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FIGS 3-5 in The first leucospid wasp from the fossil record (Hymenoptera: Leucospidae)

FIGS 3-5. Female holotype of Leucospis glaesaria; setae and sculpturing omitted. (3) Face, frontal view; antennae omitted except for the position of one scape. (4) Metasomal apex. (5) Hind leg. Scale bars= 1 mm; ep, epimerum; stippled outline indicates impunctate area on metacoxa.

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FIGS 1, 2 in The first leucospid wasp from the fossil record (Hymenoptera: Leucospidae)

FIGS 1, 2. Photomicrographs of female holotype of Leucospis glaesaria. (1) Lateral habitus. (2) Detailed lateral view showing surface sculpturing.

Fig. 2 in The Chalcidoidea bush of life: evolutionary history of a massive radiation of minute wasps

Fig. 2. Overview of the topologies obtained with the different datasets as saturation decreases. Datasets (AHE414 and UCEs) are described in Table 1, and trees are available in Fig. S1 and Appendix S1. Groups that are discussed in text are highlighted. Only IQ-TREE trees are shown. ROTO/BAEO = Rotoitidae (Baeomorphidae); CHAL = Chalcididae; EURY = Eurytomidae; GALL = gall clade (see text); MYMA = Mymaridae; PTERO = group of Pteromalid wasps (Austroterobiinae; part Colotrechninae; Miscogastrinae; part Ormocerinae; Otitesellinae; Pteromalinae; Sycoecinae; Sycoryctinae); "Tiny Wasp clade" (see text).

Fig. 3 in The Chalcidoidea bush of life: evolutionary history of a massive radiation of minute wasps

Fig. 3. Collapsed summary cladograms. Combined AHE (exonsAA) and UCE (UCEs90-25) results for 433 taxa, 2054 loci including 103 395 AA +180 711 nucleotide sites. Results of IQ-TREE concatenated analysis with one partition for each type of data (exonsAA vs. UCEs90-25). SHaLRT/UFBoot/gCF/sCF are indicated at nodes. Clades were collapsed to higher level groups (family, subfamily, tribe). Colours are meant to allow for comparisons between trees. Vertical bars represent similar clade relationships for the analyses of IQ-TREE: UCEs90-25 (UCE407), exonsAA (AHE414), AHE520AA; parsimony: combined (COM433), UCEs90-25 (UCE407), exonsAA (AHE414), AHE520AA; Munro et al. (2011; 720 taxa); Heraty et al. (2013; 300 taxa). Vertical red bars with an X were not recovered as monophyletic in that analysis. Faded colour bars represent that the clade was included but relationships alternated. P indicates paraphyletic lineages. Clades without an X or bar were supported; the lack of a bar indicates the clade was supported but the deeper relationships were not. Higher group names refer to the classification before Burks et al. (2022). Family abbreviations expanded in Table S1.

Fig. 1 in The Chalcidoidea bush of life: evolutionary history of a massive radiation of minute wasps

Fig. 1. Comparison of properties of the analysed datasets. Datasets (AHE414 and UCEs) are described in Table 1. For each panel, letters above box plots reflect pairwise comparisons of marginal means estimated from the best-fit models; distributions sharing a letter do not differ significantly. Points: raw data (Table S2a). In (e), saturation was assessed by calculating the R2 of the linear regression of uncorrected p-distances against inferred distances in individual gene trees. Highest R2 are for least saturated loci. The scale of the Y axis is reversed to better show decrease in saturation. (f) The convergence of trees as saturation decreases. The Y axis shows the relative RF distance between pairs of trees obtained with either the combined exons or the combined UCEs. The X axis show the absolute value of the difference between the medians of the R2 of the linear regression of uncorrected p-distances against inferred distances in gene trees that were combined to get the compared trees [cf. (e)]. Four comparisons were performed in each case as datasets were analysed with and without partitioning.

Fig. 5 in The Chalcidoidea bush of life: evolutionary history of a massive radiation of minute wasps

Fig. 5. Global historical biogeography of Chalcidoidea and new classification. The chronogram obtained from the complete set of ingroup taxa is illustrated. The previous classification is used to annotate tips (four letter prefixes; see also Table S1 for complete information on sampling) with successive grey and white boxes grouping the tip labels. The new familial classification from Burks et al. (2022) is shown to the right. For clarity, ancestral ranges are given only up to family level and only for the BAYEAREALIKE + J model (which was selected by AICc). All inferences of ancestral ranges are provided in Fig. S5. Inferences of ancestral ranges were conducted with only one specimen per genus as shown with brackets that connect tips. Current distribution of genera is shown with coloured boxes at tips. Sampling area of specimens is indicated in tip labels. NEO = Neotropical; NEA = Nearctic; AFR = Afrotropical; PAL = Palaearctic; ORI = Oriental; AUS = Australasian. UKN = Unknown when collection data are unavailable. Stars indicate that specimens were sampled in areas where species was introduced or not yet cited. Sampling area for the specimen used for sequencing exons is listed first, sampling area for the specimen used for sequencing UCEs is listed second; n.a. is used when no specimen was sequenced and only one sampling area is reported when exons and UCEs were obtained from specimens sampled in the same areas (or from the same specimen). Unless specified, nodes are supported by SHaLRT ≥80%, UFBoot ≥95% and sCF ≥34.3 (minimum support for a family that is well defined morphologically, Trichogrammatidae). Nodes with a grey circle are supported by SHaLRT <80% or UFBoot <95%; nodes with a black circle are supported by SHaLRT <80% and UFBoot <95%; nodes with a black triangle are supported with sCF <34.3. Images on the left of tentative family names are all at the same scale. Images on the right of tentative family names have been magnified. Photos ©K. Bolte (Baeomorphidae); ©J.-Y. Rasplus (all others).

Fig. 4 in The Chalcidoidea bush of life: evolutionary history of a massive radiation of minute wasps

Fig. 4. The Chalcidoidea bush of life. (a) IQ-TREE tree obtained from the combined exonsAA+UCEs90-25 datasets (see also Fig. S1). Monophyletic families are in grey, para- or polyphyletic families are in colour. Higher level groups/clades discussed in text are highlighted with boxes. Statistical support for backbone nodes are shown with single (SH-aLRT ≧80% or UFboot ≧95%) or double stars (SH-aLRT ≧80% and UFboot ≧95%). (b) Contribution of the exonsAA and UCEs90-25 datasets to the combined tree. Gene concordance factor (gCF); gene discordance factor due to polyphyly (gDFP); site concordance factor averaged over 100 quartets (sCF). Points: raw data (Table S2d). (c) Comparison of branch length for the backbone nodes and other ingroup nodes. Points: raw data (Table S2c). For (b) and (c), stars above box plots indicate statistical significance: ns, p> 0.05; ***, p ≤ 0.001; ****, p ≤ 0.0001. (d) Correlation between node age and sCF (outgroups excluded). Points: raw data (Table S2e); line: regression curve for the best-fit model (log linear model; p <2.2e—16).

Fig. 4 in Diversity, Host Ranges, and Potential Drivers of Speciation the Inquiline Enemies of Oak Gall Wasps (Hymenoptera: Cynipidae)

Fig. 4. Collection and emergence dates for each of the 27 clades (putative species) of Synergus in this study. Dots indicate individual Synergus emergences; left-most margins of boxes demarcate the earliest gall collection that produced Synergus in each clade. In three cases, boxes with different border styles are used to indicate collection events from galls that occur at different times during the year, and which may indicate the use of temporally distributed gall hosts. Though galls were sometimes collected in different years, each collection was standardized to the year in which the gall first formed. Dates span>1 yr because some insects did not emerge from galls until more than a year after galls were collected.

Fig. 5 in Diversity, Host Ranges, and Potential Drivers of Speciation the Inquiline Enemies of Oak Gall Wasps (Hymenoptera: Cynipidae)

Fig. 5. Collection dates, emergence dates, and gall morphologies of three species previously collected under the name Synergus laeviventris.The relationships (as implied by mtCOI) among the three species are shown in the tree at left. Red triangles denote collection dates for galls from which at least one S. laeviventris (black dots) later emerged, such that this figure allows for assessment of when S. laeviventris emerged relative to when their host galls were collected. Boxes are used to isolate distinct collection/eclosion relationships from one another, which we interpret as evidence for discrete generations. Gall wasp species are labeled above the photos of their respective galls (* indicates the photo is a cross-section). Dates are organized to reflect emergence of Synergus relative to when focal galls were formed by gall wasps, such that some Synergus emerge up to 1 yr after gall formation. Dates of eclosion are combined across the 3 yr of collection.

Fig. 1 in Diversity, Host Ranges, and Potential Drivers of Speciation the Inquiline Enemies of Oak Gall Wasps (Hymenoptera: Cynipidae)

Fig. 1. Map of gall collections that yielded Synergus samples used in this study. For a list of samples, locations, and tree and gall associations, see SuppTable 2 (online only).

Fig. 3 in Diversity, Host Ranges, and Potential Drivers of Speciation the Inquiline Enemies of Oak Gall Wasps (Hymenoptera: Cynipidae)

Fig. 3. Summary phylogenetic tree of Nearctic Synergus mtCOI sequences from this study (shown here collapsed into two clades) combined with Palearctic mtCOI sequences from Synergus and other inquilinous cynipids previously published in Ács et al. (2010). Values above the branches represent Bayesian posterior probabilities and the values below the branch are maximum likelihood bootstrap values. See Supp Figs. 48 and 49 (online only) for full trees.

Fig. 2 in Diversity, Host Ranges, and Potential Drivers of Speciation the Inquiline Enemies of Oak Gall Wasps (Hymenoptera: Cynipidae)

Fig. 2. Overview of all data used in inferring Synergus species hypotheses for this study.Left: simplified mtCOI phylogeny of Synergus included in this study (see Supp Fig. 47 [online only] for full tree). Bold branches indicate support ≥0.9. 'Clade' describes putative species assignments based on the sum of information to the right of this column. Gray bars in ABGD (conservative ['C'] and liberal ['L'] partitions) and bPTP columns indicate assignments of individuals into groups by these respective algorithms. 'Morphological ID' refers to each collection's similarity (or lack of similarity) to previously described species. 'Gall host', 'oak section', 'plant tissue(s)', and 'Host gall morphology' refer to ecological characters for Synergus in each clade, and example photos of galls are shown in Fig. 6.

Fig. 13 in Comparative anatomy of venom glands suggests a role of maternal secretions in gall induction by cynipid wasps (Hymenoptera: Cynipidae)

Fig. 13. Ultrastructure of the reproductive apparatus of Cynipini.(A, B) Volume rendered micrographs of accessory gland of the agamic generation of Disholcaspis quercusmamma confocal images stained with phalloidin (actin), revealing smooth muscles cells, and Hoescht (blue), revealing nuclei. (C)Transmission electron micrograph of a smooth muscle cell on the accessory gland of the agamic generation of Acraspis macrocarpae. (D, E) Confocal images of an accessory gland of D. quercusmamma stained with Hoescht (DNA) and Nile Red (lipid). (F) Transmission electron micrograph of the accessory gland of A. macrocarpae. (G–I) Confocal images of: (G, H) an accessory sac and (I) an oviduct of D. quercusmamma with Hoescht and Nile Red staining. Legend: N: nucleus, f: muscle fiber, m: mitochondria, lv: lipid vesicle.

Fig. 14 in Comparative anatomy of venom glands suggests a role of maternal secretions in gall induction by cynipid wasps (Hymenoptera: Cynipidae)

Fig. 14. Ovariole anatomy of various Cynipids. Ovarioles of 19 species belonging to five tribes, with insets showing the egg development stage. M: mature egg, D: developing egg, Ga.: galler species, and In.: inquiline species. Square colors correspond to tribe colors introduced in Fig. 2.

Fig. 11 in Comparative anatomy of venom glands suggests a role of maternal secretions in gall induction by cynipid wasps (Hymenoptera: Cynipidae)

Fig. 11. Relative size of the accessory apparatus in Cynipoids.Accessory sac diameter relative to metasoma length, with the name of the species with the highest ratio.The ratios of (A) accessory sac area over metasoma area and (B) accessory gland length over metasoma length for each species dissected.The dendrogram is adapted from Blaimer et al. (2020).The band indicates the species lifestyle (galler or inquiline) and the generation for Cynipini (sexual, agamic, or unknown). The dot colors in (A) correspond to the cynipoid tribes displayed in the dendrogram in (B).

Fig. 9 in Comparative anatomy of venom glands suggests a role of maternal secretions in gall induction by cynipid wasps (Hymenoptera: Cynipidae)

Fig. 9. Ultrastructure of Cynipini venom glands. (A) Transversal section of a venom gland of the agamic generation of Disholcaspis quercusmamma, obtained with confocal microscope and stained with Hoechst (DNA) and phalloidin (actin). S: nuclei of secretory cells; L: lumen; white arrow: nuclei of ductule cells. (B, C) Volume rendered micrographs of D. quercusmamma agamic generation venom gland. (B) Secretory cell nuclei (blue) and end apparati (represented by red actin canals), with a subfigure showing the proximity between the nuclei and the secretory region. (C) End apparati of secretory cells, with a subfigure showing the curvature of the secretory region.Yellow: lumen. (D, E) Surface rendered micrographs of A. erinacei agamic generation venom gland obtained by serial block face scanning electron microscopy (D: top view, E: lateral view).The subfigures show the connections between the subcellular elements. Red: end apparatus; green: duct; yellow: lumen; blue: secretory cell nuclei; and purple: ductule cell nuclei.

Fig. 10 in Comparative anatomy of venom glands suggests a role of maternal secretions in gall induction by cynipid wasps (Hymenoptera: Cynipidae)

Fig. 10. Transmission electron micrographs of the venom gland secretory region of Cynipoids. (A, A′) Disholcaspis quercusmamma agamic generation. (B, B′) Acraspis erinacei agamic generation. (C, C′) Amphibolips confluenta sexual generation. (D, D′, D") Diastrophus nebulosus. (E, E′) Antistrophus laciniatus. (F) Synergus sp. (G) Schematic summarizing the organization of the venom secretory unit inferred from TEM images. The extracellular medium of the 3 tested Cynipini contain high concentrations of nanometric particles. In D. quercusmamma, these particles (P1) are also found in secretory vesicles and seem to assemble into fibers once secreted. In A. erinacei, some secretory vesicles are observed, without particle content, whereas no vesicle are clearly visible in A. confluenta. In this last species, secretory units are surrounded by large granules. In Diastrophus nebulosus, nanometric particles seems to assemble into a 250 nm particle (P2) within secretory vesicles. Once secreted these P2 seem to form larger agglomerations. Lamellar bodies are also secreted in this species. The secretory unit is reduced and surrounded by light vesicles that do not contain any particles in the inquiline Synergus sp. An accumulation of vesicles and granules is observed around the secretory unit of A. laciniatus. In comparison, virus-like particles are secreted in Leptopilina sp. (adapted from Ferrarese et al., 2009). Legend: d: duct, e: extracellular medium, v: vesicle, mv: microvilli, g: granules, P1: 50 nm particle, P2: 250 nm particle, L: lamellar body, VLP: viruslike particle, p40: virus-like particle protein p40. Scale bar = 2 μm.

Fig. 12 in Comparative anatomy of venom glands suggests a role of maternal secretions in gall induction by cynipid wasps (Hymenoptera: Cynipidae)

Fig. 12. Reproductive apparatus anatomy of various Cynipids. Scale bar = 0.5 mm. Ovaries and accessory glands of 8 species belonging to three tribes, with insets showing the detail of the accessory sacs when necessary. Green arrow: accessory sacs, pink arrow: ovary extremity, and yellow dashed line: accessory gland.

Fig. 8 in Comparative anatomy of venom glands suggests a role of maternal secretions in gall induction by cynipid wasps (Hymenoptera: Cynipidae)

Fig. 8. Evolutionary pattern of the venom apparatus relative size in Cynipoids. Phenograms depicting the relative size of the venom gland on the left (ratio venom gland length over metasoma length) and the relative size of venom reservoir on the right (ratio venom reservoir area over metasoma area), with indication of the four gall-inducing clades.The phenograms are based on a time calibrated phylogeny of Cynipoidea (Blaimer et al. 2020, Ward et al. 2022), and phylogenetic tree tips are plotted according to their phenotypic value. A: agamic generation, S: sexual generation.

Fig. 7 in Comparative anatomy of venom glands suggests a role of maternal secretions in gall induction by cynipid wasps (Hymenoptera: Cynipidae)

Fig. 7. Boxplots of the relative size of the venom apparatus in Cynipoids in relation to taxonomic group and life-history traits. This includes (A) lifestyle, (B) tribes, (C) plant tissue attacked among gallers, (D) number of larval chambers in the induced galls (uni- or multilocular), and, among Cynipini, (E) generation. Diplolepidini were excluded due to their absence of fully developed venom apparatus. Bars marked with a different letter indicate significant difference, NS indicates a non-significant difference (Tukey post hoc test, P ≤ 0.05).