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BackgroundPolytene chromosome banding patterns have long been used by Drosophila evolutionists to infer degree of relatedness among taxa. Recently, nucleotide sequences have preempted this traditional method. We place the classical Drosophila evolutionary biology tools of polytene chromosome inversion analysis in a phylogenetic context and assess their utility in comparison to nucleotide sequences.ResultsA simultaneous analysis framework was used to examine the congruence of the chromosomal inversion data with more recent DNA sequence data in four Drosophila species groups - the melanogaster, virilis, repleta, and picture wing. Inversions and nucleotides were highly congruent with one another based on incongruence length difference and partitioned Bremer support values. Inversion phylogenies were less resolved because of fewer numbers of characters. Partitioned Bremer supports, corrected for the number of characters in each matrix, were higher for inversion matrices.ConclusionsPolytene chromosome data are highly congruent with DNA sequence data and, when placed in a simultaneous analysis framework, are shown to be more information rich than nucleotide data.

Variation in gene expression in response to the use of alternate host plants can reveal genetic and physiological mechanisms explaining why insect-host relationships vary from host specialism to generalism. Interpreting transcriptome variation relies on well-annotated genomes, making drosophilids valuable model systems, particularly those species with tractable ecological associations. Patterns of whole genome expression and alternate gene splicing in response to growth on different hosts have revealed expression of gene networks of known detoxification genes as well as novel functionally enriched genes of diverse metabolic and structural functions. Integrating trancriptomic responses with fitness differences and levels of phenotypic plasticity in response to alternate hosts will help to reveal the general nature of genotype-phenotype relationships., (Copyright © 2019. Published by Elsevier Inc.)

We assessed the effects of temperature differences on gene expression using whole-transcriptome microarrays and cuticular hydrocarbon variation in populations of cactophilic Drosophila mojavensis . Four populations from Baja California and mainland Mexico and Arizona were each reared on two different host cacti, reared to sexual maturity on laboratory media, and adults were exposed for 12 hr to 15, 25, or 35°C. Temperature differences influenced the expression of 3,294 genes, while population differences and host plants affected >2,400 each in adult flies. Enriched, functionally related groups of genes whose expression changed at high temperatures included heat response genes, as well as genes affecting chromatin structure. Gene expression differences between mainland and peninsular populations included genes involved in metabolism of secondary compounds, mitochondrial activity, and tRNA synthases. Flies reared on the ancestral host plant, pitaya agria cactus, showed upregulation of genes involved in metabolism, while flies reared on organ pipe cactus had higher expression of DNA repair and chromatin remodeling genes. Population × environment (G × E) interactions had widespread effects on the transcriptome where population × temperature interactions affected the expression of >5,000 orthologs, and there were >4,000 orthologs that showed temperature × host plant interactions. Adults exposed to 35°C had lower amounts of most cuticular hydrocarbons than those exposed to 15 or 25°C, including abundant unsaturated alkadienes. For insects adapted to different host plants and climatic regimes, our results suggest that temperature shifts associated with climate change have large and significant effects on transcriptomes of genetically differentiated natural populations.

Preadult determinants of adult fitness and behaviour have been documented in a variety of organisms with complex life cycles, but little is known about expression patterns of genes underlying these adult traits. We explored the effects of differences in egg-to-adult development time on adult transcriptome and cuticular hydrocarbon variation in order to understand the nature of the genetic correlation between preadult development time and premating isolation between populations of Drosophila mojavensis reared in different host cactus environments. Transcriptome variation was analysed separately in flies reared on each host and revealed that hundreds of genes in adults were differentially expressed (FDR P < 0.05) due to development time differences. For flies reared on pitaya agria cactus, longer preadult development times caused increased expression of genes in adults enriched for ribosome production, protein metabolism, chromatin remodelling and regulation of alternate splicing and transcription. Baja California flies reared on organ pipe cactus showed fewer differentially expressed genes in adults due to longer preadult development time, but these were enriched for ATP synthesis and the TCA cycle. Mainland flies reared on organ pipe cactus with shorter development times showed increased transcription of genes enriched for mitochondria and energy production, protein synthesis and glucose metabolism: adults with longer development times had increased expression of genes enriched for adult life span, cuticle proteins and ion binding, although most differentially expressed genes were unannotated. Differences due to population, sex, mating status and their interactions were also assessed. Adult cuticular hydrocarbon profiles also showed shifts due to egg-to-adult development time and were influenced by population and mating status. These results help to explain why preadult life history variation determines subsequent expression of the adult transcriptome along with traits involved with reproductive isolation and revealed previously undocumented connections between genetic and environmental influences over the entire life cycle in this desert insect., (© 2015 John Wiley & Sons Ltd.)

Models of speciation-with-gene-flow have shown that the reduction in recombination between alternative chromosome arrangements can facilitate the fixation of locally adaptive genes in the face of gene flow and contribute to speciation. However, it has proven frustratingly difficult to show empirically that inversions have reduced gene flow and arose during or shortly after the onset of species divergence rather than represent ancestral polymorphisms. Here, we present an analysis of whole genome data from a pair of cactophilic fruit flies, Drosophila mojavensis and D. arizonae, which are reproductively isolated in the wild and differ by several large inversions on three chromosomes. We found an increase in divergence at rearranged compared to colinear chromosomes. Using the density of divergent sites in short sequence blocks we fit a series of explicit models of species divergence in which gene flow is restricted to an initial period after divergence and may differ between colinear and rearranged parts of the genome. These analyses show that D. mojavensis and D. arizonae have experienced postdivergence gene flow that ceased around 270 KY ago and was significantly reduced in chromosomes with fixed inversions. Moreover, we show that these inversions most likely originated around the time of species divergence which is compatible with theoretical models that posit a role of inversions in speciation with gene flow., (© 2015 The Author(s). Evolution published by Wiley Periodicals, Inc. on behalf of The Society for the Study of Evolution.)

We compared whole transcriptome variation in six pre-adult stages and seven adult female ages in two populations of cactophilic Drosophila mojavensis reared on two host plants to understand how differences in gene expression influence standing life history variation. We used singular value decomposition (SVD) to identify dominant trajectories of life cycle gene expression variation, performed pairwise comparisons of stage and age differences in gene expression across the life cycle, identified when genes exhibited maximum levels of life cycle gene expression, and assessed population and host cactus effects on gene expression. Life cycle SVD analysis returned four significant components of transcriptional variation, revealing functional enrichment of genes responsible for growth, metabolic function, sensory perception, neural function, translation and ageing. Host cactus effects on female gene expression revealed population- and stage-specific differences, including significant host plant effects on larval metabolism and development, as well as adult neurotransmitter binding and courtship behaviour gene expression levels. In 3- to 6-day-old virgin females, significant upregulation of genes associated with meiosis and oogenesis was accompanied by downregulation of genes associated with somatic maintenance, evidence for a life history trade-off. The transcriptome of D. mojavensis reared in natural environments throughout its life cycle revealed core developmental transitions and genome-wide influences on life history variation in natural populations., (© 2014 John Wiley & Sons Ltd.)

Analysis of sexual selection and sexual isolation in Drosophila mojavensis and its relatives has revealed a pervasive role of rearing substrates on adult courtship behavior when flies were reared on fermenting cactus in preadult stages. Here, we assessed expression of contact pheromones comprised of epicuticular hydrocarbons (CHCs) from eclosion to 28 days of age in adults from two populations reared on fermenting tissues of two host cacti over the entire life cycle. Flies were never exposed to laboratory food and showed significant reductions in average CHC amounts consistent with CHCs of wild-caught flies. Overall, total hydrocarbon amounts increased from eclosion to 14-18 days, well past age at sexual maturity, and then declined in older flies. Most flies did not survive past 4 weeks. Baja California and mainland populations showed significantly different age-specific CHC profiles where Baja adults showed far less age-specific changes in CHC expression. Adults from populations reared on the host cactus typically used in nature expressed more CHCs than on the alternate host. MANCOVA with age as the covariate for the first six CHC principal components showed extensive differences in CHC composition due to age, population, cactus, sex, and age × population, age × sex, and age × cactus interactions. Thus, understanding variation in CHC composition as adult D. mojavensis age requires information about population and host plant differences, with potential influences on patterns of mate choice, sexual selection, and sexual isolation, and ultimately how these pheromones are expressed in natural populations. Studies of drosophilid aging in the wild are badly needed.

Pheromones play an important role in the behavior, ecology, and evolution of many organisms. The structure of many insect pheromones typically consists of a hydrocarbon backbone, occasionally modified with various functional oxygen groups. Here we show that sex-specific triacylclyerides (TAGs) are broadly conserved across the subgenus Drosophila in 11 species and represent a novel class of pheromones that has been largely overlooked. In desert-adapted drosophilids, 13 different TAGs are secreted exclusively by males from the ejaculatory bulb, transferred to females during mating, and function synergistically to inhibit courtship from other males. Sex-specific TAGs are comprised of at least one short branched tiglic acid and a long linear fatty acyl component, an unusual structural motif that has not been reported before in other natural products. The diversification of chemical cues used by desert-adapted Drosophila as pheromones may be related to their specialized diet of fermenting cacti.

Sexual selection within populations can play an important role in speciation when divergence in mating signals and their corresponding preferences occur along different coevolutionary trajectories in different populations. In insects, one potential target of sexual selection is the blend of cuticular hydrocarbons (CHCs), which often show intra- and interspecific variation, sexual dimorphism and may act as pheromones. In Drosophila montana, a cold-adapted, circumboreal member of the Drosophila virilis species group, flies from different populations have been found to show significant premating isolation as well as variation in male mating signal (song) and female preference. While the role of male courtship song in mate choice has been studied extensively, CHCs in this species have received little attention. In this study, we identified most of the CHCs found on the cuticle of D. montana and characterized population divergence and sexual dimorphism of CHC profiles among flies established from three natural populations - one European and two North American. We also studied their potential role as pheromones by analyzing CHCs of flies used in female-choice mating experiments. We report significant population×sex effects on CHC profiles, as well as significant relationships between some CHC principal components and particular mating behaviours, such as female attractiveness and male mating success, providing evidence that CHCs may play a role in mate choice in this species. The study also provides evidence for variation in the degree to which CHCs play a role in chemical communication among these populations, which may have an influence on the speciation process itself, and could be due to variation in interactions with other closely-related species that occur sympatrically with D. montana in some, but not other, parts of its distribution., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Revealing the genetic basis of traits that cause reproductive isolation, particularly premating or sexual isolation, usually involves the same challenges as most attempts at genotype-phenotype mapping and so requires knowledge of how these traits are expressed in different individuals, populations, and environments, particularly under natural conditions. Genetic dissection of speciation phenotypes thus requires understanding of the internal and external contexts in which underlying genetic elements are expressed. Gene expression is a product of complex interacting factors internal and external to the organism including developmental programs, the genetic background including nuclear-cytotype interactions, epistatic relationships, interactions among individuals or social effects, stochasticity, and prevailing variation in ecological conditions. Understanding of genomic divergence associated with reproductive isolation will be facilitated by functional expression analysis of annotated genomes in organisms with well-studied evolutionary histories, phylogenetic affinities, and known patterns of ecological variation throughout their life cycles. I review progress and prospects for understanding the pervasive role of host plant use on genetic and phenotypic expression of reproductive isolating mechanisms in cactophilic Drosophila mojavensis and suggest how this system can be used as a model for revealing the genetic basis for species formation in organisms where speciation phenotypes are under the joint influences of genetic and environmental factors., (© The American Genetic Association. 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© 2013 The Author(s). Evolution © 2013 The Society for the Study of Evolution.)

We used whole-transcriptome microarrays to assess changes in gene expression and monitored mortality rates and epicuticular hydrocarbons (CHCs) in response to desiccation stress in four natural populations of Drosophila mojavensis from Baja California and mainland Mexico. Desiccation had the greatest effect on gene expression, followed by biogeographical variation at regional and population levels. Genes involved in environmental sensing and cuticular structure were up-regulated in dry conditions, while genes involved in transcription itself were down-regulated. Flies from Baja California had higher expression of reproductive and mitochondrial genes, suggesting that these populations have greater fecundity and higher metabolic rates. Host plant differences had a surprisingly minor effect on the transcriptome. In most cases, desiccation-caused mortality was greater in flies reared on fermenting cactus tissues than that on laboratory media. Water content of adult females and males was significantly different and was lower in Baja California males. Different groups of CHCs simultaneously increased and decreased in amounts due to desiccation exposure of 9 and 18 h and were population-specific and dependent on larval rearing substrates. Overall, we observed that changes in gene expression involved a coordinated response of behavioural, cuticular and metabolic genes. Together with differential expression of cuticular hydrocarbons, this study revealed some of the mechanisms that have allowed D. mojavensis to exploit its harsh desert conditions. Certainly, for D. mojavensis that uses different host plants, population-level understanding of responses to stressors associated with future climate change in desert regions must be evaluated across geographical and local ecological scales., (© 2013 Blackwell Publishing Ltd.)

Sexual signals in cactophilic Drosophila mojavensis include cuticular hydrocarbons (CHCs), contact pheromones that mediate female discrimination of males during courtship. CHCs, along with male courtship songs, cause premating isolation between diverged populations, and are influenced by genotype × environment interactions caused by different host cacti. CHC profiles of mated and unmated adult flies from a Baja California and a mainland Mexico population of D. mojavensis reared on two host cacti were assayed to test the hypothesis that male CHCs mediate within-population female discrimination of males. In multiple choice courtship trials, mated and unmated males differed in CHC profiles, indicating that females prefer males with particular blends of CHCs. Mated and unmated females significantly differed in CHC profiles as well. Adults in the choice trials had CHC profiles that were significantly different from those in pair-mated adults from no-choice trials revealing an influence of sexual selection. Females preferred different male CHC blends in each population, but the influence of host cactus on CHC variation was significant only in the mainland population indicating population-specific plasticity in CHCs. Different groups of CHCs mediated female choice-based sexual selection in each population suggesting that geographical and ecological divergence has the potential to promote divergence in mate communication systems., (© 2012 The Authors. Journal of Evolutionary Biology © 2012 European Society For Evolutionary Biology.)

We present a revised molecular phylogeny of the Drosophila repleta group including 62 repleta group taxa and nine outgroup species based on four mitochondrial and six nuclear DNA sequence fragments. With ca. 100 species endemic to the New World, the repleta species group represents one of the major species radiations in the genus Drosophila. Most repleta group species are associated with cacti in arid or semiarid regions. Contrary to previous results, maximum likelihood and Bayesian phylogenies of the 10-gene dataset strongly support the monophyly of the repleta group. Several previously described subdivisions in the group were also recovered, despite poorly resolved relationships between these clades. Divergence time estimates suggested that the repleta group split from its sister group about 21millionyears ago (Mya), although diversification of the crown group began ca. 16Mya. Character mapping of patterns of host plant use showed that flat leaf Opuntia use is common throughout the phylogeny and that shifts in host use from Opuntia to the more chemically complex columnar cacti occurred several times independently during the history of this group. Although some species retained the use of Opuntia after acquiring the use of columnar cacti, there were multiple, phylogenetically independent instances of columnar cactus specialization with loss of Opuntia as a host. Concordant with our proposed timing of host use shifts, these dates are consistent with the suggested times when the Opuntioideae originated in South America. We discuss the generally accepted South American origin of the repleta group., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The cactophilic fly Drosophila mojavensis exhibits considerable intraspecific genetic structure across allopatric geographic regions and shows associations with different host cactus species across its range. The divergence between these populations has been studied for more than 60years, yet their exact historical relationships have not been resolved. We analysed sequence data from 15 intronic X-linked loci across populations from Baja California, mainland Sonora-Arizona and Mojave Desert regions under an isolation-with-migration model to assess multiple scenarios of divergence. We also compared the results with a pre-existing sequence data set of eight autosomal loci. We derived a population tree with Baja California placed at its base and link their isolation to Pleistocene climatic oscillations. Our estimates suggest the Baja California population diverged from an ancestral Mojave Desert/mainland Sonora-Arizona group around 230,000-270,000years ago, while the split between the Mojave Desert and mainland Sonora-Arizona populations occurred one glacial cycle later, 117,000-135,000years ago. Although we found these three populations to be effectively allopatric, model ranking could not rule out the possibility of a low level of gene flow between two of them. Finally, the Mojave Desert population showed a small effective population size, consistent with a historical population bottleneck. We show that model-based inference from multiple loci can provide accurate information on the historical relationships of closely related groups allowing us to set into historical context a classic system of incipient ecological speciation., (© 2012 Blackwell Publishing Ltd.)

Background: We characterized variation and chemical composition of epicuticular hydrocarbons (CHCs) in the seven species of the Drosophila buzzatii cluster with gas chromatography/mass spectrometry. Despite the critical role of CHCs in providing resistance to desiccation and involvement in communication, such as courtship behavior, mating, and aggregation, few studies have investigated how CHC profiles evolve within and between species in a phylogenetic context. We analyzed quantitative differences in CHC profiles in populations of the D. buzzatii species cluster in order to assess the concordance of CHC differentiation with species divergence., Results: Thirty-six CHC components were scored in single fly extracts with carbon chain lengths ranging from C29 to C39, including methyl-branched alkanes, n-alkenes, and alkadienes. Multivariate analysis of variance revealed that CHC amounts were significantly different among all species and canonical discriminant function (CDF) analysis resolved all species into distinct, non-overlapping groups. Significant intraspecific variation was found in different populations of D. serido suggesting that this taxon is comprised of at least two species. We summarized CHC variation using CDF analysis and mapped the first five CHC canonical variates (CVs) onto an independently derived period (per) gene + chromosome inversion + mtDNA COI gene for each sex. We found that the COI sequences were not phylogenetically informative due to introgression between some species, so only per + inversion data were used. Positive phylogenetic signal was observed mainly for CV1 when parsimony methods and the test for serial independence (TFSI) were used. These results changed when no outgroup species were included in the analysis and phylogenetic signal was then observed for female CV3 and/or CV4 and male CV4 and CV5. Finally, removal of divergent populations of D. serido significantly increased the amount of phylogenetic signal as up to four out of five CVs then displayed positive phylogenetic signal., Conclusions: CHCs were conserved among species while quantitative differences in CHC profiles between populations and species were statistically significant. Most CHCs were species-, population-, and sex-specific. Mapping CHCs onto an independently derived phylogeny revealed that a significant portion of CHC variation was explained by species' systematic affinities indicating phylogenetic conservatism in the evolution of these hydrocarbon arrays, presumptive waterproofing compounds and courtship signals as in many other drosophilid species.

We describe indirect genetic benefits of mate choice in two allopatric populations of cactophilic Drosophila mojavensis. By manipulating mate choice opportunity, we show that greater mate choice among sexually mature adults leads to shorter offspring egg-to-adult development times; the extent of this reduction was influenced by population origin and by host plant environment. We performed multiple-choice mating trials with individually marked flies to investigate whether differential male mating success was a consequence of female choice, male interaction, or both. We demonstrate that male copulation frequency was not random and instead, was determined by female choice. Virgin females in these trials were no less discriminating than females that had been previously exposed to males. These results suggest that there are indirect benefits of female mate choice that are population and environment specific, consistent with the hypothesis of ecologically influenced 'good genes' sexual selection., (© 2011 The Authors. Journal of Evolutionary Biology © 2011 European Society For Evolutionary Biology.)

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.

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., (© 2010 The Author(s). Evolution© 2010 The Society for the Study of Evolution.)

Understanding speciation relies critically on the identification of mechanisms responsible for maintaining species integrity (i.e., reproductive isolation) especially when closely related species are sympatric in nature. Studies of reproductive isolation in Drosophila often involve laboratory mating experiments that assume that patterns of mate choice in the laboratory are similar to those in the wild. Two sibling species, Drosophila arizonae and D. mojavensis, known to exhibit low levels of interspecific hybridization in the laboratory, but not in nature, were used in multiple-choice mating trials using various mating chamber designs as well as synthetic and natural media for developing larvae and courting adults. Sympatric populations were more sexually isolated than allopatric ones, consistent with past studies, and all experimental variables tested (chamber size, host plant presence and rearing substrates) had significant effects on levels of premating isolation between these species. Flies reared on cactus showed increased premating isolation versus those reared on synthetic laboratory food as did providing fermenting host plant tissue during mating trials. Also, surprisingly, smaller mating chambers led to an increase in premating isolation versus larger containers. The design of these types of mating trials is thus critical to understanding how mating behaviors in the laboratory are related to those in natural populations.

Perturbation of gene or chromosome frequencies in natural populations is one of the most powerful ways of demonstrating whether natural selection maintains genetic polymorphism or if other evolutionary forces are at work. Gene arrangement frequencies in two natural populations of Drosophila robusta were perturbed multiple times by releasing adult flies with contrasting karyotypes and carefully monitoring post-perturbation presence of hybrids and chromosome frequencies. In all cases, frequencies quickly returned to pre-perturbation levels, and in the following sampling periods, no evidence of the introduced chromosomes was apparent. Analysis of post-perturbation frequency changes included tests for heterogeneity among chromosome arrangements in rates of return to equilibrium values using population admixture analysis. In several cases, significant heterogeneity was detected indicating some form of natural selection was operating. Technical challenges to carrying out perturbation experiments in the wild are also discussed.

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.

We tested the hypothesis that intrademic sexual selection has caused sexual isolation between populations of geographically isolated populations of cactophilic Drosophila mojavensis, and was mediated by epicuticular hydrocarbons (EHCs), contact pheromones in this system. Sexual selection and sexual isolation were estimated using a Baja California and mainland population by comparing the number of mated and unmated males and females in each of four pairwise population mating trials. EHC profiles were significantly different in mated and unmated males in the interdemic (Bajafemale symbol x Mainlandmale symbol and Mainlandfemale symbol x Bajamale symbol), but not the intrademic mating trials. A small number of EHCs was identified that best discriminated among mated and unmated males, mostly alkadienes with 34 and 37 carbons. Females showed population-specific preferences for male EHC profiles. However, EHC profiles between mated and unmated males in the intrademic mating trials were not significantly different, consistent with undetectable sexual selection estimated directly from numbers of copulating pairs vs. unmated adults. Thus, sexual isolation among populations was much stronger than sexual selection within these populations of D. mojavensis.

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.

Low concentrations of dietary triacylglycerols including tristearin, triolein, and tripalmitolein, were assessed to determine their effects during egg to pupal stages on adult epicuticular hydrocarbon (EHC) variation in cactophilic Drosophila mojavensis. Triacylglycerols were added singly and in combination at concentrations of 1%, 3%, and 9% to a lipid-free culture medium. Control diets included Carolina Drosophila and lipid-free media. Egg to adult viability was reduced at triacylglycerol concentrations greater than 1%, except for tristearin. Both triolein and tripalmitolein increased EHC amounts to levels similar to those in combination and control diets. Tristearin caused significantly lower quantities of EHCs in adult flies than triolein or tripalmitolein, consistent with previous studies on reduced tristearin assimilation into adult EHCs. We rejected the hypothesis that unsaturated and saturated triacylglycerols were assimilated into unsaturated and saturated adult EHCs, respectively. Since these triacylglycerols comprise a fraction of known lipids in the columnar cacti used for breeding in nature, and EHCs serve as contact pheromones in D. mojavensis, these and other naturally occurring triacylglycerols may provide a direct causal link between host plant use and patterns of chemically mediated mate choice.

To what degree is the divergent adaptation responsible for life's phenotypic variety also responsible for generating the millions of species that manifest this variation? Theory predicts that ecological divergence among populations should promote reproductive isolation, and recent empirical studies provide support for this hypothesis in a limited number of specific taxa. However, the essential question of whether ecology plays a truly general role in speciation has yet to be systematically evaluated. Here we address this integral issue using an approach that adds an ecological dimension to comparative studies investigating the relationship between reproductive isolation and divergence time. Specifically, we quantify ecological divergence for >500 species pairs from eight plant, invertebrate, and vertebrate taxa and statistically isolate its association with reproductive isolation. This approach demonstrates a highly consistent and significant positive association between ecological divergence and reproductive isolation across taxa. This relationship was also observed across different aspects of ecological divergence and components of reproductive isolation. These findings are highly consistent with the hypothesis that ecological adaptation plays a fundamental and taxonomically general role in promoting reproductive isolation and speciation.

Background: Studied since the early 1940's, chromosomal polymorphisms in the deciduous woods species Drosophila robusta have been characterized by well-defined latitudinal, longitudinal, and elevational clines, but--until at least ten years ago--stable, local population frequencies. Recent biogeographical analyses indicate that D. robusta invaded North America from southeast Asia and has persisted in eastern temperate forests for at least 20-25 my without speciating. The abundant chromosome polymorphisms found across the range of D. robusta are thus likely to be relatively ancient, having accumulated over many well known climatic cycles in North America. Sufficient long-term data are now available such that we can now gauge the rate of these evolutionary changes in natural populations due to environmental change., Results: Recent local collections have revealed significant changes in the frequencies of several chromosomal forms. New data presented here extend the range of these changes to six states, three in the northeastern United States and three west of the Mississippi River. These data reinforce recent directional changes in which the frequencies of three gene arrangements have reached percentage levels typical of distant southern populations consistent with regional climatic changes. Another gene arrangement has been steadily decreasing in frequency at a number of the sites studied. Meteorological records from 1945 to 2003 indicate temperature increases at all study sites, particularly average minimum air temperatures., Conclusions: Observation of parallel genetic flux suggests that these long-term temporal frequency shifts in widely disparate populations of D. robusta are evolutionary responses to environmental change. Since these chromosomes are known to be sensitive to ambient temperature, regional climatic shifts associated with global warming are likely to be responsible.

Sequences from three gene regions from the nuclear and mitochondrial genomes were used to examine the extent and nature of hierarchical structure in the Drosophilamojavensis cluster (Drosophila arizonae, D. mojavensis and D. navojoa) of the D. repleta species group. To determine the genetic divergence of these three species, sequence data were analyzed using maximum parsimony and population aggregation analysis. Individual and combined gene genealogies indicate that D. arizonae and D. mojavensis are neither diagnosable nor monophyletic with respect to one another. Although D. navojoa has differentiated from D. arizonae and D. mojavensis, as diagnosed by nuclear gene sequences, it may have undergone a reticulation event with D. arizonae. Our results suggest that either these taxa are still undergoing differentiation at the molecular level or have experienced gene flow in the recent past.

Understanding the genetic bases of phenotypes associated with the earliest stages of divergence will reveal a great deal about species formation. I review a number of model systems, most involving plant-insect interactions, that have already revealed genetic aspects of incipient speciation. It is suggested that progress in understanding the causal forces driving mating signal evolution and incipient speciation will be expedited in model systems where; (1) ecological and evolutionary information is available, (2) different aspects of mating behaviors that function in mate and/or species recognition are known, (3) genetic analysis of single phenotypes is undertaken, (4) analysis of sexual selection and isolation is performed under natural conditions (or in the wild), and (5) comparative data from related species are available to assess phylogenetic trends.

Adult epicuticular hydrocarbon variation of 14 geographically isolated populations of cactophilic Drosophila mojavensis was assessed to further investigate mechanisms of sexual isolation. Hydrocarbon transfer experiments demonstrated that these compounds are part of the mate recognition system in this species. Sixteen of the 23 epicuticular hydrocarbon components studied differed in amounts between males and females, and 13 differed in quantity between the geographic regions encompassing Baja California and mainland Mexico (Sonora and Sinaloa). Eight hydrocarbon components, seven of which differed in quantity between sexes, showed significant sex-by-region interactions, indicating region-specific sex reversals in hydrocarbon quantities. Such regional variation in epicuticular hydrocarbon profiles suggests that these hydrocarbon differences have also evolved in D. mojavensis since this species invaded mainland Sonora and Sinaloa from Baja California by switching host plants, in addition to a number of key genetic, behavioral, and life-history characters.

Epicuticular hydrocarbon variation was investigated among the three species of the Drosophila mojavensis cluster (D. mojavensis, D. arizonae, and D. navojoa) within the large D. repleta group. Because these hydrocarbons serve as contact pheromones in adult D. mojavensis, the chemical characteristics and differences in hydrocarbon profiles in populations of these three sibling species were further investigated. Twenty-seven hydrocarbon components with chain lengths ranging from C28 to C40, including n-alkanes, methyl-branched alkanes, n-alkenes, methyl-branched alkenes, and alkadienes were observed. Hydrocarbon profiles among the three species reared on different cactus hosts were easily aligned with previously identified components in D. mojavensis. Male and female D. navojoa possessed a 31-methyldotricont-6-ene absent in both D. arizonae and D. mojavensis, while lacking the 8,24-tritricontadiene present in these two species. D. navojoa adults had far less 2-methyloctacosane than these sibling species, but the significance of this difference was obscured by the degree of variation among populations in amounts of this hydrocarbon. Mainland and Baja California populations of D. mojavensis were fixed for differences in the amounts 8,24-tritricontadiene, 9,25-pentatricontadiene, and 9,27-heptatricontadiene, consistent with all previous studies. Amounts of 18 of the 27 hydrocarbon components were greater in flies reared on Opuntia cactus. Canonical discriminant function analysis resolved all three species into distinct, nonoverlapping groups, suggesting that epicuticular hydrocarbon profiles are species-specific in the D. mojavensis cluster. Based on the amounts of interpopulation variation in hydrocarbon profiles in these three species, we hypothesize that epicuticular hydrocarbon differences may evolve early during the formation of new species.

The species in the repleta group of the genus Drosophila have been placed into five subgroups-the mulleri, hydei, mercatorum, repleta, and fasciola subgroups. Each subgroup has been further subdivided into complexes and clusters. Extensive morphological and cytological analyses of the members of this species group have formed the foundation for the proposed relationships among the members of the repleta species group. Fifty-four taxa, including 46 taxa belonging to the repleta species group, were sequenced for fragments of four genes-16S ribosomal DNA (16S), cytochrome oxidase II (COII), and nitrogen dehydrogenase 1 (ND1) of the mitochondrial genome and a region of the hunchback (hb) nuclear gene. We also generated a partial data set of elongation factor 1-alpha (Ef1alpha) sequences for a subset of taxa. Our analysis used both DNA characters and chromosomal inversion data. The phylogenetic hypothesis we obtained supports many of the traditionally accepted clades within the mulleri subgroup, but the monophyly of taxonomic groups outside of this subgroup appears not to be supported. Phylogenetic analysis revealed one well-supported, highly resolved clade that consists of closely related members of the mulleri and buzzatii complexes. The remaining taxa, a wide assortment of taxonomic groups, ranging from members of other species groups to members of several subgroups and members of three species complexes from the mulleri subgroup are found in poorly supported arrangements at the base of the tree., (Copyright 2000 Academic Press.)

Tarsal spurs play an important role in intrasexual competition for females among male wild turkeys (Meleagris gallopavo). Thus variation in spur development may have important fitness consequences. Fitness correlates of spur development were studied in a free-living population of wild turkeys and it was found that heavier males and males with longer beards had longer spurs. Males that had longer spurs spent more time on display areas during the breeding season and less time moving among these areas compared to males with shorter spurs, independently of their body mass. Otherwise ideally symmetrical spurs showed fluctuating asymmetry between left and right tarsi, the degree of absolute asymmetry decreased with spur length in adults, but not in subadults, and males that survived at least one winter had more symmetrical spurs compared to males that did not. We conclude that if the ability to produce symmetrical spurs has a genetic basis, then spur length and spur asymmetry could reliably indicate individual quality and that these traits are under directional selection for increased size and symmetry in wild turkeys.

Studies of behavioral isolation among geographically isolated populations of Drosophila mojavensis have provided an understanding of incipient speciation wherein phylogeny and ecology play a prominent role. Populations of D. mojavensis in mainland Mexico and southern Arizona exhibit low but significant premating isolation from Baja California populations in laboratory mate choice tests. These same populations have undergone considerable life-history evolution in response to use of different host plants, suggesting that behavioral isolation between populations is a pleiotropic consequence of adaptation to different environments, or Mayr's geographic speciation hypothesis. This hypothesis was tested using bidirectional artificial selection on egg-to-adult development time in replicate lines of a mainland and Baja population cultured on two host cacti for 13 generations. Response to selection was greatest in the slow lines cultured on one host, yet there was uneven response in some lines due to variation in cactus tissue quality. Realized heritabilities for development time ranged from 0.04 to 0.16, which is consistent with previous estimates from half-sib/full-sib analyses of genetic variation. In most lines that responded to selection, premating isolation decreased to near zero. Correlated responses in behavioral isolation suggest that adaptation to contrasting environments can cause secondary responses in mate recognition systems that can influence the formation of new species.

Natural populations of Drosophila robusta are polymorphic for chromosomal gene arrangements in most of its range, the deciduous forests of North America east of the Rocky Mountains. Many of the gene arrangements are the result of paracentric inversions on both arms of the metacentric second chromosome. They are frequently in linkage disequilibrium, determined in many areas largely, or entirely, by differing frequencies of cis and trans forms of the double heterokaryotypes and their component linkage combinations. Presence and degree of linkage disequilibrium in these populations varies with locality and gender, with males from southern localities exhibiting the largest deviation from equilibrium. Analysis of the extensive karyotype data encompassing the entire species range collected over the past 50 years shows that natural selection is primarily responsible for maintaining these complex polymorphisms.

The extent of host-specific genetic variation for two life-history traits, egg to adult developmental time and viability, and one morphological trait closely tied to fitness, adult thorax size, was exposed by employing a nested half-sib/full-sib breeding design with Baja and mainland populations of Drosophila mojavensis recently extracted from nature. This study was motivated by the presence of substantial variation in life histories among populations of D. mojavensis that use the fermenting tissues of particular species of columnar cacti for feeding and breeding in the Sonoran Desert. Full-sib progeny from all sire-dam crosses were split into cultures of agria cactus, Stenocereus gummosus, and organ pipe cactus, S. thurberi, to examine patterns of genotype-by-environment interaction for these fitness components. Baja flies expressed shorter egg-to-adult developmental times, higher viabilities, and smaller body sizes than mainland flies consistent with previous studies. Significant sire and dam components of variance were exposed for developmental time and thorax size. Genotype-by-environment interactions were significant at the level of dams for developmental time and nearly significant for viability (P = 0.09). Narrow- and broad-sense heritabilities were influenced by host cactus, sex, and population. No strong pattern of genetic correlation emerged among fitness components suggesting that host-range expansion has not been accompanied by formation of coadapted life histories, yet the ability to estimate genetic correlations and their standard errors was compromised by the unbalanced nature of the data set. Genetic correlations in performance across cacti were slightly positive, evidence for ecological generalism among populations explaining the observed pattern of multiple host cactus use within the species range of D. mojavensis., (© 1993 The Society for the Study of Evolution.)

A quantitative genetic analysis of nine adult fitness components was performed in two populations of cactophilic Drosophila mojavensis under natural conditions of fermenting cactus and ethanol vapour. Female progeny from 18 sires and 36 dams were treated to a range of six exposure periods to males to assess effects of remating frequency on female fitness. Lifetime fecundity increased with increasing male exposure, but longevity showed an intermediate optimum with temporary exposure to males of 2-4 days. Narrow-sense heritabilities were significant for egg production traits while broad-sense heritabilities were significant for longevity-related traits. Positive genetic correlations between components of fitness were expressed among functionally related traits, e.g. longevity was positively correlated with lifetime fecundity, the number of clutches laid, clutch size, and the number of eggs laid per day. Negative genetic correlations were detected between early and late life fecundity suggesting genetic tradeoffs among components of adult fitness.

Chromosomally polymorphic populations of Drosophila mojavensis from Baja California feed and breed on agria cactus, Stenocereus gummosus; whereas, monomorphic Arizona populations are associated exclusively with organ pipe cactus, S. thurberi. The effects of this host plant shift in expanding the kinds of feeding and breeding sites were assessed by manipulating larval density and recording differences in egg to adult development time and viability, and adult thorax size in both populations on artificially rotted substrates of both cactus species. Older agria rots increased development time but had no effect on viability. Organ pipe rots were qualitatively poorer substrates than agria rots for both monomorphic and polymorphic populations of D. mojavensis, especially at higher larval densities causing longer egg to adult development times, lower viabilities, and smaller thorax sizes than agria.The Baja population expressed shorter development times, higher viabilities, and smaller thorax sizes than the Arizona population on both cactus substrates. No evidence for cactus host race formation was found. The Baja population was less sensitive to increasing larval densities for all fitness characters studied on both cactus substrates indicating greater developmental homeostasis than in the monomorphic Arizona population. These data support the hypothesized central-marginal population structure within this species coincident with the distribution of host plants and lend insight into the process of adaptive divergence at different life history stages caused by host plant shifts.