Complexities of Inferring Symbiont Function: Paraburkholderia Symbiont Dynamics in Social Amoeba Populations and Their Impacts on the Amoeba Microbiota.

The relationship between the social amoeba Dictyostelium discoideum and its endosymbiotic bacteria Paraburkholderia provides a model system for studying the development of symbiotic relationships. Laboratory experiments have shown that any of three species of the Paraburkholderia symbiont allow D. discoideum food bacteria to persist through the amoeba life cycle and survive in amoeba spores rather than being fully digested. This phenomenon is termed "farming," as it potentially allows spores dispersed to food-poor locations to grow their own. The occurrence and impact of farming in natural populations, however, have been a challenge to measure. Here, we surveyed natural D. discoideum populations and found that only one of the three symbiont species, Paraburkholderia agricolaris, remained prevalent. We then explored the effect of Paraburkholderia on the amoeba microbiota, expecting that by facilitating bacterial food carriage, it would diversify the microbiota. Contrary to our expectations, Paraburkholderia tended to infectiously dominate the D. discoideum microbiota, in some cases decreasing diversity. Similarly, we found little evidence for Paraburkholderia facilitating the carriage of particular food bacteria. These findings highlight the complexities of inferring symbiont function in nature and suggest the possibility that Paraburkholderia could be playing multiple roles for its host. IMPORTANCE The functions of symbionts in natural populations can be difficult to completely discern. The three Paraburkholderia bacterial farming symbionts of the social amoeba Dictyostelium discoideum have been shown in the laboratory environment to allow the amoebas to carry, rather than fully digest, food bacteria. This potentially provides a fitness benefit to the amoebas upon dispersal to food-poor environments, as they could grow their food. We expected that meaningful food carriage would manifest as a more diverse microbiota. Surprisingly, we found that Paraburkholderia tended to infectiously dominate the D. discoideum microbiota rather than diversifying it. We determined that only one of the three Paraburkholderia symbionts has increased in prevalence in natural populations in the past 20 years, suggesting that this symbiont may be beneficial, however. These findings suggest that Paraburkholderia may have an alternative function for its host, which drives its prevalence in natural populations.

Characterization of the bacterial microbiomes of social amoebae and exploration of the roles of host and environment on microbiome composition.

As predators of bacteria, amoebae select for traits that allow bacteria to become symbionts by surviving phagocytosis and exploiting the eukaryotic intracellular environment. Soil-dwelling social amoebae can help us answer questions about the natural ecology of these amoeba-bacteria symbioses along the pathogen-mutualist spectrum. Our objective was to characterize the natural bacterial microbiome of phylogenetically and morphologically diverse social amoeba species using next-generation sequencing of 16S rRNA amplicons directly from amoeba fruiting bodies. We found six phyla of amoeba-associated bacteria: Proteobacteria, Bacteroidetes, Actinobacteria, Chlamydiae, Firmicutes, and Acidobacteria. The most common associates of amoebae were classified to order Chlamydiales and genus Burkholderia-Caballeronia-Paraburkholderia. These bacteria were present in multiple amoeba species across multiple locations. While there was substantial intraspecific variation, there was some evidence for host specificity and differentially abundant taxa between different amoeba hosts. Amoebae microbiomes were distinct from the microbiomes of their soil habitat, and soil pH affected amoeba microbiome diversity. Alpha-diversity was unsurprisingly lower in amoebae samples compared with soil, but beta-diversity between amoebae samples was higher than between soil samples. Further exploration of social amoebae microbiomes may help us understand the roles of bacteria, host, and environment on symbiotic interactions and microbiome formation in basal eukaryotic organisms.

The specificity of Burkholderia symbionts in the social amoeba farming symbiosis: Prevalence, species, genetic and phenotypic diversity.

The establishment of symbioses between eukaryotic hosts and bacterial symbionts in nature is a dynamic process. The formation of such relationships depends on the life history of both partners. Bacterial symbionts of amoebae may have unique evolutionary trajectories to the symbiont lifestyle, because bacteria are typically ingested as prey. To persist after ingestion, bacteria must first survive phagocytosis. In the social amoeba Dictyostelium discoideum, certain strains of Burkholderia bacteria are able to resist amoebal digestion and maintain a persistent relationship that includes carriage throughout the amoeba's social cycle that culminates in spore formation. Some Burkholderia strains allow their host to carry other bacteria, as food. This carried food is released in new environments in a trait called farming. To better understand the diversity and prevalence of Burkholderia symbionts and the traits they impart to their amoebae hosts, we first screened 700 natural isolates of D. discoideum and found 25% infected with Burkholderia. We next used a multilocus phylogenetic analysis and identified two independent transitions by Burkholderia to the symbiotic lifestyle. Finally, we tested the ability of 38 strains of Burkholderia from D. discoideum, as well as strains isolated from other sources, for traits relevant to symbiosis in D. discoideum. Only D. discoideum native isolates belonging to the Burkholderia agricolaris, B. hayleyella, and B. bonniea species were able to form persistent symbiotic associations with D. discoideum. The Burkholderia-Dictyostelium relationship provides a promising arena for further studies of the pathway to symbiosis in a unique system.

Burkholderia bacteria infectiously induce the proto-farming symbiosis of Dictyostelium amoebae and food bacteria.

Symbiotic associations can allow an organism to acquire novel traits by accessing the genetic repertoire of its partner. In the Dictyostelium discoideum farming symbiosis, certain amoebas (termed "farmers") stably associate with bacterial partners. Farmers can suffer a reproductive cost but also gain beneficial capabilities, such as carriage of bacterial food (proto-farming) and defense against competitors. Farming status previously has been attributed to amoeba genotype, but the role of bacterial partners in its induction has not been examined. Here, we explore the role of bacterial associates in the initiation, maintenance, and phenotypic effects of the farming symbiosis. We demonstrate that two clades of farmer-associated Burkholderia isolates colonize D. discoideum nonfarmers and infectiously endow them with farmer-like characteristics, indicating that Burkholderia symbionts are a major driver of the farming phenomenon. Under food-rich conditions, Burkholderia-colonized amoebas produce fewer spores than uncolonized counterparts, with the severity of this reduction being dependent on the Burkholderia colonizer. However, the induction of food carriage by Burkholderia colonization may be considered a conditionally adaptive trait because it can confer an advantage to the amoeba host when grown in food-limiting conditions. We observed Burkholderia inside and outside colonized D. discoideum spores after fruiting body formation; this observation, together with the ability of Burkholderia to colonize new amoebas, suggests a mixed mode of symbiont transmission. These results change our understanding of the D. discoideum farming symbiosis by establishing that the bacterial partner, Burkholderia, is an important causative agent of the farming phenomenon.

Macroevolutionary persistence of heritable endosymbionts: acquisition, retention and expression of adaptive phenotypes in Spiroplasma.

The phylogenetic incongruence between insects and their facultative maternally transmitted endosymbionts indicates that these infections are generally short-lived evolutionarily. Therefore, long-term persistence of many endosymbionts must depend on their ability to colonize and spread within new host species. At least 17 species of Drosophila are infected with endosymbiotic Spiroplasma that have various phenotypic effects. We transinfected five strains of Spiroplasma from three divergent clades into Drosophila neotestacea to test their capacity to spread in a novel host. A strain that causes male killing in Drosophila melanogaster (its native host) also does so in D. neotestacea, even though these host species diverged 40-60 mya. A strain native to D. neotestacea (designated sNeo) and the two other strains of the poulsonii clade of Spiroplasma confer resistance to wasp parasitism, suggesting that this trait may be ancestral within this clade of Spiroplasma. Conversely, no strain other than sNeo conferred resistance to the sterilizing effects of nematode parasitism, suggesting that nematode resistance is a recently derived condition. The apparent addition of nematode resistance to a Spiroplasma lineage that already confers resistance to wasp parasitism suggests endosymbionts can increase the repertoire of traits conducive to their spread. The capacity of an endosymbiont to undergo maternal transmission and express adaptive phenotypes in novel hosts, without requiring a period of host-symbiont co-evolution, enables the spread of such symbionts immediately after the colonization of a new host. This could be critical for the macroevolutionary persistence of facultative endosymbionts whose sojourn times within individual host species are relatively brief.

Dynamics of the continent-wide spread of a Drosophila defensive symbiont.

Facultative symbionts can represent important sources of adaptation for their insect hosts and thus have the potential for rapid spread. Drosophila neotestacea harbours a heritable symbiont, Spiroplasma, that confers protection against parasitic nematodes. We previously found a cline in Spiroplasma prevalence across central Canada, ending abruptly at the Rocky Mountains. Resampling these populations 9 years later revealed that Spiroplasma had increased substantially across the region, resembling a Fisherian wave of advance. Associations between Spiroplasma infection and host mitochondrial DNA indicate that the increase was due to local increase of Spiroplasma-infected flies. Finally, we detected Spiroplasma west of the Rocky Mountains for the first time and showed that defence against nematodes occurs in flies with a western genetic background. Because nematode infection is common throughout D. neotestacea's range, we expect Spiroplasma to spread to the Pacific coast.

Cryptic Onchocerca species infecting North American cervids, with implications for the evolutionary history of host associations in Onchocerca.

Parasites in the genus Onchocerca infect humans, ruminants, camels, horses, suids, and canids, with effects ranging from relatively benign to debilitating. In North America, Onchocerca cervipedis is the sole species known to infect cervids, while at least 5 Onchocerca species infect Eurasian cervids. In this study, we report the discovery of a cervid-parasitizing Onchocerca only distantly related to O. cervipedis. To reconstruct the phylogenetic history of the genus Onchocerca, we used newly acquired DNA sequence from O. cervipedis (from moose in Northwest Territories, Canada) and from the newly discovered species (from white-tailed deer in upstate New York), as well as previously published sequences. Ancestral host reconstructions suggest that host switches have been common throughout the evolutionary history of Onchocerca, and that bovid- and cervid-parasitizing species have been particularly important sources of descendant species. North America cervids might therefore serve as a source for Onchocerca invasions into new hosts. Given the high density of deer populations, the potential for zoonotic infections may also exist. Our discovery of a new Onchocerca species with relatively limited sampling suggests that the diversity of Onchocerca associated with cervids in North America may be greater than previously thought, and surveys utilizing molecules and morphology are necessary.

Novel Chlamydiae and Amoebophilus endosymbionts are prevalent in wild isolates of the model social amoeba Dictyostelium discoideum.

Amoebae interact with bacteria in multifaceted ways. Amoeba predation can serve as a selective pressure for the development of bacterial virulence traits. Bacteria may also adapt to life inside amoebae, resulting in symbiotic relationships. Indeed, particular lineages of obligate bacterial endosymbionts have been found in different amoebae. Here, we screened an extensive collection of Dictyostelium discoideum wild isolates for the presence of these bacterial symbionts using endosymbiont specific PCR primers. We find that these symbionts are surprisingly common, identified in 42% of screened isolates (N = 730). Members of the Chlamydiae phylum are particularly prevalent, occurring in 27% of the amoeba isolated. They are novel and phylogenetically distinct from other Chlamydiae. We also found Amoebophilus symbionts in 8% of screened isolates (N = 730). Antibiotic-cured amoebae behave similarly to their Chlamydiae or Amoebophilus-infected counterparts, suggesting that these endosymbionts do not significantly impact host fitness, at least in the laboratory. We found several natural isolates were co-infected with multiple endosymbionts, with no obvious fitness effect of co-infection under laboratory conditions. The high prevalence and novelty of amoeba endosymbiont clades in the model organism D. discoideum opens the door to future research on the significance and mechanisms of amoeba-symbiont interactions.

Endosymbiotic adaptations in three new bacterial species associated with Dictyostelium discoideum: Paraburkholderia agricolaris sp. nov., Paraburkholderia hayleyella sp. nov., and Paraburkholderia bonniea sp. nov.

Here we give names to three new species of Paraburkholderia that can remain in symbiosis indefinitely in the spores of a soil dwelling eukaryote, Dictyostelium discoideum. The new species P. agricolaris sp. nov., P. hayleyella sp. nov., and P. bonniea sp. nov. are widespread across the eastern USA and were isolated as internal symbionts of wild-collected D. discoideum. We describe these sp. nov. using several approaches. Evidence that they are each a distinct new species comes from their phylogenetic position, average nucleotide identity, genome-genome distance, carbon usage, reduced length, cooler optimal growth temperature, metabolic tests, and their previously described ability to invade D. discoideum amoebae and form a symbiotic relationship. All three of these new species facilitate the prolonged carriage of food bacteria by D. discoideum, though they themselves are not food. Further studies of the interactions of these three new species with D. discoideum should be fruitful for understanding the ecology and evolution of symbioses.

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.

Intracellular Burkholderia Symbionts induce extracellular secondary infections; driving diverse host outcomes that vary by genotype and environment.

Symbiotic associations impact and are impacted by their surrounding ecosystem. The association between Burkholderia bacteria and the soil amoeba Dictyostelium discoideum is a tractable model to unravel the biology underlying symbiont-endowed phenotypes and their impacts. Several Burkholderia species stably associate with D. discoideum and typically reduce host fitness in food-rich environments while increasing fitness in food-scarce environments. Burkholderia symbionts are themselves inedible to their hosts but induce co-infections with secondary bacteria that can serve as a food source. Thus, Burkholderia hosts are "farmers" that carry food bacteria to new environments, providing a benefit when food is scarce. We examined the ability of specific Burkholderia genotypes to induce secondary co-infections and assessed host fitness under a range of co-infection conditions and environmental contexts. Although all Burkholderia symbionts intracellularly infected Dictyostelium, we found that co-infections are predominantly extracellular, suggesting that farming benefits are derived from extracellular infection of host structures. Furthermore, levels of secondary infection are linked to conditional host fitness; B. agricolaris infected hosts have the highest level of co-infection and have the highest fitness in food-scarce environments. This study illuminates the phenomenon of co-infection induction across Dictyostelium associated Burkholderia species and exemplifies the contextual complexity of these associations.

Evaluation of the genomic extent of effects of fixed inversion differences on intraspecific variation and interspecific gene flow in Drosophila pseudoobscura and D. persimilis.

There is increasing evidence that chromosomal inversions may facilitate the formation or persistence of new species by allowing genetic factors conferring species-specific adaptations or reproductive isolation to be inherited together and by reducing or eliminating introgression. However, the genomic domain of influence of the inverted regions on introgression has not been carefully studied. Here, we present a detailed study on the consequences that distance from inversion breakpoints has had on the inferred level of gene flow and divergence between Drosophila pseudoobscura and D. persimilis. We identified the locations of the inversion breakpoints distinguishing D. pseudoobscura and D. persimilis in chromosomes 2, XR, and XL. Population genetic data were collected at specific distances from the inversion breakpoints of the second chromosome and at two loci inside the XR and XL inverted regions. For loci outside the inverted regions, we found that distance from the nearest inversion breakpoint had a significant effect on several measures of divergence and gene flow between D. pseudoobscura and D. persimilis. The data fitted a logarithmic relationship, showing that the suppression of crossovers in inversion heterozygotes also extends to loci located outside the inversion but close to it (within 1-2 Mb). Further, we detected a significant reduction in nucleotide variation inside the inverted second chromosome region of D. persimilis and near one breakpoint, consistent with a scenario in which this inversion arose and was fixed in this species by natural selection.

Diversity of Free-Living Environmental Bacteria and Their Interactions With a Bactivorous Amoeba.

A small subset of bacteria in soil interact directly with eukaryotes. Which ones do so can reveal what is important to a eukaryote and how eukaryote defenses might be breached. Soil amoebae are simple eukaryotic organisms and as such could be particularly good for understanding how eukaryote microbiomes originate and are maintained. One such amoeba, Dictyostelium discoideum, has both permanent and temporary associations with bacteria. Here we focus on culturable bacterial associates in order to interrogate their relationship with D. discoideum. To do this, we isolated over 250 D. discoideum fruiting body samples from soil and deer feces at Mountain Lake Biological Station. In one-third of the wild D. discoideum we tested, one to six bacterial species were found per fruiting body sorus (spore mass) for a total of 174 bacterial isolates. The remaining two-thirds of D. discoideum fruiting body samples did not contain culturable bacteria, as is thought to be the norm. A majority (71.4%) of the unique bacterial haplotypes are in Proteobacteria. The rest are in either Actinobacteria, Bacteriodetes, or Firmicutes. The highest bacterial diversity was found in D. discoideum fruiting bodies originating from deer feces (27 OTUs), greater than either of those originating in shallow (11 OTUs) or in deep soil (4 OTUs). Rarefaction curves and the Chao1 estimator for species richness indicated the diversity in any substrate was not fully sampled, but for soil it came close. A majority of the D. discoideum-associated bacteria were edible by D. discoideum and supported its growth (75.2% for feces and 81.8% for soil habitats). However, we found several bacteria genera were able to evade phagocytosis and persist in D. discoideum cells through one or more social cycles. This study focuses not on the entire D. discoideum microbiome, but on the culturable subset of bacteria that have important eukaryote interactions as prey, symbionts, or pathogens. These eukaryote and bacteria interactions may provide fertile ground for investigations of bacteria using amoebas to gain an initial foothold in eukaryotes and of the origins of symbiosis and simple microbiomes.

Functional equivalence of morphologies enables morphological and ecological diversity.

Diversity in organismal forms among taxa is thought to reflect distinct selection pressures across environments. The central assumption underlying this expectation is that taxa experiencing similar selection have similar response to that selection. However, because selection acts on trait function, taxa similarity in selection response depends crucially on the relationship between function and morphology. Further, when a trait consists of multiple parts, changes in function in response to selection can result from modification of different parts, and adaptation to the same environment might result in functional but not morphological similarity. Here, we address the extent to which functional and morphological diversity in masticatory apparatus of soricid shrews reflects a shared ecological characteristic of their diet type. We examine the factors limiting morphological variation across shrew species by assessing the relative contribution of trait function (biomechanics of the jaw), ecology, and phylogeny to species similarity in mandibular traits. We found that species that shared diet type were functionally but not morphologically similar. The presence of multiple semi-independently varying traits enabled functional equivalence of composite foraging morphologies and resulted in variable response to selection exerted by similar diet. We show that functional equivalence of multiple morphologies enabled persistence of differences in habitat use (e.g., habitat moisture and coverage) among species that specialize on the same diet. We discuss the importance of developmental and functional integration among traits for evolutionary diversification of morphological structures that generate equivalent functions.

Age-specific metabolic rates and mortality rates in the genus Drosophila.

Early theories of aging suggested that organisms with relatively high metabolic rates would live shorter lives. Despite widespread tests of this 'rate of living' theory of aging, there is little empirical evidence to support the idea. A more fine-grained approach that examined age-related changes in metabolic rate over the life span could provide valuable insight into the relationship between metabolic rate and aging. Here we compare age-related metabolic rate (measured as CO2 production per hour) and age-related mortality rate among five species in the genus Drosophila. We find no evidence that longer-lived species have lower metabolic rates. In all five species, there is no clear evidence of an age-related metabolic decline. Metabolic rates are strikingly constant throughout the life course, with the exception of females of D. hydei, in which metabolic rates show an increase over the first third of the life span and then decline. We argue that some physiological traits may have been shaped by such strong selection over evolutionary time that they are relatively resistant to the decline in the force of selection that occurs within the life time of a single individual. We suggest that comparisons of specific traits that do not show signs of aging with those traits that do decline with age could provide insight into the aging process.

Density dynamics of diverse Spiroplasma strains naturally infecting different species of Drosophila.

Facultative heritable bacterial endosymbionts can have dramatic effects on their hosts, ranging from mutualistic to parasitic. Within-host bacterial endosymbiont density plays a critical role in maintenance of a symbiotic relationship, as it can affect levels of vertical transmission and expression of phenotypic effects, both of which influence the infection prevalence in host populations. Species of genus Drosophila are infected with Spiroplasma, whose characterized phenotypic effects range from that of a male-killing reproductive parasite to beneficial defensive endosymbiont. For many strains of Spiroplasma infecting at least 17 species of Drosophila, however, the phenotypic effects are obscure. The infection prevalence of these Spiroplasma vary within and among Drosophila species, and little is known about the within-host density dynamics of these diverse strains. To characterize the patterns of Spiroplasma density variation among Drosophila we used quantitative PCR to assess bacterial titer at various life stages of three species of Drosophila naturally-infected with two different types of Spiroplasma. For naturally infected Drosophila species we found that non-male-killing infections had consistently lower densities than the male-killing infection. The patterns of Spiroplasma titer change during aging varied among Drosophila species infected with different Spiroplasma strains. Bacterial density varied within and among populations of Drosophila, with individuals from the population with the highest prevalence of infection having the highest density. This density variation underscores the complex interaction of Spiroplasma strain and host genetic background in determining endosymbiont density.

Infectious adaptation: potential host range of a defensive endosymbiont in Drosophila.

Maternally transmitted symbionts persist over macroevolutionary timescales by undergoing occasional lateral transfer to new host species. To invade a new species, a symbiont must survive and reproduce in the new host, undergo maternal transmission, and confer a selective benefit sufficient to overcome losses due to imperfect maternal transmission. Drosophila neotestacea is naturally infected with a strain of Spiroplasma that restores fertility to nematode-parasitized females, which are otherwise sterilized by parasitism. We experimentally transferred Spiroplasma from D. neotestacea to four other species of mycophagous Drosophila that vary in their ability to resist and/or tolerate nematode parasitism. In all four species, Spiroplasma achieved within-host densities and experienced rates of maternal transmission similar to that in D. neotestacea. Spiroplasma restored fertility to nematode-parasitized females in one of these novel host species. Based on estimates of maternal transmission fidelity and the expected benefit of Spiroplasma infection in the wild, we conclude that Spiroplasma has the potential to spread and become abundant within Drosophila putrida, which is broadly sympatric with D. neotestacea and in which females are rendered completely sterile by nematode parasitism. Thus, a major adaptation within D. putrida could arise via lateral transmission of a heritable symbiont from D. neotestacea.

The Spiroplasma heritable bacterial endosymbiont of Drosophila.

Since the discovery of the small, gram-positive bacteria, Spiroplasma, as a sex-ratio distorting agent in Drosophila over 50 years ago, substantial progress has been made in understanding the relationship of this bacteria with its insect host. Thus far, Spiroplasma have been found as heritable endosymbionts in sixteen different species of Drosophila. In some species these bacteria cause a male-killing phenotype, where the males die during embryogenesis. In other species, however, Spiroplasma does not cause male-killing, and its fitness effects are unclear. Though recent research has identified multiple factors that affect the prevalence and transmission of Spiroplasma in Drosophila populations, much work remains to fully characterize this symbiosis. Spiroplasma is the only identified heritable bacterial endosymbiont of Drosophila, other than Wolbachia, and can serve as a useful as model for elucidating the nature of insect/bacterial interactions.

Variable incidence of Spiroplasma infections in natural populations of Drosophila species.

Spiroplasma is widespread as a heritable bacterial symbiont in insects and some other invertebrates, in which it sometimes acts as a male-killer and causes female-biased sex ratios in hosts. Besides Wolbachia, it is the only heritable bacterium known from Drosophila, having been found in 16 of over 200 Drosophila species screened, based on samples of one or few individuals per species. To assess the extent to which Spiroplasma infection varies within and among species of Drosophila, intensive sampling consisting of 50-281 individuals per species was conducted for natural populations of 19 Drosophila species. Infection rates varied among species and among populations of the same species, and 12 of 19 species tested negative for all individuals. Spiroplasma infection never was fixed, and the highest infection rates were 60% in certain populations of D. hydei and 85% in certain populations of D. mojavensis. In infected species, infection rates were similar for males and females, indicating that these Spiroplasma infections do not confer a strong male-killing effect. These findings suggest that Spiroplasma has other effects on hosts that allow it to persist, and that environmental or host variation affects transmission or persistence leading to differences among populations in infection frequencies.

Molecular phylogeography of common garter snakes (Thamnophis sirtalis) in western North America: implications for regional historical forces.

Complete ND2 and partial ND4 and cytochrome b mitochondrial DNA (mtDNA) sequences were analysed to evaluate the phylogeographic patterns of common garter snakes (Thamnophis sirtalis) in western North America. This species is widely distributed throughout North America, and exhibits extensive phenotypic variation in the westernmost part of its range. The overall phylogeographic pattern based on mtDNA sequences is concordant with results from studies of other species in this region, implicating historical vicariant processes during the Pleistocene and indicating bottleneck effects of recent dispersal into postglacial habitat. Indeed, the topology is statistically consistent with the hypothesis of both southern (Great Basin and California) and northern (Haida Gwaii) refugia. Specifically, we identified genetic breaks among three major clades: Northwest Coastal populations, Intermountain populations, and all California populations. The California clade contained the only other well-supported branching patterns detected; relationships among populations within the two northern clades were indistinguishable. These molecular splits contrast sharply with all prior geographical analyses of phenotypic variation in T. sirtalis in this region. Our results suggest that the extensive phenotypic variation in western T. sirtalis has been shaped more by local evolutionary forces than by shared common ancestry. Consequently, we consider all morphologically based subspecies designations of T. sirtalis in this region invalid because they do not reflect reciprocal monophyly of the mtDNA sequences.