Host Preference Among Populations of Drosophila mojavensis (Diptera: Drosophilidae) that Use Different Host Cacti

Previous studies have suggested that all populations of cactophilic Drosophila mojavensis prefer pitaya agria cactus, Stenocereus gummosus, over all other potential hosts for feeding and breeding, including populations that inhabit areas where no agria grows. We sampled five geographically isolated populations of D. mojavensis from nature to assess host choice within and between populations. Host choice tests were performed in a laboratory olfactometer by allowing adult D. mojavensis to choose between plumes of synthetic volatile cocktails of two widespread host cacti. Overall, each population showed significant preference for agria volatiles with one exception: a mainland Sonora population that uses organ pipe cactus in nature exhibited preference for organ pipe volatiles, suggesting a possible shift in host preference. The degree of preference for agria volatiles was greatest in a population from southern California that use California barrel cactus as a host. Since southern Californian populations of D. mojavensis are thought to be derived from those in Baja California, preference for agria volatiles is considered a retained ancestral trait. Three populations from Baja California and mainland Mexico that use agria in the wild expressed lower, but similar preferences for agria volatiles. Because populations of D. mojavensis are ancestral to those in mainland Mexico, Arizona, and California, the shift from agria to alternate hosts has not been accompanied by strong changes in host preference behavior.     

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.     

Resource availability and population size in cactophilic Drosophila

1. Four species of Drosophila, Drosophila nigrospiracula ( Patterson & Wheeler 1942 ) , Drosophila mettleri ( Heed 1977 ) , Drosophila pachea ( Patterson & Wheeler 1942 ) , and Drosophila mojavensis ( Patterson & Crow 1940 ) , are endemic to the Sonoran Desert of North America and breed in different species of necrotic columnar cacti. Differences in resource availability have been suggested to explain the interspecific variability in fly population biology, but resource availability for these species has not been quantitatively assessed thoroughly in either spatial or temporal terms. The resource availability was quantified quarterly at three sites for 3 years and population sizes for each Drosophila species were estimated.
2. Spatial and temporal availability of resources differed significantly among species of host cacti, with organpipe cactus ( Stenocereus thurberi ) being the least abundant and senita ( Lophocereus schottii ) the most abundant spatially.
3. Drosophila species differed significantly in population size. The largest population sizes were found for D. nigrospiracula and D. mojavensis and smallest for D. pachea . Populations of D. mettleri were intermediate to these.
4. Population size was greatest for fly species utilizing host species having the largest and longest lasting necroses.
5. Resource availability does not explain the reduction of fly populations in the summer. Necroses were most abundant when flies were absent.     

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.     

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.     

Evolution of the mojavensis cluster of cactophilic Drosophila with descriptions of two new species

The mojavensis cluster of the repleta species group of Drosophila (Drosophilidae: Diptera) consists of three species. One is newly described as D. navojoa. A second species, described here as D. arizonae, replaces D. arizonensis, which has become a junior subjective synonym for D. mojavensis, the third species in the cluster. A phylogeny of the three species is presented, based on chromosomal inversions, morphology, and the ability to produce hybrids. Breakage points are assigned for all inversions, and male genitalia are figured; 186 crosses were made from 225 possible combinations among 15 geographic strains from the southwestern United States, Mexico, and Guatemala. It is confirmed that D. mojavensis and D. arizonae are very closely related and shown that D. navojoa is more distantly related in regard to all criteria. This relationship is supported by the geographical positions of the ancestral gene sequences in each species, which show a sequential northwest movement (D. navojoa----D. arizonae----D. mojavensis) from southern Mexico to southern California and northern Arizona. The relationship is also supported by the fact that D. navojoa breeds in Opuntia cactus, an ancestral behavior, whereas the other two species breed chiefly in Stenocereus cacti, a derived behavior. The possible role of this host plant shift in speciation is discussed.     

Sequence variation of alcohol dehydrogenase (Adh) paralogs in cactophilic Drosophila

This study focuses on the population genetics of alcohol dehydrogenase (Adh) in cactophilic Drosophila. Drosophila mojavensis and D. arizonae utilize cactus hosts, and each host contains a characteristic mixture of alcohol compounds. In these Drosophila species there are two functional Adh loci, an adult form (Adh-2) and a larval and ovarian form (Adh-1). Overall, the greater level of variation segregating in D. arizonae than in D. mojavensis suggests a larger population size for D. arizonae. There are markedly different patterns of variation between the paralogs across both species. A 16-bp intron haplotype segregates in both species at Adh-2, apparently the product of an ancient gene conversion event between the paralogs, which suggests that there is selection for the maintenance of the intron structure possibly for the maintenance of pre-mRNA structure. We observe a pattern of variation consistent with adaptive protein evolution in the D. mojavensis lineage at Adh-1, suggesting that the cactus host shift that occurred in the divergence of D. mojavensis from D. arizonae had an effect on the evolution of the larval expressed paralog. Contrary to previous work we estimate a recent time for both the divergence of D. mojavensis and D. arizonae (2.4 +/- 0.7 MY) and the age of the gene duplication (3.95 +/- 0.45 MY).     

Dispersal in cactophilic Drosophila

Three species of Drosophila each breed in necrotic tissue of specific columnar cacti endemic to the Sonoran Desert. Drosophila pachea breeds in senita ( Lophocereus schottii) , D. nigrospiracula breeds in saguaro ( Carnegiea gigantea ) or cardón ( Pachycereus pringlei ), and D. mojavensis uses organ pipe ( Stenocereus thurberi ) in Sonora, Mexico and southern Arizona. Patches of these three host cacti have very different spatial distributions, with those of senita being quite frequent and close together, while those of the other hosts are much father apart. Testing all three species simultaneously, we used capture-mark-release-recapture methods to ask if dispersal differed in these species and if differences were those predicted by the spatial availability of the host patches. D. pachea dispersed the shortest distance in all experiments. Furthermore, D. pachea was the only species showing sex-biased dispersal, with male flies exhibiting the greater propensity to disperse. The observations suggest that across similar spatial scales, D. pachea should show greater population genetic structure than the other two species, and that mitochondrial DNA, because of its maternal inheritance, might show greater evidence of structure than nuclear markers.     

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.     

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.     

Host plant adaptation in Drosophila mettleri populations

The process of local adaptation creates diversity among allopatric populations, and may eventually lead to speciation. Plant-feeding insect populations that specialize on different host species provide an excellent opportunity to evaluate the causes of ecological specialization and the subsequent consequences for diversity. In this study, we used geographically separated Drosophila mettleri populations that specialize on different host cacti to examine oviposition preference for and larval performance on an array of natural and non-natural hosts (eight total). We found evidence of local adaptation in performance on saguaro cactus (Carnegiea gigantea) for populations that are typically associated with this host, and to chemically divergent prickly pear species (Opuntia spp.) in a genetically isolated population on Santa Catalina Island. Moreover, each population exhibited reduced performance on the alternative host. This finding is consistent with trade-offs associated with adaptation to these chemically divergent hosts, although we also discuss alternative explanations for this pattern. For oviposition preference, Santa Catalina Island flies were more likely to oviposit on some prickly pear species, but all populations readily laid eggs on saguaro. Experiments with non-natural hosts suggest that factors such as ecological opportunity may play a more important role than host plant chemistry in explaining the lack of natural associations with some hosts.     

Experimental Evolution of Alkaloid Tolerance in Sibling <Emphasis Type="Italic">Drosophila</Emphasis> Species with Different Degrees of Specialization

Drosophila buzzatii and Drosophila koepferae are sibling species with marked ecological differences related to their patterns of host exploitation. D. buzzatii is a polyphagous species with a sub-cosmopolitan distribution, while D. koepferae is endemic to the mountain plateaus of the Andes, where it exploits alkaloidiferous columnar cacti as primary hosts. We use experimental evolution to study the phenotypic response of these cactophilic Drosophila when confronting directional selection to cactus chemical defenses for 20 generations. Flies adapted to cactus diets also experienced higher viability on alkaloid-enriched media, suggesting the selection of adaptive genetic variation for chemical-stress tolerance. The more generalist species D. buzzatii showed a rapid adaptive response to moderate levels of secondary metabolites, whereas the columnar cacti specialist D. koepferae tended to maximize fitness under harder conditions. The evolutionary dynamic of fitness-related traits suggested the implication of metabolic efficiency as a key mediator in the adaptive response to chemical stress. Although we found no evidence of adaptation costs accompanying specialization, our results suggest the involvement of compensatory evolution. Overall, our study proposes that differential adaptation to secondary metabolites may contribute to varying degrees of host specialization, favoring niche partitioning among these closely related species.     

Behavioral evolution accompanying host shifts in cactophilic Drosophila larvae

For plant utilizing insects, the shift to a novel host is generally accompanied by a complex set of phenotypic adaptations. Many such adaptations arise in response to differences in plant chemistry, competitive environment, or abiotic conditions. One less well‐understood factor in the evolution of phytophagous insects is the selective environment provided by plant shape and volume. Does the physical structure of a new plant host favor certain phenotypes? Here, we use cactophilic Drosophila, which have colonized the necrotic tissues of cacti with dramatically different shapes and volumes, to examine this question. Specifically, we analyzed two behavioral traits in larvae, pupation height, and activity that we predicted might be related to the ability to utilize variably shaped hosts. We found that populations of D. mojavensis living on lengthy columnar or barrel cactus hosts have greater activity and pupate higher in a laboratory environment than populations living on small and flat prickly pear cactus cladodes. Crosses between the most phenotypically extreme populations suggest that the genetic architectures of these behaviors are distinct. A comparison of activity in additional cactophilic species that are specialized on small and large cactus hosts shows a consistent trend. Thus, we suggest that greater motility and an associated tendency to pupate higher in the laboratory are potential larval adaptations for life on a large plant where space is more abundant and resources may be more sparsely distributed.     

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.     

Elemental stoichiometry of Drosophila and their hosts

1. Nitrogen (N) and phosphorus (P) availabilities are important ecological determinants of resource use in nature. Despite the wide range of hosts used by species of the genus Drosophila , elemental composition of natural resources of these flies has never been investigated.
2. Total body N and P contents were determined in seven species of wild-caught Drosophila , their natural hosts, and artificial diets routinely used to rear these flies in the laboratory. The flies tested included D. hydei, D. arizonae, D. simulans and D. pseudoobscura collected from rotting fruit (melons), and the cactophilic D. nigrospiracula, D. mojavensis and D. pachea collected from their specific host plants, Saguaro, Organpipe and Senita cactus, respectively.
3. Natural hosts varied in elemental composition, with fruit showing higher N (2·8–4·3% dry mass) and P (0·50–0·67%) levels compared with cacti (0·5–1·6% N; 0·01–0·29% P). No consistent differences in N and P levels were found between healthy and necrotic cactus tissue.
4. Total body N and P also varied among Drosophila species. This variation mirrored the levels of N and P found in the respective hosts and laboratory diets. N:P ratios were consistently lower in female flies compared with conspecific males suggesting phosphorus demands during oogenesis are high.
5. Potential mechanisms by which Drosophila deal with N or P limitation in nature are discussed.     

Activity variation in alcohol dehydrogenase paralogs is associated with adaptation to cactus host use in cactophilic Drosophila

Drosophila mojavensis and Drosophila arizonae are species of cactophilic flies that share a recent duplication of the alcohol dehydrogenase (Adh) locus. One paralog (Adh-2) is expressed in adult tissues and the other (Adh-1) in larvae and ovaries. Enzyme activity measurements of the ADH-2 amino acid polymorphism in D. mojavensis suggest that the Fast allozyme allele has a higher activity on 2-propanol than 1-propanol. The Fast allele was found at highest frequency in populations that utilize hosts with high proportions of 2-propanol, while the Slow allele is most frequent in populations that utilize hosts with high proportions of 1-propanol. This suggests that selection for ADH-2 allozyme alleles with higher activity on the most abundant alcohols is occurring in each D. mojavensis population. In the other paralog, ADH-1, significant differences between D. mojavensis and D. arizonae are associated with a previously shown pattern of adaptive protein evolution in D. mojavensis. Examination of protein sequences showed that a large number of amino acid fixations between the paralogs have occurred in catalytic residues. These changes are potentially responsible for the significant difference in substrate specificity between the paralogs. Both functional and sequence variation within and between paralogs suggests that Adh has played an important role in the adaptation of D. mojavensis and D. arizonae to their cactophilic life.