Evolutionary adaptation under climate change: Aedes sp. demonstrates potential to adapt to warming.

Climate warming is expected to shift the distributions of mosquitoes and mosquito-borne diseases, facilitating expansions at cool range edges and contractions at warm range edges. However, whether mosquito populations could maintain their warm edges through evolutionary adaptation remains unknown. Here, we investigate the potential for thermal adaptation in Aedes sierrensis, a congener of the major disease vector species that experiences large thermal gradients in its native range, by assaying tolerance to prolonged and acute heat exposure, and its genetic basis in a diverse, field-derived population. We found pervasive evidence of heritable genetic variation in acute heat tolerance, which phenotypically trades off with tolerance to prolonged heat exposure. A simple evolutionary model based on our data shows that the estimated maximum rate of evolutionary adaptation in mosquito heat tolerance typically exceeds that of projected climate warming under idealized conditions. Our findings indicate that natural mosquito populations may have the potential to track projected warming via genetic adaptation. Prior climate-based projections may thus underestimate the range of mosquito and mosquito-borne disease distributions under future climate conditions.

Unraveling the genomic diversity and admixture history of captive tigers in the United States.

Genomic studies of endangered species have primarily focused on describing diversity patterns and resolving phylogenetic relationships, with the overarching goal of informing conservation efforts. However, few studies have investigated genomic diversity housed in captive populations. For tigers (Panthera tigris), captive individuals vastly outnumber those in the wild, but their diversity remains largely unexplored. Privately owned captive tiger populations have remained an enigma in the conservation community, with some believing that these individuals are severely inbred, while others believe they may be a source of now-extinct diversity. Here, we present a large-scale genetic study of the private (non-zoo) captive tiger population in the United States, also known as "Generic" tigers. We find that the Generic tiger population has an admixture fingerprint comprising all six extant wild tiger subspecies. Of the 138 Generic individuals sequenced for the purpose of this study, no individual had ancestry from only one subspecies. We show that the Generic tiger population has a comparable amount of genetic diversity relative to most wild subspecies, few private variants, and fewer deleterious mutations. We observe inbreeding coefficients similar to wild populations, although there are some individuals within both the Generic and wild populations that are substantially inbred. Additionally, we develop a reference panel for tigers that can be used with imputation to accurately distinguish individuals and assign ancestry with ultralow coverage (0.25×) data. By providing a cost-effective alternative to whole-genome sequencing (WGS), the reference panel provides a resource to assist in tiger conservation efforts for both ex- and in situ populations.

Single-fly genome assemblies fill major phylogenomic gaps across the Drosophilidae Tree of Life.

Long-read sequencing is driving rapid progress in genome assembly across all major groups of life, including species of the family Drosophilidae, a longtime model system for genetics, genomics, and evolution. We previously developed a cost-effective hybrid Oxford Nanopore (ONT) long-read and Illumina short-read sequencing approach and used it to assemble 101 drosophilid genomes from laboratory cultures, greatly increasing the number of genome assemblies for this taxonomic group. The next major challenge is to address the laboratory culture bias in taxon sampling by sequencing genomes of species that cannot easily be reared in the lab. Here, we build upon our previous methods to perform amplification-free ONT sequencing of single wild flies obtained either directly from the field or from ethanol-preserved specimens in museum collections, greatly improving the representation of lesser studied drosophilid taxa in whole-genome data. Using Illumina Novaseq X Plus and ONT P2 sequencers with R10.4.1 chemistry, we set a new benchmark for inexpensive hybrid genome assembly at US $150 per genome while assembling genomes from as little as 35 ng of genomic DNA from a single fly. We present 183 new genome assemblies for 179 species as a resource for drosophilid systematics, phylogenetics, and comparative genomics. Of these genomes, 62 are from pooled lab strains and 121 from single adult flies. Despite the sample limitations of working with small insects, most single-fly diploid assemblies are comparable in contiguity (>1 Mb contig N50), completeness (>98% complete dipteran BUSCOs), and accuracy (>QV40 genome-wide with ONT R10.4.1) to assemblies from inbred lines. We present a well-resolved multi-locus phylogeny for 360 drosophilid and 4 outgroup species encompassing all publicly available (as of August 2023) genomes for this group. Finally, we present a Progressive Cactus whole-genome, reference-free alignment built from a subset of 298 suitably high-quality drosophilid genomes. The new assemblies and alignment, along with updated laboratory protocols and computational pipelines, are released as an open resource and as a tool for studying evolution at the scale of an entire insect family.

Enrichment of hard sweeps on the X chromosome compared to autosomes in six Drosophila species.

The X chromosome, being hemizygous in males, is exposed one-third of the time increasing the visibility of new mutations to natural selection, potentially leading to different evolutionary dynamics than autosomes. Recently, we found an enrichment of hard selective sweeps over soft selective sweeps on the X chromosome relative to the autosomes in a North American population of Drosophila melanogaster. To understand whether this enrichment is a universal feature of evolution on the X chromosome, we analyze diversity patterns across 6 commonly studied Drosophila species. We find an increased proportion of regions with steep reductions in diversity and elevated homozygosity on the X chromosome compared to autosomes. To assess if these signatures are consistent with positive selection, we simulate a wide variety of evolutionary scenarios spanning variations in demography, mutation rate, recombination rate, background selection, hard sweeps, and soft sweeps and find that the diversity patterns observed on the X are most consistent with hard sweeps. Our findings highlight the importance of sex chromosomes in driving evolutionary processes and suggest that hard sweeps have played a significant role in shaping diversity patterns on the X chromosome across multiple Drosophila species.

Decoupled evolution of the Sex Peptide gene family and Sex Peptide Receptor in Drosophilidae.

Across internally fertilising species, males transfer ejaculate proteins that trigger wide-ranging changes in female behaviour and physiology. Much theory has been developed to explore the drivers of ejaculate protein evolution. The accelerating availability of high-quality genomes now allows us to test how these proteins are evolving at fine taxonomic scales. Here, we use genomes from 264 species to chart the evolutionary history of Sex Peptide (SP), a potent regulator of female post-mating responses in Drosophila melanogaster. We infer that SP first evolved in the Drosophilinae subfamily and has since followed markedly different evolutionary trajectories in different lineages. Outside of the Sophophora-Lordiphosa, SP exists largely as a single-copy gene with independent losses in several lineages. Within the Sophophora-Lordiphosa, the SP gene family has repeatedly and independently expanded. Up to seven copies, collectively displaying extensive sequence variation, are present in some species. Despite these changes, SP expression remains restricted to the male reproductive tract. Alongside, we document considerable interspecific variation in the presence and morphology of seminal microcarriers that, despite the critical role SP plays in microcarrier assembly in D. melanogaster, appears to be independent of changes in the presence/absence or sequence of SP. We end by providing evidence that SP's evolution is decoupled from that of its receptor, Sex Peptide Receptor, in which we detect no evidence of correlated diversifying selection. Collectively, our work describes the divergent evolutionary trajectories that a novel gene has taken following its origin and finds a surprisingly weak coevolutionary signal between a supposedly sexually antagonistic protein and its receptor.

Single-fly assemblies fill major phylogenomic gaps across the Drosophilidae Tree of Life.

Long-read sequencing is driving rapid progress in genome assembly across all major groups of life, including species of the family Drosophilidae, a longtime model system for genetics, genomics, and evolution. We previously developed a cost-effective hybrid Oxford Nanopore (ONT) long-read and Illumina short-read sequencing approach and used it to assemble 101 drosophilid genomes from laboratory cultures, greatly increasing the number of genome assemblies for this taxonomic group. The next major challenge is to address the laboratory culture bias in taxon sampling by sequencing genomes of species that cannot easily be reared in the lab. Here, we build upon our previous methods to perform amplification-free ONT sequencing of single wild flies obtained either directly from the field or from ethanol-preserved specimens in museum collections, greatly improving the representation of lesser studied drosophilid taxa in whole-genome data. Using Illumina Novaseq X Plus and ONT P2 sequencers with R10.4.1 chemistry, we set a new benchmark for inexpensive hybrid genome assembly at US $150 per genome while assembling genomes from as little as 35 ng of genomic DNA from a single fly. We present 183 new genome assemblies for 179 species as a resource for drosophilid systematics, phylogenetics, and comparative genomics. Of these genomes, 62 are from pooled lab strains and 121 from single adult flies. Despite the sample limitations of working with small insects, most single-fly diploid assemblies are comparable in contiguity (>1Mb contig N50), completeness (>98% complete dipteran BUSCOs), and accuracy (>QV40 genome-wide with ONT R10.4.1) to assemblies from inbred lines. We present a well-resolved multi-locus phylogeny for 360 drosophilid and 4 outgroup species encompassing all publicly available (as of August 2023) genomes for this group. Finally, we present a Progressive Cactus whole-genome, reference-free alignment built from a subset of 298 suitably high-quality drosophilid genomes. The new assemblies and alignment, along with updated laboratory protocols and computational pipelines, are released as an open resource and as a tool for studying evolution at the scale of an entire insect family.

Genome Report: chromosome-scale genome assembly of the African spiny mouse (Acomys cahirinus).

There is increasing interest in the African spiny mouse (Acomys cahirinus) as a model organism because of its ability for regeneration of tissue after injury in skin, muscle, and internal organs such as the kidneys. A high-quality reference genome is needed to better understand these regenerative properties at the molecular level. Here, we present an improved reference genome for A. cahirinus generated from long Nanopore sequencing reads. We confirm the quality of our annotations using RNA sequencing data from 4 different tissues. Our genome is of higher contiguity and quality than previously reported genomes from this species and will facilitate ongoing efforts to better understand the regenerative properties of this organism.

Rapid evolutionary diversification of the flamenco locus across simulans clade Drosophila species.

Suppression of transposable elements (TEs) is paramount to maintain genomic integrity and organismal fitness. In D. melanogaster, the flamenco locus is a master suppressor of TEs, preventing the mobilization of certain endogenous retrovirus-like TEs from somatic ovarian support cells to the germline. It is transcribed by Pol II as a long (100s of kb), single-stranded, primary transcript, and metabolized into ~24-32 nt Piwi-interacting RNAs (piRNAs) that target active TEs via antisense complementarity. flamenco is thought to operate as a trap, owing to its high content of recent horizontally transferred TEs that are enriched in antisense orientation. Using newly-generated long read genome data, which is critical for accurate assembly of repetitive sequences, we find that flamenco has undergone radical transformations in sequence content and even copy number across simulans clade Drosophilid species. Drosophila simulans flamenco has duplicated and diverged, and neither copy exhibits synteny with D. melanogaster beyond the core promoter. Moreover, flamenco organization is highly variable across D. simulans individuals. Next, we find that D. simulans and D. mauritiana flamenco display signatures of a dual-stranded cluster, with ping-pong signals in the testis and/or embryo. This is accompanied by increased copy numbers of germline TEs, consistent with these regions operating as functional dual-stranded clusters. Overall, the physical and functional diversity of flamenco orthologs is testament to the extremely dynamic consequences of TE arms races on genome organization, not only amongst highly related species, but even amongst individuals.

Decoupled evolution of the Sex Peptide gene family and Sex Peptide Receptor in Drosophilidae.

Across internally fertilising species, males transfer ejaculate proteins that trigger wide-ranging changes in female behaviour and physiology. Much theory has been developed to explore the drivers of ejaculate protein evolution. The accelerating availability of high-quality genomes now allows us to test how these proteins are evolving at fine taxonomic scales. Here, we use genomes from 264 species to chart the evolutionary history of Sex Peptide (SP), a potent regulator of female post-mating responses in Drosophila melanogaster. We infer that SP first evolved in the Drosophilinae subfamily and has followed markedly different evolutionary trajectories in different lineages. Outside of the Sophophora-Lordiphosa, SP exists largely as a single-copy gene with independent losses in several lineages. Within the Sophophora-Lordiphosa, the SP gene family has repeatedly and independently expanded. Up to seven copies, collectively displaying extensive sequence variation, are present in some species. Despite these changes, SP expression remains restricted to the male reproductive tract. Alongside, we document considerable interspecific variation in the presence and morphology of seminal microcarriers that, despite the critical role SP plays in microcarrier assembly in D. melanogaster, appear to be independent of changes in the presence/absence or sequence of SP. We end by providing evidence that SP's evolution is decoupled from that of its receptor, SPR, in which we detect no evidence of correlated diversifying selection. Collectively, our work describes the divergent evolutionary trajectories that a novel gene has taken following its origin and finds a surprisingly weak coevolutionary signal between a supposedly sexually antagonistic protein and its receptor.

GENOME REPORT: Chromosome-scale genome assembly of the African spiny mouse ( Acomys cahirinus ).

There is increasing interest in the African spiny mouse ( Acomys cahirinus ) as a model organism because of its ability for regeneration of tissue after injury in skin, muscle, and internal organs such as the kidneys. A high-quality reference genome is needed to better understand these regenerative properties at the molecular level. Here, we present an improved reference genome for A. cahirinus generated from long Nanopore sequencing reads. We confirm the quality of our annotations using RNA sequencing data from four different tissues. Our genome is of higher contiguity and quality than previously reported genomes from this species and will facilitate ongoing efforts to better understand the regenerative properties of this organism.

Antigenic diversity in malaria parasites is maintained on extrachromosomal DNA.

Sequence variation among antigenic var genes enables Plasmodium falciparum malaria parasites to evade host immunity. Using long sequence reads from haploid clones from a mutation accumulation experiment, we detect var diversity inconsistent with simple chromosomal inheritance. We discover putatively circular DNA that is strongly enriched for var genes, which exist in multiple alleles per locus separated by recombination and indel events. Extrachromosomal DNA likely contributes to rapid antigenic diversification in P. falciparum.

Evolution and development of male-specific leg brushes in Drosophilidae.

The origin, diversification, and secondary loss of sexually dimorphic characters are common in animal evolution. In some cases, structurally and functionally similar traits have evolved independently in multiple lineages. Prominent examples of such traits include the male-specific grasping structures that develop on the front legs of many dipteran insects. In this report, we describe the evolution and development of one of these structures, the male-specific "sex brush." The sex brush is composed of densely packed, irregularly arranged modified bristles and is found in several distantly related lineages in the family Drosophilidae. Phylogenetic analysis using 250 genes from over 200 species provides modest support for a single origin of the sex brush followed by many secondary losses; however, independent origins of the sex brush cannot be ruled out completely. We show that sex brushes develop in very similar ways in all brush-bearing lineages. The dense packing of brush hairs is explained by the specification of bristle precursor cells at a near-maximum density permitted by the lateral inhibition mechanism, as well as by the reduced size of the surrounding epithelial cells. In contrast to the female and the ancestral male condition, where bristles are arranged in stereotypical, precisely spaced rows, cell migration does not contribute appreciably to the formation of the sex brush. The complex phylogenetic history of the sex brush can make it a valuable model for investigating coevolution of sex-specific morphology and mating behavior.

Secondary reversion to sexual monomorphism associated with tissue-specific loss of doublesex expression.

Animal evolution is characterized by frequent turnover of sexually dimorphic traits-new sex-specific characters are gained, and some ancestral sex-specific characters are lost, in many lineages. In insects, sexual differentiation is predominantly cell autonomous and depends on the expression of the doublesex (dsx) transcription factor. In most cases, cells that transcribe dsx have the potential to undergo sex-specific differentiation, while those that lack dsx expression do not. Consistent with this mode of development, comparative research has shown that the origin of new sex-specific traits can be associated with the origin of new spatial domains of dsx expression. In this report, we examine the opposite situation-a secondary loss of the sex comb, a male-specific grasping structure that develops on the front legs of some drosophilid species. We show that while the origin of the sex comb is linked to an evolutionary gain of dsx expression in the leg, sex comb loss in a newly identified species of Lordiphosa (Drosophilidae) is associated with a secondary loss of dsx expression. We discuss how the developmental control of sexual dimorphism affects the mechanisms by which sex-specific traits can evolve.

Widespread introgression across a phylogeny of 155 Drosophila genomes.

Genome-scale sequence data have invigorated the study of hybridization and introgression, particularly in animals. However, outside of a few notable cases, we lack systematic tests for introgression at a larger phylogenetic scale across entire clades. Here, we leverage 155 genome assemblies from 149 species to generate a fossil-calibrated phylogeny and conduct multilocus tests for introgression across 9 monophyletic radiations within the genus Drosophila. Using complementary phylogenomic approaches, we identify widespread introgression across the evolutionary history of Drosophila. Mapping gene-tree discordance onto the phylogeny revealed that both ancient and recent introgression has occurred across most of the 9 clades that we examined. Our results provide the first evidence of introgression occurring across the evolutionary history of Drosophila and highlight the need to continue to study the evolutionary consequences of hybridization and introgression in this genus and across the tree of life.

Two new hybrid populations expand the swordtail hybridization model system.

Natural hybridization events provide unique windows into the barriers that keep species apart as well as the consequences of their breakdown. Here, we characterize hybrid populations formed between the northern swordtail fish Xiphophorus cortezi and Xiphophorus birchmanni from collection sites on two rivers. We use simulations and new genetic reference panels to develop sensitive and accurate local ancestry calling in this novel system. Strikingly, we find that hybrid populations on both rivers consist of two genetically distinct subpopulations: a cluster of pure X. birchmanni individuals and one of phenotypically intermediate hybrids that derive ∼85-90% of their genome from X. cortezi. Simulations suggest that initial hybridization occurred ∼150 generations ago at both sites, with little evidence for contemporary gene flow between subpopulations. This population structure is consistent with strong assortative mating between individuals of similar ancestry. The patterns of population structure uncovered here mirror those seen in hybridization between X. birchmanni and its sister species, Xiphophorus malinche, indicating an important role for assortative mating in the evolution of hybrid populations. Future comparisons will provide a window into the shared mechanisms driving the outcomes of hybridization not only among independent hybridization events between the same species but also across distinct species pairs.

Highly contiguous assemblies of 101 drosophilid genomes.

Over 100 years of studies in Drosophila melanogaster and related species in the genus Drosophila have facilitated key discoveries in genetics, genomics, and evolution. While high-quality genome assemblies exist for several species in this group, they only encompass a small fraction of the genus. Recent advances in long-read sequencing allow high-quality genome assemblies for tens or even hundreds of species to be efficiently generated. Here, we utilize Oxford Nanopore sequencing to build an open community resource of genome assemblies for 101 lines of 93 drosophilid species encompassing 14 species groups and 35 sub-groups. The genomes are highly contiguous and complete, with an average contig N50 of 10.5 Mb and greater than 97% BUSCO completeness in 97/101 assemblies. We show that Nanopore-based assemblies are highly accurate in coding regions, particularly with respect to coding insertions and deletions. These assemblies, along with a detailed laboratory protocol and assembly pipelines, are released as a public resource and will serve as a starting point for addressing broad questions of genetics, ecology, and evolution at the scale of hundreds of species.

Identification and Characterization of Breakpoints and Mutations on Drosophila melanogaster Balancer Chromosomes.

Balancers are rearranged chromosomes used in Drosophila melanogaster to maintain deleterious mutations in stable populations, preserve sets of linked genetic elements and construct complex experimental stocks. Here, we assess the phenotypes associated with breakpoint-induced mutations on commonly used third chromosome balancers and show remarkably few deleterious effects. We demonstrate that a breakpoint in p53 causes loss of radiation-induced apoptosis and a breakpoint in Fucosyltransferase A causes loss of fucosylation in nervous and intestinal tissue-the latter study providing new markers for intestinal cell identity and challenging previous conclusions about the regulation of fucosylation. We also describe thousands of potentially harmful mutations shared among X or third chromosome balancers, or unique to specific balancers, including an Ankyrin2 mutation present on most TM3 balancers, and reiterate the risks of using balancers as experimental controls. We used long-read sequencing to confirm or refine the positions of two inversions with breakpoints lying in repetitive sequences and provide evidence that one of the inversions, In(2L)Cy, arose by ectopic recombination between foldback transposon insertions and the other, In(3R)C, cleanly separates subtelomeric and telomeric sequences and moves the subtelomeric sequences to an internal chromosome position. In addition, our characterization of In(3R)C shows that balancers may be polymorphic for terminal deletions. Finally, we present evidence that extremely distal mutations on balancers can add to the stability of stocks whose purpose is to maintain homologous chromosomes carrying mutations in distal genes. Overall, these studies add to our understanding of the structure, diversity and effectiveness of balancer chromosomes.

A community-maintained standard library of population genetic models.

The explosion in population genomic data demands ever more complex modes of analysis, and increasingly, these analyses depend on sophisticated simulations. Recent advances in population genetic simulation have made it possible to simulate large and complex models, but specifying such models for a particular simulation engine remains a difficult and error-prone task. Computational genetics researchers currently re-implement simulation models independently, leading to inconsistency and duplication of effort. This situation presents a major barrier to empirical researchers seeking to use simulations for power analyses of upcoming studies or sanity checks on existing genomic data. Population genetics, as a field, also lacks standard benchmarks by which new tools for inference might be measured. Here, we describe a new resource, stdpopsim, that attempts to rectify this situation. Stdpopsim is a community-driven open source project, which provides easy access to a growing catalog of published simulation models from a range of organisms and supports multiple simulation engine backends. This resource is available as a well-documented python library with a simple command-line interface. We share some examples demonstrating how stdpopsim can be used to systematically compare demographic inference methods, and we encourage a broader community of developers to contribute to this growing resource.

Intraspecific Variation in Growth and Reproduction of the Marine Leech, Heptacyclus buthi.

Heptacyclus buthi was harvested from fish hosts in rocky intertidal zones of Sonoma and Marin Counties, California, in October 2008 (n = 162) and October 2010 (n = 51). The size of the leeches was quantified using a method that approximated the sagittal cross-section of each specimen. Size-frequency curves were modeled to estimate the number of size-class cohorts in each year. If H. buthi is an annual species like many of its relatives, the single cohort modeled for in 2010 and the comparable "older" cohort in 2008, both with a broad range of sizes, may represent 1 component of its reproductive life history. A second, younger, more-numerous, less-variable cohort modeled from the 2008 sample may represent a second reproductive bout during that year that was prevented in the subsequent La Niña period of 2010-2011.