Restriction-site associated DNA markers provide new insights into the evolutionary history of the bark beetle genus Dendroctonus.

The bark beetle genus Dendroctonus contains some of the most economically important pests of conifers worldwide. Despite many attempts, there is no agreement today on the phylogenetic relationships within the genus, which limits our understanding of its evolutionary history. Here, using restriction-site associated DNA (RAD) markers from 70 specimens representing 17 species (85% of the known diversity) we inferred the phylogeny of the genus, its time of origin and biogeographic history, as well as the evolution of key ecological traits (host plants, larval behavior and adults' attack strategies). For all combinations of tested parameters (from 6444 to 23,570 RAD tags analyzed), the same, fully resolved topology was inferred. Our analyses suggest that the most recent common ancestor (mrca) of all extant Dendroctonus species was widely distributed across eastern Palearctic and western Nearctic during the early Miocene, from where species dispersed to other Holarctic regions. A first main inter-continental vicariance event occurred during early Miocene isolating the ancestors of D. armandi in the Palearctic, which was followed by the radiation of the main Dendroctonus lineages in North America. During the Late Miocene, the ancestor of the 'rufipennis' species group colonized north-east Palearctic regions from western North America, which was followed by a second main inter-continental vicariance event isolating Pleistocene populations in Asia (D. micans) and western North America (D. murrayanae and D. punctatus). The present study supports previous hypotheses explaining intercontinental range disjunctions across the Northern Hemisphere by the fragmentation of a continuous distribution due to climatic cooling, host range fragmentation and geological changes during the late Cenozoic. The reconstruction of ancestral ecological traits indicates that the mrca bored individual galleries and mass attacked the boles of pines. The gregarious feeding behavior of the larvae as well as the individual attack of the base of trees have apparently independently evolved twice in North America (in the 'rufipennis' and the 'valens' species groups), which suggests a higher adaptive potential than previously thought and may be of interest for plant protection and biodiversity conservation in a rapidly changing world.

Torymidae (Hymenoptera, Chalcidoidea) revised: molecular phylogeny, circumscription and reclassification of the family with discussion of its biogeography and evolution of life-history traits.

A phylogeny of the Torymidae (Chalcidoidea) is estimated using 4734 nucleotides from five genes. Twelve outgroups and 235 ingroup taxa are used, representing about 70% of the recognized genera. Our analyses do not recover Torymidae as monophyletic and we recognize instead two families: Megastigmidae (stat. rev.) and Torymidae s.s. (stat. rev.). Within Torymidae s.s., we recognize six subfamilies and six tribes, including Chalcimerinae, Glyphomerinae and Microdontomerinae (subf. nov.), and two new tribes: Boucekinini and Propalachiini (trib. nov.). Seven unclassified genera (i.e. Cryptopristus, Echthrodape, Exopristoides, Exopristus, part of Glyphomerus, Thaumatorymus, Zaglyptonotus) are assigned to tribes within our new classification. Five genera are restored from synonymy-Ameromicrus and Didactyliocerus from under Torymoides (stat. rev.), Iridophaga and Iridophagoides from under Podagrionella (stat. rev.) and Nannocerus from under Torymus (stat. rev.)-and three genera are synonymized-Allotorymus under Torymussyn. nov., Ditropinotus under Eridontomerussyn. nov. and Pseuderimerus under Erimerussyn. nov. A Palaearctic or Eurasian origin for Torymidae is proposed. The ancestral area of Megastigmidae is indicated as the Australian region. The most probable ancestral life strategy for Torymidae s.s. is ectoparasitism on gall-forming Cynipidae. The life strategy and putative hosts of the common ancestor of Megastigmidae remain uncertain.

Evolution, systematics and historical biogeography of sand flies of the subgenus Paraphlebotomus (Diptera, Psychodidae, Phlebotomus) inferred using restriction-site associated DNA markers.

Phlebotomine sand flies are the main natural vectors of Leishmania, which cause visceral and tegumentary tropical diseases worldwide. However, their taxonomy and evolutionary history remain poorly studied. Indeed, as for many human disease vectors, their small size is a challenge for morphological and molecular works. Here, we successfully amplified unbiased copies of whole genome to sequence thousands of restriction-site associated DNA (RAD) markers from single specimens of phlebotomines. RAD markers were used to infer a fully resolved phylogeny of the subgenus Paraphlebotomus (11 species + 5 outgroups, 32 specimens). The subgenus was not recovered as monophyletic and we describe a new subgenus Artemievus subg. nov. Depaquit for Phlebotomus alexandri. We also confirm the validity of Ph. riouxi which is reinstated as valid species. Our analyses suggest that Paraphlebotomus sensu nov. originated ca 12.9-8.5 Ma and was possibly largely distributed from peri-Mediterranean to Irano-Turanian regions. Its biogeographical history can be summarized into three phases: i) a first split between Ph. riouxi + Ph. chabaudi and other species that may have resulted from the rise of the Saharan belt ca 8.5 Ma; ii) a Messinian vicariant event (7.3-5.3 Ma) during which the prolonged drought could have resulted in the divergence of main lineages; iii) a recent radiation event (3-2 Ma) that correspond to cycles of wet and dry periods in the Middle East and the East African subregions during the Pleistocene. Interestingly these cycles are also hypothetical drivers of the diversification of rodents, in the burrows of which Paraphlebotomus larvae develop. By meeting the challenge of sequencing pangenomics markers from single, minute phlebotomines, this work opens new avenues for improving our understanding of the epidemiology of leishmaniases and possibly other human diseases transmitted by arthropod vectors.

Ooctonus vulgatus (Hymenoptera, Mymaridae), a potential biocontrol agent to reduce populations of Philaenus spumarius (Hemiptera, Aphrophoridae) the main vector of Xylella fastidiosa in Europe.

As a vector of Xylella fastidiosa (Wells, 1987) in Europe, the meadow spittlebug Philaenus spumarius (Linnaeus, 1758) (Hemiptera, Aphrophoridae) is a species of major concern. Therefore, tools and agents to control this ubiquitous insect that develops and feeds on hundreds of plant species are wanted. We conducted a field survey of P. spumarius eggs in Corsica and provide a first report of Ooctonus vulgatus Haliday, 1833 (Hymenoptera, Mymaridae) as a potential biocontrol agent of P. spumarius in Europe. To allow species identification, we summarized the main characters distinguishing O. vulgatus from other European species of Ooctonus and generated COI DNA barcodes. Parasitism rates were variable in the four localities included in the survey but could reach 69% (for an average number of eggs that hatched per locality of 109). Based on the geographic occurrences of O. vulgatus obtained from the literature, we calibrated an ecological niche model to assess its potential distribution in the Holarctic. Obviously, several questions need to be addressed to determine whether O. vulgatus could become an effective biocontrol agent of P. spumarius in Europe. So far, O. vulgatus has been reared only from P. spumarius eggs, but its exact host-range should be evaluated to ensure efficiency and avoid non-target effect. The top-down impact of the parasitoid on vector populations should also be assessed on large data sets. Finally, the feasibility of mass rearing should be tested. We hope this report serves as a starting point to initiate research on this parasitoid wasp to assess whether it could contribute to reduce the spread and impact of X. fastidiosa in Europe.

Pushing the limits of whole genome amplification: successful sequencing of RADseq library from a single microhymenopteran (Chalcidoidea, Trichogramma).

A major obstacle to high-throughput genotyping of microhymenoptera is their small size. As species are difficult to discriminate, and because complexes may exist, the sequencing of a pool of specimens is hazardous. Thus, one should be able to sequence pangenomic markers (e.g., RADtags) from a single specimen. To date, whole genome amplification (WGA) prior to library construction is still a necessity as at most 10 ng of DNA can be obtained from single specimens (sometimes less). However, this amount of DNA is not compatible with manufacturer's requirements for commercial kits. Here we test the accuracy of the GenomiPhi kit V2 on Trichogramma wasps by comparing RAD libraries obtained from the WGA of single specimens (F0 and F1 generation, about1 ng input DNA for the WGA (0.17-2.9 ng)) and a biological amplification of genomic material (the pool of the progeny of the F1 generation). Globally, we found that 99% of the examined loci (up to 48,189 for one of the crosses, 109 bp each) were compatible with the mode of reproduction of the studied model (haplodiploidy) and Mendelian inheritance of alleles. The remaining 1% (0.01% of the analysed nucleotides) could represent WGA bias or other experimental/analytical bias. This study shows that the multiple displacement amplification method on which the GenomiPhi kit relies, could also be of great help for the high-throughput genotyping of microhymenoptera used for biological control, or other organisms from which only a very small amount of DNA can be extracted, such as human disease vectors (e.g.,  sandflies, fleas, ticks etc.).