Jacob E. Crawford

De Novo Transcriptome Sequencing in Anopheles funestus Using Illumina RNA-Seq Technology

Background Anopheles funestus is one of the primary vectors of human malaria, which causes a million deaths each year in sub-Saharan Africa. Few scientific resources are available to facilitate studies of this mosquito species and relatively little is known about its basic biology and evolution, making development and implementation of novel disease control efforts more difficult. The An. funestus genome has not been sequenced, so in order to facilitate genome-scale experimental biology, we have sequenced the adult female transcriptome of An. funestus from a newly founded colony in Burkina Faso, West Africa, using the Illumina GAIIx next generation sequencing platform. Methodology/Principal Findings We assembled short Illumina reads de novo using a novel approach involving iterative de novo assemblies and “target-based” contig clustering. We then selected a conservative set of 15,527 contigs through comparisons to four Dipteran transcriptomes as well as multiple functional and conserved protein domain databases. Comparison to the Anopheles gambiae immune system identified 339 contigs as putative immune genes, thus identifying a large portion of the immune system that can form the basis for subsequent studies of this important malaria vector. We identified 5,434 1∶1 orthologues between An. funestus and An. gambiae and found that among these 1∶1 orthologues, the protein sequence of those with putative immune function were significantly more diverged than the transcriptome as a whole. Short read alignments to the contig set revealed almost 367,000 genetic polymorphisms segregating in the An. funestus colony and demonstrated the utility of the assembled transcriptome for use in RNA-seq based measurements of gene expression. Conclusions/Significance We developed a pipeline that makes de novo transcriptome sequencing possible in virtually any organism at a very reasonable cost (

A genomic history of Aboriginal Australia

6,300 in sequencing costs in our case). We anticipate that our approach could be used to develop genomic resources in a diversity of systems for which full genome sequence is currently unavailable. Our An. funestus contig set and analytical results provide a valuable resource for future studies in this non-model, but epidemiologically critical, vector insect.

Diabolical survival in Death Valley: recent pupfish colonization, gene flow and genetic assimilation in the smallest species range on earth.

The population history of Aboriginal Australians remains largely uncharacterized. Here we generate high-coverage genomes for 83 Aboriginal Australians (speakers of Pama–Nyungan languages) and 25 Papuans from the New Guinea Highlands. We find that Papuan and Aboriginal Australian ancestors diversified 25–40 thousand years ago (kya), suggesting pre-Holocene population structure in the ancient continent of Sahul (Australia, New Guinea and Tasmania). However, all of the studied Aboriginal Australians descend from a single founding population that differentiated ~10–32 kya. We infer a population expansion in northeast Australia during the Holocene epoch (past 10,000 years) associated with limited gene flow from this region to the rest of Australia, consistent with the spread of the Pama–Nyungan languages. We estimate that Aboriginal Australians and Papuans diverged from Eurasians 51–72 kya, following a single out-of-Africa dispersal, and subsequently admixed with archaic populations. Finally, we report evidence of selection in Aboriginal Australians potentially associated with living in the desert.

Uncovering the genetic history of the present-day Greenlandic population.

One of the most endangered vertebrates, the Devils Hole pupfish Cyprinodon diabolis, survives in a nearly impossible environment: a narrow subterranean fissure in the hottest desert on earth, Death Valley. This species became a conservation icon after a landmark 1976 US Supreme Court case affirming federal groundwater rights to its unique habitat. However, one outstanding question about this species remains unresolved: how long has diabolis persisted in this hellish environment? We used next-generation sequencing of over 13 000 loci to infer the demographic history of pupfishes in Death Valley. Instead of relicts isolated 2–3 Myr ago throughout repeated flooding of the entire region by inland seas as currently believed, we present evidence for frequent gene flow among Death Valley pupfish species and divergence after the most recent flooding 13 kyr ago. We estimate that Devils Hole was colonized by pupfish between 105 and 830 years ago, followed by genetic assimilation of pelvic fin loss and recent gene flow into neighbouring spring systems. Our results provide a new perspective on an iconic endangered species using the latest population genomic methods and support an emerging consensus that timescales for speciation are overestimated in many groups of rapidly evolving species.

Assessing the Accuracy and Power of Population Genetic Inference from Low-Pass Next-Generation Sequencing Data

Because of past limitations in samples and genotyping technologies, important questions about the history of the present-day Greenlandic population remain unanswered. In an effort to answer these questions and in general investigate the genetic history of the Greenlandic population, we analyzed ∼200,000 SNPs from more than 10% of the adult Greenlandic population (n = 4,674). We found that recent gene flow from Europe has had a substantial impact on the population: more than 80% of the Greenlanders have some European ancestry (on average ∼25% of their genome). However, we also found that the amount of recent European gene flow varies across Greenland and is far smaller in the more historically isolated areas in the north and east and in the small villages in the south. Furthermore, we found that there is substantial population structure in the Inuit genetic component of the Greenlanders and that individuals from the east, west, and north can be distinguished from each other. Moreover, the genetic differences in the Inuit ancestry are consistent with a single colonization wave of the island from north to west to south to east. Although it has been speculated that there has been historical admixture between the Norse Vikings who lived in Greenland for a limited period ∼600–1,000 years ago and the Inuit, we found no evidence supporting this hypothesis. Similarly, we found no evidence supporting a previously hypothesized admixture event between the Inuit in East Greenland and the Dorset people, who lived in Greenland before the Inuit.

Detecting adaptive trait loci in nonmodel systems: divergence or admixture mapping?

Next-generation sequencing (NGS) technologies have made it possible to address population genetic questions in almost any system, but high error rates associated with such data can introduce significant biases into downstream analyses, necessitating careful experimental design and interpretation in studies based on short-read sequencing. Exploration of population genetic analyses based on NGS has revealed some of the potential biases, but previous work has emphasized parameters relevant to human population genetics and further examination of parameters relevant to other systems is necessary, including situations where sample sizes are small and genetic variation is high. To assess experimental power to address several principal objectives of population genetic studies under these conditions, we simulated population samples under selective sweep, population growth, and population subdivision models and tested the power to accurately infer population genetic parameters from sequence polymorphism data obtained through simulated 4×, 8×, and 15× read depth sequence data. We found that estimates of population genetic differentiation and population growth parameters were systematically biased when inference was based on 4× sequencing, but biases were markedly reduced at even 8× read depth. We also found that the power to identify footprints of positive selection depends on an interaction between read depth and the strength of selection, with strong selection being recovered consistently at all read depths, but weak selection requiring deeper read depths for reliable detection. Although we have explored only a small subset of the many possible experimental designs and population genetic models, using only one SNP-calling approach, our results reveal some general patterns and provide some assessment of what biases could be expected under similar experimental structures.

Evidence for Population-Specific Positive Selection on Immune Genes of Anopheles gambiae

Mapping adaptive trait loci (ATL) underlying ecological divergence is an essential step towards understanding the processes that generate phenotypic diversity. Technological advances have made it possible to sequence exomes in nonmodel systems, providing an efficient means of analysing functional genetic variants. Divergence scans of genetic markers for outlier loci, or ‘divergence mapping’, have been used to map locally adapted genes, but this approach is likely to be underpowered when background divergence is elevated. Genotype–phenotype association tests in admixed populations, or ‘admixture mapping’, may provide a useful approach for mapping locally adapted loci when neutral divergence is high. To determine the power and limits of divergence mapping, we simulated exomes containing a single ATL across two parental populations of varying neutral divergence, estimated divergence and quantified the power to identify the ATL. We found that divergence mapping had very high power when background FST is <0.2, but decreased dramatically above this level. To evaluate the utility of admixture mapping, we simulated exomes from admixed populations, then simulated phenotypes, conducted genotype–phenotype association tests and found that even two generations of random mating after admixture could provide high mapping power in scenarios where pure divergence mapping was ineffective (FST = 0.35). Moreover, admixture mapping had high power across all levels of divergence after 20 generations since admixture. Together with high‐throughput exome sequencing, admixture mapping could be used to map ATL in systems such as Heliconius butterflies or Gryllus crickets when experimental design and analytical approach are chosen accordingly.

Population genomics reveals that an anthropophilic population of Aedes aegypti mosquitoes in West Africa recently gave rise to American and Asian populations of this major disease vector

Host-pathogen interactions can be powerful drivers of adaptive evolution, shaping the patterns of molecular variation at the genes involved. In this study, we sequenced alleles from 28 immune-related loci in wild samples of multiple genetic subpopulations of the African malaria mosquito Anopheles gambiae, obtaining unprecedented sample sizes and providing the first opportunity to contrast patterns of molecular evolution at immune-related loci in the recently discovered GOUNDRY population to those of the indoor-resting M and S molecular forms. In contrast to previous studies that focused on immune genes identified in laboratory studies, we centered our analysis on genes that fall within a quantitative trait locus associated with resistance to Plasmodium falciparum in natural populations of A. gambiae. Analyses of haplotypic and genetic diversity at these 28 loci revealed striking differences among populations in levels of genetic diversity and allele frequencies in coding sequence. Putative signals of positive selection were identified at 11 loci, but only one was shared by two subgroups of A. gambiae. Striking patterns of linkage disequilibrium were observed at several loci. We discuss these results with respect to ecological differences among these strata as well as potential implications for disease transmission.

Reticulate Speciation and Barriers to Introgression in the Anopheles gambiae Species Complex

BackgroundThe mosquito Aedes aegypti is the main vector of dengue, Zika, chikungunya and yellow fever viruses. This major disease vector is thought to have arisen when the African subspecies Ae. aegypti formosus evolved from being zoophilic and living in forest habitats into a form that specialises on humans and resides near human population centres. The resulting domestic subspecies, Ae. aegypti aegypti, is found throughout the tropics and largely blood-feeds on humans.ResultsTo understand this transition, we have sequenced the exomes of mosquitoes collected from five populations from around the world. We found that Ae. aegypti specimens from an urban population in Senegal in West Africa were more closely related to populations in Mexico and Sri Lanka than they were to a nearby forest population. We estimate that the populations in Senegal and Mexico split just a few hundred years ago, and we found no evidence of Ae. aegypti aegypti mosquitoes migrating back to Africa from elsewhere in the tropics. The out-of-Africa migration was accompanied by a dramatic reduction in effective population size, resulting in a loss of genetic diversity and rare genetic variants.ConclusionsWe conclude that a domestic population of Ae. aegypti in Senegal and domestic populations on other continents are more closely related to each other than to other African populations. This suggests that an ancestral population of Ae. aegypti evolved to become a human specialist in Africa, giving rise to the subspecies Ae. aegypti aegypti. The descendants of this population are still found in West Africa today, and the rest of the world was colonised when mosquitoes from this population migrated out of Africa. This is the first report of an African population of Ae. aegypti aegypti mosquitoes that is closely related to Asian and American populations. As the two subspecies differ in their ability to vector disease, their existence side by side in West Africa may have important implications for disease transmission.

Asian wild rice is a hybrid swarm with extensive gene flow and feralization from domesticated rice

Abstract Speciation as a process remains a central focus of evolutionary biology, but our understanding of the genomic architecture and prevalence of speciation in the face of gene flow remains incomplete. The Anopheles gambiae species complex of malaria mosquitoes is a radiation of ecologically diverse taxa. This complex is well-suited for testing for evidence of a speciation continuum and genomic barriers to introgression because its members exhibit partially overlapping geographic distributions as well as varying levels of divergence and reproductive isolation. We sequenced 20 genomes from wild A. gambiae s.s., Anopheles coluzzii, Anopheles arabiensis, and compared these with 12 genomes from the “GOUNDRY” subgroup of A. gambiae s.l. Amidst a backdrop of strong reproductive isolation, we find strong evidence for a speciation continuum with introgression of autosomal chromosomal regions among species and subgroups. The X chromosome, however, is strongly differentiated among all taxa, pointing to a disproportionately large effect of X chromosome genes in driving speciation among anophelines. Strikingly, we find that autosomal introgression has occurred from contemporary hybridization between A. gambiae and A. arabiensis despite strong divergence (∼5× higher than autosomal divergence) and isolation on the X chromosome. In addition to the X, we find strong evidence that lowly recombining autosomal regions, especially pericentromeric regions, serve as barriers to introgression secondarily to the X. We show that speciation with gene flow results in genomic mosaicism of divergence and introgression. Such a reticulate gene pool connecting vector taxa across the speciation continuum has important implications for malaria control efforts.