Rapid host switching of Wolbachia and even more rapid turnover of their phages and incompatibility-causing loci.

About half of all insect species carry maternally inherited Wolbachia alphaproteobacteria, making Wolbachia the most common endosymbionts known in nature. Often Wolbachia spread to high frequencies within populations due to cytoplasmic incompatibility (CI), a Wolbachia-induced sperm modification caused by prophage-associated genes (cifs) that kill embryos without Wolbachia. Several Wolbachia variants also block viruses, including wMel from Drosophila melanogaster when transinfected into the mosquito Aedes aegypti. CI enables the establishment and stable maintenance of pathogen-blocking wMel in natural Ae. aegypti populations. These transinfections are reducing dengue disease incidence on multiple continents. While it has long been known that closely related Wolbachia occupy distantly related hosts, the timing of Wolbachia host switching and molecular evolution has not been widely quantified. We provide a new, conservative calibration for Wolbachia chronograms based on examples of co-divergence of Wolbachia and their insect hosts. Synthesizing publicly available and new genomic data, we use our calibration to demonstrate that wMel-like variants separated by only about 370,000 years have naturally colonized holometabolous dipteran and hymenopteran insects that diverged approximately 350 million years ago. Data from Wolbachia variants closely related to those currently dominant in D. melanogaster and D. simulans illustrate that cifs are rapidly acquired and lost among Wolbachia genomes, on a time scale of 104-105 years. This turnover occurs with and without the Wovirus prophages that contain them, with closely related cifs found in distantly related phages and distantly related cifs found in closely related phages. We present evidence for purifying selection on CI rescue function and on particular Cif protein domains. Our results quantify the tempo and mode of rapid host switching and horizontal gene transfer that underlie the spread and diversity of Wolbachia sampled from diverse host species. The wMel variants we highlight from hosts in different climates may offer new options for broadening Wolbachia-based biocontrol of diseases and pests.

Why Wolbachia-induced cytoplasmic incompatibility is so common.

Cytoplasmic incompatibility (CI) is the most common reproductive manipulation produced by Wolbachia, obligately intracellular alphaproteobacteria that infect approximately half of all insect species. Once infection frequencies within host populations approach 10%, intense CI can drive Wolbachia to near fixation within 10 generations. However, natural selection among Wolbachia variants within individual host populations does not favor enhanced CI. Indeed, variants that do not cause CI but increase host fitness or are more reliably maternally transmitted are expected to spread if infected females remain protected from CI. Nevertheless, approximately half of analyzed Wolbachia infections cause detectable CI. Why? The frequency and persistence of CI are more plausibly explained by preferential spread to new host species (clade selection) rather than by natural selection among variants within host populations. CI-causing Wolbachia lineages preferentially spread into new host species because 1) CI increases equilibrium Wolbachia frequencies within host populations, and 2) CI-causing variants can remain at high frequencies within populations even when conditions change so that initially beneficial Wolbachia infections become harmful. An epidemiological model describing Wolbachia acquisition and loss by host species and the loss of CI-induction within Wolbachia lineages yields simple expressions for the incidence of Wolbachia infections and the fraction of those infections causing CI. Supporting a determinative role for differential interspecific spread in maintaining CI, many Wolbachia infections were recently acquired by their host species, many show evidence for contemporary spatial spread or retreat, and rapid evolution of CI-inducing loci, especially degradation, is common.

Why did the Wolbachia transinfection cross the road? drift, deterministic dynamics, and disease control.

Maternally inherited Wolbachia transinfections are being introduced into natural mosquito populations to reduce the transmission of dengue, Zika, and other arboviruses. Wolbachia-induced cytoplasmic incompatibility provides a frequency-dependent reproductive advantage to infected females that can spread transinfections within and among populations. However, because transinfections generally reduce host fitness, they tend to spread within populations only after their frequency exceeds a critical threshold. This produces bistability with stable equilibrium frequencies at both 0 and 1, analogous to the bistability produced by underdominance between alleles or karyotypes and by population dynamics under Allee effects. Here, we analyze how stochastic frequency variation produced by finite population size can facilitate the local spread of variants with bistable dynamics into areas where invasion is unexpected from deterministic models. Our exemplar is the establishment of wMel Wolbachia in the Aedes aegypti population of Pyramid Estates (PE), a small community in far north Queensland, Australia. In 2011, wMel was stably introduced into Gordonvale, separated from PE by barriers to A. aegypti dispersal. After nearly 6 years during which wMel was observed only at low frequencies in PE, corresponding to an apparent equilibrium between immigration and selection, wMel rose to fixation by 2018. Using analytic approximations and statistical analyses, we demonstrate that the observed fixation of wMel at PE is consistent with both stochastic transition past an unstable threshold frequency and deterministic transformation produced by steady immigration at a rate just above the threshold required for deterministic invasion. The indeterminacy results from a delicate balance of parameters needed to produce the delayed transition observed. Our analyses suggest that once Wolbachia transinfections are established locally through systematic introductions, stochastic "threshold crossing" is likely to only minimally enhance spatial spread, providing a local ratchet that slightly-but systematically-aids area-wide transformation of disease-vector populations in heterogeneous landscapes.

A phylogeny for the Drosophila montium species group: A model clade for comparative analyses.

The Drosophila montium species group is a clade of 94 named species, closely related to the model species D. melanogaster. The montium species group is distributed over a broad geographic range throughout Asia, Africa, and Australasia. Species of this group possess a wide range of morphologies, mating behaviors, and endosymbiont associations, making this clade useful for comparative analyses. We use genomic data from 42 available species to estimate the phylogeny and relative divergence times within the montium species group, and its relative divergence time from D. melanogaster. To assess the robustness of our phylogenetic inferences, we use 3 non-overlapping sets of 20 single-copy coding sequences and analyze all 60 genes with both Bayesian and maximum likelihood methods. Our analyses support monophyly of the group. Apart from the uncertain placement of a single species, D. baimaii, our analyses also support the monophyly of all seven subgroups proposed within the montium group. Our phylograms and relative chronograms provide a highly resolved species tree, with discordance restricted to estimates of relatively short branches deep in the tree. In contrast, age estimates for the montium crown group, relative to its divergence from D. melanogaster, depend critically on prior assumptions concerning variation in rates of molecular evolution across branches, and hence have not been reliably determined. We discuss methodological issues that limit phylogenetic resolution - even when complete genome sequences are available - as well as the utility of the current phylogeny for understanding the evolutionary and biogeographic history of this clade.

Quantitative methods for assessing local and bodywide contributions to Wolbachia titer in maternal germline cells of Drosophila.

BACKGROUND: Little is known about how bacterial endosymbionts colonize host tissues. Because many insect endosymbionts are maternally transmitted, egg colonization is critical for endosymbiont success. Wolbachia bacteria, carried by approximately half of all insect species, provide an excellent model for characterizing endosymbiont infection dynamics. To date, technical limitations have precluded stepwise analysis of germline colonization by Wolbachia. It is not clear to what extent titer-altering effects are primarily mediated by growth rates of Wolbachia within cell lineages or migration of Wolbachia between cells. RESULTS: The objective of this work is to inform mechanisms of germline colonization through use of optimized methodology. The approaches are framed in terms of nutritional impacts on Wolbachia. Yeast-rich diets in particular have been shown to suppress Wolbachia titer in the Drosophila melanogaster germline. To determine the extent of Wolbachia sensitivity to diet, we optimized 3-dimensional, multi-stage quantification of Wolbachia titer in maternal germline cells. Technical and statistical validation confirmed the identity of Wolbachia in vivo, the reproducibility of Wolbachia quantification and the statistical power to detect these effects. The data from adult feeding experiments demonstrated that germline Wolbachia titer is distinctly sensitive to yeast-rich host diets in late oogenesis. To investigate the physiological basis for these nutritional impacts, we optimized methodology for absolute Wolbachia quantification by real-time qPCR. We found that yeast-rich diets exerted no significant effect on bodywide Wolbachia titer, although ovarian titers were significantly reduced. This suggests that host diets affects Wolbachia distribution between the soma and late stage germline cells. Notably, relative qPCR methods distorted apparent wsp abundance, due to altered host DNA copy number in yeast-rich conditions. This highlights the importance of absolute quantification data for testing mechanistic hypotheses. CONCLUSIONS: We demonstrate that absolute quantification of Wolbachia, using well-controlled cytological and qPCR-based methods, creates new opportunities to determine how bacterial abundance within the germline relates to bacterial distribution within the body. This methodology can be applied to further test germline infection dynamics in response to chemical treatments, genetic conditions, new host/endosymbiont combinations, or potentially adapted to analyze other cell and tissue types.

Evolutionary Ecology of Wolbachia Releases for Disease Control.

Wolbachia is an endosymbiotic Alphaproteobacteria that can suppress insect-borne diseases through decreasing host virus transmission (population replacement) or through decreasing host population density (population suppression). We contrast natural Wolbachia infections in insect populations with Wolbachia transinfections in mosquitoes to gain insights into factors potentially affecting the long-term success of Wolbachia releases. Natural Wolbachia infections can spread rapidly, whereas the slow spread of transinfections is governed by deleterious effects on host fitness and demographic factors. Cytoplasmic incompatibility (CI) generated by Wolbachia is central to both population replacement and suppression programs, but CI in nature can be variable and evolve, as can Wolbachia fitness effects and virus blocking. Wolbachia spread is also influenced by environmental factors that decrease Wolbachia titer and reduce maternal Wolbachia transmission frequency. More information is needed on the interactions between Wolbachia and host nuclear/mitochondrial genomes, the interaction between invasion success and local ecological factors, and the long-term stability of Wolbachia-mediated virus blocking.

Wolbachia Acquisition by Drosophila yakuba-Clade Hosts and Transfer of Incompatibility Loci Between Distantly Related Wolbachia.

Maternally transmitted Wolbachia infect about half of insect species, yet the predominant mode(s) of Wolbachia acquisition remains uncertain. Species-specific associations could be old, with Wolbachia and hosts codiversifying (i.e., cladogenic acquisition), or relatively young and acquired by horizontal transfer or introgression. The three Drosophila yakuba-clade hosts [(D. santomea, D. yakuba) D. teissieri] diverged ∼3 MYA and currently hybridize on the West African islands Bioko and São Tomé. Each species is polymorphic for nearly identical Wolbachia that cause weak cytoplasmic incompatibility (CI)-reduced egg hatch when uninfected females mate with infected males. D. yakuba-clade Wolbachia are closely related to wMel, globally polymorphic in D. melanogaster We use draft Wolbachia and mitochondrial genomes to demonstrate that D. yakuba-clade phylogenies for Wolbachia and mitochondria tend to follow host nuclear phylogenies. However, roughly half of D. santomea individuals, sampled both inside and outside of the São Tomé hybrid zone, have introgressed D. yakuba mitochondria. Both mitochondria and Wolbachia possess far more recent common ancestors than the bulk of the host nuclear genomes, precluding cladogenic Wolbachia acquisition. General concordance of Wolbachia and mitochondrial phylogenies suggests that horizontal transmission is rare, but varying relative rates of molecular divergence complicate chronogram-based statistical tests. Loci that cause CI in wMel are disrupted in D. yakuba-clade Wolbachia; but a second set of loci predicted to cause CI are located in the same WO prophage region. These alternative CI loci seem to have been acquired horizontally from distantly related Wolbachia, with transfer mediated by flanking Wolbachia-specific ISWpi1 transposons.

Revisiting a Key Innovation in Evolutionary Biology: Felsenstein's "Phylogenies and the Comparative Method".

The comparative method has long been a fundamental exploratory tool in evolutionary biology, but this venerable approach was revolutionized in 1985, when Felsenstein published "Phylogenies and the Comparative Method" in The American Naturalist. This article forced comparative biologists to start thinking phylogenetically when conducting statistical analyses of correlated trait evolution rather than simply applying conventional statistical methods that ignore evolutionary relationships. It did so by introducing a novel analytical method (phylogenetically "independent contrasts") that required a phylogenetic topology with branch lengths and that assumed a Brownian motion model of trait evolution. Independent contrasts enabled comparative biologists to avoid the statistical dilemma of nonindependence of species values, arising from shared ancestry, but came at the cost of needing a detailed phylogeny and of accepting a specific model of character change. Nevertheless, this article not only revitalized comparative biology but even encouraged studies aimed at estimating phylogenies. Felsenstein's characteristically lucid and concise statement of the problem (illustrated with powerful graphics), coupled with an oncoming flood of new molecular data and techniques for estimating phylogenies, led Felsenstein's 1985 article to become the second most cited article in the history of this journal. Here we present a personal review of comparative biology before, during, and after Joe's article. For historical context, we append a perspective written by Joe himself that describes how his article evolved, unedited transcripts of reviews of his submitted manuscript, and a guide to some nontrivial calculations. These additional materials help emphasize that the process of science does not always occur gradually or predictably.

Loss of cytoplasmic incompatibility and minimal fecundity effects explain relatively low Wolbachia frequencies in Drosophila mauritiana.

Maternally transmitted Wolbachia bacteria infect about half of all insect species. Many Wolbachia cause cytoplasmic incompatibility (CI) and reduced egg hatch when uninfected females mate with infected males. Although CI produces a frequency-dependent fitness advantage that leads to high equilibrium Wolbachia frequencies, it does not aid Wolbachia spread from low frequencies. Indeed, the fitness advantages that produce initial Wolbachia spread and maintain non-CI Wolbachia remain elusive. wMau Wolbachia infecting Drosophila mauritiana do not cause CI, despite being very similar to CI-causing wNo from Drosophila simulans (0.068% sequence divergence over 682,494 bp), suggesting recent CI loss. Using draft wMau genomes, we identify a deletion in a CI-associated gene, consistent with theory predicting that selection within host lineages does not act to increase or maintain CI. In the laboratory, wMau shows near-perfect maternal transmission; but we find no significant effect on host fecundity, in contrast to published data. Intermediate wMau frequencies on the island of Mauritius are consistent with a balance between unidentified small, positive fitness effects and imperfect maternal transmission. Our phylogenomic analyses suggest that group-B Wolbachia, including wMau and wPip, diverged from group-A Wolbachia, such as wMel and wRi, 6-46 million years ago, more recently than previously estimated.

Rapid Global Spread of wRi-like Wolbachia across Multiple Drosophila.

Maternally transmitted Wolbachia, Spiroplasma, and Cardinium bacteria are common in insects [1], but their interspecific spread is poorly understood. Endosymbionts can spread rapidly within host species by manipulating host reproduction, as typified by the global spread of wRi Wolbachia observed in Drosophila simulans [2, 3]. However, because Wolbachia cannot survive outside host cells, spread between distantly related host species requires horizontal transfers that are presumably rare [4-7]. Here, we document spread of wRi-like Wolbachia among eight highly diverged Drosophila hosts (10-50 million years) over only about 14,000 years (5,000-27,000). Comparing 110 wRi-like genomes, we find ≤0.02% divergence from the wRi variant that spread rapidly through California populations of D. simulans. The hosts include both globally invasive species (D. simulans, D. suzukii, and D. ananassae) and narrowly distributed Australian endemics (D. anomalata and D. pandora) [8]. Phylogenetic analyses that include mtDNA genomes indicate introgressive transfer of wRi-like Wolbachia between closely related species D. ananassae, D. anomalata, and D. pandora but no horizontal transmission within species. Our analyses suggest D. ananassae as the Wolbachia source for the recent wRi invasion of D. simulans and D. suzukii as the source of Wolbachia in its sister species D. subpulchrella. Although six of these wRi-like variants cause strong cytoplasmic incompatibility, two cause no detectable reproductive effects, indicating that pervasive mutualistic effects [9, 10] complement the reproductive manipulations for which Wolbachia are best known. "Super spreader" variants like wRi may be particularly useful for controlling insect pests and vector-borne diseases with Wolbachia transinfections [11].

Genome comparisons indicate recent transfer of wRi-like Wolbachia between sister species Drosophila suzukii and D. subpulchrella.

Wolbachia endosymbionts may be acquired by horizontal transfer, by introgression through hybridization between closely related species, or by cladogenic retention during speciation. All three modes of acquisition have been demonstrated, but their relative frequency is largely unknown. Drosophila suzukii and its sister species D. subpulchrella harbor Wolbachia, denoted wSuz and wSpc, very closely related to wRi, identified in California populations of D. simulans. However, these variants differ in their induced phenotypes: wRi causes significant cytoplasmic incompatibility (CI) in D. simulans, but CI has not been detected in D. suzukii or D. subpulchrella. Our draft genomes of wSuz and wSpc contain full-length copies of 703 of the 734 single-copy genes found in wRi. Over these coding sequences, wSuz and wSpc differ by only 0.004% (i.e., 28 of 704,883 bp); they are sisters relative to wRi, from which each differs by 0.014%-0.015%. Using published data from D. melanogaster, Nasonia wasps and Nomada bees to calibrate relative rates of Wolbachia versus host nuclear divergence, we conclude that wSuz and wSpc are too similar-by at least a factor of 100-to be plausible candidates for cladogenic transmission. These three wRi-like Wolbachia, which differ in CI phenotype in their native hosts, have different numbers of orthologs of genes postulated to contribute to CI; and the CI loci differ at several nucleotides that may account for the CI difference. We discuss the general problem of distinguishing alternative modes of Wolbachia acquisition, focusing on the difficulties posed by limited knowledge of variation in absolute and relative rates of molecular evolution for host nuclear genomes, mitochondria, and Wolbachia.

Commentary: Fisher's infinitesimal model: A story for the ages.

Mendel (1866) suggested that if many heritable "factors" contribute to a trait, near-continuous variation could result. Fisher (1918) clarified the connection between Mendelian inheritance and continuous trait variation by assuming many loci, each with small effect, and by informally invoking the central limit theorem. Barton et al. (2017) rigorously analyze the approach to a multivariate Gaussian distribution of the genetic effects for descendants of parents who may be related. This commentary distinguishes three nested approximations, referred to as "infinitesimal genetics," "Gaussian descendants" and "Gaussian population," each plausibly called "the infinitesimal model." The first and most basic is Fisher's "infinitesimal" approximation of the underlying genetics - namely, many loci, each making a small contribution to the total variance. As Barton et al. (2017) show, in the limit as the number of loci increases (with enough additivity), the distribution of genotypic values for descendants approaches a multivariate Gaussian, whose variance-covariance structure depends only on the relatedness, not the phenotypes, of the parents (or whether their population experiences selection or other processes such as mutation and migration). Barton et al. (2017) call this rigorously defensible "Gaussian descendants" approximation "the infinitesimal model." However, it is widely assumed that Fisher's genetic assumptions yield another Gaussian approximation, in which the distribution of breeding values in a population follows a Gaussian - even if the population is subject to non-Gaussian selection. This third "Gaussian population" approximation, is also described as the "infinitesimal model." Unlike the "Gaussian descendants" approximation, this third approximation cannot be rigorously justified, except in a weak-selection limit, even for a purely additive model. Nevertheless, it underlies the two most widely used descriptions of selection-induced changes in trait means and genetic variances, the "breeder's equation" and the "Bulmer effect." Future generations may understand why the "infinitesimal model" provides such useful approximations in the face of epistasis, linkage, linkage disequilibrium and strong selection.

Local introduction and heterogeneous spatial spread of dengue-suppressing Wolbachia through an urban population of Aedes aegypti.

Dengue-suppressing Wolbachia strains are promising tools for arbovirus control, particularly as they have the potential to self-spread following local introductions. To test this, we followed the frequency of the transinfected Wolbachia strain wMel through Ae. aegypti in Cairns, Australia, following releases at 3 nonisolated locations within the city in early 2013. Spatial spread was analysed graphically using interpolation and by fitting a statistical model describing the position and width of the wave. For the larger 2 of the 3 releases (covering 0.97 km2 and 0.52 km2), we observed slow but steady spatial spread, at about 100-200 m per year, roughly consistent with theoretical predictions. In contrast, the smallest release (0.11 km2) produced erratic temporal and spatial dynamics, with little evidence of spread after 2 years. This is consistent with the prediction concerning fitness-decreasing Wolbachia transinfections that a minimum release area is needed to achieve stable local establishment and spread in continuous habitats. Our graphical and likelihood analyses produced broadly consistent estimates of wave speed and wave width. Spread at all sites was spatially heterogeneous, suggesting that environmental heterogeneity will affect large-scale Wolbachia transformations of urban mosquito populations. The persistence and spread of Wolbachia in release areas meeting minimum area requirements indicates the promise of successful large-scale population transformation.

Deploying dengue-suppressing Wolbachia : Robust models predict slow but effective spatial spread in Aedes aegypti.

A novel strategy for controlling the spread of arboviral diseases such as dengue, Zika and chikungunya is to transform mosquito populations with virus-suppressing Wolbachia. In general, Wolbachia transinfected into mosquitoes induce fitness costs through lower viability or fecundity. These maternally inherited bacteria also produce a frequency-dependent advantage for infected females by inducing cytoplasmic incompatibility (CI), which kills the embryos produced by uninfected females mated to infected males. These competing effects, a frequency-dependent advantage and frequency-independent costs, produce bistable Wolbachia frequency dynamics. Above a threshold frequency, denoted pˆ, CI drives fitness-decreasing Wolbachia transinfections through local populations; but below pˆ, infection frequencies tend to decline to zero. If pˆ is not too high, CI also drives spatial spread once infections become established over sufficiently large areas. We illustrate how simple models provide testable predictions concerning the spatial and temporal dynamics of Wolbachia introductions, focusing on rate of spatial spread, the shape of spreading waves, and the conditions for initiating spread from local introductions. First, we consider the robustness of diffusion-based predictions to incorporating two important features of wMel-Aedes aegypti biology that may be inconsistent with the diffusion approximations, namely fast local dynamics induced by complete CI (i.e., all embryos produced from incompatible crosses die) and long-tailed, non-Gaussian dispersal. With complete CI, our numerical analyses show that long-tailed dispersal changes wave-width predictions only slightly; but it can significantly reduce wave speed relative to the diffusion prediction; it also allows smaller local introductions to initiate spatial spread. Second, we use approximations for pˆ and dispersal distances to predict the outcome of 2013 releases of wMel-infected Aedes aegypti in Cairns, Australia, Third, we describe new data from Ae. aegypti populations near Cairns, Australia that demonstrate long-distance dispersal and provide an approximate lower bound on pˆ for wMel in northeastern Australia. Finally, we apply our analyses to produce operational guidelines for efficient transformation of vector populations over large areas. We demonstrate that even very slow spatial spread, on the order of 10-20 m/month (as predicted), can produce area-wide population transformation within a few years following initial releases covering about 20-30% of the target area.

Wolbachia in the Drosophila yakuba Complex: Pervasive Frequency Variation and Weak Cytoplasmic Incompatibility, but No Apparent Effect on Reproductive Isolation.

Three hybridizing species-the clade [(Drosophila yakuba, D. santomea), D. teissieri]-comprise the yakuba complex in the D. melanogaster subgroup. Their ranges overlap on Bioko and São Tomé, islands off west Africa. All three species are infected with Wolbachia-maternally inherited, endosymbiotic bacteria, best known for manipulating host reproduction to favor infected females. Previous analyses reported no cytoplasmic incompatibility (CI) in these species. However, we discovered that Wolbachia from each species cause intraspecific and interspecific CI. In D teissieri, analyses of F1 and backcross genotypes show that both host genotype and Wolbachia variation modulate CI intensity. Wolbachia-infected females seem largely protected from intraspecific and interspecific CI, irrespective of Wolbachia and host genotypes. Wolbachia do not affect host mating behavior or female fecundity, within or between species. The latter suggests little apparent effect of Wolbachia on premating or gametic reproductive isolation (RI) between host species. In nature, Wolbachia frequencies varied spatially for D. yakuba in 2009, with 76% (N = 155) infected on São Tomé, and only 3% (N = 36) infected on Bioko; frequencies also varied temporally in D. yakuba and D. santomea on São Tomé between 2009 and 2015. These temporal frequency fluctuations could generate asymmetries in interspecific mating success, and contribute to postzygotic RI. However, the fluctuations in Wolbachia frequencies that we observe also suggest that asymmetries are unlikely to persist. Finally, we address theoretical questions that our empirical findings raise about Wolbachia persistence when conditions fluctuate, and about the stable coexistence of Wolbachia and host variants that modulate Wolbachia effects.

Persistence of a Wolbachia infection frequency cline in Drosophila melanogaster and the possible role of reproductive dormancy.

Field populations of arthropods are often polymorphic for Wolbachia but the factors maintaining intermediate Wolbachia frequencies are generally not understood. In Drosophila melanogaster, Wolbachia frequencies are highly variable across the globe. We document the persistence of a Wolbachia infection frequency cline in D. melanogaster populations from eastern Australia across at least 20 years, with frequencies generally high in the tropics but lower in cool temperate regions. The results are interpreted using a model of frequency dynamics incorporating cytoplasmic incompatibility (CI), imperfect maternal transmission and Wolbachia effects on fitness. Clinal variation is less pronounced in eastern North America which may reflect annual recolonization at higher latitudes. Limited samples from Africa from latitudes matching our tropical and subtropical samples from Australia and North America show comparably high infection frequencies, but some equatorial samples show lower frequencies. Adult dormancy across cold periods may contribute to the Australian Wolbachia cline. Infected flies exposed to cold conditions for an extended period had reduced fecundity and viability, an effect not evident in unexposed controls. These fitness costs may contribute to the relatively low Wolbachia frequencies in Australian temperate areas; whereas different processes, including CI induced by young males, may contribute to higher frequencies in tropical locations.

Strange little flies in the big city: exotic flower-breeding drosophilidae (Diptera) in urban Los Angeles.

Urban landscapes are commonly considered too mundane and corrupted to be biotically interesting. Recent insect surveys employing 29 Malaise traps throughout Los Angeles, California, however, have uncovered breeding populations of two unexpected species of one of the most studied and familiar groups of organisms, Drosophila "fruit" flies. Unlike most introduced species of drosophilids, which breed in fresh or decaying fruits, these are specialized flower-breeders. A common species in the survey was Drosophila (Drosophila) gentica Wheeler and Takada, previously collected only once, in El Salvador. It belongs to the flavopilosa species group, all species of which have been known until now from central Chile, Argentina and Uruguay, to Veracruz, Mexico and the Caribbean, breeding in flowers of Cestrum ("jessamine") and Sessea (Solanaceae). The Los Angeles populations are probably breeding in a native and/or introduced Cestrum; in addition, populations in San Luis Obispo County were visiting ornamental Cestrum. Drosophila gentica occurs as far north as San Francisco, where it was found breeding in Cestrum aurantiacum. D. gentica is redescribed and figured in detail for diagnostic and identification purposes. Specimens from Jamaica previously identified as D. gentica are a distinct species but are not formally described in lieu of complete male specimens. Rare in the Malaise traps was Drosophila (Sophophora) flavohirta Malloch, a common species in Australia on the blossoms of native Myrtaceae, found on introduced Eucalyptus in South Africa and both Eucalyptus and Syzygium in Madagascar; adults feed on myrtaceous pollen and nectar, larvae breed in the flowers. It is also redescribed in detail, including its unusual egg. This is the first New World report of this species; DNA sequences confirm it is a morphologically highly aberrant member of the D. melanogaster species group. This study reveals how intensive field sampling can uncover remarkable biodiversity in even the most urbanized areas.

Wolbachia do not live by reproductive manipulation alone: infection polymorphism in Drosophila suzukii and D. subpulchrella.

Drosophila suzukii recently invaded North America and Europe. Populations in Hawaii, California, New York and Nova Scotia are polymorphic for Wolbachia, typically with <20% infection frequency. The Wolbachia in D. suzukii, denoted wSuz, is closely related to wRi, the variant prevalent in continental populations of D. simulans. wSuz is also nearly identical to Wolbachia found in D. subpulchrella, plausibly D. suzukii's sister species. This suggests vertical Wolbachia transmission through cladogenesis ('cladogenic transmission'). The widespread occurrence of 7-20% infection frequencies indicates a stable polymorphism. wSuz is imperfectly maternally transmitted, with wild infected females producing on average 5-10% uninfected progeny. As expected from its low frequency, wSuz produces no cytoplasmic incompatibility (CI), that is, no increased embryo mortality when infected males mate with uninfected females, and no appreciable sex-ratio distortion. The persistence of wSuz despite imperfect maternal transmission suggests positive fitness effects. Assuming a balance between selection and imperfect transmission, we expect a fitness advantage on the order of 20%. Unexpectedly, Wolbachia-infected females produce fewer progeny than do uninfected females. We do not yet understand the maintenance of wSuz in D. suzukii. The absence of detectable CI in D. suzukii and D. subpulchrella makes it unlikely that CI-based mechanisms could be used to control this species without transinfection using novel Wolbachia. Contrary to their reputation as horizontally transmitted reproductive parasites, many Wolbachia infections are acquired through introgression or cladogenesis and many cause no appreciable reproductive manipulation. Such infections, likely to be mutualistic, may be central to understanding the pervasiveness of Wolbachia among arthropods.