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Establishing causal links between adaptive mutations and ecologically relevant phenotypes is key to understanding the process of adaptation, which is a central goal in evolutionary biology with applications for conservation, medicine, and agriculture. Yet despite recent progress, the number of identified causal adaptive mutations remains limited. Linking genetic variation to fitness-related effects is complicated by gene-by-gene and gene-by-environment interactions, among other processes. Transposable elements, which are often ignored in the quest for the genetic basis of adaptive evolution, are a genome-wide source of regulatory elements across organisms that can potentially result in adaptive phenotypes. In this work, we combine gene expression, in vivo reporter assays, CRISPR/Cas9 genome editing, and survival experiments to characterize in detail the molecular and phenotypic consequences of a natural Drosophila melanogaster transposable element insertion: the roo solo-LTR FBti0019985. This transposable element provides an alternative promoter to the transcription factor Lime, involved in cold- and immune-stress responses. We found that the effect of FBti0019985 on Lime expression depends on the interplay between the developmental stage and environmental condition. We further establish a causal link between the presence of FBti0019985 and increased survival to cold- and immune-stress. Our results exemplify how several developmental stages and environmental conditions need to be considered to characterize the molecular and functional effects of a genetic variant, and add to the growing body of evidence that transposable elements can induce complex mutations with ecologically relevant effects.

[Summary] We present a protocol for generating a precise deletion, without altering the genetic background of the strain, of a transposable element (TE) in a natural population of Drosophila melanogaster using two steps of CRISPR-Cas9 homology-directed repair. We describe steps for replacing the TE by a fluorescent marker and for subsequent marker removal using single-guide RNAs, repair plasmids, and microinjection. We also detail steps for screening the deletion of the TE and generating a homozygous mutant strain.

[Background] Climate change is one of the main factors shaping the distribution and biodiversity of organisms, among others by greatly altering water availability, thus exposing species and ecosystems to harsh desiccation conditions. However, most of the studies so far have focused on the effects of increased temperature. Integrating transcriptomics and physiology is key to advancing our knowledge on how species cope with desiccation stress, and these studies are still best accomplished in model organisms., [Results] Here, we characterized the natural variation of European D. melanogaster populations across climate zones and found that strains from arid regions were similar or more tolerant to desiccation compared with strains from temperate regions. Tolerant and sensitive strains differed not only in their transcriptomic response to stress but also in their basal expression levels. We further showed that gene expression changes in tolerant strains correlated with their physiological response to desiccation stress and with their cuticular hydrocarbon composition, and functionally validated three of the candidate genes identified. Transposable elements, which are known to influence stress response across organisms, were not found to be enriched nearby differentially expressed genes. Finally, we identified several tRNA-derived small RNA fragments that differentially targeted genes in response to desiccation stress., [Conclusions] Overall, our results showed that basal gene expression differences across individuals should be analyzed if we are to understand the genetic basis of differential stress survival. Moreover, tRNA-derived small RNA fragments appear to be relevant across stress responses and allow for the identification of stress-response genes not detected at the transcriptional level.

The advent of long-read sequencing technologies has allowed the generation of multiple high-quality de novo genome assemblies for multiple species, including well-known model species such as Drosophila melanogaster. Genome assemblies for multiple individuals of the same species are key to discover the genetic diversity present in natural populations, especially the one generated by transposable elements, the most common type of structural variant. Despite the availability of multiple genomic data sets for D. melanogaster populations, we lack an efficient visual tool to display different genome assemblies simultaneously. In this work, we present DrosOmics, a population genomic-oriented browser currently containing 52 high-quality reference genomes of D. melanogaster, including annotations from a highly reliable set of transposable elements, and functional transcriptomics and epigenomics data for 26 genomes. DrosOmics is based on JBrowse 2, a highly scalable platform, which allows the visualization of multiple assemblies at once, key to unraveling structural and functional features of D. melanogaster natural populations. DrosOmics is an open access browser and is freely available at http://gonzalezlab.eu/drosomics.

Transcriptomes are dynamic, with cells, tissues, and body parts expressing particular sets of transcripts. Transposable elements (TEs) are a known source of transcriptome diversity; however, studies often focus on a particular type of chimeric transcript, analyze single body parts or cell types, or are based on incomplete TE annotations from a single reference genome. In this work, we have implemented a method based on de novo transcriptome assembly that minimizes the potential sources of errors while identifying a comprehensive set of gene-TE chimeras. We applied this method to the head, gut, and ovary dissected from five Drosophila melanogaster natural strains, with individual reference genomes available. We found that ∼19% of body part-specific transcripts are gene-TE chimeras. Overall, chimeric transcripts contribute a mean of 43% to the total gene expression, and they provide protein domains for DNA binding, catalytic activity, and DNA polymerase activity. Our comprehensive data set is a rich resource for follow-up analysis. Moreover, because TEs are present in virtually all species sequenced to date, their role in spatially restricted transcript expression is likely not exclusive to the species analyzed in this work.

Drug–drug salts are a kind of pharmaceutical multicomponent solid in which the two co-existing components are active pharmaceutical ingredients (APIs) in their ionized forms. This novel approach has attracted great interest in the pharmaceutical industry since it not only allows concomitant formulations but also has proved potential to improve the pharmacokinetics of the involved APIs. This is especially interesting for those APIs that have relevant dose-dependent secondary effects, such as non-steroidal anti-inflammatory drugs (NSAIDs). In this work, six multidrug salts involving six different NSAIDs and the antibiotic ciprofloxacin are reported. The novel solids were synthesized using mechanochemical methods and comprehensively characterized in the solid state. Moreover, solubility and stability studies, as well as bacterial inhibition assays, were performed. Our results suggest that our drug–drug formulations enhanced the solubility of NSAIDs without affecting the antibiotic efficacy.

Transposable elements (TEs), also known as transposons, are repetitive DNA sequences, present in virtually all organisms, that can move from one genomic position to another. TEs can be a source of mutations with important consequences for organisms. Despite their interest, its repetitive nature has made their study very challenging. However, the emergence of new sequencing technologies that allow obtaining long-read sequences, has improved the in silico de novo detection and annotation of TEs. The de novo annotation of TEs has already been performed in several organisms including the fruit fly Drosophila melanogaster. Yet, experimental validation can be used to confirm the presence of TEs in specific D. melanogaster natural populations. Here, we present a step-by-step protocol to experimentally validate by polymerase chain reaction (PCR) the presence and/or absence of TEs in natural populations of D. melanogaster. This detailed protocol has been implemented in the participant high schools of the Citizen Fly Lab activity that is part of the international citizen science project Melanogaster: Catch the Fly! (https://melanogaster.eu). Specifically, the students collaborate with the scientists of the European Drosophila Population Genomics Consortium (DrosEU) in the experimental validation of new genetic variants, previously identified using bioinformatic techniques.

High quality reference genomes are crucial to understanding genome function, structure and evolution. The availability of reference genomes has allowed us to start inferring the role of genetic variation in biology, disease, and biodiversity conservation. However, analyses across organisms demonstrate that a single reference genome is not enough to capture the global genetic diversity present in populations. In this work, we generate 32 high-quality reference genomes for the well-known model species D. melanogaster and focus on the identification and analysis of transposable element variation as they are the most common type of structural variant. We show that integrating the genetic variation across natural populations from five climatic regions increases the number of detected insertions by 58%. Moreover, 26% to 57% of the insertions identified using long-reads were missed by short-reads methods. We also identify hundreds of transposable elements associated with gene expression variation and new TE variants likely to contribute to adaptive evolution in this species. Our results highlight the importance of incorporating the genetic variation present in natural populations to genomic studies, which is essential if we are to understand how genomes function and evolve.

Anopheles coluzzii is one of the primary vectors of human malaria in sub-Saharan Africa. Recently, it has spread into the main cities of Central Africa threatening vector control programs. The adaptation of An. coluzzii to urban environments partly results from an increased tolerance to organic pollution and insecticides. Some of the molecular mechanisms for ecological adaptation are known, but the role of transposable elements (TEs) in the adaptive processes of this species has not been studied yet. As a first step toward assessing the role of TEs in rapid urban adaptation, we sequenced using long reads six An. coluzzii genomes from natural breeding sites in two major Central Africa cities. We de novo annotated TEs in these genomes and in an additional high-quality An. coluzzii genome, and we identified 64 new TE families. TEs were nonrandomly distributed throughout the genome with significant differences in the number of insertions of several superfamilies across the studied genomes. We identified seven putatively active families with insertions near genes with functions related to vectorial capacity, and several TEs that may provide promoter and transcription factor binding sites to insecticide resistance and immune-related genes. Overall, the analysis of multiple high-quality genomes allowed us to generate the most comprehensive TE annotation in this species to date and identify several TE insertions that could potentially impact both genome architecture and the regulation of functionally relevant genes. These results provide a basis for future studies of the impact of TEs on the biology of An. coluzzii.

Drosophila melanogaster is a leading model in population genetics and genomics, and a growing number of whole-genome datasets from natural populations of this species have been published over the last years. A major challenge is the integration of disparate datasets, often generated using different sequencing technologies and bioinformatic pipelines, which hampers our ability to address questions about the evolution of this species. Here we address these issues by developing a bioinformatics pipeline that maps pooled sequencing (Pool-Seq) reads from D. melanogaster to a hologenome consisting of fly and symbiont genomes and estimates allele frequencies using either a heuristic (PoolSNP) or a probabilistic variant caller (SNAPE-pooled). We use this pipeline to generate the largest data repository of genomic data available for D. melanogaster to date, encompassing 271 previously published and unpublished population samples from over 100 locations in > 20 countries on four continents. Several of these locations have been sampled at different seasons across multiple years. This dataset, which we call Drosophila Evolution over Space and Time (DEST), is coupled with sampling and environmental meta-data. A web-based genome browser and web portal provide easy access to the SNP dataset. We further provide guidelines on how to use Pool-Seq data for model-based demographic inference. Our aim is to provide this scalable platform as a community resource which can be easily extended via future efforts for an even more extensive cosmopolitan dataset. Our resource will enable population geneticists to analyze spatio-temporal genetic patterns and evolutionary dynamics of D. melanogaster populations in unprecedented detail.

From the reaction of Co(II) hydroxy-carbonates and N-p-(R)-phenethyliminodiacetic acids (Hpheida, R = H and HMOpheida, R = CHO) two binary complexes have been obtained in aqueous media. The crystal pattern of [Co(pheida)(HO)]·1.5HO (1) (monoclinic, space group C2/c) differs from the related non-hydrated crystal of [Co(MOpheida)(HO)] (2) (monoclinic, space group P2/c). Both Co(II) complexes display distorted octahedral geometry imposed by 3d electronic configuration. Our crystallographic results reveal that the metal chelates have a molecular structure and the used methoxy-substituent on the pheida skeleton yield different structural features. The iminodiacetic acid-arms [IDA] of pheida-like ligands adopt fac-NO conformation. For both chelators 1 and 2, single-point energy was calculated using restricted and/or unrestricted Hartree–Fock/BP/B3LYP functional. The analytical frequency, electronic absorption, and HOMO–LUMO energy gap were calculated using B3LYP functional with orbital basis set def2-SVP or def2-TZVP (for 2) along with the auxiliary basis set def2/J. The quantum chemical calculated geometry parameters are compared with their corresponding X-ray crystallographic data. The optical band gap (E ) arises due to the electronic transitions. The direct and indirect band gap energy measured 3.26, 3.19 eV for 1 and 3.39, 3.35 eV for 2, respectively, reflecting their semi-conducting nature. Crystal structure for HMOpheida acid also reported herein.

Transposable elements (TEs) are not randomly distributed in the genome. A genome-wide analysis of the D. melanogaster genome found that differences in TE density across 50 kb genomic regions was due both to transposition and duplication. At smaller genomic scales, promoter regions of hsp genes and the promoter region of CG18446 have been shown to accumulate TE insertions. In this work, we have further analyzed the promoter region of CG18446. We screened 218 strains collected in 15 natural populations, and we found that the CG18446 promoter region contains 20 independent roo insertions. Based on phylogenetic analysis, we suggest that the presence of multiple roo insertions in this region is likely to be the result of several bursts of transposition. Moreover, we found that the roo insertional cluster in the CG18446 promoter region is unique: no other promoter region in the genome contains a similar number of roo insertions. We found that, similar to hsp gene promoters, chromatin accessibility could be one of the factors explaining the recurrent insertions of roo elements in CG18446 promoter region.

The synthetic nucleoside acyclovir is considered an outstanding model of the natural nucleoside guanosine. With the purpose of deepening on the influence and nature of non-covalent interactions regarding molecular recognition patterns, three novel Cu(II) complexes, involving acyclovir (acv) and the ligand receptor N-(2-hydroxyethyl)ethylenediamine (hen), have been synthesized and thoroughly characterized. The three novel compounds introduce none, one or two acyclovir molecules, respectively. Molecular recognition has been evaluated using single crystal X-ray diffraction. Furthermore, theoretical calculations and other physical methods such as thermogravimetric analysis, infrared and UV-Vis spectroscopy, electron paramagnetic resonance and magnetic measurements have been used. Theoretical calculations are in line with experimental results, supporting the relevance of the [metal-N7(acv) + H-bond] molecular recognition pattern. It was also shown that (hen)O-H group is used as preferred H-donor when it is found within the basal coordination plane, since the higher polarity of the terminal (hen)O-H versus the N-H group favours its implication. Otherwise, when (hen)O-H occupies the distal coordination site, (hen)N-H groups can take over

Increases in industrialisation and anthropogenic activity have resulted in an increase in pollutants released into the environment. Of these pollutants, heavy metals such as copper are particularly concerning due to their bio-accumulative nature. Due to its highly heterogeneous distribution and its dual nature as both an essential micronutrient and toxic element, the genetic basis of copper tolerance is likely shaped by a complex interplay of physiological and environmental factors. Drosophila melanogaster, a long-standing sentinel of environmental toxins, is uniquely suited for the study of copper tolerance in arthropods and other more diverse species. In this study, we utilized the natural variation present in multiple populations of D. melanogaster collected across Europe to screen for variation in copper tolerance, which we found to be highly variable both within and between locations. While these collection locations covered a wide range of atmospheric and soil pollution levels, the degree of urbanization at the collection sites, rather than any other combination of environmental factors, was linked to copper tolerance. Moreover, differential expression analysis revealed that metabolism, reproduction, and protease induction contribute to copper response in tolerant and sensitive lines to different degrees. Additionally, the greatest transcriptomic and physiological responses to copper toxicity were seen in the midgut; where preservation of gut acidity is strongly linked to greater tolerance. Overall, our study provides a unique perspective on the genetic and environmental factors that shape copper tolerance in natural D. melanogaster populations and identifies new genes and physiological traits involved in this complex phenotype.

Lignin in soils is a result of organic matter input and depletion, which is regulated by various factors, such as environmental factors like climate change (temperature, moisture, etc.) [1]. Therefore, lignin is considered an indicator of soil organic carbon storage and dynamics. Given its chemical structure, hence recalcitrance, lignin could be used as a biomarker of processes such as stabilization, mineralization, or biodegradation of soil organic matter (SOM) [2]. The study of C and H isotopes may certainly result in a comprehensive approach to estimate the fate of organic compounds, and to better understand the link between H and C cycles in the soil. The use of C stable isotopes is widely used to investigate sources, evolution, and dynamics of SOM, whilst H stable isotopes provide geographical information and insight into the water dynamics in soils [3]. This communication describes a methodology based on analytical pyrolysis for the direct measure of lignin-derived phenolic compounds specific ¿13C and ¿2H isotope composition (Py-CSIA). Based on this methodology, we aim to evaluate the ¿13C and ¿2H isotope composition of lignin-derived phenols as biomarkers of changes in SOM dynamics driven by climatic factors. Composite dehesa surface (0-10 cm) soil samples (Pozoblanco, Córdoba, Spain) were taken from four forced climatic treatment plots representing warming (W), drought (D), its combination (W+D), and control (D), in two distinct habitats: under evergreen oak canopy and in open pasture. The samples were analysed in triplicate for chemical structural characterization by analytical pyrolysis (Py-GC/MS) and in parallel for ¿13C and ¿2H (Py-CSIA) using the same chromatographic conditions. The ¿13C average CSIA values ranged from -27.15 ¿ to -33.45 ¿. Significant differences are reportedly driven by the differences in habitat: more depleted values were found in lignin produced in the open pasture than in tree habitat. In addition to the differences reported for ha

[Background] Anopheles coluzzii is one of the primary vectors of human malaria in sub-Saharan Africa. Recently, it has colonized the main cities of Central Africa threatening vector control programs. The adaptation of An. coluzzii to urban environments is partly due to an increased tolerance to organic pollution and insecticides. While some of the molecular mechanisms for ecological adaptation, including chromosome rearrangements and introgressions, are known, the role of transposable elements (TEs) in the adaptive processes of this species has not been studied yet. To assess the role of TEs in rapid urban adaptation, the first step is to accurately annotate TE insertions in the genomes of natural populations collected in urban settings., [Results] We sequenced using long-reads six An. coluzzii genomes from natural breeding sites in two major Central Africa cities. We de novo annotated the complete set of TEs in these genomes and in an additional high quality An. coluzzii genome available and identified 64 previously undescribed TE families. TEs were non-randomly distributed throughout the genome with significant differences in the number of insertions of several superfamilies across the studied genomes. We identified seven putatively active families with insertions near genes with functions related to vectorial capacity. Moreover, we identified several TE insertions providing promoter and transcription factor binding sites to insecticide resistance and immune-related genes., [Conclusions] The analysis of multiple genomes sequenced using long-read technologies allowed us to generate the most comprehensive TE annotations in this species to date. We identified several TE insertions that could potentially impact both genome architecture and the regulation of functionally relevant genes in An. coluzzii. These results provide a basis for future studies of the impact of TEs on the biology of An. coluzzii.

Drosophila melanogaster is an important model for antiviral immunity in arthropods, but very few DNA viruses have been described from the family Drosophilidae. This deficiency limits our opportunity to use natural host-pathogen combinations in experimental studies, and may bias our understanding of the Drosophila virome. Here, we report fourteen DNA viruses detected in a metagenomic analysis of 6668 pool-sequenced Drosophila, sampled from forty-seven European locations between 2014 and 2016. These include three new nudiviruses, a new and divergent entomopoxvirus, a virus related to Leptopilina boulardi filamentous virus, and a virus related to Musca domestica salivary gland hypertrophy virus. We also find an endogenous genomic copy of galbut virus, a double-stranded RNA partitivirus, segregating at very low frequency. Remarkably, we find that Drosophila Vesanto virus, a small DNA virus previously described as a bidnavirus, may be composed of up to twelve segments and thus represent a new lineage of segmented DNA viruses. Two of the DNA viruses, Drosophila Kallithea nudivirus and Drosophila Vesanto virus are relatively common, found in 2 per cent or more of wild flies. The others are rare, with many likely to be represented by a single infected fly. We find that virus prevalence in Europe reflects the prevalence seen in publicly available datasets, with Drosophila Kallithea nudivirus and Drosophila Vesanto virus the only ones commonly detectable in public data from wild-caught flies and large population cages, and the other viruses being rare or absent. These analyses suggest that DNA viruses are at lower prevalence than RNA viruses in D.melanogaster, and may be less likely to persist in laboratory cultures. Our findings go some way to redressing an earlier bias toward RNA virus studies in Drosophila, and lay the foundation needed to harness the power of Drosophila as a model system for the study of DNA viruses.

[Background] Variation in gene expression underlies interindividual variability in relevant traits including immune response. However, the genetic variation responsible for these gene expression changes remains largely unknown. Among the non-coding variants that could be relevant, transposable element insertions are promising candidates as they have been shown to be a rich and diverse source of cis-regulatory elements., [Results] In this work, we use a population genetics approach to identify transposable element insertions likely to increase the tolerance of Drosophila melanogaster to bacterial infection by affecting the expression of immune-related genes. We identify 12 insertions associated with allele-specific expression changes in immune-related genes. We experimentally validate three of these insertions including one likely to be acting as a silencer, one as an enhancer, and one with a dual role as enhancer and promoter. The direction in the change of gene expression associated with the presence of several of these insertions is consistent with an increased survival to infection. Indeed, for one of the insertions, we show that this is the case by analyzing both natural populations and CRISPR/Cas9 mutants in which the insertion is deleted from its native genomic context., [Conclusions] We show that transposable elements contribute to gene expression variation in response to infection in D. melanogaster and that this variation is likely to affect their survival capacity. Because the role of transposable elements as regulatory elements is not restricted to Drosophila, transposable elements are likely to play a role in immune response in other organisms as well.

To advance our understanding of adaptation to temporally varying selection pressures, we identified signatures of seasonal adaptation occurring in parallel among Drosophila melanogaster populations. Specifically, we estimated allele frequencies genome-wide from flies sampled early and late in the growing season from 20 widely dispersed populations. We identified parallel seasonal allele frequency shifts across North America and Europe, demonstrating that seasonal adaptation is a general phenomenon of temperate fly populations. Seasonally fluctuating polymorphisms are enriched in large chromosomal inversions, and we find a broad concordance between seasonal and spatial allele frequency change. The direction of allele frequency change at seasonally variable polymorphisms can be predicted by weather conditions in the weeks prior to sampling, linking the environment and the genomic response to selection. Our results suggest that fluctuating selection is an important evolutionary force affecting patterns of genetic variation in Drosophila.

Drosophila melanogaster is a leading model in population genetics and genomics, and a growing number of whole-genome datasets from natural populations of this species have been published over the last years. A major challenge is the integration of disparate datasets, often generated using different sequencing technologies and bioinformatic pipelines, which hampers our ability to address questions about the evolution of this species. Here we address these issues by developing a bioinformatics pipeline that maps pooled sequencing (Pool-Seq) reads from D. melanogaster to a hologenome consisting of fly and symbiont genomes and estimates allele frequencies using either a heuristic (PoolSNP) or a probabilistic variant caller (SNAPE-pooled). We use this pipeline to generate the largest data repository of genomic data available for D. melanogaster to date, encompassing 271 previously published and unpublished population samples from over 100 locations in > 20 countries on four continents. Several of these locations have been sampled at different seasons across multiple years. This dataset, which we call Drosophila Evolution over Space and Time (DEST), is coupled with sampling and environmental meta-data. A web-based genome browser and web portal provide easy access to the SNP dataset. We further provide guidelines on how to use Pool-Seq data for model-based demographic inference. Our aim is to provide this scalable platform as a community resource which can be easily extended via future efforts for an even more extensive cosmopolitan dataset. Our resource will enable population geneticists to analyze spatio-temporal genetic patterns and evolutionary dynamics of D. melanogaster populations in unprecedented detail.

Population genomics is a fast-developing discipline with promising applications in a growing number of life sciences fields. Advances in sequencing technologies and bioinformatics tools allow population genomics to exploit genome-wide information to identify the molecular variants underlying traits of interest and the evolutionary forces that modulate these variants through space and time. However, the cost of genomic analyses of multiple populations is still too high to address them through individual genome sequencing. Pooling individuals for sequencing can be a more effective strategy in Single Nucleotide Polymorphism (SNP) detection and allele frequency estimation because of a higher total coverage. However, compared to individual sequencing, SNP calling from pools has the additional difficulty of distinguishing rare variants from sequencing errors, which is often avoided by establishing a minimum threshold allele frequency for the analysis. Finding an optimal balance between minimizing information loss and reducing sequencing costs is essential to ensure the success of population genomics studies. Here, we have benchmarked the performance of SNP callers for Pool-seq data, based on different approaches, under different conditions, and using computer simulations and real data. We found that SNP callers performance varied for allele frequencies up to 0.35. We also found that SNP callers based on Bayesian (SNAPE-pooled) or maximum likelihood (MAPGD) approaches outperform the two heuristic callers tested (VarScan and PoolSNP), in terms of the balance between sensitivity and FDR both in simulated and sequencing data. Our results will help inform the selection of the most appropriate SNP caller not only for large-scale population studies but also in cases where the Pool-seq strategy is the only option, such as in metagenomic or polyploid studies.

While several studies in a diverse set of species have shed light on the genes underlying adaptation, our knowledge on the selective pressures that explain the observed patterns lags behind. Drosophila melanogaster is a valuable organism to study environmental adaptation because this species originated in Southern Africa and has recently expanded worldwide, and also because it has a functionally well-annotated genome. In this study, we aimed to decipher which environmental variables are relevant for adaptation of D. melanogaster natural populations in Europe and North America. We analysed 36 whole-genome pool-seq samples of D. melanogaster natural populations collected in 20 European and 11 North American locations. We used the BayPass software to identify single nucleotide polymorphisms (SNPs) and transposable elements (TEs) showing signature of adaptive differentiation across populations, as well as significant associations with 59 environmental variables related to temperature, rainfall, evaporation, solar radiation, wind, daylight hours, and soil type. We found that in addition to temperature and rainfall, wind related variables are also relevant for D. melanogaster environmental adaptation. Interestingly, 23%–51% of the genes that showed significant associations with environmental variables were not found overly differentiated across populations. In addition to SNPs, we also identified 10 reference transposable element insertions associated with environmental variables. Our results showed that genome-environment association analysis can identify adaptive genetic variants that are undetected by population differentiation analysis while also allowing the identification of candidate environmental drivers of adaptation.