Preliminary Characterization of Phage-Like Particles from the Male-Killing Mollicute Spiroplasma poulsonii (an Endosymbiont of Drosophila).

Bacteriophages are vastly abundant, diverse, and influential, but with few exceptions (e.g. the Proteobacteria genera Wolbachia and Hamiltonella), 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 mollicute Spiroplasma associated with Drosophila flies, genomic sequences of such phage are lacking, and their effects on the Spiroplasma-Drosophila interaction have not been comprehensively characterized. We used a density step gradient to isolate phage-like particles from the male-killing bacterium Spiroplasma poulsonii (strains NSRO and MSRO-Br) harbored by Drosophila 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 in Drosophila-associated Spiroplasma strains. 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 known Spiroplasma toxin (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 the Spiroplasma poulsonii clade.

Genetic identification and diversity of stocks of the African bonytongue, Heterotis niloticus (Osteoglossiformes: Arapaiminae), in Nigeria, West Africa.

Inland fisheries are an important source of protein and income for people in Africa. Their sustainable management can greatly benefit from identification of regional genetic stocks and characterization of their genetic diversity, but such information is lacking for most African freshwater fisheries. The African bonytongue, Heterotis niloticus, is an important component of inland fisheries in West 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 genetic 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 southern 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 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.

Species delimitation and mitonuclear discordance within a species complex of biting midges.

The inability to distinguish between species can be a serious problem in groups responsible for pathogen transmission. Culicoides biting midges transmit many pathogenic agents infecting wildlife and livestock. In North America, the C. variipennis species complex contains three currently recognized species, only one of which is a known vector, but limited species-specific characters have hindered vector surveillance. Here, genomic data were used to investigate population structure and genetic differentiation within this species complex. Single nucleotide polymorphism data were generated for 206 individuals originating from 17 locations throughout the United States and Canada. Clustering analyses suggest the occurrence of two additional cryptic species within this complex. All five species were significantly differentiated in both sympatry and allopatry. Evidence of hybridization was detected in three different species pairings indicating incomplete reproductive isolation. Additionally, COI sequences were used to identify the hybrid parentage of these individuals, which illuminated discordance between the divergence of the mitochondrial and nuclear datasets.

Population genetics and independently replicated evolution of predator-associated burst speed ecophenotypy in mosquitofish.

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.

DROP: Molecular voucher database for identification of Drosophila parasitoids.

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 (Drosophila parasitoids). 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, 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 six 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 catalyse 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.

Differential gene expression in a tripartite interaction: Drosophila, Spiroplasma and parasitic wasps.

BACKGROUND: Several facultative bacterial symbionts of insects protect their hosts against natural enemies. Spiroplasma poulsonii strain sMel (hereafter Spiroplasma), a male-killing heritable symbiont of Drosophila melanogaster, confers protection against some species of parasitic wasps. Several lines of evidence suggest that Spiroplasma-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. METHODS: Here we used RNA-seq to evaluate the response of D. melanogaster to infection by Spiroplasma and parasitism by the Spiroplasma-susceptible wasp Leptopilina heterotoma, and the Spiroplasma-resistant wasp Ganaspis sp. In addition, we used quantitative (q)PCR to evaluate the transcript levels of the Spiroplasma-encoded Ribosomal inactivation protein (RIP) genes. RESULTS: In the absence of Spiroplasma infection, we found evidence of Drosophila immune activation by Ganaspis sp., but not by L. 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 the Spiroplasma-infected treatments of genes known to have male-biased expression. We detected very few genes whose expression patterns appeared to be influenced by the Spiroplasma-L. heterotoma interaction, and these genes are not known to be associated with immune response. For most of these genes, parasitism by L. heterotoma (in the absence of Spiroplasma) caused an expression change that was at least partly reversed when both L. heterotoma and Spiroplasma were present. It is unclear whether such genes are involved in the Spiroplasma-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 of Spiroplasma on the development time of L. heterotoma-parasitized flies. One of the five RIP genes (RIP2) was consistently highly expressed independently of wasp parasitism, in two substrains of sMel. Finally, the RNAseq data revealed evidence consistent with RIP-induced damage in the ribosomal (r)RNA of the Spiroplasma-susceptible, but not the Spiroplasma-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 the Spiroplasma-mediated protection against wasps, and that the mechanism by which Ganaspis sp. resists/tolerates Spiroplasma does not involve inhibition of RIP transcription.

Rapid molecular evolution of Spiroplasma symbionts of Drosophila.

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.

Wolbachia pipientis Associated With Tephritid Fruit Fly Pests: From Basic Research to Applications.

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.

A new species of the live-bearing fish genus Poeciliopsis from northern Mexico (Cyprinodontiformes, Poeciliidae).

Poeciliopsis jackschultzi sp. nov., is described based on seven specimens (17.9-26.7 mm SL) from the Río Concepción (also known as Río Magdalena), Sonora, Mexico. The new species belongs to the Leptorhaphis species group and can be distinguished from other members of this group by features of the skeleton and colouration. The new species is sympatric with P. occidentalis, a hybridogenetic all-female biotype P. monacha-occidentalis, and hybrids between P. monacha-occidentalis females and P. jackschultzi males. The distribution of P. jackschultzi is highly restricted, and the main habitat, spring-fed marshy streams and pools, is susceptible to loss and degradation in a desert environment with increasing human water demand.

Population genetics and historical demographic inferences of the blue crab Callinectes sapidus in the US based on microsatellites.

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.

Effect of heritable symbionts on maternally-derived embryo transcripts.

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.

A multilocus phylogeny of the fish genus Poeciliopsis: Solving taxonomic uncertainties and preliminary evidence of reticulation.

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.

Evolution of African barbs from the Lake Victoria drainage system, Kenya.

The Lake Victoria drainage basin (LVD) in Kenya is home to ten nominal species of small barbs (Enteromius) and one of large barbs (Labeobarbus altianalis). A recent molecular study genetically characterized small barbs in this region and found evidence of introgression between certain species, complicating the taxonomy and species identification of these fishes. This study aimed to extend our understanding on the evolution of these fishes by: (1) determining whether putatively pure individuals of Enteromius cercops are found in the Kenyan LVD, as the previous study only found hybrid individuals of this species in this region; (2) testing the sister relationship between Enteromius profundus, endemic to Lake Victoria, and Enteromius radiatus, also found in Lake Victoria, which had been previously synonymized; (3) examining the phylogenetic relationships of small barbs of the Kenyan LVD with those reported from other ichthyological provinces of Africa; and (4) examining the phylogenetic relationships of Labeobarbus altianalis with other Labeobarbus species. To this end, we obtained mitochondrial Cytochrome b and nuclear Growth Hormone (GH) intron 2 gene sequences of nine Enteromius species from the LVD in Kenya, as well as cytochrome b sequences for L. altianalis. We conducted Maximum likelihood and Bayesian phylogenetic analyses to establish their evolutionary relationships in relation to many other barbs specimens from Africa. Phylogenetic analyses did not reveal instances of hybridization/introgression among the individuals sequenced by us. A sister relationship between E. profundus and E. radiatus was not found. This latter species shows instead a sister relationship with a lineage comprised of two species from West Africa. Other sister relationships between taxa from the East coast and other ecoregions from Africa are observed, suggesting that past drainage connections and vicariant events contributed to the diversification of Enteromius. Finally, only a single haplotype was recovered among the L. altianalis individuals examined, which is most similar to a specimen from Lake Edward in Uganda.

Out of Asia: mitochondrial evolutionary history of the globally introduced supralittoral isopod Ligia exotica.

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.

Phylogeographic patterns of a lower intertidal isopod in the Gulf of California and the Caribbean and comparison with other intertidal isopods.

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.

Phylogenetic analyses of the subgenus Mollienesia (Poecilia, Poeciliidae, Teleostei) reveal taxonomic inconsistencies, cryptic biodiversity, and spatio-temporal aspects of diversification in Middle America.

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.

Conflicting genomic signals affect phylogenetic inference in four species of North American pines.

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.

Independent origins of resistance or susceptibility of parasitic wasps to a defensive symbiont.

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.

Constrained body shape among highly genetically divergent allopatric lineages of the supralittoral isopod Ligia occidentalis (Oniscidea).

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.

Multiple transisthmian divergences, extensive cryptic diversity, occasional long-distance dispersal, and biogeographic patterns in a marine coastal isopod with an amphi-American distribution.

Excirolana braziliensis is a coastal intertidal isopod with a broad distribution spanning the Atlantic and Pacific tropical and temperate coasts of the American continent. Two separate regional studies (one in Panama and one in Chile) revealed the presence of highly genetically divergent lineages, implying that this taxon constitutes a cryptic species complex. The relationships among the lineages found in these two different regions and in the rest of the distribution, however, remain unknown. To better understand the phylogeographic patterns of E. braziliensis, we conducted phylogenetic analyses of specimens from much of its entire range. We obtained DNA sequences for fragments of four mitochondrial genes (16S rDNA, 12S rDNA, COI, and Cytb) and also used publicly available sequences. We conducted maximum likelihood and Bayesian phylogenetic reconstruction methods. Phylogeographic patterns revealed the following: (1) new highly divergent lineages of E. braziliensis; (2) three instances of Atlantic-Pacific divergences, some of which appear to predate the closure of the Isthmus of Panama; (3) the distributional limit of highly divergent lineages found in Brazil coincides with the boundary between two major marine coastal provinces; (4) evidence of recent long-distance dispersal in the Caribbean; and (5) populations in the Gulf of California have closer affinities with lineages further south in the Pacific, which contrasts with the closer affinity with the Caribbean reported for other intertidal organisms. The high levels of cryptic diversity detected also bring about challenges for the conservation of this isopod and its fragile environment, the sandy shores. Our findings underscore the importance of comprehensive geographic sampling for phylogeographic and taxonomical studies of broadly distributed putative species harboring extensive cryptic diversity.