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Capturing phylogenetic signal from a massive radiation can be daunting. The superfamily Chalcidoidea is an excellent example of a hyperdiverse group that has remained recalcitrant to phylogenetic resolution. Chalcidoidea are mostly parasitoid wasps that until now included 27 families, 87 subfamilies and as many as 500,000 estimated species. We combined 1007 exons obtained with Anchored Hybrid Enrichment with 1048 Ultra-Conserved Elements (UCEs) for 433 taxa including all extant families, over 95% of all subfamilies and 356 genera chosen to represent the vast diversity of the superfamily. Going back and forth between molecular results and our collective morphological and biological knowledge, we detected insidious bias driven by the saturation of nucleotide data and highlighted morphological convergences. Our final results are based on a concatenated analysis of the least saturated exons and UCE data sets (2054 loci, 284,106 sites). Our analyses support a sister relationship with Mymarommatoidea. Seven of the previously recognized families were not monophyletic, so foundations for a new classification are discussed. Biology appears potentially more informative than morphology, as illustrated by the elucidation of a clade of plant gall associates and a clade of taxa with planidial first-instar larvae. The phylogeny suggests a shift from smaller soft-bodied wasps to larger and more heavily sclerotized wasps. Deep divergences in Chalcidoidea coincide with an increase in insect families in the fossil record, and an early shift to phytophagy corresponds with the beginning of the “Angiosperm Terrestrial Revolution”. Our dating analyses suggest a Middle Jurassic origin of 174 Ma (167.3-180.5 Ma) and a crown age of 162.2 Ma (153.9–169.8 Ma) for Chalcidoidea. During the Cretaceous, Chalcidoidea underwent a rapid radiation in southern Gondwana with subsequent dispersals to the Northern Hemisphere. This scenario is discussed with regard to knowledge about host taxa of chalcid wasps, their fossil record, and Earth’s paleogeographic history.

Monitoring of plant pathogens is usually carried out by analyzing symptomatic plant material. However when plants are frequently asymptomatic, which is the case for Xylella fastidiosa (Xf), this approach is problematic because it limits our ability to early detect the bacterium hence to understand and control its spread. To complement plant survey and facilitate large-scale monitoring, we propose to generalize the use of insect vectors as sentinels. We tested this approach in Corsica using Philaenus spumarius, an ubiquitous insect vector, as sentinel. We will present a high throughput method based on a two-step PCR approach followed by Illumina sequencing. Two genes of the MLST of Xf were targeted to identify the three main subspecies of Xf (multiplex, pauca or fastidiosa). This approach enables larger screening than classic (nested) PCR approaches followed by Sanger sequencing. We used this approach to monitor the spatio-temporal prevalence of Xf within vector populations (27 populations of 30 specimens). Our results show that Xf occurs throughout Corsica with a prevalence in insects up to 43% depending on the region and the year of sampling. Moreover, we show that Xf is present in ecosystems assumed uncontaminated on the basis of plant monitoring. No spatial, temporal or vegetation-related pattern of Xf prevalence in insects seem to emerge from our first data. Our results confirm that Xf introduction in Corsica likely predates 2015, it suggests ecological resilience of ecosystems and argues in favor of using insect vectors for Xf survey at large scale in Europe., FR; PPT; pauline.farigoule@inrae.fr

Philaenus spumarius (Hemiptera: Aphrophoridae) is a major vector of Xylella fastidiosa (Xf) in Europe. However, the drivers influencing its abundance are still poorly known. In Corsica from 2018 to 2020, we monitored the abundance of P. spumarius three times a year in 64 permanent plots distributed along vegetation and altitudinal (0-600m) gradients. The vegetation gradient ranged from shrubland to oak forest and accounted for the density of Cistus monspeliensis on which P. spumarius usually develops and feed in Corsica. Abundance of P. spumarius was estimated through visual counting of foams and sweep netting of adults. Both vegetation composition and climate descriptors were recorded at each sampling location. We used partial least squares path modelling and generalized linear mixed models to unravel the relationships between vegetation / climate descriptors and P. spumarius abundance. Philaenus spumarius abundance significantly decreases with increasing average temperature. Abundance of P. spumarius was significantly and positively correlated with C. monspeliensis density, confirming difference in host plant preference between Corsica and mainland France. A significant effect of vegetation structure was also found with forests hosting lower P. spumarius abundance. The strong association observed between P. spumarius and C. monspeliensis suggests that Xf expansion may have been facilitated by firebreaks or disturbed habitats where C. monspeliensis is abundant as an early colonizer. Managing C. monspeliensis in the vicinity of agricultural areas may constitute an essential component of prophylaxis against Xf in Corsica.

Identification of adult vectors of Xylella fastidiosa (Xf) based on morphological characters can be difficult for non-specialists. The identification of eggs or larvae is even more difficult and sometimes impossible. To circumvent these difficulties and enable large survey of populations of insect vectors in Europe we have developed a web-interface database of COI barcodes. COI barcodes were generated with a high-throughput approach involving two PCR steps to target COI and index samples followed by Illumina sequencing. Multiple quality controls were implemented including i) non-destructive DNA extraction that allows vouchering and morphological re-examination by taxonomists; ii) bioinformatic controls to discards possible contaminants, PCR or sequencing errors, NUMTs and heteroplasms. All barcodes have been uploaded in a web-interface database (Arthemis DB@se: http://arthemisdb.supagro.inra.fr/DefaultInfo.aspx?Page=Home). Metadata associated with all records are available online, as well as biological data on vectors and pictures. Users can identify a query sequence through BLAST comparison against our reference library. Currently, the database hosts 636 barcodes for 72 species of potential vectors of Xf: 490 sequences representing 43 European species and 145 sequences representing 29 non-European species. All frequent species have been barcoded. Missing species are rare or localized and do not represent an important threat for European agriculture. COI allows reliable identification for 80% of the species, but we observed issues for the remaining 20%. The most important issue concerns Philaenus spumarius and its close relative P. tesselatus. These two species are hardly distinguishable even with male genitalia and share the same COI sequence. It was also difficult to cross validate morphological and molecular results for some species within the genera Neophilaenus and Aphrophora., FR; PPT; jean-claude.streito@inrae.fr

In Europe, many potential and confirmed vectors of Xylella fastidiosa (Xf) are polyphagous but appear to aggregate on their preferred host plants at local scales. For example, Philaenus spumarius is found most often on Asteraceae on the Californian coast, or on Apiaceae and Rubiaceae in the meadows of central Italy. However, vectors keep the ability to exploit multiple host-plants, which can be crucial in primary Xf transmission to crops. In this study, we assessed the habitat preferences of spittlebugs on and in the vicinity of Corsican clementine and olive groves, focusing on four habitats: Cistus monspeliensis border, Dittrichia viscosa cover, crop foliage and grove ground vegetation. Spittlebug abundance was assessed three times a year during two years on five to nine clementine and olive organic groves. Nymph and adult abundances were quantified based on spittle mass counts and nymph identification in April and sweep net sampling in June and October. Habitat preferences were inferred based on generalized linear mixed models. Overall, 6647 spittle masses and 1714 adults belonging to four species were found. Philaenus spumarius had a significant preference for Cistus monspeliensis, and to a lesser extent for Dittrichia viscosa. Neophilaenus campestris mostly occurred on ground vegetation of the grove and on Dittrichia viscosa. Lepyronia coleoptrata was less abundant on crop foliage than on ground vegetation and Aphrophora alni showed an even abundance in all habitats. This study demonstrates the specialization of P. spumarius on C. monspeliensis in the diversified Corsican landscape as well as strong habitat preferences for N. campestris. All recorded species were able to colonize clementine or olive foliage, but rarely, and in similar abundancies for all species. Should they be proven equivalent effective vectors, this suggests similar roles of these four species in Xf transmission to crops.

Fastq paired reads for analyzed samples are available as a NCBI Sequence Read Archive (ID#PRJNA606284). Custom script to detect cross-contamination is available from https://github.com/DNAdiversity/UCE-Cross-Contamination-Check.Datasets (morphological matrix, concatenated UCEs) and trees have been uploaded on Zenodo (https://doi.org/10.5281/zenodo.3666638).; International audience; Recent technical advances combined with novel computational approaches have promised the acceleration of our understanding of the tree of life. However, when it comes to hyperdiverse and poorly known groups of invertebrates, studies are still scarce. As published phylogenies will be rarely challenged by future taxonomists, careful attention must be paid to potential analytical bias. We present the first molecular phylogenetic hypothesis for the family Chalcididae, a group of parasitoid wasps, with a representative sampling (144 ingroups and seven outgroups) that covers all described subfamilies and tribes, and 82% of the known genera. Analyses of 538 Ultra-Conserved Elements (UCEs) with supermatrix (RAxML and IQTREE) and gene tree reconciliation approaches (ASTRAL, ASTRID) resulted in highly supported topologies in overall agreement with morphology but reveal conflicting topologies for some of the deepest nodes. To resolve these conflicts, we explored the phylogenetic tree space with clustering and gene genealogy interrogation methods, analyzed marker and taxon properties that could bias inferences and performed a thorough morphological analysis (130 characters encoded for 40 taxa representative of the diversity). This joint analysis reveals that UCEs enable attainment of resolution between ancestry and convergent/divergent evolution when morphology is not informative enough, but also shows that a systematic exploration of bias with different analytical methods and a careful analysis of morphological features is required to prevent publication of artifactual results. We highlight a GC content bias for maximum-likelihood approaches, an artifactual mid-point rooting of the ASTRAL tree and a deleterious effect of high percentage of missing data (>85% missing UCEs) on gene tree reconciliation methods. Based on the results we propose a new classification of the family into eight subfamilies and ten tribes that lay the foundation for future studies on the evolutionary history of Chalcididae.

In Europe, many potential and confirmed vectors of Xylella fastidiosa (Xf) are polyphagous but appear to aggregate on their preferred host plants at local scales. For example, Philaenus spumarius is found most often on Asteraceae on the Californian coast, or on Apiaceae and Rubiaceae in the meadows of central Italy. However, vectors keep the ability to exploit multiple host-plants, which can be crucial in primary Xf transmission to crops. In this study, we assessed the habitat preferences of spittlebugs on and in the vicinity of Corsican clementine and olive groves, focusing on four habitats: Cistus monspeliensis border, Dittrichia viscosa cover, crop foliage and grove ground vegetation. Spittlebug abundance was assessed three times a year during two years on five to nine clementine and olive organic groves. Nymph and adult abundances were quantified based on spittle mass counts and nymph identification in April and sweep net sampling in June and October. Habitat preferences were inferred based on generalized linear mixed models. Overall, 6647 spittle masses and 1714 adults belonging to four species were found. Philaenus spumarius had a significant preference for Cistus monspeliensis, and to a lesser extent for Dittrichia viscosa. Neophilaenus campestris mostly occurred on ground vegetation of the grove and on Dittrichia viscosa. Lepyronia coleoptrata was less abundant on crop foliage than on ground vegetation and Aphrophora alni showed an even abundance in all habitats. This study demonstrates the specialization of P. spumarius on C. monspeliensis in the diversified Corsican landscape as well as strong habitat preferences for N. campestris. All recorded species were able to colonize clementine or olive foliage, but rarely, and in similar abundancies for all species. Should they be proven equivalent effective vectors, this suggests similar roles of these four species in Xf transmission to crops., FR; PPT; xavier.mesmin@inrae.fr

Philaenus spumarius (Hemiptera: Aphrophoridae) is a major vector of Xylella fastidiosa (Xf) in Europe. However, the drivers influencing its abundance are still poorly known. In Corsica from 2018 to 2020, we monitored the abundance of P. spumarius three times a year in 64 permanent plots distributed along vegetation and altitudinal (0-600m) gradients. The vegetation gradient ranged from shrubland to oak forest and accounted for the density of Cistus monspeliensis on which P. spumarius usually develops and feed in Corsica. Abundance of P. spumarius was estimated through visual counting of foams and sweep netting of adults. Both vegetation composition and climate descriptors were recorded at each sampling location. We used partial least squares path modelling and generalized linear mixed models to unravel the relationships between vegetation / climate descriptors and P. spumarius abundance. Philaenus spumarius abundance significantly decreases with increasing average temperature. Abundance of P. spumarius was significantly and positively correlated with C. monspeliensis density, confirming difference in host plant preference between Corsica and mainland France. A significant effect of vegetation structure was also found with forests hosting lower P. spumarius abundance. The strong association observed between P. spumarius and C. monspeliensis suggests that Xf expansion may have been facilitated by firebreaks or disturbed habitats where C. monspeliensis is abundant as an early colonizer. Managing C. monspeliensis in the vicinity of agricultural areas may constitute an essential component of prophylaxis against Xf in Corsica., FR; PPT; marguerite.chartois@inrae.fr