Transcriptome-wide expression variation associated with environmental plasticity and mating success in cactophilic Drosophila mojavensis.

Ecological speciation occurs with the adaptation of populations to different environments and concurrent evolution of reproductive isolation. Phenotypic plasticity might influence both ecological adaptation and reproductive traits. We examined environment-specific gene expression and male mating success in cactophilic Drosophila mojavensis using transcriptome sequencing. This species exhibits cactus-dependent mating success across different species of host plants, with genotype-by-environment interactions for numerous traits. We cultured flies from egg to eclosion on two natural cactus hosts and surveyed gene expression in adult males that were either successful or unsuccessful in achieving copulation in courtship trials. We identified gene expression differences that included functions involved with metabolism, most likely related to chemical differences between host cactus species. Several epigenetic-related functions were identified that might play a role in modulating gene expression in adults due to host cactus effects on larvae, and mating success. Cactus-dependent mating success involved expression differences of genes implicated in translation, transcription, and nervous system development. This suggests a role of neurological function genes in the mating success of D. mojavensis males. Together, these results suggest that the influence of environmental variation on mating success via regulation of gene expression might be an important aspect of ecological speciation.

Male-specific transfer and fine scale spatial differences of newly identified cuticular hydrocarbons and triacylglycerides in a Drosophila species pair.

We analyzed epicuticular hydrocarbon variation in geographically isolated populations of D. mojavensis cultured on different rearing substrates and a sibling species, D. arizonae, with ultraviolet laser desorption/ionization mass spectrometry (UV-LDI MS). Different body parts, i.e. legs, proboscis, and abdomens, of both species showed qualitatively similar hydrocarbon profiles consisting mainly of long-chain monoenes, dienes, trienes, and tetraenes. However, D. arizonae had higher amounts of most hydrocarbons than D. mojavensis and females of both species exhibited greater hydrocarbon amounts than males. Hydrocarbon profiles of D. mojavensis populations were significantly influenced by sex and rearing substrates, and differed between body parts. Lab food-reared flies had lower amounts of most hydrocarbons than flies reared on fermenting cactus substrates. We discovered 48 male- and species-specific hydrocarbons ranging in size from C(22) to C(50) in the male anogenital region of both species, most not described before. These included several oxygen-containing hydrocarbons in addition to high intensity signals corresponding to putative triacylglycerides, amounts of which were influenced by larval rearing substrates. Some of these compounds were transferred to female cuticles in high amounts during copulation. This is the first study showing that triacylglycerides may be a separate class of courtship-related signaling molecules in drosophilids. This study also extends the kind and number of epicuticular hydrocarbons in these species and emphasizes the role of larval ecology in influencing amounts of these compounds, many of which mediate courtship success within and between species.

Genetics of incipient speciation in Drosophila mojavensis. III. Life-history divergence in allopatry and reproductive isolation.

We carried out a three-tiered genetic analysis of egg-to-adult development time and viability in ancestral and derived populations of cactophilic Drosophila mojavensis to test the hypothesis that evolution of these life-history characters has shaped premating reproductive isolation in this species. First, a common garden experiment with 11 populations from Baja California and mainland Mexico and Arizona reared on two host species revealed significant host plant X region and population interactions for viability and development time, evidence for host plant adaptation. Second, replicated line crosses with flies reared on both hosts revealed autosomal, X chromosome, cytoplasmic, and autosome X cactus influences on development time. Viability differences were influenced by host plants, autosomal dominance, and X chromosomal effects. Many of the F(1) , F(2) , and backcross generations showed evidence of heterosis for viability. Third, a QTL analysis of male courtship song and epicuticular hydrocarbon variation based on 1688 Baja × mainland F(2) males also revealed eight QTL influencing development time differences. Mainland alleles at six of these loci were associated with longer development times, consistent with population-level differences. Eight G × E interactions were also detected caused by longer development times of mainland alleles expressed on a mainland host with smaller differences among Baja genotypes reared on the Baja host plant. Four QTL influenced both development time and epicuticular hydrocarbon differences associated with courtship success, and there was a significant QTL-based correlation between development time and cuticular hydrocarbon variation. Thus, the regional shifts in life histories that evolved once D. mojavensis invaded mainland Mexico from Baja California by shifting host plants were genetically correlated with variation in cuticular hydrocarbon-based mate preferences.

Genetics of incipient speciation in Drosophila mojavensis: II. Host plants and mating status influence cuticular hydrocarbon QTL expression and G x E interactions.

We performed a quantitative trait locus (QTL) analysis of epicuticular hydrocarbon variation in 1650 F(2) males from crosses of Baja California and mainland Mexico populations of Drosophila mojavensis cultured on two major host cacti. Principal component (PC) analysis revealed five PCs that accounted for 82% of the total epicuticular hydrocarbon variation. Courtship trials with mainland females were used to characterize hydrocarbon profiles of mated and unmated F(2) males, and logistic regression analysis showed that cactus substrates, two PCs, and a PC by cactus interaction were associated with mating success. Multiple QTLs were detected for each hydrocarbon PC and seven G x E (cactus) interactions were uncovered for the X, second, and fourth chromosomes. Males from the courtship trials and virgins were used, so "exposure to females" was included as a factor in QTL analyses. "Exposed" males expressed significantly different hydrocarbon profiles than virgins for most QTLs, particularly for the two PCs associated with mating success. Ten QTLs showed G x E (exposure) interactions with most resulting from mainland genotypes expressing altered hydrocarbon amounts when exposed to females compared to Baja genotypes. Many cactus x exposure interaction terms detected across QTL and all PCs confirmed that organ pipe-reared males expressed significantly lower hydrocarbon amounts when exposed to females than when reared on agria cactus. Epicuticular hydrocarbon variation in D. mojavensis is therefore a multigenic trait with some epistasis, multiple QTLs exhibited pleiotropy, correlated groups of hydrocarbons and cactus substrates determined courtship success, and males altered their hydrocarbon profiles in response to females.

Genetics of incipient speciation in Drosophila mojavensis. I. Male courtship song, mating success, and genotype x environment interactions.

Few studies have examined genotype by environment (GxE) effects on premating reproductive isolation and associated behaviors, even though such effects may be common when speciation is driven by adaptation to different environments. In this study, mating success and courtship song differences among diverging populations of Drosophila mojavensis were investigated in a two-environment quantitative trait locus (QTL) analysis. Baja California and mainland Mexico populations of D. mojavensis feed and breed on different host cacti, so these host plants were used to culture F2 males to examine host-specific QTL effects and GxE interactions influencing mating success and courtship songs. Linear selection gradient analysis showed that mainland females mated with males that produced songs with significantly shorter L(long)-IPIs, burst durations, and interburst intervals. Twenty-one microsatellite loci distributed across all five major chromosomes were used to localize effects of mating success, time to copulation, and courtship song components. Male courtship success was influenced by a single detected QTL, the main effect of cactus, and four GxE interactions, whereas time to copulation was influenced by three different QTLs on the fourth chromosome. Multiple-locus restricted maximum likelihood (REML) analysis of courtship song revealed consistent effects linked with the same fourth chromosome markers that influenced time to copulation, a number of GxE interactions, and few possible cases of epistasis. GxE interactions for mate choice and song can maintain genetic variation in populations, but alter outcomes of sexual selection and isolation, so signal evolution and reproductive isolation may be slowed in diverging populations. Understanding the genetics of incipient speciation in D. mojavensis clearly depends on cactus-specific expression of traits associated with courtship behavior and sexual isolation.