Genetic and neural mechanisms that inhibit Drosophila from mating with other species.

Genetically hard-wired neural mechanisms must enforce behavioral reproductive isolation because interspecies courtship is rare even in sexually naïve animals of most species. We find that the chemoreceptor Gr32a inhibits male D. melanogaster from courting diverse fruit fly species. Gr32a recognizes nonvolatile aversive cues present on these reproductively dead-end targets, and activity of Gr32a neurons is necessary and sufficient to inhibit interspecies courtship. Male-specific Fruitless (Fru(M)), a master regulator of courtship, also inhibits interspecies courtship. Gr32a and Fru(M) are not coexpressed, but Fru(M) neurons contact Gr32a neurons, suggesting that these genes influence a shared neural circuit that inhibits interspecies courtship. Gr32a and Fru(M) also suppress within-species intermale courtship, but we show that distinct mechanisms preclude sexual displays toward conspecific males and other species. Although this chemosensory pathway does not inhibit interspecies mating in D. melanogaster females, similar mechanisms appear to inhibit this behavior in many other male drosophilids.

Reverse and forward engineering of Drosophila corneal nanocoatings.

Insect eyes have an anti-reflective coating, owing to nanostructures on the corneal surface creating a gradient of refractive index between that of air and that of the lens material1,2. These nanocoatings have also been shown to provide anti-adhesive functionality3. The morphology of corneal nanocoatings are very diverse in arthropods, with nipple-like structures that can be organized into arrays or fused into ridge-like structures4. This diversity can be attributed to a reaction-diffusion mechanism4 and patterning principles developed by Alan Turing5, which have applications in numerous biological settings6. The nanocoatings on insect corneas are one example of such Turing patterns, and the first known example of nanoscale Turing patterns4. Here we demonstrate a clear link between the morphology and function of the nanocoatings on Drosophila corneas. We find that nanocoatings that consist of individual protrusions have better anti-reflective properties, whereas partially merged structures have better anti-adhesion properties. We use biochemical analysis and genetic modification techniques to reverse engineer the protein Retinin and corneal waxes as the building blocks of the nanostructures. In the context of Turing patterns, these building blocks fulfil the roles of activator and inhibitor, respectively. We then establish low-cost production of Retinin, and mix this synthetic protein with waxes to forward engineer various artificial nanocoatings with insect-like morphology and anti-adhesive or anti-reflective function. Our combined reverse- and forward-engineering approach thus provides a way to economically produce functional nanostructured coatings from biodegradable materials.

The evolutionary novelty of insect defensins: from bacterial killing to toxin neutralization.

Insect host defense comprises two complementary dimensions, microbial killing-mediated resistance and microbial toxin neutralization-mediated resilience, both jointly providing protection against pathogen infections. Insect defensins are a class of effectors of innate immunity primarily responsible for resistance to Gram-positive bacteria. Here, we report a newly originated gene from an ancestral defensin via genetic deletion following gene duplication in Drosophila virilis, which confers an enhanced resilience to Gram-positive bacterial infection. This gene encodes an 18-mer arginine-rich peptide (termed DvirARP) with differences from its parent gene in its pattern of expression, structure and function. DvirARP specifically expresses in D. virilis female adults with a constitutive manner. It adopts a novel fold with a 310 helix and a two CXC motif-containing loop stabilized by two disulfide bridges. DvirARP exhibits no activity on the majority of microorganisms tested and only a weak activity against two Gram-positive bacteria. DvirARP knockout flies are viable and have no obvious defect in reproductivity but they are more susceptible to the DvirARP-resistant Staphylococcus aureus infection than the wild type files, which can be attributable to its ability in neutralization of the S. aureus secreted toxins. Phylogenetic distribution analysis reveals that DvirARP is restrictedly present in the Drosophila subgenus, but independent deletion variations also occur in defensins from the Sophophora subgenus, in support of the evolvability of this class of immune effectors. Our work illustrates for the first time how a duplicate resistance-mediated gene evolves an ability to increase the resilience of a subset of Drosophila species against bacterial infection.

Topologically associating domains and their role in the evolution of genome structure and function in Drosophila.

Topologically associating domains (TADs) were recently identified as fundamental units of three-dimensional eukaryotic genomic organization, although our knowledge of the influence of TADs on genome evolution remains preliminary. To study the molecular evolution of TADs in Drosophila species, we constructed a new reference-grade genome assembly and accompanying high-resolution TAD map for D. pseudoobscura Comparison of D. pseudoobscura and D. melanogaster, which are separated by ∼49 million years of divergence, showed that ∼30%-40% of their genomes retain conserved TADs. Comparative genomic analysis of 17 Drosophila species revealed that chromosomal rearrangement breakpoints are enriched at TAD boundaries but depleted within TADs. Additionally, genes within conserved TADs show lower expression divergence than those located in nonconserved TADs. Furthermore, we found that a substantial proportion of long genes (>50 kbp) in D. melanogaster (42%) and D. pseudoobscura (26%) constitute their own TADs, implying transcript structure may be one of the deterministic factors for TAD formation. By using structural variants (SVs) identified from 14 D. melanogaster strains, its three closest sibling species from the D. simulans species complex, and two obscura clade species, we uncovered evidence of selection acting on SVs at TAD boundaries, but with the nature of selection differing between SV types. Deletions are depleted at TAD boundaries in both divergent and polymorphic SVs, suggesting purifying selection, whereas divergent tandem duplications are enriched at TAD boundaries relative to polymorphism, suggesting they are adaptive. Our findings highlight how important TADs are in shaping the acquisition and retention of structural mutations that fundamentally alter genome organization.

The contribution of mutation and selection to multivariate quantitative genetic variance in an outbred population of Drosophila serrata.

Genetic variance is not equal for all multivariate combinations of traits. This inequality, in which some combinations of traits have abundant genetic variation while others have very little, biases the rate and direction of multivariate phenotypic evolution. However, we still understand little about what causes genetic variance to differ among trait combinations. Here, we investigate the relative roles of mutation and selection in determining the genetic variance of multivariate phenotypes. We accumulated mutations in an outbred population of Drosophila serrata and analyzed wing shape and size traits for over 35,000 flies to simultaneously estimate the additive genetic and additive mutational (co)variances. This experimental design allowed us to gain insight into the phenotypic effects of mutation as they arise and come under selection in naturally outbred populations. Multivariate phenotypes associated with more (less) genetic variance were also associated with more (less) mutational variance, suggesting that differences in mutational input contribute to differences in genetic variance. However, mutational correlations between traits were stronger than genetic correlations, and most mutational variance was associated with only one multivariate trait combination, while genetic variance was relatively more equal across multivariate traits. Therefore, selection is implicated in breaking down trait covariance and resulting in a different pattern of genetic variance among multivariate combinations of traits than that predicted by mutation and drift. Overall, while low mutational input might slow evolution of some multivariate phenotypes, stabilizing selection appears to reduce the strength of evolutionary bias introduced by pleiotropic mutation.

The evolution of sex-biased gene expression in the Drosophila brain.

Genes with sex-biased expression in Drosophila are thought to underlie sexually dimorphic phenotypes and have been shown to possess unique evolutionary properties. However, the forces and constraints governing the evolution of sex-biased genes in the somatic tissues of Drosophila are largely unknown. By using population-scale RNA sequencing data, we show that sex-biased genes in the Drosophila brain are highly enriched on the X Chromosome and that most are biased in a species-specific manner. We show that X-linked male-biased genes, and to a lesser extent female-biased genes, are enriched for signatures of directional selection at the gene expression level. By examining the evolutionary properties of gene-flanking regions on the X Chromosome, we find evidence that adaptive cis-regulatory changes are more likely to drive the expression evolution of X-linked male-biased genes than other X-linked genes. Finally, we examine whether constraint owing to broad expression across multiple tissues and genetic constraint owing to the largely shared male and female genomes could be responsible for the observed patterns of gene expression evolution. We find that expression breadth does not constrain the directional evolution of gene expression in the brain. Additionally, we find that the shared genome between males and females imposes a substantial constraint on the expression evolution of sex-biased genes. Overall, these results significantly advance our understanding of the patterns and forces shaping the evolution of sexual dimorphism in the Drosophila brain.

Behavioral and environmental contributions to drosophilid social networks.

Animals interact with each other in species-specific reproducible patterns. These patterns of organization are captured by social network analysis, and social interaction networks (SINs) have been described for a wide variety of species including fish, insects, birds, and mammals. The aim of this study is to understand the evolution of social organization in Drosophila Using a comparative ecological, phylogenetic, and behavioral approach, the different properties of SINs formed by 20 drosophilids were compared. We investigate whether drosophilid network structures arise from common ancestry, a response to the species' past climate, other social behaviors, or a combination of these factors. This study shows that differences in past climate predicted the species' current SIN properties. The drosophilid phylogeny offered no value to predicting species' differences in SINs through phylogenetic signal tests. This suggests that group-level social behaviors in drosophilid species are shaped by divergent climates. However, we find that the social distance at which flies interact correlated with the drosophilid phylogeny, indicating that behavioral elements of SINs have remained largely unchanged in their evolutionary history. We find a significant correlation of leg length to social distance, outlining the interdependence of anatomy and complex social structures. Although SINs display a complex evolutionary relationship across drosophilids, this study suggests that the ecology, and not common ancestry, contributes to diversity in social structure in Drosophila.

A major role for noncoding regulatory mutations in the evolution of enzyme activity.

The quantitative evolution of protein activity is a common phenomenon, yet we know little about any general mechanistic tendencies that underlie it. For example, an increase (or decrease) in enzyme activity may evolve from changes in protein sequence that alter specific activity, or from changes in gene expression that alter the amount of protein produced. The latter in turn could arise via mutations that affect gene transcription, posttranscriptional processes, or copy number. Here, to determine the types of genetic changes underlying the quantitative evolution of protein activity, we dissected the basis of ecologically relevant differences in Alcohol dehydrogenase (Adh) enzyme activity between and within several Drosophila species. By using recombinant Adh transgenes to map the functional divergence of ADH enzyme activity in vivo, we find that amino acid substitutions explain only a minority (0 to 25%) of between- and within-species differences in enzyme activity. Instead, noncoding substitutions that occur across many parts of the gene (enhancer, promoter, and 5' and 3' untranslated regions) account for the majority of activity differences. Surprisingly, one substitution in a transcriptional Initiator element has occurred in parallel in two species, indicating that core promoters can be an important natural source of the tuning of gene activity. Furthermore, we show that both regulatory and coding substitutions contribute to fitness (resistance to ethanol toxicity). Although qualitative changes in protein specificity necessarily derive from coding mutations, these results suggest that regulatory mutations may be the primary source of quantitative changes in protein activity, a possibility overlooked in most analyses of protein evolution.

The exon junction complex is required for definition and excision of neighboring introns in Drosophila.

Splicing of pre-mRNAs results in the deposition of the exon junction complex (EJC) upstream of exon-exon boundaries. The EJC plays crucial post-splicing roles in export, translation, localization, and nonsense-mediated decay of mRNAs. It also aids faithful splicing of pre-mRNAs containing large introns, albeit via an unknown mechanism. Here, we show that the core EJC plus the accessory factors RnpS1 and Acinus aid in definition and efficient splicing of neighboring introns. This requires prior deposition of the EJC in close proximity to either an upstream or downstream splicing event. If present in isolation, EJC-dependent introns are splicing-defective also in wild-type cells. Interestingly, the most affected intron belongs to the piwi locus, which explains the reported transposon desilencing in EJC-depleted Drosophila ovaries. Based on a transcriptome-wide analysis, we propose that the dependency of splicing on the EJC is exploited as a means to control the temporal order of splicing events.

Heterochromatin formation in Drosophila requires genome-wide histone deacetylation in cleavage chromatin before mid-blastula transition in early embryogenesis.

Su(var) mutations define epigenetic factors controlling heterochromatin formation and gene silencing in Drosophila. Here, we identify SU(VAR)2-1 as a novel chromatin regulator that directs global histone deacetylation during the transition of cleavage chromatin into somatic blastoderm chromatin in early embryogenesis. SU(VAR)2-1 is heterochromatin-associated in blastoderm nuclei but not in later stages of development. In larval polytene chromosomes, SU(VAR)2-1 is a band-specific protein. SU(VAR)2-1 directs global histone deacetylation by recruiting the histone deacetylase RPD3. In Su(var)2-1 mutants H3K9, H3K27, H4K8 and H4K16 acetylation shows elevated levels genome-wide and heterochromatin displays aberrant histone hyper-acetylation. Whereas H3K9me2- and HP1a-binding appears unaltered, the heterochromatin-specific H3K9me2S10ph composite mark is impaired in heterochromatic chromocenters of larval salivary polytene chromosomes. SU(VAR)2-1 contains an NRF1/EWG domain and a C2HC zinc-finger motif. Our study identifies SU(VAR)2-1 as a dosage-dependent, heterochromatin-initiating SU(VAR) factor, where the SU(VAR)2-1-mediated control of genome-wide histone deacetylation after cleavage and before mid-blastula transition (pre-MBT) is required to enable heterochromatin formation.

A Burst of Genetic Innovation in Drosophila Actin-Related Proteins for Testis-Specific Function.

Many cytoskeletal proteins perform fundamental biological processes and are evolutionarily ancient. For example, the superfamily of actin-related proteins (Arps) specialized early in eukaryotic evolution for diverse cellular roles in the cytoplasm and the nucleus. Despite its strict conservation across eukaryotes, we find that the Arp superfamily has undergone dramatic lineage-specific diversification in Drosophila. Our phylogenomic analyses reveal four independent Arp gene duplications that occurred in the common ancestor of the obscura group of Drosophila and have been mostly preserved in this lineage. All four obscura-specific Arp paralogs are predominantly expressed in the male germline and have evolved under positive selection. We focus our analyses on the divergent Arp2D paralog, which arose via a retroduplication event from Arp2, a component of the Arp2/3 complex that polymerizes branched actin networks. Computational modeling analyses suggest that Arp2D can replace Arp2 in the Arp2/3 complex and bind actin monomers. Together with the signature of positive selection, our findings suggest that Arp2D may augment Arp2's functions in the male germline. Indeed, we find that Arp2D is expressed during and following male meiosis, where it localizes to distinct locations such as actin cones-specialized cytoskeletal structures that separate bundled spermatids into individual mature sperm. We hypothesize that this unprecedented burst of genetic innovation in cytoskeletal proteins may have been driven by the evolution of sperm heteromorphism in the obscura group of Drosophila.

A phylogeny for the Drosophila montium species group: A model clade for comparative analyses.

The Drosophila montium species group is a clade of 94 named species, closely related to the model species D. melanogaster. The montium species group is distributed over a broad geographic range throughout Asia, Africa, and Australasia. Species of this group possess a wide range of morphologies, mating behaviors, and endosymbiont associations, making this clade useful for comparative analyses. We use genomic data from 42 available species to estimate the phylogeny and relative divergence times within the montium species group, and its relative divergence time from D. melanogaster. To assess the robustness of our phylogenetic inferences, we use 3 non-overlapping sets of 20 single-copy coding sequences and analyze all 60 genes with both Bayesian and maximum likelihood methods. Our analyses support monophyly of the group. Apart from the uncertain placement of a single species, D. baimaii, our analyses also support the monophyly of all seven subgroups proposed within the montium group. Our phylograms and relative chronograms provide a highly resolved species tree, with discordance restricted to estimates of relatively short branches deep in the tree. In contrast, age estimates for the montium crown group, relative to its divergence from D. melanogaster, depend critically on prior assumptions concerning variation in rates of molecular evolution across branches, and hence have not been reliably determined. We discuss methodological issues that limit phylogenetic resolution - even when complete genome sequences are available - as well as the utility of the current phylogeny for understanding the evolutionary and biogeographic history of this clade.

An investigation of Y chromosome incorporations in 400 species of Drosophila and related genera.

Y chromosomes are widely believed to evolve from a normal autosome through a process of massive gene loss (with preservation of some male genes), shaped by sex-antagonistic selection and complemented by occasional gains of male-related genes. The net result of these processes is a male-specialized chromosome. This might be expected to be an irreversible process, but it was found in 2005 that the Drosophila pseudoobscura Y chromosome was incorporated into an autosome. Y chromosome incorporations have important consequences: a formerly male-restricted chromosome reverts to autosomal inheritance, and the species may shift from an XY/XX to X0/XX sex-chromosome system. In order to assess the frequency and causes of this phenomenon we searched for Y chromosome incorporations in 400 species from Drosophila and related genera. We found one additional large scale event of Y chromosome incorporation, affecting the whole montium subgroup (40 species in our sample); overall 13% of the sampled species (52/400) have Y incorporations. While previous data indicated that after the Y incorporation the ancestral Y disappeared as a free chromosome, the much larger data set analyzed here indicates that a copy of the Y survived as a free chromosome both in montium and pseudoobscura species, and that the current Y of the pseudoobscura lineage results from a fusion between this free Y and the neoY. The 400 species sample also showed that the previously suggested causal connection between X-autosome fusions and Y incorporations is, at best, weak: the new case of Y incorporation (montium) does not have X-autosome fusion, whereas nine independent cases of X-autosome fusions were not followed by Y incorporations. Y incorporation is an underappreciated mechanism affecting Y chromosome evolution; our results show that at least in Drosophila it plays a relevant role and highlight the need of similar studies in other groups.

Supervised machine learning reveals introgressed loci in the genomes of Drosophila simulans and D. sechellia.

Hybridization and gene flow between species appears to be common. Even though it is clear that hybridization is widespread across all surveyed taxonomic groups, the magnitude and consequences of introgression are still largely unknown. Thus it is crucial to develop the statistical machinery required to uncover which genomic regions have recently acquired haplotypes via introgression from a sister population. We developed a novel machine learning framework, called FILET (Finding Introgressed Loci via Extra-Trees) capable of revealing genomic introgression with far greater power than competing methods. FILET works by combining information from a number of population genetic summary statistics, including several new statistics that we introduce, that capture patterns of variation across two populations. We show that FILET is able to identify loci that have experienced gene flow between related species with high accuracy, and in most situations can correctly infer which population was the donor and which was the recipient. Here we describe a data set of outbred diploid Drosophila sechellia genomes, and combine them with data from D. simulans to examine recent introgression between these species using FILET. Although we find that these populations may have split more recently than previously appreciated, FILET confirms that there has indeed been appreciable recent introgression (some of which might have been adaptive) between these species, and reveals that this gene flow is primarily in the direction of D. simulans to D. sechellia.

Karyotype Description of Two Andean Species of the guarani Group of Drosophila (Díptera: Drosophilidae) and Cytological Notes.

The guarani group of Drosophila genus (Diptera: Drosophilidae) is formed by 24 species however the relationship of these species is not clear. In the present study are described the karyotypes of Drosophila sachapuyu Peñafiel and Rafael, 2018 and Drosophila zamorana Peñafiel and Rafael, 2018, two Andean species members of the guarani group. Mitotic chromosomes from cerebral ganglia of third stand larval were obtained by thermal shock and cell suspension techniques. The karyotype of D. sachapuyu, presents 2n = 10 (4R, 1V; X = R, Y = R) while D. zamorana exhibits karyotype 2n = 12 (5R, 1V; X = V, Y = R).

A Continuum of Evolving De Novo Genes Drives Protein-Coding Novelty in Drosophila.

Orphan genes, lacking detectable homologs in outgroup species, typically represent 10-30% of eukaryotic genomes. Efforts to find the source of these young genes indicate that de novo emergence from non-coding DNA may in part explain their prevalence. Here, we investigate the roots of orphan gene emergence in the Drosophila genus. Across the annotated proteomes of twelve species, we find 6297 orphan genes within 4953 taxon-specific clusters of orthologs. By inferring the ancestral DNA as non-coding for between 550 and 2467 (8.7-39.2%) of these genes, we describe for the first time how de novo emergence contributes to the abundance of clade-specific Drosophila genes. In support of them having functional roles, we show that de novo genes have robust expression and translational support. However, the distinct nucleotide sequences of de novo genes, which have characteristics intermediate between intergenic regions and conserved genes, reflect their recent birth from non-coding DNA. We find that de novo genes encode more disordered proteins than both older genes and intergenic regions. Together, our results suggest that gene emergence from non-coding DNA provides an abundant source of material for the evolution of new proteins. Following gene birth, gradual evolution over large evolutionary timescales moulds sequence properties towards those of conserved genes, resulting in a continuum of properties whose starting points depend on the nucleotide sequences of an initial pool of novel genes.

Origin and evolution of the CYP4G subfamily in insects, cytochrome P450 enzymes involved in cuticular hydrocarbon synthesis.

The large and diverse P450 (CYP) superfamily encodes enzymes with a wide spectrum of monooxygenase and related activities. Insect P450 enzymes of the CYP4G subfamily are known to catalyze the synthesis of cuticular hydrocarbons that serve multiple functions from desiccation resistance to chemical communication. These functions are essential for survival. In order to understand the evolution of insect CYP4G genes, 368 sequences from 24 insect orders and 167 species were mined and analyzed. The genomes of most species of Neoptera carry at least two CYP4G genes that are paralogs of the two Drosophila CYP4G genes. The duplication of the original CYP4G is basal to Neoptera and no CYP4G is found in Paleoptera, or beyond the class Insecta. The sequences of CYP4G and particularly their active site have been highly conserved over 400 MY, but all CYP4G sequences are characterized by a +44 residue insertion between the G and H helices, which protrudes from the globular structure of the enzyme distally from the membrane anchor. Although it is generally considered that genes with highly conserved sequence and function are evolutionarily "stable", the evidence from the CYP4G subfamily shows that since their initial duplication over 400 MYA, these genes have experienced many gene births and deaths. The CYP4G1 homolog has been lost several times, and is missing in five orders of insects. These losses are both ancient, as in all Hemiptera and Thysanoptera, and more recent as in honey bees. Serial duplications leading to CYP4G gene clusters have also been observed, as in house flies and in fireflies. The detailed evolutionary history of CYP4G genes does not support the "stability" of these essential genes, but rather a "revolving door" pattern where their essential function is maintained despite an apparently random birth and death process. The dual function of cuticular hydrocarbons, in desiccation resistance achieved mainly by the quantity of hydrocarbons produced and in chemical communication, achieved by the blend of hydrocarbons produced, may explain the apparently paradoxical evolution of CYP4G genes.

Variation in mobility and exercise adaptations between Drosophila species.

Locomotion and mobility have been studied extensively in Drosophila melanogaster but less is known about the locomotor capacity of other Drosophila species, while the response to chronic exercise in other species has yet to be examined. We have shown that adult male D. melanogaster adapt to exercise training with improved running endurance, climbing speed, and flight ability compared to unexercised flies. Here, we examine baseline mobility of D. sechellia, D. simulans, and D. virilis, and their response to chronic exercise training. We found significant interspecific differences in mobility and in the response to exercise. Although there is a significant sex difference in exercise adaptations in D. melanogaster, intraspecific analysis reveals few sex differences in other Drosophila species. As octopamine has been shown to be important for exercise adaptations in D. melanogaster, we also asked if any observed differences could be attributed to baseline octopamine levels. We find that octopamine and tyramine levels have the same rank order as baseline climbing speed and endurance in males, but do not predict the response to chronic exercise in males or females. Future research should focus on determining the mechanisms responsible for the inter- and intraspecific differences in mobility and the response to exercise.

Nascent-seq indicates widespread cotranscriptional RNA editing in Drosophila.

The RNA editing enzyme ADAR chemically modifies adenosine (A) to inosine (I), which is interpreted by the ribosome as a guanosine. Here we assess cotranscriptional A-to-I editing in Drosophila by isolating nascent RNA from adult fly heads and subjecting samples to high throughput sequencing. There are a large number of edited sites within nascent exons. Nascent RNA from an ADAR-null strain was also sequenced, indicating that almost all A-to-I events require ADAR. Moreover, mRNA editing levels correlate with editing levels within the cognate nascent RNA sequence, indicating that the extent of editing is set cotranscriptionally. Surprisingly, the nascent data also identify an excess of intronic over exonic editing sites. These intronic sites occur preferentially within introns that are poorly spliced cotranscriptionally, suggesting a link between editing and splicing. We conclude that ADAR-mediated editing is more widespread than previously indicated and largely occurs cotranscriptionally.

Desiccation resistance and pigmentation variation reflects bioclimatic differences in the Drosophila americana species complex.

BACKGROUND: Disentangling the selective factors shaping adaptive trait variation is an important but challenging task. Many studies-especially in Drosophila-have documented trait variation along latitudinal or altitudinal clines, but frequently lack resolution about specific environmental gradients that could be causal selective agents, and often do not investigate covariation between traits simultaneously. Here we examined variation in multiple macroecological factors across geographic space and their associations with variation in three physiological traits (desiccation resistance, UV resistance, and pigmentation) at both population and species scales, to address the role of abiotic environment in shaping trait variation. RESULTS: Using environmental data from collection locations of three North American Drosophila species-D. americana americana, D. americana texana and D. novamexicana-we identified two primary axes of macroecological variation; these differentiated species habitats and were strongly loaded for precipitation and moisture variables. In nine focal populations (three per species) assayed for each trait, we detected significant species-level variation for both desiccation resistance and pigmentation, but not for UV resistance. Species-level trait variation was consistent with differential natural selection imposed by variation in habitat water availability, although patterns of variation differed between desiccation resistance and pigmentation, and we found little evidence for pleiotropy between traits. CONCLUSIONS: Our multi-faceted approach enabled us to identify potential agents of natural selection and examine how they might influence the evolution of multiple traits at different evolutionary scales. Our findings highlight that environmental factors influence functional trait variation in ways that can be complex, and point to the importance of studies that examine these relationships at both population- and species-levels.