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Bacteriophages are vastly abundant, diverse, and influential, but with few exceptions (e.g. the Proteobacteria generaWolbachiaandHamiltonella), the role of phages in heritable bacteria-arthropod interactions, which are ubiquitous and diverse, remains largely unexplored. Despite prior studies documenting phage-like particles in the mollicuteSpiroplasmaassociated withDrosophilaflies, genomic sequences of such phage are lacking, and their effects on theSpiroplasma-Drosophilainteraction have not been comprehensively characterized. We used a density step gradient to isolate phage-like particles from the male-killing bacteriumSpiroplasma poulsonii(strains NSRO and MSRO-Br) harbored byDrosophila melanogaster. Isolated particles were subjected to DNA sequencing, assembly, and annotation. Several lines of evidence suggest that we recovered phage-like particles of similar features (shape, size, DNA content) to those previously reported inDrosophila-associatedSpiroplasmastrains. We recovered three ∼19 kb phage-like contigs (two in NSRO and one in MSRO-Br) containing 21–24 open reading frames, a read-alignment pattern consistent with circular permutation, and terminal redundancy (at least in NSRO). Although our results do not allow us to distinguish whether these phage-like contigs represent infective phage-like particles capable of transmitting their DNA to new hosts, their encoding of several typical phage genes suggests that they are at least remnants of functional phage. We also recovered two smaller non-phage-like contigs encoding a knownSpiroplasmatoxin (Ribosome Inactivating Protein; RIP), and an insertion element, suggesting that they are packaged into particles. Substantial homology of our particle-derived contigs was found in the genome assemblies of members of theSpiroplasma poulsoniiclade.

Many species show replicated ecophenotypy due to recurring patterns of natural selection. Based on the presence or absence of pursuit predators, at least 17 species of fish repeatedly differentiated in body shape in a manner that increases burst swimming speed and the likelihood of predator escape. The predator-associated burst speed (PABS) ecophenotype is characterized by a small head and trunk and enlarged caudal region. Mechanisms promoting replicated phenotype-environment association include selection (without evolution), a single instance of adaptive evolution followed by biased habitat occupation, repeated instances of local adaptation, or adaptive phenotypic plasticity. Common garden rearing of mosquitofish, Gambusia affinis, demonstrated a likely heritable basis for PABS phenotypy, but it is unknown whether populations are otherwise genetically distinct or whether replicated ecophenotypy represents a single or replicated instances of adaptation. To genetically characterize the populations and test hypotheses of single or multiple adaptations, we characterized variation in 12 polymorphic DNA microsatellites in the previously studied G. affinis populations. Populations were genetically distinct by multilocus analysis, exhibited high allelic diversity, and were heterozygote deficient, which effects were attributed to G. affinis's shoaling nature and habitat patchiness. Genetic and phenotypic distances among populations were correlated for non-PABS but not PABS morphology. Multilocus analysis demonstrated ecophenotype polyphyly and scattered multivariate genetic structure which support only the replicated-adaptation model. As all of the diverse tests performed demonstrated lack of congruence between patterns of molecular genetic and PABS differentiation, it is likely that divergent natural selection drove multiple instances of adaptive evolution.

Inland fisheries are an important source of protein and income for people in Africa, and sustainable fisheries management requires knowledge of regional stocks. Presently, information regarding genetic diversity of African freshwater fishes is very limited. Here we present findings from a spatial analysis of genetic patterns in the African bonytongue, Heterotis niloticus, an important component of inland fisheries in West Africa. The species is common throughout Western and Central Africa and the Nile Basin; and has been introduced for aquaculture in locations across Africa. Nigeria has the largest fishery for African bonytongues, representing ~86% of the global total. Recent declines in yields at some Nigerian locations, however, suggest current levels of exploitation may be unsustainable. Habitat degradation also may be impacting some stocks. Despite its commercial and nutritional importance, the African bonytongue has been the subject of scant research to support management. We examined patterns of genetic diversity in natural populations of H. niloticus at four locations in Nigeria, including Kainji Lake, a reservoir on the Niger River in north-central Nigeria, and three southtern localities (Ethiope River, Igbokoda River, and Epe Lagoon), as well fish from the Ouémé River delta near Porto Novo, Benin. Eighty-five specimens were genotyped for nine microsatellite-loci. Genetic diversity estimates were highest at Kainji Lake, and substantially lower at southern localities. High levels of genetic differentiation were detected between samples from Kainji Lake and those from the southern localities. Low, yet significant FST values were observed among samples from southern Nigerian localities that were more differentiated from the sample from nearby coastal Benin. We thus recommend that African bonytongues from the five locations be considered distinct genetic stocks and managed accordingly.

The fish genus Poeciliopsis constitutes a valuable research system for evolutionary ecology, whose phylogenetic relationships have not been fully elucidated. We conducted a multilocus phylogenetic study of the genus based on seven nuclear and two mitochondrial loci with a thorough set of analytical approaches, that is, concatenated (also known as super‐matrix), species trees, and phylogenetic networks. Although several relationships remain unresolved, the overall results uncovered phylogenetic affinities among several members of this genus. A population previously considered of undetermined taxonomic status could be unequivocally assigned to P. scarlli; revealing a relatively recent dispersal event across the Trans‐Mexican Volcanic Belt (TMVB) or Pacific Ocean, which constitute a strong barrier to north–south dispersal of many terrestrial and freshwater taxa. The closest relatives of P. balsas, a species distributed south of the TMVB, are distributed in the north; representing an additional north–south split in the genus. An undescribed species of Poeciliopsis, with a highly restricted distribution (i.e., a short stretch of the Rio Concepcion; just south of the US‐Mexico border), falls within the Leptorhaphis species complex. Our results are inconsistent with the hypothesis that this species originated by “breakdown” of an asexual hybrid lineage. On the other hand, network analyses suggest one or more possible cases of reticulation within the genus that require further evaluation with genome‐wide marker representation and additional analytical tools. The most strongly supported case of reticulation occurred within the subgenus Aulophallus (restricted to Central America), and implies a hybrid origin for P. retropinna (i.e., between P. paucimaculata and P. elongata). We consider that P. balsas and P. new species are of conservation concern.

Molecular identification is increasingly used to speed up biodiversity surveys and laboratory experiments. However, many groups of organisms cannot be reliably identified using standard databases such as GenBank or BOLD due to lack of sequenced voucher specimens identified by experts. Sometimes a large number of sequences are available, but with too many errors to allow identification. Here we address this problem for parasitoids of Drosophila by introducing a curated open-access molecular reference database, DROP (Drosophilaparasitoids). Identifying Drosophila parasitoids is challenging and poses a major impediment to realize the full potential of this model system in studies ranging from molecular mechanisms to food webs, and in biological control of Drosophila suzukii. In DROP (http://doi.org/10.5281/zenodo.4519656), genetic data are linked to voucher specimens and, where possible, the voucher specimens are identified by taxonomists and vetted through direct comparison with primary type material. To initiate DROP, we curated 154 laboratory strains, 856 vouchers, 554 DNA sequences, 16 genomes, 14 transcriptomes, and 6 proteomes drawn from a total of 183 operational taxonomic units (OTUs): 114 described Drosophila parasitoid species and 69 provisional species. We found species richness of Drosophila parasitoids to be heavily underestimated and provide an updated taxonomic catalogue for the community. DROP offers accurate molecular identification and improves cross-referencing between individual studies that we hope will catalyze research on this diverse and fascinating model system. Our effort should also serve as an example for researchers facing similar molecular identification problems in other groups of organisms.

The level of gene flow between diverging lineages ultimately determines the outcome of a speciation event. If secondary contact occurs before this process is complete, reproductive isolation barriers must exist or evolve to prevent hybridization. The selective pressures facilitating and maintaining genetic divergence do not always involve an observable phenotypic response, thus cryptic species form. The inability to distinguish between sibling species can be a particularly serious problem in groups responsible for pathogen transmission. Culicoides biting midges occur almost world-wide and vector many disease-causing pathogens that affect wildlife and livestock. In North America, the C. variipennis species complex contains three currently recognized species, only one of which is a vector, and limited molecular and morphological differences have hindered vector surveillance. Here, genomic methods were used to investigate speciation and genetic structure within this complex. Single nucleotide polymorphism (SNP) data were generated using ddRAD sequencing for 206 individuals originating from 17 locations throughout the United States and Canada. Clustering analyses consistently suggest the occurrence of five putative species with significant differentiation occurring in both sympatric and allopatric populations. Evidence of hybridization was detected in three different species pairings, indicating a lack of pre-zygotic reproductive isolation within the complex. Mitochondrial genes were used to trace the hybrid parentage of these individuals, which illuminated discordance with the SNP data. In this study, we highlight the potential role of geographic, ecological, and behavioral isolation in speciation and in maintaining species boundaries, despite hybridization and long range dispersal.

Spiroplasma is a genus of Mollicutes whose members include plant pathogens, insect pathogens and endosymbionts of animals. Spiroplasma phenotypes have been repeatedly observed to be spontaneously lost in Drosophila cultures, and several studies have documented a high genomic turnover in Spiroplasma symbionts and plant pathogens. These observations suggest that Spiroplasma evolves quickly in comparison to other insect symbionts. Here, we systematically assess evolutionary rates and patterns of Spiroplasma poulsonii , a natural symbiont of Drosophila. We analysed genomic evolution of sHy within flies, and sMel within in vitro culture over several years. We observed that S. poulsonii substitution rates are among the highest reported for any bacteria, and around two orders of magnitude higher compared with other inherited arthropod endosymbionts. The absence of mismatch repair loci mutS and mutL is conserved across Spiroplasma , and likely contributes to elevated substitution rates. Further, the closely related strains sMel and sHy (>99.5 % sequence identity in shared loci) show extensive structural genomic differences, which potentially indicates a higher degree of host adaptation in sHy, a protective symbiont of Drosophila hydei. Finally, comparison across diverse Spiroplasma lineages confirms previous reports of dynamic evolution of toxins, and identifies loci similar to the male-killing toxin Spaid in several Spiroplasma lineages and other endosymbionts. Overall, our results highlight the peculiar nature of Spiroplasma genome evolution, which may explain unusual features of its evolutionary ecology.

Several facultative bacterial symbionts of insects protect their hosts against natural enemies.Spiroplasma poulsoniistrainsMel, a male-killing heritable symbiont ofDrosophila melanogaster, confers protection against some species of parasitic wasps. Several lines of evidence suggest thatSpiroplasma-encoded ribosome inactivating proteins (RIPs) are involved in the protection mechanism, but the potential contribution of the fly-encoded functions has not been deeply explored. Here we used RNA-seq to evaluate the response ofD. melanogasterto infection bySpiroplasmaand parasitism by theSpiroplasma-susceptible waspLeptopilina heterotoma, and theSpiroplasma-resistant waspGanaspis hookeri. In the absence ofSpiroplasmainfection, we found evidence ofDrosophilaimmune activation byG. hookeri, but not byL. heterotoma, which in turn negatively influenced functions associated with male gonad development. As expected for a symbiont that kills males, we detected extensive downregulation in theSpiroplasma-infected treatments of genes known to have male-biased expression. We detected very few genes whose expression was influenced by theSpiroplasma-L. heterotomainteraction, and they do not appear to be related to immune response. For most of them, parasitism byL. heterotoma(in the absence ofSpiroplasma) caused an expression change that was at least partly reversed whenSpiroplasmawas also present. It is unclear whether such genes are involved in theSpiroplasma-mediated mechanism that leads to wasp death or fly rescue. Nonetheless, the expression pattern of some of these genes, which reportedly undergo expression shifts during the larva-to-pupa transition, is suggestive of an influence ofSpiroplasmaon the development time ofL. heterotoma-parasitized flies. In addition, we used the RNAseq data and quantitative (q)PCR to evaluate the transcript levels of theSpiroplasma-encoded RIP genes. One of the five RIP genes (RIP2) was consistently highly expressed independently of wasp parasitism, in two substrains ofsMel. Finally, the RNAseq data revealed evidence consistent with RIP-induced damage in the ribosomal (r)RNA of theSpiroplasma-susceptible, but not theSpiroplasma-resistant, wasp. We conclude that immune priming is unlikely to contribute to theSpiroplasma-mediated protection against wasps, and that the mechanism by whichG. hookeriresists/toleratesSpiroplasmadoes not involve inhibition of RIP transcription.

Spiroplasma are a group of Mollicutes whose members include plant pathogens, insect pathogens, and endosymbionts of animals. Spiroplasma phenotypes have been repeatedly observed to be spontaneously lost in Drosophila cultures, and several studies have documented a high genomic turnover in Spiroplasma symbionts and plant pathogens. These observations suggest that Spiroplasma evolves quickly in comparison to other insect symbionts. Here, we systematically assess evolutionary rates and patterns of Spiroplasma poulsonii, a natural symbiont of Drosophila. We analysed genomic evolution of sHy within flies, and sMel within in vitro culture over several years. We observed that S. poulsonii substitution rates are among the highest reported for any bacteria, and around two orders of magnitude higher compared with other inherited arthropod endosymbionts. The absence of mismatch repair loci mutS and mutL is conserved across Spiroplasma and likely contributes to elevated substitution rates. Further, the closely related strains sMel and sHy (>99.5% sequence identity in shared loci) show extensive structural genomic differences, which potentially indicates a higher degree of host adaptation in sHy, a protective symbiont of Drosophila hydei. Finally, comparison across diverse Spiroplasma lineages confirms previous reports of dynamic evolution of toxins, and identifies loci similar to the male-killing toxin Spaid in several Spiroplasma lineages and other endosymbionts. Overall, our results highlight the peculiar nature of Spiroplasma genome evolution, which may explain unusual features of its evolutionary ecology.

Members of the true fruit flies (family Tephritidae) are among the most serious agricultural pests worldwide, whose control and management demands large and costly international efforts. The need for cost-effective and environmentally friendly integrated pest management (IPM) has led to the development and implementation of autocidal control strategies. These approaches include the widely used sterile insect technique and the incompatible insect technique (IIT). IIT relies on maternally transmitted bacteria (namely Wolbachia) to cause a conditional sterility in crosses between released mass-reared Wolbachia-infected males and wild females, which are either uninfected or infected with a different Wolbachia strain (i.e., cytoplasmic incompatibility; CI). Herein, we review the current state of knowledge on Wolbachia-tephritid interactions including infection prevalence in wild populations, phenotypic consequences, and their impact on life history traits. Numerous pest tephritid species are reported to harbor Wolbachia infections, with a subset exhibiting high prevalence. The phenotypic effects of Wolbachia have been assessed in very few tephritid species, due in part to the difficulty of manipulating Wolbachia infection (removal or transinfection). Based on recent methodological advances (high-throughput DNA sequencing) and breakthroughs concerning the mechanistic basis of CI, we suggest research avenues that could accelerate generation of necessary knowledge for the potential use of Wolbachia-based IIT in area-wide integrated pest management (AW-IPM) strategies for the population control of tephritid pests. Instituto de Genética Fil: Mateos, Mariana. Texas A&M University. Departments of Ecology and Conservation Biology, and Wildlife and Fisheries Sciences; Estados Unidos Fil: Martinez Montoya, Humberto. Universidad Autónoma de Tamaulipas. Unidad Académica Multidisciplinaria Reynosa Aztlan. Laboratorio de Genética y Genómica Comparativa; México Fil: Lanzavecchia, Silvia Beatriz. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética; Argentina Fil: Conte, Claudia Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética; Argentina Fil: Guillén, Karina. El Colegio de la Frontera Sur; México Fil: Morán-Aceves, Brenda M. El Colegio de la Frontera Sur; México Fil: Toledo, Jorge. El Colegio de la Frontera Sur; México Fil: Liedo, Pablo. El Colegio de la Frontera Sur; México Fil: Asimakis, Elias D. University of Patras. Department of Environmental Engineering; Grecia Fil: Doudoumis, Vangelis. University of Patras. Department of Environmental Engineering; Grecia Fil: Kyritsis, Georgios A. University of Thessaly. Department of Agriculture Crop Production and Rural Environment. Laboratory of Entomology and Agricultural Zoology; Grecia Fil: Papadopoulos, Nikos T. University of Thessaly. Department of Agriculture Crop Production and Rural Environment. Laboratory of Entomology and Agricultural Zoology; Grecia Fil: Augustinos, Antonios A. Hellenic Agricultural Organization. Institute of Industrial and Forage Crops. Department of Plant Protection; Grecia Fil: Segura, Diego Fernando. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética. Laboratorio de Genética de Insectos de Importancia Económica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Tsiamis, George. University of Patras. Department of Environmental Engineering; Grecia

Maternally-transmitted endosymbiotic bacteria are ubiquitous in insects. Among other influential phenotypes, many heritable symbionts of arthropods are notorious for manipulating host reproduction through one of four reproductive syndromes, which are generally exerted during early developmental stages of the host: male feminization; parthenogenesis induction; male killing; and cytoplasmic incompatibility (CI). Major advances have been achieved in understanding mechanisms and identifying symbiont factors involved in reproductive manipulation, particularly male killing and cytoplasmic incompatibility. Nonetheless, whether cytoplasmically-transmitted bacteria influence the maternally-loaded components of the egg or early embryo has not been examined. In the present study, we investigated whether heritable endosymbionts that cause different reproductive phenotypes in Drosophila melanogaster influence the mRNA transcriptome of early embryos. We used mRNA-seq to evaluate differential expression in Drosophila embryos lacking endosymbionts (control) to those harbouring the male-killing Spiroplasma poulsonii strain MSRO-Br, the CI-inducing Wolbachia strain wMel, or Spiroplasma poulsonii strain Hyd1; a strain that lacks a reproductive phenotype and is naturally associated with Drosophila hydei. We found no consistent evidence of influence of symbiont on mRNA composition of early embryos, suggesting that the reproductive manipulation mechanism does not involve alteration of maternally-loaded transcripts. In addition, we capitalized on several available mRNA-seq datasets derived from Spiroplasma-infected Drosophila melanogaster embryos, to search for signals of depurination of rRNA, consistent with the activity of Ribosome Inactivating Proteins (RIPs) encoded by Spiroplasma poulsonii. We found small but statistically significant signals of depurination of Drosophila rRNA in the Spiroplasma treatments (both strains), but not in the symbiont-free control or Wolbachia treatment, consistent with the action of RIPs. The depurination signal was slightly stronger in the treatment with the male-killing strain. This result supports a recent report that RIP-induced damage contributes to male embryo death.

BackgroundSeveral facultative bacterial symbionts of insects protect their hosts against natural enemies.Spiroplasma poulsoniistrainsMel (hereafterSpiroplasma), a male-killing heritable symbiont ofDrosophila melanogaster, confers protection against some species of parasitic wasps. Several lines of evidence suggest thatSpiroplasma-encoded ribosome inactivating proteins (RIPs) are involved in the protection mechanism, but the potential contribution of the fly-encoded functions (e.g., immune response), has not been deeply explored.MethodsHere we used RNA-seq to evaluate the response ofD. melanogasterto infection bySpiroplasmaand parasitism by theSpiroplasma-susceptible waspLeptopilina heterotoma, and theSpiroplasma-resistant waspGanaspissp. In addition, we used quantitative (q)PCR to evaluate the transcript levels of theSpiroplasma-encoded Ribosomal inactivation protein (RIP) genes.ResultsIn the absence ofSpiroplasmainfection, we found evidence ofDrosophilaimmune activation byGanaspissp., but not byL. heterotoma, which in turn negatively influenced functions associated with male gonad development. As expected for a symbiont that kills males, we detected extensive downregulation in theSpiroplasma-infected treatments of genes known to have male-biased expression. We detected very few genes whose expression patterns appeared to be influenced by theSpiroplasma-L. heterotomainteraction, and these genes are not known to be associated with immune response. For most of these genes, parasitism byL. heterotoma(in the absence ofSpiroplasma) caused an expression change that was at least partly reversed when bothL. heterotomaandSpiroplasmawere present. It is unclear whether such genes are involved in theSpiroplasma-mediated mechanism that leads to wasp death and/or fly rescue. Nonetheless, the expression pattern of some of these genes, which reportedly undergo expression shifts during the larva-to-pupa transition, is suggestive of an influence ofSpiroplasmaon the development time ofL. heterotoma-parasitized flies. One of the five RIP genes (RIP2) was consistently highly expressed independently of wasp parasitism, in two substrains ofsMel. Finally, the RNAseq data revealed evidence consistent with RIP-induced damage in the ribosomal (r)RNA of theSpiroplasma-susceptible, but not theSpiroplasma-resistant, wasp. Acknowledging the caveat that we lacked adequate power to detect the majority of DE genes with fold-changes lower than 3, we conclude that immune priming is unlikely to contribute to theSpiroplasma-mediated protection against wasps, and that the mechanism by whichGanaspis sp. resists/toleratesSpiroplasmadoes not involve inhibition of RIP transcription.

A growing body of knowledge on the diversity and evolution of intertidal isopods across different regions worldwide has enhanced our understanding on biological diversification at the poorly studied, yet vast, sea–land interface. High genetic divergences among numerous allopatric lineages have been identified within presumed single broadly distributed species. Excirolana mayana is an intertidal isopod that is commonly found in sandy beaches throughout the Gulf of California. Its distribution in the Pacific extends from this basin to Colombia and in the Atlantic from Florida to Venezuela. Despite its broad distribution and ecological importance, its evolutionary history has been largely neglected. Herein, we examined phylogeographic patterns of E. mayana in the Gulf of California and the Caribbean, based on maximum‐likelihood and Bayesian phylogenetic analyses of DNA sequences from four mitochondrial genes (16S rDNA, 12S rDNA, cytochrome oxidase I gene, and cytochrome b gene). We compared the phylogeographic patterns of E. mayana with those of the coastal isopods Ligia and Excirolana braziliensis (Gulf of California and Caribbean) and Tylos (Gulf of California). We found highly divergent lineages in both, the Gulf of California and Caribbean, suggesting the presence of multiple species. We identified two instances of Atlantic–Pacific divergences. Some geographical structuring among the major clades found in the Caribbean is observed. Haplotypes from the Gulf of California form a monophyletic group sister to a lineage found in Venezuela. Phylogeographic patterns of E. mayana in the Gulf of California differ from those observed in Ligia and Tylos in this region. Nonetheless, several clades of E. mayana have similar distributions to clades of these two other isopod taxa. The high levels of cryptic diversity detected in E. mayana also pose challenges for the conservation of this isopod and its fragile environment, the sandy shores.

Insect microbe associations are diverse, widespread, and influential. Among the fitness effects of microbes on their hosts, defense against natural enemies is increasingly recognized as ubiquitous, particularly among those associations involving heritable, yet facultative, bacteria. Protective mutualisms generate complex ecological and coevolutionary dynamics that are only beginning to be elucidated. These depend in part on the degree to which symbiont‐mediated protection exhibits specificity to one or more members of the natural enemy community. Recent findings in a well‐studied defensive mutualism system (i.e., aphids, bacteria, parasitoid wasps) reveal repeated instances of evolution of susceptibility or resistance to defensive bacteria by parasitoids. This study searched for similar patterns in an emerging model system for defensive mutualisms: the interaction of Drosophila, bacteria in the genus Spiroplasma, and wasps that parasitize larval stages of Drosophila. Previous work indicated that three divergent species of parasitic wasps are strongly inhibited by the presence of Spiroplasma in three divergent species of Drosophila, including D. melanogaster. The results of this study uncovered two additional wasp species that are susceptible to Spiroplasma and two that are unaffected by Spiroplasma, implying at least two instances of loss or gain of susceptibility to Spiroplasma among larval parasitoids of Drosophila.

Multiple highly divergent lineages have been identified within Ligia occidentalis sensu lato, a rocky supralittoral isopod distributed along a ~3000 km latitudinal gradient that encompasses several proposed marine biogeographic provinces and ecoregions in the eastern Pacific. Highly divergent lineages have nonoverlapping geographic distributions, with distributional limits that generally correspond with sharp environmental changes. Crossbreeding experiments suggest postmating reproductive barriers exist among some of them, and surveys of mitochondrial and nuclear gene markers do not show evidence of hybridization. Populations are highly isolated, some of which appear to be very small; thus, the effects of drift are expected to reduce the efficiency of selection. Large genetic divergences among lineages, marked environmental differences in their ranges, reproductive isolation, and/or high isolation of populations may have resulted in morphological differences in L. occidentalis, not detected yet by traditional taxonomy. We used landmark‐based geometric morphometric analyses to test for differences in body shape among highly divergent lineages of L. occidentalis, and among populations within these lineages. We analyzed a total of 492 individuals from 53 coastal localities from the southern California Bight to Central Mexico, including the Gulf of California. We conducted discriminant function analyses (DFAs) on body shape morphometrics to assess morphological variation among genetically differentiated lineages and their populations. We also tested for associations between phylogeny and morphological variation, and whether genetic divergence is correlated to multivariate morphological divergence. We detected significant differences in body shape among highly divergent lineages, and among populations within these lineages. Nonetheless, neither lineages nor populations can be discriminated on the basis of body shape, because correct classification rates of cross‐validated DFAs were low. Genetic distance and phylogeny had weak to no effect on body shape variation. The supralittoral environment appears to exert strong stabilizing selection and/or strong functional constraints on body shape in L. occidentalis, thereby leading to morphological stasis in this isopod.

The native range of the blue crab Callinectes sapidus spans Nova Scotia to northern Argentina. In the US, it constitutes a keystone species in estuarine habitats of the Atlantic coast and Gulf of Mexico (GOM), serving as both predator and prey to other species, and also has historically represented a multi-billion dollar fishery. Knowledge relevant to effective management and monitoring of this ecologically and economically important species, such as levels of population genetic differentiation and genetic diversity, is necessary. Although several population genetics studies have attempted to address these questions in one or more parts of its distribution, conflicting results and potential problems with the markers used, as well as other issues, have obscured our understanding on them. In this study, we examined large-scale genetic connectivity of the blue crab in the US, using 16 microsatellites, and genotyped individuals from Chesapeake Bay, in the US Atlantic, and from nine localities along the US GOM coast. Consistent with the high long-distance dispersal potential of this species, very low levels of genetic differentiation were detected for the blue crab among the ten US localities examined, suggesting it constitutes a large panmictic population within this region. Estimations of genetic diversity for the blue crab appear to be high in the US, and provide a baseline for monitoring temporal changes in this species. Demographic analyses indicate a recent range expansion of the US population, probably during the Holocene. In addition, capitalizing on published microsatellite data from southern Brazil, our analyses detected high genetic differentiation between localities in the US and Brazil. These results point to the need for examination of genetic diversity and differentiation along the area spanning the US to southern Brazil.

The native ranges and invasion histories of many marine species remain elusive due to a dynamic dispersal process via marine vessels. Molecular markers can aid in identification of native ranges and elucidation of the introduction and establishment process. The supralittoral isopod Ligia exotica has a wide tropical and subtropical distribution, frequently found in harbors and ports around the globe. This isopod is hypothesized to have an Old World origin, from where it was unintentionally introduced to other regions via wooden ships and solid ballast. Its native range, however, remains uncertain. Recent molecular studies uncovered the presence of two highly divergent lineages of L. exotica in East Asia, and suggest this region is a source of nonindigenous populations. In this study, we conducted phylogenetic analyses (Maximum Likelihood and Bayesian) of a fragment of the mitochondrial 16S ribosomal (r)DNA gene using a dataset of this isopod that greatly expanded previous representation from Asia and putative nonindigenous populations around the world. For a subset of samples, sequences of 12S rDNA and NaK were also obtained and analyzed together with 16S rDNA. Our results show that L. exotica is comprised of several highly divergent genetic lineages, which probably represent different species. Most of the 16S rDNA genetic diversity (48 haplotypes) was detected in East and Southeast Asia. Only seven haplotypes were observed outside this region (in the Americas, Hawai’i, Africa and India), which were identical or closely related to haplotypes found in East and Southeast Asia. Phylogenetic patterns indicate the L. exotica clade originated and diversified in East and Southeast Asia, and only members of one of the divergent lineages have spread out of this region, recently, suggesting the potential to become invasive is phylogenetically constrained.

Maternally transmitted endosymbionts of insects are ubiquitous in nature and play diverse roles in the ecology and evolution of their hosts. To persist in host lineages, many symbionts manipulate host reproduction to their advantage (e.g. cytoplasmic incompatibility and male-killing), or confer fitness benefits to their hosts (e.g. metabolic provisioning and defense against natural enemies). Recent studies suggest that strains of the bacterial genus Spiroplasma protect their host (flies in the genus Drosophila ) against parasitoid attack. The Spiroplasma -conferred protection is partial and flies surviving a wasp attack have reduced adult longevity and fecundity. Therefore, it is unclear whether protection against wasps alone can counter Spiroplasma loss by imperfect maternal transmission and any possible fitness costs to harboring Spiroplasma . To address this question, we conducted a population cage study comparing Spiroplasma frequencies over time (host generations) under conditions of high wasp pressure and no wasp pressure. A dramatic increase of Spiroplasma prevalence was observed under high wasp pressure. In contrast, Spiroplasma prevalence in the absence of wasps did not change significantly over time; a pattern consistent with random drift. Thus, the defensive mechanism may contribute to the high prevalence of Spiroplasma in host populations despite imperfect vertical transmission.

Maternally transmitted associations between endosymbiotic bacteria and insects are diverse and widespread in nature. Owing to imperfect vertical transmission, many heritable microbes have evolved compensational mechanisms to enhance their persistence in host lineages, such as manipulating host reproduction and conferring fitness benefits to host. Symbiont-mediated defense against natural enemies of hosts is increasingly recognized as an important mechanism by which endosymbionts enhance host fitness. Members of the genus Spiroplasma associated with distantly related Drosophila hosts are known to engage in either reproductive parasitism (i.e., male killing) or defense against natural enemies (the parasitic wasp Leptopilina heterotoma and a nematode). A male-killing strain of Spiroplasma (strain Melanogaster Sex Ratio Organism (MSRO)) co-occurs with Wolbachia (strain wMel) in certain wild populations of the model organism Drosophila melanogaster. We examined the effects of Spiroplasma MSRO and Wolbachia wMel on Drosophila survival against parasitism by two common wasps, Leptopilina heterotoma and Leptopilina boulardi, that differ in their host ranges and host evasion strategies. The results indicate that Spiroplasma MSRO prevents successful development of both wasps, and confers a small, albeit significant, increase in larva-to-adult survival of flies subjected to wasp attacks. We modeled the conditions under which defense can contribute to Spiroplasma persistence. Wolbachia also confers a weak, but significant, survival advantage to flies attacked by L. heterotoma. The host protective effects exhibited by Spiroplasma and Wolbachia are additive and may provide the conditions for such cotransmitted symbionts to become mutualists. Occurrence of Spiroplasma-mediated protection against distinct parasitoids in divergent Drosophila hosts suggests a general protection mechanism.

Aim The Point Conception (PC) biogeographical boundary is defined by a transition between cold northern and warm southern water masses, accompanied by shifts in numerous ecological and environmental parameters. While these factors can potentially contribute to the genetic differentiation of lineages whose distributions span this boundary, few organisms exhibit genetic signatures of restricted gene flow across this region. We examine the effects of the PC boundary and other potential dispersal barriers on phylogeographical patterns in the rocky supralittoral isopod Ligia occidentalis. This isopod has a limited dispersal potential, its distribution spans the PC region, and it exhibits high levels of allopatric genetic differentiation south of PC. Location Eastern Pacific coastline between southern Oregon and the Baja California Peninsula. Methods We conducted a thorough sampling of L. occidentalis from the PC area to its northern range limit in southern Oregon, and increased previous sampling coverage south of PC. We obtained sequences of the mitochondrial genes cytochrome c oxidase subunit I (COI) and 16S rDNA for the new localities and combined them with a previously published data set, resulting in a total of 58 sampling locations. We also obtained sequences of the nuclear gene sodium–potassium ATPase a-subunit (NaK). We conducted phylogenetic and population-genetic analyses. Results The geographical limit between the two most divergent clades of L. occidentalis (20–25% divergence for COI) distributed between southern Oregon and the Baja California Peninsula occurs at the PC biogeographical boundary. Levels of allopatric genetic divergences were greatly reduced north of PC (≤ 3.0% divergence for COI). Main conclusions The geographical limit between the two main L. occidentalis clades in California largely reflects the changes in sea surface temperature that define the PC biogeographical boundary. Reduced levels of genetic divergence among L. occidentalis lineages found north of PC suggest a recent expansion in the northern range of this isopod.

Maternally transmitted endosymbiotic bacteria of the genus Spiroplasma associate with numerous insect species, including the genus Drosophila. Among the Spiroplasma strains associated with Drosophila, several manipulate their host’s reproduction by killing the male offspring of the infected females. Although the male-killing mechanism is not well understood, previous studies of non-native strains transferred to D. melanogaster (strain Oregon-R) indicate that the male-killing strain achieves higher densities than two non-male-killing strains. Whether this pattern of higher male-killing strain densities occurs in other host-symbiont strain combinations is not known. Herein, we used quantitative PCR to examine infection densities of one non-male-killing strain native to D. hydei (Hyd1), and two male-killing strains; one native to D. nebulosa (NSRO), and one native to D. melanogaster (MSRO; recently discovered), upon artificial transfer to D. melanogaster (strain Canton-S). Infection densities were examined at four weekly intervals in adult flies, across three consecutive generations following artificial transfer. Infection densities of the non-male-killing strain were significantly lower than those of the two male killers immediately after adult emergence. At later time points, however, the non-male-killing strain (Hyd1) is capable of proliferating to densities similar to those of the two male-killing strains (NSRO and MSRO) in D. melanogaster (Canton-S). We also examined the effect of co-infection by the heritable bacterium Wolbachia, on Spiroplasma densities and male-killing ability. Wolbachia had little to no effect of Spiroplasma densities, but the male-killing ability of MSRO was lower in the presence of Wolbachia. Generation post-infection had little effect on Spiroplasma densities, but affected the male-killing ability.

We investigated replicated ecological speciation in the livebearing fish Poecilia mexicana and P. sulphuraria (Poeciliidae), which inhabit freshwater habitats and have also colonized multiple sulfidic springs in southern Mexico. These springs exhibit extreme hypoxia and high concentrations of hydrogen sulfide, which is lethal to most metazoans. We used phylogenetic analyses to test whether springs were independently colonized, performed phenotypic assessments of body and gill morphology variation to identify convergent patterns of trait differentiation, and conducted an eco-toxicological experiment to detect differences in sulfide tolerances among ecotypes. Our results indicate that sulfidic springs were colonized by three different lineages, two within P. mexicana and one representing P. sulphuraria. Colonization occurred earlier in P. sulphuraria, whereas invasion of sulfidic springs in P. mexicana was more recent, such that each population is more closely related to neighboring populations from adjacent nonsulfidic habitats. Sulfide spring fish also show divergence from nonsulfidic phenotypes and a phenotypic convergence toward larger heads, larger gills, and increased tolerance to H(2) S. Together with previous studies that indicated significant reproductive isolation between fish from sulfidic and nonsulfidic habitats, this study provides evidence for repeated ecological speciation in the independent sulfide spring populations of P. mexicana and P. sulphuraria.

Natural hybrid zones provide opportunities to study a range of evolutionary phenomena from speciation to the genetic basis of fitness-related traits. We show that widespread hybridization has occurred between two neo-tropical stream fishes with partial reproductive isolation. Phylogenetic analyses of mitochondrial sequence data showed that the swordtail fish Xiphophorus birchmanni is monophyletic and that X. malinche is part of an independent monophyletic clade with other species. Using informative single nucleotide polymorphisms in one mitochondrial and three nuclear intron loci, we genotyped 776 specimens collected from twenty-three sites along seven separate stream reaches. Hybrid zones occurred in replicated fashion in all stream reaches along a gradient from high to low elevation. Genotyping revealed substantial variation in parental and hybrid frequencies among localities. Tests of F(IS) and linkage disequilibrium (LD) revealed generally low F(IS) and LD except in five populations where both parental species and hybrids were found suggesting incomplete reproductive isolation. In these locations, heterozygote deficiency and LD were high, which suggests either selection against early generation hybrids or assortative mating. These data lay the foundation to study the adaptive basis of the replicated hybrid zone structure and for future integration of behaviour and genetics to determine the processes that lead to the population genetic patterns observed in these hybrid zones.

The subgenus Mollienesia is a diverse group of freshwater fishes, including species that have served as important models across multiple biological disciplines. Nonetheless, the taxonomic history of this group has been conflictive and convoluted, in part because the evolutionary relationships have not been rigorously resolved. We conducted a comprehensive molecular phylogenetic analysis of the subgenus Mollienesia to identify taxonomic discrepancies and potentially identify undescribed species, estimate ancestral areas of origin and estimate dates of divergence, as well as explore biogeographical patterns. Our findings confirm the presence of three main clades composed of the P. latipinna, P. sphenops, and P. mexicana species complexes. Unlike previously hypothesized morphology-based analyses, species found on the Caribbean Islands are not part of Mollienesia, but are more closely related to species of the subgenus Limia. Our study also revealed several taxonomic inconsistencies and distinct lineages in the P. mexicana species complex that may represent undescribed species. The diversity in the subgenus Mollienesia is a result of dynamic geologic activity leading to vicariant events, dispersal across geologic blocks, and ecological speciation.

Eleven nuclear genes and multiple phylogenetic techniques were used to study relationships among four of North American pines from subsection Australes (genus Pinus). Support for phylogenies reconstructed based on all data was generally weak and inconsistent among methods. Subsequent analysis revealed that certain topologies were supported by genes with common putative functionalities. Therefore, each set of genes was analysed independently. Recovered alternative topologies were highly supported and the results were consistent among methods. Multiple evolutionary hypotheses could potentially explain the observed patterns, although incomplete lineage sorting seems to be the simplest one., Adaptive evolutionary processes in plants may be accompanied by episodes of introgression, parallel evolution and incomplete lineage sorting that pose challenges in untangling species evolutionary history. Genus Pinus (pines) is one of the most abundant and most studied groups among gymnosperms, and a good example of a lineage where these phenomena have been observed. Pines are among the most ecologically and economically important plant species. Some, such as the pines of the southeastern USA (southern pines in subsection Australes), are subjects of intensive breeding programmes. Despite numerous published studies, the evolutionary history of Australes remains ambiguous and often controversial. We studied the phylogeny of four major southern pine species: shortleaf (Pinus echinata), slash (P. elliottii), longleaf (P. palustris) and loblolly (P. taeda), using sequences from 11 nuclear loci and maximum likelihood and Bayesian methods. Our analysis encountered resolution difficulties similar to earlier published studies. Although incomplete lineage sorting and introgression are two phenomena presumptively underlying our results, the phylogenetic inferences seem to be also influenced by the genes examined, with certain topologies supported by sets of genes sharing common putative functionalities. For example, genes involved in wood formation supported the clade echinata–taeda, genes linked to plant defence supported the clade echinata–elliottii and genes linked to water management properties supported the clade echinata–palustris. The support for these clades was very high and consistent across methods. We discuss the potential factors that could underlie these observations, including incomplete lineage sorting, hybridization and parallel or adaptive evolution. Our results likely reflect the relatively short evolutionary history of the subsection that is thought to have begun during the middle Miocene and has been influenced by climate fluctuations.