GENETICS OF INCIPIENT SPECIATION IN DROSOPHILA MOJAVENSIS: II. HOST PLANTS AND MATING STATUS INFLUENCE CUTICULAR HYDROCARBON QTL EXPRESSION AND G × E INTERACTIONS

We performed a quantitative trait locus (QTL) analysis of epicuticular hydrocarbon variation in 1650 F₂ 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 F₂ 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 × 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 × E (exposure) interactions with most resulting from mainland genotypes expressing altered hydrocarbon amounts when exposed to females compared to Baja genotypes. Many cactus × 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.     

Sensitivity to larval density in populations of Drosophila mojavensis: Influences of host plant variation on components of fitness

Summary 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.     

Population genetics and geographic variation of alcohol dehydrogenase (Adh) paralogs and glucose-6-phosphate dehydrogenase (G6pd) in Drosophila mojavensis

Populations of Drosophila mojavensis from the deserts of the Baja California peninsula and mainland Mexico utilize different cactus hosts with different alcohol contents. The enzyme alcohol dehydrogenase (ADH) has been proposed to play an important role in the adaptation of Drosophila species to their environment. This study investigates the role of ADH in the adaptation of the cactophilic D. mojavensis to its cactus host. In D. mojavensis and its sibling species, D. arizonae, the Adh gene has duplicated, giving rise to a larval/ovarian form (Adh-1) and an adult form (Adh-2). Studies of sequence variation presented here indicate that the Adh paralogs have followed different evolutionary trajectories. Adh-1 exhibits an excess of fixed amino acid replacements, suggesting adaptive evolution, which could have been a result of several host shifts that occurred during the divergence of D. mojavensis. A 17-bp intron haplotype polymorphism segregates in Adh-2 and has markedly different frequencies in the Baja and mainland populations. The presence of the intron polymorphism suggests possible selection for the maintenance of pre-mRNA structure. Finally, this study supports the proposed Baja California origination of D. mojavensis and subsequent colonization of the mainland accompanied by a host shift.     

Pre-mating isolation is determined by larval rearing substrates in cactophilicDrosophila mojavensis. II. Effects of larval substrates on time to copulation,mate choice and mating propensity

Summary It has been hypothesized that reproductive character displacement has evolved in mainland Sonora, Mexico populations of cactophilicD. mojavensis due to the presence of a sympatric sibling speciesD. arizonae. In laboratory tests using ancestral Baja California populations and derived, sympatric mainland populations, asymmetrical sexual isolation has been observed among populations ofD. mojavensis where mainland females discriminate against Baja males. Effects of different pre-adult rearing environments on adult mating behaviour were assessed by comparing fermenting cactus tissues like those used in nature for breeding with laboratory media because previous studies have employed synthetic growth media for fly growth and development. Significant behavioural isolation was evident in all cases when larvae were reared on laboratory food, but was non-significant when flies were reared on fermenting cactus, except for the cactus used by most mainland populations, consistent with previous studies. Time to copulation of Baja females was greater than mainland females over all substrates, but male time to copulation did not differ between populations. Time to copulation for both sexes was significantly greater when flies were reared on laboratory food with one exception. The degree of behavioural isolation was weakly correlated with time to copulation across food types (Spearman rank correlation = 0.58,p = 0.099). Therefore, use of laboratory media in this and previous studies exaggerated adult pre-mating isolation and time to copulation in comparison to natural breeding substrates. These experiments suggest that a change in host substrates by saprophagous insects (where chemical differences exist between hosts) may have subtle effects on mating behaviour in a manner which promotes low levels of sexual isolation as a by-product of their utilization of a particular substrate during larval development. ForD. mojavensis, these results suggest that over evolutionary time, radiation into a new environment (from Baja to the mainland) allowed utilization of new host plants that may have incidentally promoted the sexual isolation patterns that have been observed within this species.See Etges (1992) for the first paper in this series.     

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 F₁, F₂, 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 F₂ 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 HOST-CACTUS RESPONSE AND LIFE-HISTORY EVOLUTION AMONG ANCESTRAL AND DERIVED POPULATIONS OF CACTOPHILIC DROSOPHILA MOJAVENSIS

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.     

Functional genomics of cactus host shifts in Drosophila mojavensis

Understanding the genetic basis of adaptation to novel environments remains one of the major challenges confronting evolutionary biologists. While newly developed genomic approaches hold considerable promise for addressing this overall question, the relevant tools have not often been available in the most ecologically interesting organisms. Our study organism, Drosophila mojavensis, is a cactophilic Sonoran Desert endemic utilizing four different cactus hosts across its geographical range. Its well-known ecology makes it an attractive system in which to study the evolution of gene expression during adaptation. As a cactophile, D. mojavensis oviposits in the necrotic tissues of cacti, therefore exposing larvae and even adults to the varied and toxic compounds of rotting cacti. We have developed a cDNA microarray of D. mojavensis to examine gene expression associated with cactus host use. Using a population from the Baja California population we examined gene expression differences of third instar larvae when reared in two chemically distinct cactus hosts, agria (Stenocereus gummosus, native host) vs. organpipe (Stenocereus thurberi, alternative host). We have observed differential gene expression associated with cactus host use in genes involved in metabolism and detoxification.     

Premating isolation is determined by larval rearing substrates in cactophilic Drosophila mojavensis. IV. Correlated responses in behavioral isolation to artificial selection on a life-history trait

Abstract 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.     

Population differences in host plant preference and the importance of yeast and plant substrate to volatile composition

Divergent selection between environments can result in changes to the behavior of an organism. In many insects, volatile compounds are a primary means by which host plants are recognized and shifts in plant availability can result in changes to host preference. Both the plant substrate and microorganisms can influence this behavior, and host plant choice can have an impact on the performance of the organism. In Drosophila mojavensis, four geographically isolated populations each use different cacti as feeding and oviposition substrates and identify those cacti by the composition of the volatile odorants emitted. Behavioral tests revealed D. mojavensis populations vary in their degree of preference for their natural host plant. Females from the Mojave population show a marked preference for their host plant, barrel cactus, relative to other cactus choices. When flies were given a choice between cacti that were not their host plant, the preference for barrel and organ pipe cactus relative to agria and prickly pear cactus was overall lower for all populations. Volatile headspace composition is influenced by the cactus substrate, microbial community, and substrate‐by‐microorganism interactions. Differences in viability, developmental time, thorax length, and dry body weight exist among populations and depend on cactus substrate and population‐by‐cactus interactions. However, no clear association between behavioral preference and performance was observed. This study highlights a complex interplay between the insect, host plant, and microbial community and the factors mediating insect host plant preference behavior.     

Model-based comparisons of phylogeographic scenarios resolve the intraspecific divergence of cactophilic Drosophila mojavensis

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.     

Premating isolation is determined by larval rearing substrates in cactophilic Drosophila mojavensis. IX. Host plant and population specific epicuticular hydrocarbon expression influences mate choice and sexual selection

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.     

Alcohol dehydrogenase polymorphism in barrel cactus populations of Drosophila mojavensis

Starch gel electrophoresis revealed that the alcohol dehydrogenase (ADH-2) locus was polymorphic in two populations (from Agua Caliente, California and the Grand Canyon, Arizona) of cactophilic Drosophila mojavensis that utilize barrel cactus (Ferocactus acanthodes) as a host plant. Electromorphs representing products of a slow (S) and a fast (F) allele were found in adult flies. The frequency of the slow allele was 0.448 in flies from Agua Caliente and 0.659 in flies from the Grand Canyon. These frequencies were intermediate to those of the low (Baja California peninsula, Mexico) and high (Sonora, Mexico and southern Arizona) frequency Adh-2^S populations of D. mojavensis that utilize different species of host cacti.     

Premating isolation is determined by larval-rearing substrates in cactophilic Drosophila mojavensis. V. Deep geographic variation in epicuticular hydrocarbons among isolated populations

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.     

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.     

The relationship of terpene composition,morphology, and distribution of populations of Bursera microphylla (Burseraceae)

On the basis of terpene composition, populations ofB. microphylla show north to south affinities on both the Baja California peninsula and the northwest coast of Mexico. The lack of east to west population affinities corresponds with the presumed absence of land connections between the peninsula and mainland throughout the history of this taxon. Morphological similarities between populations in an east-west direction are explained on the basis of contemporary parallel selective influences and not by the closeness of relationship.     

Evolution of developmental homeostasis inDrosophila mojavensis

Summary Variation in life histories among populations of cactophilicDrosophila mojavensis has been hypothesized to be a by-product of a shift to one of two alternate host plants. When cultured on the ancestral and a secondary host cactus, a Baja population expressed shorter development times and smaller thorax sizes than a mainland population, but viability did not differ. Comparisons with all reciprocal F₁ and F₂ crosses between populations revealed that genetic differences in development time and thorax size were largely additive. Homeostasis in these life history traits was population specific, except for viability. Homeostasis in development time was greater in the Baja population than in the other crosses, suggesting dominance for decreased homeostasis in the mainland population. Underdominance in viability homeostasis of the F₁ hybrids suggested some incompatibility between populations. Homeostasis in thorax size was greater in females than in males and differed among parental populations. Maintenance of heritable differences and genetic variation for homeostasis in these traits suggested a role for cactus-specific differences in environmental uncertainty caused by variation in breeding site duration and abundance in nature.     

Premating isolation is determined by larval rearing substrates in cactophilic Drosophila mojavensis. X. Age‐specific dynamics of adult epicuticular hydrocarbon expression in response to different host plants

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.     

Genetic differentiation and demographic history in Drosophila pachea from the Sonoran Desert

Genetic variation at six microsatellite DNA loci and a segment of the mitochondrial cytochrome oxidase subunit I (COI) locus was used to estimate gene flow, population structure, and demographic history in the cactophilic Drosophila pachea from the Sonoran Desert of North America, a species that shows a strict association with its senita host cactus (genus Lophocereus). For microsatellite analyses, thirteen populations of D. pachea were sampled, five in mainland Mexico and the southwestern USA, and eight on the Baja California (Baja) peninsula, covering essentially the entire range of the species. Analysis of molecular variance (AMOVA) of microsatellite data revealed that populations from both the mainland and the Baja peninsula generally showed little structure, although there were a few exceptions, suggesting some local differentiation and restriction of gene flow within both regions. Pairwise comparisons of F(ST) among each of the mainland and Baja populations showed evidence of both panmixia and population subdivision. AMOVA performed on grouped populations from both the mainland and Baja, however, revealed significant partitioning of genetic variation among the two regions, but no partitioning among localities within each region. Bayesian skyline analyses of the COI data set, consisting of four mainland and seven peninsular populations, revealed population expansions dating to the Pleistocene or late Pliocene in D. pachea from both regions, although regional differences were seen in the estimated timing of the expansions and in changes in effective population size over time.