Marco Gebiola

Integration of molecular, ecological, morphological and endosymbiont data for species delimitation within the Pnigalio soemius complex (Hymenoptera: Eulophidae)

Integrative taxonomy is a recently developed approach that uses multiple lines of evidence such as molecular, morphological, ecological and geographical data to test species limits, and it stands as one of the most promising approaches to species delimitation in taxonomically difficult groups. The Pnigalio soemius complex (Hymenoptera: Eulophidae) represents an interesting taxonomical and ecological study case, as it is characterized by a lack of informative morphological characters, deep mitochondrial divergence, and is susceptible to infection by parthenogenesis‐inducing Rickettsia. We tested the effectiveness of an integrative taxonomy approach in delimiting species within the P. soemius complex. We analysed two molecular markers (COI and ITS2) using different methods, performed multivariate analysis on morphometric data and exploited ecological data such as host–plant system associations, geographical separation, and the prevalence, type and effects of endosymbiont infection. The challenge of resolving different levels of resolution in the data was met by setting up a formal procedure of data integration within and between conflicting independent lines of evidence. An iterative corroboration process of multiple sources of data eventually indicated the existence of several cryptic species that can be treated as stable taxonomic hypotheses. Furthermore, the integrative approach confirmed a trend towards host specificity within the presumed polyphagous P. soemius and suggested that Rickettsia could have played a major role in the reproductive isolation and genetic diversification of at least two species.

Genetic Diversity of the Invasive Gall Wasp Leptocybe invasa (Hymenoptera: Eulophidae) and of its Rickettsia Endosymbiont, and Associated Sex-Ratio Differences.

The blue-gum chalcid Leptocybe invasa Fisher & LaSalle (Hymenoptera: Eulophidae) is a gall wasp pest of Eucalyptus species, likely native to Australia. Over the past 15 years it has invaded 39 countries on all continents where eucalypts are grown. The worldwide invasion of the blue gum chalcid was attributed to a single thelytokous morphospecies formally described in 2004. Subsequently, however, males have been recorded in several countries and the sex ratio of field populations has been found to be highly variable in different areas. In order to find an explanation for such sex ratio differences, populations of L. invasa from a broad geographical area were screened for the symbionts currently known as reproductive manipulators, and both wasps and symbionts were genetically characterized using multiple genes. Molecular analyses suggested that L. invasa is in fact a complex of two cryptic species involved in the rapid and efficient spread of the wasp, the first recovered from the Mediterranean region and South America, the latter from China. All screened specimens were infected by endosymbiotic bacteria belonging to the genus Rickettsia. Two closely related Rickettsia strains were found, each infecting one of the two putative cryptic species of L. invasa and associated with different average sex ratios. Rickettsia were found to be localized in the female reproductive tissues and transovarially transmitted, suggesting a possible role of Rickettsia as the causal agent of thelytokous parthenogenesis in L. invasa. Implications for the variation of sex ratio and for the management of L. invasa are discussed.

Cophylogenetic relationships between Anicetus parasitoids (Hymenoptera: Encyrtidae) and their scale insect hosts (Hemiptera: Coccidae)

BackgroundNumerous studies have investigated cospeciation between parasites and their hosts, but there have been few studies concerning parasitoids and insect hosts. The high diversity and host specialization observed in Anicetus species suggest that speciation and adaptive radiation might take place with species diversification in scale insect hosts. Here we examined the evolutionary history of the association between Anicetus species and their scale insect hosts via distance-based and tree-based methods.ResultsA total of 94 Anicetus individuals (nine parasitoid species) and 113 scale insect individuals (seven host species) from 14 provinces in China were collected in the present study. DNA sequence data from a mitochondrial gene (COI) and a nuclear ribosomal gene (28S D2 region) were used to reconstruct the phylogenies of Anicetus species and their hosts. The distance-based analysis showed a significant fit between Anicetus species and their hosts, but tree-based analyses suggested that this significant signal could be observed only when the cost of host-switching was high, indicating the presence of parasite sorting on related host species.ConclusionsThis study, based on extensive rearing of parasitoids and species identification, provides strong evidence for a prevalence of sorting events and high host specificity in the genus Anicetus, offering insights into the diversification process of Anicetus species parasitizing scale insects.

A revision of the Encarsia pergandiella species complex (Hymenoptera: Aphelinidae) shows cryptic diversity in parasitoids of whitefly pests

Encarsia pergandiella Howard, described from North America (USA), and Encarsia tabacivora Viggiani, described from South America (Brazil) (Hymenoptera: Aphelinidae), are two formally recognized taxonomic entities, that have been treated by several authors as synonyms due to lack of strong diagnostic characters. Taxonomy of these species is further complicated because several populations, geographically separated and differing in their biology, have been included under the concept of E. pergandiella. Among these, a population originally collected in Brazil and introduced to North America reproduces by thelytokous parthenogenesis and is infected by the symbiont Cardinium, while a morphologically indistinguishable population, naturally occurring in Texas, is biparental and infected by a related strain of Cardinium that induces cytoplasmic incompatibility. A third population known from California and introduced to the Old World is biparental and uninfected by intracellular symbionts. While adult females of the first two populations have entirely light yellow bodies and pupate face up (light form), those of the third population have largely brown bodies and pupate face down (dark form). Other dark form populations are known from Texas, Florida and New York. Because these parasitoids are economically important biological control agents of cosmopolitan whitefly pests, it is critical to characterize them correctly. In this study, we integrated molecular and morphometric analyses to substantiate observed differences in biological traits, and resolve the complicated taxonomy of this species complex. We sequenced the mitochondrial cytochrome c oxidase subunit I gene and the D2 region of the ribosomal 28S gene for individuals of both light form (from Texas and Brazil) and dark form (from California, Texas, Italy and Canary Islands) originating from laboratory cultures or collected in the field. Phylogenetic analysis unambiguously distinguished three well‐supported groups corresponding to the Texas light form, the Brazil light form and the dark form. Individuals of these three groups, in combination with all available type material (E. pergandiella, its synonym Encarsia versicolor Girault and E. tabacivora) and additional museum specimens of the dark form from New York and Italy, were subjected to multivariate morphometric analyses using Burnaby principal component analysis followed by a linear discriminant analysis, and multivariate ratio analysis. Overall, the analyses showed that: (i) E. pergandiella and E. tabacivora are two distinct species; (ii) the thelytokous Brazil light form corresponds to E. tabacivora; (iii) the biparental Texas light form is a new species formally described here as Encarsia suzannae sp.n.; (iv) two new biparental species can be referred to the dark form, one described as Encarsia gennaroi sp.n. including the populations sampled in California, Texas, Italy and Canary Islands, and the other corresponding to the population from New York described as Encarsia marthae sp.n. A dichotomous key for both sexes of the species of the E. pergandiella complex is provided for identification.

“Darwin's corollary” and cytoplasmic incompatibility induced by Cardinium may contribute to speciation in Encarsia wasps (Hymenoptera: Aphelinidae)

The potential importance of cytoplasmic incompatibility (CI)‐inducing bacterial symbionts in speciation of their arthropod hosts has been debated. Theoretical advances have led to a consensus that a role is plausible when CI is combined with other isolating barriers. However, the insect model systems Nasonia and Drosophila are the only two experimental examples documented. Here, we analyzed the components of reproductive isolation between the parasitoid wasp Encarsia suzannae, which is infected by the CI‐inducing symbiont Cardinium, and its uninfected sibling species Encarsia gennaroi. Laboratory crosses demonstrated that: (1) sexual isolation is incomplete; (2) hybrid offspring production is greatly reduced in the interspecific CI cross; (3) viable hybrids may be produced by curing E. suzannae males of Cardinium with antibiotics; (4) hybrid offspring production in the reciprocal cross is greatly reduced by hybrid inviability due to genetic incompatibilities; (5) hybrid sterility is nearly complete in both directions at the F1 stage. Thus, asymmetrical hybrid incompatibilities and CI act as complementary isolating mechanisms. We propose a new model for contributions of CI symbionts to speciation, with CI reducing gene flow between species in one direction, and in the other, a symbiont sweep resulting in accelerated mtDNA evolution, negative cytonuclear interactions, and hybrid incompatibilities.

Description of Synergus castaneus n. sp. (Hymenoptera: Cynipidae: Synergini) Associated with an Unknown Gall on Castanea spp. (Fagaceae) in China

ABSTRACT A species of the tribe Synergini (Hymenoptera: Cynipidae) was reared from a gall collected in China on Castanea mollissima Blume and Castanea seguinii Dode. The morphological features of this gall wasp indicate that it is an undescribed species belonging to the genus Synergus and is herein described as a new species, Synergus castaneus Pujade-Villar, Bernardo et Viggiani sp. nov. (Hymenoptera Cynipidae: Synergini). This is the first known instance of a species of Synergus emerging from Castanea galls (Fagaceae). Data on the diagnosis, distribution, and biology of the new species are given. Morphological features of this species are discussed and illustrated. No other known gall-inducing species emerged from the collected galls, whereas tens of specimens of parasitoids and adults of the inquiline micromoth Andrioplecta pulverula Meyrick (Lepidoptera: Tortricidae) did. 28S-D2 and COI sequences confirmed that S. castaneus is a distinct species belonging to the genus Synergus.

Cytological analysis of cytoplasmic incompatibility induced by Cardinium suggests convergent evolution with its distant cousin Wolbachia

Cytoplasmic incompatibility (CI) is a conditional sterility in numerous arthropods that is caused by inherited, intracellular bacteria such as Wolbachia. Matings between males carrying CI-inducing Wolbachia and uninfected females, or between males and females infected with different Wolbachia strains, result in progeny that die during very early embryogenesis. Multiple studies in diploid (Drosophila) and haplodiploid (Nasonia) insects have shown that CI-Wolbachia cause a failure of the paternally derived chromatin from resolving into distinct chromosomes. This leads to the formation of chromatin bridges and other mitotic defects as early as the first mitotic division, and to early mitotic arrest. It is currently unknown if CI-inducing symbionts other than Wolbachia affect similar cellular processes. Here, we investigated CI caused by an unrelated bacterium, Cardinium, which naturally infects a parasitic wasp, Encarsia suzannae. CI crosses in this host–symbiont system resulted in early mitotic defects including asynchrony of paternal and maternal chromosome sets as they enter mitosis, chromatin bridges and improper chromosome segregation that spanned across multiple mitotic divisions, triggering embryonic death through accumulated aneuploidy. We highlight small differences with CI-Wolbachia, which could be due to the underlying CI mechanism or host-specific effects. Our results suggest a convergence of CI-related cellular phenotypes between these two unrelated symbionts.

Life inside a gall: closeness does not favour horizontal transmission of Rickettsia between a gall wasp and its parasitoid

The incidence of horizontal transmission as a route for spreading symbiont infections is still being debated, but a common view is that horizontal transfers require intimate between-species relationships. Here we study a system that meets ideal requirements for horizontal transmission: the gall wasp Leptocybe invasa and its parasitoid Quadrastichus mendeli (Hymenoptera: Eulophidae). These wasps belong to the same subfamily, spend most of their lives inside the same minute gall and are both infected by Rickettsia, a maternally inherited endosymbiotic bacteria that infects several arthropods, sometimes manipulating their reproduction, like inducing thelytokous parthenogenesis in L. invasa. Despite intimate contact, close phylogenetic relationship and the parasitoids host specificity, we show that host and parasitoid do not share the same Rickettsia. We provide indirect evidence that Rickettsia infecting Q. mendeli may be inducing thelytokous parthenogenesis, as the symbiont is densely present in the reproductive apparatus and is vertically transmitted. Phylogenetic analyses based on 16S and gltA placed this symbiont in the leech group. The confirmed and presumed parthenogenesis-inducing Rickettsia discovered so far only infect eulophid wasps, and belong to three different groups, suggesting multiple independent evolution of the parthenogenesis inducing phenotype. We also show some degree of cospeciation between Rickettsia and their eulophid hosts.

No evidence of parthenogenesis‐inducing bacteria involved in Thripoctenus javae thelytoky: an unusual finding in Chalcidoidea

All Hymenoptera have a haplodiploid mode of sex determination. Although most species reproduce by arrhenotokous parthenogenesis, there are many thelytokous species, in which unfertilized eggs develop into diploid females. Thelytoky can be genetic or due to microbial infection. In the large Chalcidoidea superfamily, thelytokous parthenogenesis is almost always associated with infection of endosymbionts of the genera Wolbachia, Cardinium, and Rickettsia. Thripoctenus javae (Girault) (Hymenoptera: Eulophidae) is a larval parasitoid of the greenhouse thrips Heliothrips haemorrhoidalis (Bouché) (Thysanoptera: Thripidae), an important worldwide pest. Both the host and its parasitoid reproduce by thelytokous parthenogenesis. The main goal of this study was to test whether endosymbiotic bacteria, either those known to induce thelytokous parthenogenesis or other sex‐manipulators, are responsible for thelytoky of two geographically distinct populations of T. javae. We used sequencing of ribosomal ITS2 and 28S‐D2 and mitochondrial COI genes to molecularly characterize the two populations, antibiotic and heat treatments, and FISH of ovaries, for thelytoky studies. It was impossible to revert thelytokous individuals back to sexual reproduction and no evidence of bacterial infection was found in parthenogenetic T. javae females. This makes T. javae the second chalcidoid in which thelytokous reproduction appears not to be associated with the presence of bacterial endosymbionts.

Molecular phylogenetic analyses and morphological variation point to taxonomic problems among four genera of parasitoid doryctine wasps (Hymenoptera : Braconidae)

Abstract. Doryctinae (Hymenoptera : Braconidae) is a large and diverse subfamily of parasitic wasps that has received much attention recently, with new species and genera described and phylogenies based on morphological and/or molecular data that have improved higher-level classification and species delimitation. However, the status of several genera is still unresolved, if not controversial. Here we focus on two related groups of such genera, Dendrosoter Wesmael–Caenopachys Foerster and Ecphylus Foerster–Sycosoter Picard & Lichtenstein. We integrated morphological and molecular (COI and 28S–D2 genes) evidence to highlight, by phylogenetic analyses (maximum likelihood and Bayesian) and a posteriori morphological examination, previously overlooked variation, which is here illustrated and discussed. Monophyly of Dendrosoter and Caenopachys and the presence of synapomorphic morphological characters support synonymy of Caenopachys under Dendrosoter. Low genetic differentiation and high variability for putatively diagnostic morphological characters found in both C. hartigii (Ratzeburg) and C. caenopachoides (Ruschka) supports synonymy of D. caenopachoides under D. hartigii, syn. nov. Morphological and molecular evidence together also indicate independent generic status for Sycosoter, stat. rev., which is here resurrected. This work represents a further advancement in the framework of the ongoing effort to improve systematics and classification of the subfamily Doryctinae.