Cactophilic Drosophila Research Articles

Divergent visual ecology of Drosophila species drives object-tracking strategies matched to landscape sparsity

Maintaining stable gaze while tracking moving objects is commonplace across animal taxa, yet how diverse ecological needs impact these processes is poorly understood. During flight, the fruit-eating fly Drosophila melanogaster maintains course by making smooth steering adjustments to fixate the image of the distant visual background on the retina, while executing body saccades to investigate nearby objects such as food sources. Cactophilic Drosophila mojavensis live where there is no canopy; rather, the flora forming visual "background" and "objects" are one and the same. We tested whether D.mojavensis have adapted their flight control strategies for a visually sparse landscape. We used a magnetic tether that allows free movement in the yaw axis. In response to a textured bar moving across a similarly textured stationary background, D.melanogaster fixates the background, thereby stabilizing gaze while integrating bar dynamics to trigger tracking saccades. By contrast, two mojavensis subspecies in the repleta subgroup and one species in the melanogaster subgroup steer to smoothly fixate the bar, seemingly ignoring the stationary surround. Desert flies execute frequent bar-tracking saccades, but theirs are triggered when rotational velocity lags the bar. Thus, D.melanogaster, which lives in visually cluttered cosmopolitan habitats, leverages the optical disparities between nearby objects and distant foliage for a hybrid control strategy: "ground-fixate, object-saccade." Flies in distant phylogenetic subgroups with similar visual ecology use a "fixate-and-saccade" strategy, which would be adaptive in a visually sparse environment where individual landscape features are both approached and used to maintain a straight course.

Fundamental Patterns of Structural Evolution Revealed by Chromosome-Length Genomes of Cactophilic Drosophila.

A thorough understanding of adaptation and speciation requires model organisms with both a history of ecological and phenotypic study as well as a complete set of genomic resources. In particular, high-quality genome assemblies of ecological model organisms are needed to assess the evolution of genome structure and its role in adaptation and speciation. Here, we generate new genomes of cactophilic Drosophila, a crucial model clade for understanding speciation and ecological adaptation in xeric environments. We generated chromosome-level genome assemblies and complete annotations for seven populations across Drosophila mojavensis, Drosophila arizonae, and Drosophila navojoa. We use these data first to establish the most robust phylogeny for this clade to date, and to assess patterns of molecular evolution across the phylogeny, showing concordance with a priori hypotheses regarding adaptive genes in this system. We then show that structural evolution occurs at constant rate across the phylogeny, varies by chromosome, and is correlated with molecular evolution. These results advance the understanding of the D. mojavensis clade by demonstrating core evolutionary genetic patterns and integrating those patterns to generate new gene-level hypotheses regarding adaptation. Our data are presented in a new public database (cactusflybase.arizona.edu), providing one of the most in-depth resources for the analysis of inter- and intraspecific evolutionary genomic data. Furthermore, we anticipate that the patterns of structural evolution identified here will serve as a baseline for future comparative studies to identify the factors that influence the evolution of genome structure across taxa.

Evidences of differential methylation in the genome during development in the cactophilic Drosophila species.

DNA methylation with 5-methylcytosine (5mC) has been reported in the genome of several eukaryotes, with marked differences between vertebrates and invertebrates. DNA methylation is poorly understood as its role in evolution in insects. Drosophila gouveai (cluster Drosophila buzzatii) presents larvae that develop obligatorily in necrotic tissues of cacti in nature, with the distribution of populations in South America, and plasticity of phenotypes in insect-plant interaction. We characterize organisms at developmental stages and analyze variations at multiple methylation-sensitive loci in pupae, and adult flies using methylation sensitive amplification polymorphism. We obtained 326 loci with CCGG targets in the genome of D. gouveai. Genomic regions with molecular lengths from 100 to 700 pb were most informative about methylation states. Multiple loci show differences in methylation-sensitive sites (MSL) concerning developmental stages, such as in pupae (MSL = 40), female reproductive tissue (MSL = 76), and male reproductive tissues (MSL = 58). Our results are the first evidence of genome-wide methylation in D. gouveai organisms.

Genetic variation in disease resistance in Drosophila spp. is mitigated in Drosophila sechellia by specialization to a toxic host

We found that Drosophila species vary in their susceptibility to the broad-spectrum entomopathogen, Metarhizium anisopliae (strain Ma549). Generalist species were generally more resistant than dietary specialists, with the cactophilic Drosophila buzzatii and Drosophila sechellia, a specialist of the Morinda citrifolia (Morinda) fruit, being most susceptible. Morinda fruit is reported to be toxic to most herbivores because it contains Octanoic Acid (OA). We confirmed that OA is toxic to Drosophila spp., other than D. sechellia, and we also found that OA is highly toxic to entomopathogenic fungi including Ma549 and Beauveria bassiana. Drosophilasechellia fed a diet containing OA, even at levels much less than found in Morinda fruit, had greatly reduced susceptibility to Ma549. This suggests that specializing to Morinda may have provided an enemy-free space, reducing adaptive prioritization on a strong immune response. Our results demonstrate that M. anisopliae and Drosophila species with divergent lifestyles provide a versatile model system for understanding the mechanisms of host–pathogen interactions at different scales and in environmental context.

Physiological and life history changes associated with seasonal adaptation in the cactophilic Drosophila mojavensis.

Many insects inhabiting temperate climates are faced with changing environmental conditions throughout the year. Depending on the species, these environmental fluctuations can be experienced within a single generation or across multiple generations. Strategies for dealing with these seasonal changes vary across populations. Drosophila mojavensis is a cactophilic Drosophila species endemic to the Sonoran Desert. The Sonoran Desert regularly reaches temperatures of 50°C in the summer months. As individuals of this population are rare to collect in the summer months, we simulated the cycling temperatures experienced by D. mojavensis in the Sonoran Desert from April to July (four generations) in a temperature- and light-controlled chamber, to understand the physiological and life history changes that allow this population to withstand these conditions. In contrast to our hypothesis of a summer aestivation, we found that D. mojavensis continue to reproduce during the summer months, albeit with lower viability, but the adult survivorship of the population is highly reduced during this period. As expected, stress resistance increased during the summer months in both the adult and the larval stages. This study examines several strategies for withstanding the Sonoran Desert summer conditions which may be informative in the study of other desert endemic species.

P450 gene family mediates allelochemical detoxification and tolerance to alkaloids in cactophilic Drosophila

AbstractCacti are characterized by the extensive production of a broad variety of toxic metabolites as an anti‐herbivore strategy but nevertheless cactophilic Drosophila spp. (Diptera: Drosophilidae) feed and breed on decaying cacti. The family of cytochrome P450 is a group of enzymes involved in the detoxification metabolism, which are critical for the adaptation of phytophagous insects, especially populations of Drosophila exploiting cacti. In this context, Drosophila buzzatii Patterson & Wheeler and Drosophila koepferae Fontdevila & Wasserman (Drosophilini), constitute an excellent model for the study of the genetic basis of tolerance to cacti allelochemicals. These closely related species allow testing to what extent a relatively small set of genes can explain host use patterns and their role in the divergent evolution of specialization. We evaluated the role of the P450 genes in the adaptation of these species to the use of one of their hosts, the columnar cactus Trichocereus terscheckii (Parm.) Friedrich & GD Rowley (Cactaceae). Experimentally evolved strains exhibiting different degrees of cactus specialization were exposed to artificial rearing media enriched with cactus allelochemicals and piperonyl butoxide (PBO), an inhibitor of P450 family activity. Our analyses showed a significant viability reduction in the presence of PBO, in the non‐tolerant strains of both species as well as in the strain historically subjected to mild concentrations of allelochemicals (the soft strain) of D. koepferae. We confirmed the key role of P450 genes for the evolution of cactus specialization but also found evidence for the evolution of other detoxification pathways.

Ortholog genes from cactophilic Drosophila provide insight into human adaptation to hallucinogenic cacti

Cultural transformations of lifestyles and dietary practices have been key drivers of human evolution. However, while most of the evidence of genomic adaptations is related to the hunter-gatherer transition to agricultural societies, little is known on the influence of other major cultural manifestations. Shamanism is considered the oldest religion that predominated throughout most of human prehistory and still prevails in many indigenous populations. Several lines of evidence from ethno-archeological studies have demonstrated the continuity and importance of psychoactive plants in South American cultures. However, despite the well-known importance of secondary metabolites in human health, little is known about its role in the evolution of ethnic differences. Herein, we identified candidate genes of adaptation to hallucinogenic cactus in Native Andean populations with a long history of shamanic practices. We used genome-wide expression data from the cactophilic fly Drosophila buzzatii exposed to a hallucinogenic columnar cactus, also consumed by humans, to identify ortholog genes exhibiting adaptive footprints of alkaloid tolerance. Genomic analyses in human populations revealed a suite of ortholog genes evolving under recent positive selection in indigenous populations of the Central Andes. Our results provide evidence of selection in genetic variants related to alkaloids toxicity, xenobiotic metabolism, and neuronal plasticity in Aymara and Quechua populations, suggesting a possible process of gene-culture coevolution driven by religious practices.

Chromosome-level hybrid de novo genome assemblies as an attainable option for nonmodel insects.

The emergence of third-generation sequencing (3GS; long-reads) is bringing closer the goal of chromosome-size fragments in de novo genome assemblies. This allows the exploration of new and broader questions on genome evolution for a number of nonmodel organisms. However, long-read technologies result in higher sequencing error rates and therefore impose an elevated cost of sufficient coverage to achieve high enough quality. In this context, hybrid assemblies, combining short-reads and long-reads, provide an alternative efficient and cost-effective approach to generate de novo, chromosome-level genome assemblies. The array of available software programs for hybrid genome assembly, sequence correction and manipulation are constantly being expanded and improved. This makes it difficult for nonexperts to find efficient, fast and tractable computational solutions for genome assembly, especially in the case of nonmodel organisms lacking a reference genome or one from a closely related species. In this study, we review and test the most recent pipelines for hybrid assemblies, comparing the model organism Drosophila melanogaster to a nonmodel cactophilic Drosophila, D. mojavensis. We show that it is possible to achieve excellent contiguity on this nonmodel organism using the dbg2olc pipeline.

Genomic analysis of the four ecologically distinct cactus host populations of Drosophila mojavensis

BackgroundRelationships between an organism and its environment can be fundamental in the understanding how populations change over time and species arise. Local ecological conditions can shape variation at multiple levels, among these are the evolutionary history and trajectories of coding genes. This study examines the rate of molecular evolution at protein-coding genes throughout the genome in response to host adaptation in the cactophilic Drosophila mojavensis. These insects are intimately associated with cactus necroses, developing as larvae and feeding as adults in these necrotic tissues. Drosophila mojavensis is composed of four isolated populations across the deserts of western North America and each population has adapted to utilize different cacti that are chemically, nutritionally, and structurally distinct.ResultsHigh coverage Illumina sequencing was performed on three previously unsequenced populations of D. mojavensis. Genomes were assembled using the previously sequenced genome of D. mojavensis from Santa Catalina Island (USA) as a template. Protein coding genes were aligned across all four populations and rates of protein evolution were determined for all loci using a several approaches.ConclusionsLoci that exhibited elevated rates of molecular evolution tend to be shorter, have fewer exons, low expression, be transcriptionally responsive to cactus host use and have fixed expression differences across the four cactus host populations. Fast evolving genes were involved with metabolism, detoxification, chemosensory reception, reproduction and behavior. Results of this study give insight into the process and the genomic consequences of local ecological adaptation.

Protein–alkaloid interaction in larval diet affects fitness in cactophilic Drosophila (Diptera: Drosophilidae)

Protein–alkaloid interaction in larval diet affects fitness in cactophilic Drosophila (Diptera: Drosophilidae)

Divergent patterns of correlated evolution in primary and secondary sexual traits of cactophilic Drosophila

The rapid diversification of sexual traits is a common phenomenon accompanying the evolution of reproductive isolation, yet the evolutionary mechanisms driving such diversification are often unknown. Based on experimentally evolved strains of two sister species of cactophilic Drosophila, we investigated the correlated evolution of primary and secondary sexual traits to semi-natural environments enriched in secondary metabolites. We compared patterns of morphological evolution in the size and shape of male wing and genitalia of Drosophila buzzatii and Drosophila koepferae selected for different levels of alkaloid intensities for 20 generations. We found similar modes of selection operating among organs but not among species. The evolution of these traits in D. koepferae were compatible with patterns of stabilizing selection, while in D. buzzatii were characterized by directional changes. We also found that allometric variation was an important component of genital shape evolution, whereas changes in the wing morphology were less pronounced and mostly non-allometric. Overall, our data suggest that the diversification of sexual traits in this species pair is related to the evolution of dissimilar genetic architectures and reinforced by divergent ecological responses.

An Improved Genome Assembly for Drosophila navojoa, the Basal Species in the mojavensis Cluster.

Three North American cactophilic Drosophila species, D. mojavensis, D. arizonae, and D. navojoa, are of considerable evolutionary interest owing to the shift from breeding in Opuntia cacti to columnar species. The 3 species form the “mojavensis cluster” of Drosophila. The genome of D. mojavensis was sequenced in 2007 and the genomes of D. navojoa and D. arizonae were sequenced together in 2016 using the same technology (Illumina) and assembly software (AllPaths-LG). Yet, unfortunately, the D. navojoa genome was considerably more fragmented and incomplete than its sister species, rendering it less useful for evolutionary genetic studies. The D. navojoa read dataset does not fully meet the strict insert size required by the assembler used (AllPaths-LG) and this incompatibility might explain its assembly problems. Accordingly, when we re-assembled the genome of D. navojoa with the SPAdes assembler, which does not have the strict AllPaths-LG requirements, we obtained a substantial improvement in all quality indicators such as N50 (from 84 kb to 389 kb) and BUSCO coverage (from 77% to 97%). Here we share a new, improved reference assembly for D. navojoa genome, along with a RNAseq transcriptome. Given the basal relationship of the Opuntia breeding D. navojoa to the columnar breeding D. arizonae and D. mojavensis, the improved assembly and annotation will allow researchers to address a range of questions associated with the genomics of host shifts, chromosomal rearrangements and speciation in this group.

Developmental and Transcriptomal Responses to Seasonal Dietary Shifts in the Cactophilic Drosophila mojavensis of North America.

Drosophila mojavensis normally breeds in necrotic columnar cactus, but they also feed and breed in Opuntia fruit (prickly pear) which serves as a seasonal resource. The prickly pear fruits (PPFs) are much different chemically from cacti, mainly in their free sugars and lipid content, raising the question of the effects of this seasonal shift on fitness and on gene expression. Here we reared 3 isofemale strains of D. mojavensis collected from different parts of the species' range on semi-natural medium of either cactus or PPF and measured the development time, survival, body weights, and desiccation resistance. All these parameters were affected by diet and by interaction with strain and or sex. Interestingly, however, there appear to be tradeoffs: flies developed faster in prickly pear and the emerging adults were heavier, but those having grown in cactus were more resistant to desiccation. We also evaluated the gene expression of emerging male and female adult flies using RNA-Seq. While more genes were down-regulated in PPF than up-regulated in both sexes, the sexes did differ in expression patterns. The majority of the genes that were preferentially expressed comparing PPF versus cactus underlie metabolism. Genes involved with carbohydrate and lipid metabolism, as well as with the amino acid serine, and their relationship to growth and development reflect the ways in which these dietary differences affect the flies.

Genetics and Distribution Modeling: The Demographic History of the Cactophilic Drosophila buzzatii Species Cluster in Open Areas of South America.

Biodiversity is the result of historical and recurrent events acting on populations and species. The Drosophila buzzatii species cluster is distributed along a diagonal of open areas in South America. Combining genetic analyses with species distribution models we evaluated the influence of climatic changes in the demography history of this cluster. We performed a Bayesian Skyline analysis and reconstructed the ancestral areas based on mitochondrial cytochrome oxidase I (mtCOI) gene. We modeled the current distribution and projected it to past (mid-Holocene and Last Glacial Maximum) and future. Our results demonstrate that climate change plays a critical role in historical demography and in defining the current and future geographic ranges of these species. The inter-Andean dry valleys and the Chiquitano Seasonally Dry Tropical Forests (SDTF) in Bolivia are considered the ancestral area for the D. buzzatii cluster. From this area, the migration route was through a west-east corridor from central Andes, throughout Bolivia, Paraguay and Argentina toward eastern and northeastern Brazil, along the edges of rain forest. The responses of the species to the climatic changes differ from the dominant assumptions of expansion during dry/cold weather and contraction during wet/warm weather that characterized the glacial cycles. We suggest that the influence of ecology and the potential responsiveness of each taxon to the environmental dynamics should be considered as well. Predictions for the future suggest a decline of suitable areas for the cluster, threatening biodiversity of these habitats. This work showed the importance of an integrative analysis of genetics and geography information to improve the inferences about demographic history hypotheses for the cluster.

Positive selection at sites of chemosensory genes is associated with the recent divergence and local ecological adaptation in cactophilic Drosophila

BackgroundAdaptation to new hosts in phytophagous insects often involves mechanisms of host recognition by genes of sensory pathways. Most often the molecular evolution of sensory genes has been explained in the context of the birth-and-death model. The role of positive selection is less understood, especially associated with host adaptation and specialization. Here we aim to contribute evidence for this latter hypothesis by considering the case of Drosophila mojavensis, a species with an evolutionary history shaped by multiple host shifts in a relatively short time scale, and its generalist sister species, D. arizonae.ResultsWe used a phylogenetic and population genetic analysis framework to test for positive selection in a subset of four chemoreceptor genes, one gustatory receptor (Gr) and three odorant receptors (Or), for which their expression has been previously associated with host shifts. We found strong evidence of positive selection at several amino acid sites in all genes investigated, most of which exhibited changes predicted to cause functional effects in these transmembrane proteins. A significant portion of the sites identified as evolving positively were largely found in the cytoplasmic region, although a few were also present in the extracellular domains.ConclusionsThe pattern of substitution observed suggests that some of these changes likely had an effect on signal transduction as well as odorant recognition and protein-protein interactions. These findings support the role of positive selection in shaping the pattern of variation at chemosensory receptors, both during the specialization onto one or a few related hosts, but as well as during the evolution and adaptation of generalist species into utilizing several hosts.

Genome-Wide Patterns of Sequence Divergence of Protein-Coding Genes Between Drosophila buzzatii and D. mojavensis.

Evolutionary rates for protein-coding genes are determined not only by natural selection but also by multiple genomic factors including mutation rates, recombination, gene expression levels, and chromosomal location. To investigate the joint effects of different genomic determinants on protein evolution, we compared the coding sequences of 9017 single-copy orthologs between 2 cactophilic species from the Drosophila subgenus, Drosophila mojavensis and D. buzzatii, whose genomes have been previously sequenced. We assessed the impact of 7 genomic determinants, that is, chromosome type, recombination, chromosomal inversions, expression breadth, expression level, gene length, and the number of exons, on divergence rates of protein-coding genes to understand patterns of evolutionary variation. Integrative analysis of these factors revealed that 1) X-linked and autosomal genes evolve at significantly different rates in agreement with the faster-X hypothesis, 2) genes located on the dot chromosome and pericentromeric regions have higher divergence rates, 3) genes located at chromosomes with more fixed inversions have higher pairwise divergence than those located at nearly collinear chromosomes, and 4) gene expression patterns can be considered the strongest determinant of protein evolution. In addition, the number of exons and protein length had a significant effect on pairwise divergence at synonymous sites. All in all, our results show the relative importance of each genomic factor on the rates of protein evolution and functional constraint in these 2 cactophilic Drosophila 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.

Genetic Diversity and Demographic History in the Cactophilic Drosophila repleta Species Group (Diptera: Drosophilidae) in North America Inferred from Mitochondrial DNA Barcodes.

Genetic diversity in mitochondrial DNA barcodes, comprising a segment of the cytochrome c oxidase subunit I (COI) gene, was used to infer demographic histories in selected taxa of the cactophilic Drosophila repleta species group in North America. Haplotype and nucleotide diversities were determined in 16 taxa based on both previously published and new sequences. Haplotype diversity (h) differed dramatically in different taxa, varying from h = 0 in Drosophila eremophila, Drosophila hexastigma, and Drosophila bifurca to h = 0.99 in Drosophila hamatofila. Genetic diversity indices and sample sizes were sufficient to infer demographic histories from mismatch distribution analysis and Bayesian skyline plots for 9 taxa: Drosophila mojavensis baja, Drosophila mojavensis sonorensis, Drosophila arizonae, Drosophila aldrichi, D. hamatofila, Drosophila spenceri, Drosophila mainlandi, Drosophila mettleri, and Drosophila nigrospiracula. Evidence was found for both population expansions and relatively stable populations in these species. Demographic history varied dramatically in subspecies of D. mojavensis, showing a relatively stable population size over time in D. m. sonorensis from the mainland Sonoran Desert whereas a large population expansion was evident in D. m. baja from the Baja California Peninsula, providing support for the hypothesis that the split of sister species D. mojavensis and D. arizonae from a common ancestor occurred on the mainland rather than the peninsula as proposed by others. No evidence was found for a causal relationship between a stable or expanding population and host plant shifts from prickly-pear cactus to columnar cacti, which has occurred independently in many taxa of the repleta group.

The influence of developmental environment on courtship song in cactophilic Drosophila.

Closely related species often differ in the signals involved in sexual communication and mate recognition. Determining the factors influencing signal quality (i.e. signal's content and conspicuousness) provides an important insight into the potential pathways by which these interspecific differences evolve. Host specificity could bias the direction of the evolution of sexual communication and the mate recognition system, favouring sensory channels that work best in the different host conditions. In this study, we focus on the cactophilic sibling species Drosophila buzzatii and D.koepferae that have diverged not only in the sensory channel used for sexual communication and mate recognition but also in the cactus species that use as primary hosts. We evaluate the role of the developmental environment in generating courtship song variation using an isofemale line design. Our results show that host environment during development induces changes in the courtship song of D.koepferae males, but not in D.buzzatii males. Moreover, we report for the first time that host rearing environment affects the conspicuousness of courtship song (i.e. song volume). Our results are mainly discussed in the context of the sensory drive hypothesis.

Experimental Evolution of Alkaloid Tolerance in Sibling Drosophila 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.