A Distinctive and Host-Restricted Gut Microbiota in Populations of a Cactophilic Drosophila Species.

Almost all animals possess gut microbial communities, but the nature of these communities varies immensely. For example, in social bees and mammals, the composition is relatively constant within species and is dominated by specialist bacteria that do not live elsewhere; in laboratory studies and field surveys of Drosophila melanogaster, however, gut communities consist of bacteria that are ingested with food and that vary widely among individuals and localities. We addressed whether an ecological specialist in its natural habitat has a microbiota dominated by gut specialists or by environmental bacteria. Drosophila nigrospiracula is a species that is endemic to the Sonoran Desert and is restricted to decaying tissues of two giant columnar cacti, Pachycereus pringlei (cardón cactus) and Carnegiea gigantea (saguaro cactus). We found that the D. nigrospiracula microbiota differs strikingly from that of the cactus tissue on which the flies feed. The most abundant bacteria in the flies are rare or completely absent in the cactus tissue and are consistently abundant in flies from different cacti and localities. Several of these fly-associated bacterial groups, such as the bacterial order Orbales and the genera Serpens and Dysgonomonas, have been identified in prior surveys of insects from the orders Hymenoptera, Coleoptera, Lepidoptera, and Diptera, including several Drosophila species. Although the functions of these bacterial groups are mostly unexplored, Orbales species studied in bees are known to break down plant polysaccharides and use the resulting sugars. Thus, these bacterial groups appear to be specialized to the insect gut environment, where they may colonize through direct host-to-host transmission in natural settings.IMPORTANCE Flies in the genus Drosophila have become laboratory models for microbiota research, yet the bacteria commonly used in these experiments are rarely found in wild-caught flies and instead represent bacteria also present in the food. This study shows that an ecologically specialized Drosophila species possesses a distinctive microbiome, composed of bacterial types absent from the flies' natural food but widespread in other wild-caught insects. This study highlights the importance of fieldwork-informed microbiota research.

Enhanced fertility and chill tolerance after cold-induced reproductive arrest in females of temperate species of the Drosophila buzzatii complex.

Long-term exposure to low temperatures during adult maturation might decrease fertility after cold recovery as a consequence of carry-over effects on reproductive tissues. This pattern should be more pronounced in tropical than in temperate species as protective mechanisms against chilling injuries are expected to be more effective in the latter. We initially determined the lower thermal thresholds to induce ovarian maturation in four closely related Drosophila species, two inhabiting temperate regions and the other two tropical areas of South America. As expected, only temperate species regularly experience cold-inducing conditions for reproductive arrest during winter in their natural environment. Subsequently, we exposed reproductively arrested and mature females to cold-inducing conditions for reproductive arrest over a long period. Following cold exposure, tropical species exhibited a dramatic fertility decline, irrespective of reproductive status. In contrast, not only were temperate females fecund and fertile but also fertility was superior in females that underwent cold-induced reproductive arrest, suggesting that it might act as a protecting mechanism ensuring fertility after cold recovery. Based on these findings, we decided to evaluate the extent to which reproductive status affects cold tolerance and energy metabolism at low temperature. We found a lower metabolic rate and a higher cold tolerance in reproductively arrested females, although only temperate species attained high levels of chill tolerance. These findings highlight the role of cold-induced reproductive arrest as part of an integrated mechanism of cold adaptation that could potentially contribute to the spread of temperate species into higher latitudes or altitudes.

Postmating transcriptional changes in reproductive tracts of con- and heterospecifically mated Drosophila mojavensis females.

In internally fertilizing organisms, mating involves a series of highly coordinated molecular interactions between the sexes that occur within the female reproductive tract. In species where females mate multiply, traits involved in postcopulatory interactions are expected to evolve rapidly, potentially leading to postmating-prezygotic (PMPZ) reproductive isolation between diverging populations. Here, we investigate the postmating transcriptional response of the lower reproductive tract of Drosophila mojavensis females following copulation with either conspecific or heterospecific (Drosophila arizonae) males at three time points postmating. Relatively few genes (15 total) were differentially regulated in the female lower reproductive tract in response to conspecific mating. Heterospecifically mated females exhibited significant perturbations in the expression of the majority of these genes, and also down-regulated transcription of a number of others, including several involved in mitochondrial function. These striking regulatory differences indicate failed postcopulatory molecular interactions between the sexes consistent with the strong PMPZ isolation observed for this cross. We also report the transfer of male accessory-gland protein (Acp) transcripts from males to females during copulation, a finding with potentially broad implications for understanding postcopulatory molecular interactions between the sexes.

Population genetics and phylogenetic relationships of beetles (Coleoptera: Histeridae and Staphylinidae) from the Sonoran Desert associated with rotting columnar cacti.

Dozens of arthropod species are known to feed and breed in the necrotic tissues (rots) of columnar cacti in the Sonoran Desert. Because the necrotic patches are ephemeral, the associated arthropods must continually disperse to new cacti and therefore the populations of any given species are expected to show very little local genetic differentiation. While this has been found to be true for the cactophilic Drosophila, the evolutionary histories and characteristics of other arthropods inhabiting the same necrotic patches, especially the beetles, have yet to be examined. Here we used nucleotide sequence data from segments of the mitochondrial 16S rRNA and cytochrome c oxidase subunit I (COI) genes to examine population structure and demographic history of three sympatric beetle species (Coleoptera: Histeridae and Staphylinidae) collected on senita cactus (Lophocereus schottii) from six widely-separated localities on the Baja California peninsula of northwestern Mexico. Two histerids, Iliotona beyeri and Carcinops gilensis, and an unidentified staphylinid, Belonuchus sp., showed little or no population structure over a broad geographic area on the peninsula, consistent with the prediction that these beetles should show high dispersal ability. Demographic tests revealed varying levels of historical population expansion among the beetle species analyzed, which are discussed in light of their ecologies and concurrent biogeographic events. Additionally, phylogenetic analyses of COI sequences in Carcinops collected on a variety of columnar cacti from both peninsular and mainland Mexico localities revealed several species-level partitions, including a putative undescribed peninsular species that occurred sympatrically with C. gilensis on senita.

Evolution of genes and genomes on the Drosophila phylogeny.

Comparative analysis of multiple genomes in a phylogenetic framework dramatically improves the precision and sensitivity of evolutionary inference, producing more robust results than single-genome analyses can provide. The genomes of 12 Drosophila species, ten of which are presented here for the first time (sechellia, simulans, yakuba, erecta, ananassae, persimilis, willistoni, mojavensis, virilis and grimshawi), illustrate how rates and patterns of sequence divergence across taxa can illuminate evolutionary processes on a genomic scale. These genome sequences augment the formidable genetic tools that have made Drosophila melanogaster a pre-eminent model for animal genetics, and will further catalyse fundamental research on mechanisms of development, cell biology, genetics, disease, neurobiology, behaviour, physiology and evolution. Despite remarkable similarities among these Drosophila species, we identified many putatively non-neutral changes in protein-coding genes, non-coding RNA genes, and cis-regulatory regions. These may prove to underlie differences in the ecology and behaviour of these diverse species.

Genetic diversity and population genetics of mosquitoes (Diptera: Culicidae: Culex spp.) from the Sonoran Desert of North America.

The population genetics and phylogenetic relationships of Culex mosquitoes inhabiting the Sonoran Desert region of North America were studied using mitochondrial DNA and microsatellite molecular markers. Phylogenetic analyses of mitochondrial cytochrome c oxidase subunit I (COI) from mosquitoes collected over a wide geographic area, including the Baja California peninsula, and mainland localities in southern Arizona, USA and Sonora, Mexico, showed several well-supported partitions corresponding to Cx. quinquefasciatus, Cx. tarsalis, and two unidentified species, Culex sp. 1 and sp. 2. Culex quinquefasciatus was found at all localities and was the most abundant species collected. Culex tarsalis was collected only at Tucson, Arizona and Guaymas, Sonora. The two unidentified species of Culex were most abundant at Navojoa in southern Sonora. Haplotype and nucleotide diversities in the COI gene segment were substantially lower in Cx. quinquefasciatus compared with the other three species. Analysis of molecular variance revealed little structure among seven populations of Cx. quinquefasciatus, whereas significant structure was found between the two populations of Cx. tarsalis. Evidence for an historical population expansion beginning in the Pleistocene was found for Cx. tarsalis. Possible explanations for the large differences in genetic diversity between Cx. quinquefasciatus and the other species of Culex are presented.

Dietary protein and sugar differentially affect development and metabolic pools in ecologically diverse Drosophila.

We examined the effects of 3 diets differing in their relative levels of sugar and protein on development and metabolic pools (protein, TG, and glycogen) among sets of isofemale lines of 2 ecologically distinct Drosophila species, D. melanogaster and D. mojavensis. Our high protein:sugar ratio diet contained 7.1% protein and 17.9% carbohydrate, the EPS diet was 4.3% protein and 21.2% carbohydrate, and the LPS was only 2.5% protein and 24.6% carbohydrate. Larvae of D. melanogaster, a generalist fruit breeder, were able to survive on all 3 diets, although all 3 metabolic pools responded with significant diet and diet × line interactions. Development was delayed by the diet with the most sugar relative to protein. The other species, D. mojavensis, a cactus breeder ecologically unaccustomed to encountering simple sugars, completely failed to survive when fed the diet with the highest sugar and showed very poor survival even with the diet with equal parts of protein and sugar. Furthermore, the D. mojavensis adult metabolic pools of protein, TG, and glycogen significantly differed from those of D. melanogaster adults fed the identical diet. Thus, considerable within- and between-species differences exist in how diets are metabolized. Given that the genomes of both of these Drosophila species have been sequenced, these differences and their genetic underpinnings hold promise for understanding human responses to nutrition and for developing strategies for dealing with metabolic disease.

A de novo transcriptional atlas in Danaus plexippus reveals variability in dosage compensation across tissues.

A detailed knowledge of gene function in the monarch butterfly is still lacking. Here we generate a genome assembly from a Mexican nonmigratory population and used RNA-seq data from 14 biological samples for gene annotation and to construct an atlas portraying the breadth of gene expression during most of the monarch life cycle. Two thirds of the genes show expression changes, with long noncoding RNAs being particularly finely regulated during adulthood, and male-biased expression being four times more common than female-biased. The two portions of the monarch heterochromosome Z, one ancestral to the Lepidoptera and the other resulting from a chromosomal fusion, display distinct association with sex-biased expression, reflecting sample-dependent incompleteness or absence of dosage compensation in the ancestral but not the novel portion of the Z. This study presents extended genomic and transcriptomic resources that will facilitate a better understanding of the monarch's adaptation to a changing environment.

Duplication, selection and gene conversion in a Drosophila mojavensis female reproductive protein family.

Protein components of the Drosophila male ejaculate, several of which evolve rapidly, are critical modulators of reproductive success. Recent studies of female reproductive tract proteins indicate they also are extremely divergent between species, suggesting that reproductive molecules may coevolve between the sexes. Our current understanding of intersexual coevolution, however, is severely limited by the paucity of genetic and evolutionary studies on the female molecules involved. Physiological evidence of ejaculate-female coadaptation, paired with a promiscuous mating system, makes Drosophila mojavensis an exciting model system in which to study the evolution of reproductive proteins. Here we explore the evolutionary dynamics of a five-paralog gene family of female reproductive proteases within populations of D. mojavensis and throughout the repleta species group. We show that the proteins have experienced ongoing gene duplication and adaptive evolution and further exhibit dynamic patterns of pseudogenation, copy number variation, gene conversion, and selection within geographically isolated populations of D. mojavensis. The integration of these patterns in a single gene family has never before been documented in a reproductive protein.

Mitochondrial DNA evidence for deep genetic divergences in allopatric populations of the rocky intertidal isopod Ligia occidentalis from the eastern Pacific.

Nucleotide sequences from the cytochrome c oxidase subunit I (COI) gene were used to test for genetic differentiation in the rocky intertidal isopod crustacean, Ligia occidentalis (Ligiidae), from the eastern Pacific. Phylogenetic analyses showed that individuals of L. occidentalis from southern California, USA to Manzanillo, Colima, Mexico partitioned into 15 highly-divergent clades. Mean Kimura 2-parameter (K2P) genetic distances among clades ranged from 13.2% to 26.7%. These values are similar to interspecific genetic distances found in a wide variety of crustaceans, including Ligia spp., suggesting that the taxon L. occidentalis represents a complex of cryptic species.

The genus Drosophila as a model for testing tree- and character-based methods of species identification using DNA barcoding.

DNA barcoding has recently been proposed as a promising tool for the (1) rapid assignment of unknown samples to described species by non-expert workers and (2) a potential method of new species discovery based on degree of DNA sequence divergence. Two broad methods have been used, one based on degree of DNA sequence variation, within and between species and another requiring the recovery of species as discrete clades (monophyly) on a phylogenetic tree. An alternative method relies on the identification of a set of specific diagnostic nucleotides for a given species (characters). The genus Drosophila has long served as a model system in genetics, development, ecology and evolutionary biology. As a result of this work, species boundaries within this genus are quite well delimited, with most taxa being defined by morphological characters and also conforming to a biological species concept (e.g., partial or complete reproductive isolation has used to erect and define species). In addition, some of the species in this group have also been subjected to phylogenetic analysis, yielding cases where taxa both conform and conflict with a phylogenetic species concept. Here, we analyzed 1058 COI sequences belonging to 68 species belonging to Drosophila and its allied genus Zaprionus and with more than a single representative to assess the performance of the three DNA barcoding methods. 26% of the species could not be defined using distance methods, i.e. had a barcoding gap of ≤ 0, and 23% were not monophyletic. We focused then on four groups of closely-related species whose taxonomy is well-established on non-molecular basis (e.g., morphology, geography, reproductive isolation) and to which most of the problematic species belonged. We showed that characters performed better than other approaches in the case of paraphyletic species, but all methods failed in the case of polyphyletic species. For these polyphyletic species, other sources of evidence (e.g., morphology, geography, reproductive isolation) are more relevant than COI sequences, highlighting the limitation of DNA barcoding and the needs for integrative taxonomy approaches. In conclusion, DNA barcoding of Drosophila shows no reason to alter the 250 years old tradition of character-based taxonomy, and many reasons to shy away from the alternatives.

Phylogenetic relationships of Sonoran Desert cactus beetles in the tribe Hololeptini (Coleoptera: Histeridae: Histerinae), with comments on the taxonomic status of Iliotona beyeri.

Nucleotide sequences from 16S rRNA and cytochrome c oxidase subunit I (COI) were used to examine phylogenetic relationships and evolution of beetles from the tribe Hololeptini (Coleoptera: Histeridae: Histerinae) that inhabit necrotic tissue of columnar cacti in the Sonoran Desert. Phylogenetic and morphological analyses revealed the presence of seven separate lineages, three representing species in the genus Iliotona, including I. beyeri stat. nov., and four species belonging to the genus Hololepta (sensu lato). The possible roles of historical vicariance and host plant associations on the evolution of the Hololeptini from the Sonoran Desert are discussed.

Gene duplication and adaptive evolution of digestive proteases in Drosophila arizonae female reproductive tracts.

It frequently has been postulated that intersexual coevolution between the male ejaculate and the female reproductive tract is a driving force in the rapid evolution of reproductive proteins. The dearth of research on female tracts, however, presents a major obstacle to empirical tests of this hypothesis. Here, we employ a comparative EST approach to identify 241 candidate female reproductive proteins in Drosophila arizonae, a repleta group species in which physiological ejaculate-female coevolution has been documented. Thirty-one of these proteins exhibit elevated amino acid substitution rates, making them candidates for molecular coevolution with the male ejaculate. Strikingly, we also discovered 12 unique digestive proteases whose expression is specific to the D. arizonae lower female reproductive tract. These enzymes belong to classes most commonly found in the gastrointestinal tracts of a diverse array of organisms. We show that these proteases are associated with recent, lineage-specific gene duplications in the Drosophila repleta species group, and exhibit strong signatures of positive selection. Observation of adaptive evolution in several female reproductive tract proteins indicates they are active players in the evolution of reproductive tract interactions. Additionally, pervasive gene duplication, adaptive evolution, and rapid acquisition of a novel digestive function by the female reproductive tract points to a novel coevolutionary mechanism of ejaculate-female interaction.

Polytene chromosomal maps of 11 Drosophila species: the order of genomic scaffolds inferred from genetic and physical maps.

The sequencing of the 12 genomes of members of the genus Drosophila was taken as an opportunity to reevaluate the genetic and physical maps for 11 of the species, in part to aid in the mapping of assembled scaffolds. Here, we present an overview of the importance of cytogenetic maps to Drosophila biology and to the concepts of chromosomal evolution. Physical and genetic markers were used to anchor the genome assembly scaffolds to the polytene chromosomal maps for each species. In addition, a computational approach was used to anchor smaller scaffolds on the basis of the analysis of syntenic blocks. We present the chromosomal map data from each of the 11 sequenced non-Drosophila melanogaster species as a series of sections. Each section reviews the history of the polytene chromosome maps for each species, presents the new polytene chromosome maps, and anchors the genomic scaffolds to the cytological maps using genetic and physical markers. The mapping data agree with Muller's idea that the majority of Drosophila genes are syntenic. Despite the conservation of genes within homologous chromosome arms across species, the karyotypes of these species have changed through the fusion of chromosomal arms followed by subsequent rearrangement events.

Multiple introductions of the Spiroplasma bacterial endosymbiont into Drosophila.

Bacterial endosymbionts are common in insects and can have dramatic effects on their host's evolution. So far, the only heritable symbionts found in Drosophila have been Wolbachia and Spiroplasma. While the incidence and effects of Wolbachia have been studied extensively, the prevalence and significance of Spiroplasma infections in Drosophila are less clear. These small, gram-positive, helical bacteria infect a diverse array of plant and arthropod hosts, conferring a variety of fitness effects. Male-killing Spiroplasma are known from certain Drosophila species; however, in others, Spiroplasma appear not to affect sex ratio. Previous studies have identified different Spiroplasma haplotypes in Drosophila populations, although no extensive surveys have yet been reported. We used a multilocus sequence analysis to reconstruct a robust Spiroplasma endosymbiont phylogeny, assess genetic diversity, and look for evidence of recombination. Six loci were sequenced from over 65 Spiroplasma-infected individuals from nine different Drosophila species. Analysis of these sequences reveals at least five separate introductions of four phylogenetically distinct Spiroplasma haplotypes, indicating that more extensive sampling will likely reveal an even greater Spiroplasma endosymbiont diversity. Patterns of variation in Drosophila mitochondrial haplotypes in Spiroplasma-infected and uninfected flies imply imperfect vertical transmission in host populations and possible horizontal transmission.

Genetic diversification and demographic history of the cactophilic pseudoscorpion Dinocheirus arizonensis from the Sonoran Desert.

Sequence data from a segment of the mitochondrial cytochrome c oxidase subunit I (COI) gene were used to examine phylogenetic relationships, estimate gene flow and infer demographic history of the cactophilic chernetid pseudoscorpion, Dinocheirus arizonensis (Banks), from the Sonoran Desert. Phylogenetic trees resolved two clades of D. arizonensis, one from mainland Sonora, Mexico and southern Arizona (clade I) and the other from the Baja California peninsula and southern Arizona (clade II). The two clades were separated by a mean genetic distance (d) of approximately 2.6%. Hierarchical analysis of molecular variance indicated highly significant population structuring in D. arizonensis (overall Phi(ST)=0.860; P<0.0001), with 80% of the genetic variation distributed among the two clades. Most pairwise comparisons of Phi(ST) among populations within each clade, however, were not significant. The results suggest that phoretic dispersal on vagile cactophilic insects such as the neriid cactus fly Odontoloxozus longicornis (Coquillett) provides sufficient gene flow to offset the accumulation of unique haplotypes within each clade of the non-vagile pseudoscorpion. Preliminary results on dispersal capability of O. longicornis were consistent with this conclusion. Tests designed to reconstruct demographic history from sequence data indicated that both clades of D. arizonensis, as well as O. longicornis, have experienced historical population expansions. Potential barriers to gene flow that may have led to genetic isolation and diversification in clades I and II of D. arizonensis are discussed.

Analysis of Drosophila species genome size and satellite DNA content reveals significant differences among strains as well as between species.

The size of eukaryotic genomes can vary by several orders of magnitude, yet genome size does not correlate with the number of genes nor with the size or complexity of the organism. Although "whole"-genome sequences, such as those now available for 12 Drosophila species, provide information about euchromatic DNA content, they cannot give an accurate estimate of genome sizes that include heterochromatin or repetitive DNA content. Moreover, genome sequences typically represent only one strain or isolate of a single species that does not reflect intraspecies variation. To more accurately estimate whole-genome DNA content and compare these estimates to newly assembled genomes, we used flow cytometry to measure the 2C genome values, relative to Drosophila melanogaster. We estimated genome sizes for the 12 sequenced Drosophila species as well as 91 different strains of 38 species of Drosophilidae. Significant differences in intra- and interspecific 2C genome values exist within the Drosophilidae. Furthermore, by measuring polyploid 16C ovarian follicle cell underreplication we estimated the amount of satellite DNA in each of these species. We found a strong correlation between genome size and amount of satellite underreplication. Addition and loss of heterochromatin satellite repeat elements appear to have made major contributions to the large differences in genome size observed in the Drosophilidae.

Molecular evolution and population genetics of two Drosophila mettleri cytochrome P450 genes involved in host plant utilization.

Understanding the genetic basis of adaptation is one of the primary goals of evolutionary biology. The evolution of xenobiotic resistance in insects has proven to be an especially suitable arena for studying the genetics of adaptation, and resistant phenotypes are known to result from both coding and regulatory changes. In this study, we examine the evolutionary history and population genetics of two Drosophila mettleri cytochrome P450 genes that are putatively involved in the detoxification of alkaloids present in two of its cactus hosts: saguaro (Carnegiea gigantea) and senita (Lophocereus schottii). Previous studies demonstrated that Cyp28A1 was highly up-regulated following exposure to rotting senita tissue while Cyp4D10 was highly up-regulated following exposure to rotting saguaro tissue. Here, we show that a subset of sites in Cyp28A1 experienced adaptive evolution specifically in the D. mettleri lineage. Moreover, neutrality tests in several populations were also consistent with a history of selection on Cyp28A1. In contrast, we did not find evidence for positive selection on Cyp4D10, although this certainly does not preclude its involvement in host plant use. A surprising result that emerged from our population genetic analyses was the presence of significant genetic differentiation between flies collected from different host plant species (saguaro and senita) at Organ Pipe National Monument, Arizona, USA. This preliminary evidence suggests that D. mettleri may have evolved into distinctive host races that specialize on different hosts, a possibility that warrants further investigation.

Heritable endosymbionts of Drosophila.

Although heritable microorganisms are increasingly recognized as widespread in insects, no systematic screens for such symbionts have been conducted in Drosophila species (the primary insect genetic models for studies of evolution, development, and innate immunity). Previous efforts screened relatively few Drosophila lineages, mainly for Wolbachia. We conducted an extensive survey of potentially heritable endosymbionts from any bacterial lineage via PCR screens of mature ovaries in 181 recently collected fly strains representing 35 species from 11 species groups. Due to our fly sampling methods, however, we are likely to have missed fly strains infected with sex ratio-distorting endosymbionts. Only Wolbachia and Spiroplasma, both widespread in insects, were confirmed as symbionts. These findings indicate that in contrast to some other insect groups, other heritable symbionts are uncommon in Drosophila species, possibly reflecting a robust innate immune response that eliminates many bacteria. A more extensive survey targeted these two symbiont types through diagnostic PCR in 1225 strains representing 225 species from 32 species groups. Of these, 19 species were infected by Wolbachia while only 3 species had Spiroplasma. Several new strains of Wolbachia and Spiroplasma were discovered, including ones divergent from any reported to date. The phylogenetic distribution of Wolbachia and Spiroplasma in Drosophila is discussed.

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.