmental heterogeneities. For mosquito species in the Anopheles gambiae complex that are significant vectors of human malaria, paracentric inversion polymorphisms are abundant and are associated with ecologically and epidemiologically important phenotypes. Improved understanding of these traits relies on determining mosquito karyotype, which currently depends upon laborious cytogenetic methods whose application is limited both by the requirement for specialized expertise and for properly preserved adult females at specific gonotrophic stages. To overcome this limitation, we developed sets of tag single nucleotide polymorphisms (SNPs) inside inversions whose biallelic genotype is strongly correlated with inversion genotype. We leveraged 1,347 fully sequenced An. gambiae and Anopheles coluzzii genomes in the Ag1000G database of natural variation. Beginning with principal components analysis (PCA) of population samples, applied to windows of the genome containing individual chromosomal rearrangements, we classified samples into three inversion genotypes, distinguishing homozygous inverted and homozygous uninverted groups by inclusion of the small subset of specimens in Ag1000G that are associated with cytogenetic metadata. We then assessed the correlation between candidate tag SNP genotypes and PCA-based inversion genotypes in our training sets, selecting those candidates with >80% agreement. Our initial tests both in held-back validation samples from Ag1000G and in data independent of Ag1000G suggest that when used for in silico inversion genotyping of sequenced mosquitoes, these tags perform better than traditional cytogenetics, even for specimens where only a small subset of the tag SNPs can be successfully ascertained.

BackgroundDe novo reference assemblies that are affordable, practical to produce, and of sufficient quality for most downstream applications, remain an unattained goal for many taxa. Insects, which may yield too little DNA from individual specimens for long-read sequencing library construction and often have highly heterozygous genomes, can be particularly hard to assemble using inexpensive short-read sequencing data. The large number of insect species with medical or economic importance makes this a critical problem to address.ResultsUsing the assembler DISCOVAR de novo, we assembled the genome of the African malaria mosquito Anopheles arabiensis using 250 bp reads from a single library. The resulting assembly had a contig N50 of 22,433 bp, and recovered the gene set nearly as well as the ALLPATHS-LG AaraD1 An. arabiensis assembly produced with reads from three sequencing libraries and much greater resources. DISCOVAR de novo appeared to perform better than ALLPATHS-LG in regions of low complexity.ConclusionsDISCOVAR de novo performed well assembling the genome of an insect of medical importance, using simpler sequencing input than previous anopheline assemblies. We have shown that this program is a viable tool for cost-effective assembly of a modestly-sized insect genome.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2531-7) contains supplementary material, which is available to authorized users.

The molecular mechanisms and genetic architecture that facilitate adaptive radiation of lineages remain elusive. Polymorphic chromosomal inversions, due to their recombination-reducing effect, are proposed instruments of ecotypic differentiation. Here we study an ecologically diversifying lineage of An. gambiae, known as the Bamako chromosomal form based on its unique complement of three chromosomal inversions, to explore the impact of these inversions on ecotypic differentiation. We used pooled and individual genome sequencing of Bamako, typical (non-Bamako) An. gambiae, and the sister species An. coluzzii to investigate evolutionary relationships and genome-wide patterns of nucleotide diversity and differentiation among lineages. Despite extensive shared polymorphism and limited differentiation from the other taxa, Bamako clusters apart from the other taxa, and forms a maximally supported clade in neighbor-joining trees based on whole genome data (including inversions) or solely on collinear regions. Nevertheless, FST outlier analysis reveals that the majority of differentiated regions between Bamako and typical An. gambiae are located inside chromosomal inversions, consistent with their role in the ecological isolation of Bamako. Exceptionally differentiated genomic regions were enriched for genes implicated in nervous system development and signaling. Candidate genes associated with a selective sweep unique to Bamako contain substitutions not observed in sympatric samples of the other taxa, and several insecticide resistance gene alleles shared between Bamako and other taxa segregate at sharply different frequencies in these samples. Bamako represents a useful window into the initial stages of ecological and genomic differentiation from sympatric populations in this important group of malaria vectors.

29Chromosomal inversion polymorphisms play an important role in adaptation to 30 environmental heterogeneities. For mosquito species in the Anopheles gambiae 31 complex that are significant vectors of human malaria, paracentric inversion 32 polymorphisms are abundant and are associated with ecologically and epidemiologically 33 important phenotypes. Improved understanding of these traits relies on determining 34 mosquito karyotype, which currently depends upon laborious cytogenetic methods 35 whose application is limited both by the requirement for specialized expertise and for 36 properly preserved adult females at specific gonotrophic stages. To overcome this 37 limitation, we developed sets of tag SNPs inside inversions whose biallelic genotype is 38 strongly correlated with inversion genotype. We leveraged 1,347 fully sequenced An. 39 gambiae and Anopheles coluzzii genomes in the Ag1000G database of natural 40 variation. Beginning with principal components analysis (PCA) of population samples, 41 applied to windows of the genome containing individual chromosomal rearrangements, 42 we classified samples into three inversion genotypes, distinguishing homozygous 43 inverted and homozygous uninverted groups by inclusion of the small subset of 44 specimens in Ag1000G that are associated with cytogenetic metadata. We then 45 assessed the correlation between candidate tag SNP genotypes and PCA-based 46 inversion genotypes in our training sets, selecting those candidates with >80% 47 for specimens where only a small subset of the tag SNPs can be successfully 51 ascertained. 52 53 54 A chromosomal inversion originates when a chromosome segment reverses end to end. 55 Inversions maintained in plant and animal populations as structural polymorphisms tend 56 to be large (several megabases) and contain hundreds of genes (reviewed in 57 Wellenreuther and Bernatchez 2018). Long-term balancing selection can maintain 58 these polymorphisms through millions of generations and multiple species radiations 59 (Wellenreuther and Bernatchez 2018). Because recombination is greatly reduced 60 between opposite orientations in inversion heterozygotes, inversions preserve 61 selectively advantageous combinations of alleles despite homogenizing gene flow in 62 collinear regions. Theory and mounting evidence implicate inversions in local 63 adaptation, adaptive divergence, and range expansion, though the precise molecular 64 mechanisms are rarely known (Hoffmann et al.