Astrid Cruaud

An Extreme Case of Plant-Insect Codiversification: Figs and Fig-Pollinating Wasps

It is thought that speciation in phytophagous insects is often due to colonization of novel host plants, because radiations of plant and insect lineages are typically asynchronous. Recent phylogenetic comparisons have supported this model of diversification for both insect herbivores and specialized pollinators. An exceptional case where contemporaneous plant-insect diversification might be expected is the obligate mutualism between fig trees (Ficus species, Moraceae) and their pollinating wasps (Agaonidae, Hymenoptera). The ubiquity and ecological significance of this mutualism in tropical and subtropical ecosystems has long intrigued biologists, but the systematic challenge posed by >750 interacting species pairs has hindered progress toward understanding its evolutionary history. In particular, taxon sampling and analytical tools have been insufficient for large-scale cophylogenetic analyses. Here, we sampled nearly 200 interacting pairs of fig and wasp species from across the globe. Two supermatrices were assembled: on an average, wasps had sequences from 77% of 6 genes (5.6 kb), figs had sequences from 60% of 5 genes (5.5 kb), and overall 850 new DNA sequences were generated for this study. We also developed a new analytical tool, Jane 2, for event-based phylogenetic reconciliation analysis of very large data sets. Separate Bayesian phylogenetic analyses for figs and fig wasps under relaxed molecular clock assumptions indicate Cretaceous diversification of crown groups and contemporaneous divergence for nearly half of all fig and pollinator lineages. Event-based cophylogenetic analyses further support the codiversification hypothesis. Biogeographic analyses indicate that the present-day distribution of fig and pollinator lineages is consistent with a Eurasian origin and subsequent dispersal, rather than with Gondwanan vicariance. Overall, our findings indicate that the fig-pollinator mutualism represents an extreme case among plant-insect interactions of coordinated dispersal and long-term codiversification. [Biogeography; coevolution; cospeciation; host switching; long-branch attraction; phylogeny.].

A molecular phylogeny of the Chalcidoidea (Hymenoptera).

Chalcidoidea (Hymenoptera) are extremely diverse with more than 23,000 species described and over 500,000 species estimated to exist. This is the first comprehensive phylogenetic analysis of the superfamily based on a molecular analysis of 18S and 28S ribosomal gene regions for 19 families, 72 subfamilies, 343 genera and 649 species. The 56 outgroups are comprised of Ceraphronoidea and most proctotrupomorph families, including Mymarommatidae. Data alignment and the impact of ambiguous regions are explored using a secondary structure analysis and automated (MAFFT) alignments of the core and pairing regions and regions of ambiguous alignment. Both likelihood and parsimony approaches are used to analyze the data. Overall there is no impact of alignment method, and few but substantial differences between likelihood and parsimony approaches. Monophyly of Chalcidoidea and a sister group relationship between Mymaridae and the remaining Chalcidoidea is strongly supported in all analyses. Either Mymarommatoidea or Diaprioidea are the sister group of Chalcidoidea depending on the analysis. Likelihood analyses place Rotoitidae as the sister group of the remaining Chalcidoidea after Mymaridae, whereas parsimony nests them within Chalcidoidea. Some traditional family groups are supported as monophyletic (Agaonidae, Eucharitidae, Encyrtidae, Eulophidae, Leucospidae, Mymaridae, Ormyridae, Signiphoridae, Tanaostigmatidae and Trichogrammatidae). Several other families are paraphyletic (Perilampidae) or polyphyletic (Aphelinidae, Chalcididae, Eupelmidae, Eurytomidae, Pteromalidae, Tetracampidae and Torymidae). Evolutionary scenarios discussed for Chalcidoidea include the evolution of phytophagy, egg parasitism, sternorrhynchan parasitism, hypermetamorphic development and heteronomy.

Empirical assessment of RAD sequencing for interspecific phylogeny.

Next-generation sequencing opened up new possibilities in phylogenetics; however, choosing an appropriate method of sample preparation remains challenging. Here, we demonstrate that restriction-site-associated DNA sequencing (RAD-seq) generates useful data for phylogenomics. Analysis of our RAD library using current bioinformatic and phylogenetic tools produced 400× more sites than our Sanger approach (2,262,825 nt/species), fully resolving relationships between 18 species of ground beetles (divergences up to 17 My). This suggests that RAD-seq is promising to infer phylogeny of eukaryotic species, though potential biases need to be evaluated and new methodologies developed to take full advantage of such data.

Laying the foundations for a new classification of Agaonidae (Hymenoptera: Chalcidoidea), a multilocus phylogenetic approach

A phylogeny of the Agaonidae (Chalcidoidea) in their restricted sense, pollinators of Ficus species (Moraceae), is estimated using 4182 nucleotides from six genes, obtained from 101 species representing 19 of the 20 recognized genera, and four outgroups. Data analysed by parsimony and Bayesian inference methods demonstrate that Agaonidae are monophyletic and that the previous classification is not supported. Agaonidae are partitioned into four groups: (i) Tetrapus, (ii) Ceratosolen + Kradibia, (iii) some Blastophaga + Wiebesia species, and (iv) all genera associated with monoecious figs and a few Blastophaga and Wiebesia. The latter group is subdivided into subgroups: (i) Pleistodontes, (ii) Blastophaga psenes and neocaledonian Dolichoris, (iii) some Blastophaga and Wiebesia species, and (iv) Platyscapa, all afrotropical genera and all genera associated with section Conosycea. Eleven genera were recovered as monophyletic, six were para‐ or polyphyletic, and two cannot be tested with our data set. Based on our phylogeny we propose a new classification for the Agaonidae. Two new subfamilies are proposed: Tetrapusiinae for the genus Tetrapus, and Kradibiinae for Ceratosolen + Kradibia. Liporrhopalum is synonymized with Kradibia and the subgenus Valisia of Blastophaga is elevated to generic rank. These changes resulted in 36 new combinations. Finally, we discuss the hypothesis of co‐speciation between the pollinators and their host species by comparing the two phylogenies.

Identification of molecular markers for DNA barcoding in the Aphidiinae (Hym. Braconidae)

Reliable identification of Aphidiinae species (Braconidae) is a prerequisite for conducting studies on aphid–parasitoid interactions at the community level. However, morphological identification of Aphidiinae species remains problematic even for specialists and is almost impossible with larval stages. Here, we compared the efficiency of two molecular markers [mitochondrial cytochrome c oxydase I (COI) and nuclear long wavelength rhodopsin (LWRh)] that could be used to accurately identify about 50 species of Aphidiinae that commonly occur in aphid–parasitoid networks in northwestern Europe. We first identified species on a morphological basis and then assessed the consistency of genetic and morphological data. Probably because of mitochondrial introgression, Aphidius ervi and A. microlophii were indistinguishable on the basis of their COI sequences, whereas LWRh sequences discriminated these species. Conversely, because of its lower variability, LWRh failed to discriminate two pairs of species (Aphidius aquilus, Aphidius salicis, Lysiphlebus confusus and Lysiphlebus fabarum). Our study showed that no unique locus but a combination of two genes should be used to accurately identify members of Aphidiinae.

Phylogeny and evolution of life-history strategies in the Sycophaginae non-pollinating fig wasps (Hymenoptera, Chalcidoidea)

BackgroundNon-pollinating Sycophaginae (Hymenoptera, Chalcidoidea) form small communities within Urostigma and Sycomorus fig trees. The species show differences in galling habits and exhibit apterous, winged or dimorphic males. The large gall inducers oviposit early in syconium development and lay few eggs; the small gall inducers lay more eggs soon after pollination; the ostiolar gall-inducers enter the syconium to oviposit and the cleptoparasites oviposit in galls induced by other fig wasps. The systematics of the group remains unclear and only one phylogeny based on limited sampling has been published to date. Here we present an expanded phylogeny for sycophagine fig wasps including about 1.5 times the number of described species. We sequenced mitochondrial and nuclear markers (4.2 kb) on 73 species and 145 individuals and conducted maximum likelihood and Bayesian phylogenetic analyses. We then used this phylogeny to reconstruct the evolution of Sycophaginae life-history strategies and test if the presence of winged males and small brood size may be correlated.ResultsThe resulting trees are well resolved and strongly supported. With the exception of Apocrytophagus, which is paraphyletic with respect to Sycophaga, all genera are monophyletic. The Sycophaginae are divided into three clades: (i) Eukoebelea; (ii) Pseudidarnes, Anidarnes and Conidarnes and (iii) Apocryptophagus, Sycophaga and Idarnes. The ancestral states for galling habits and male morphology remain ambiguous and our reconstructions show that the two traits are evolutionary labile.ConclusionsThe three main clades could be considered as tribes and we list some morphological characters that define them. The same biologies re-evolved several times independently, which make Sycophaginae an interesting model to test predictions on what factors will canalize the evolution of a particular biology. The ostiolar gall-inducers are the only monophyletic group. In 15 Myr, they evolved several morphological adaptations to enter the syconia that make them strongly divergent from their sister taxa. Sycophaginae appears to be another example where sexual selection on male mating opportunities favored winged males in species with small broods and wingless males in species with large broods. However, some species are exceptional in that they lay few eggs but exhibit apterous males, which we hypothesize could be due to other selective pressures selecting against the re-appearance of winged morphs.

Floral volatiles, pollinator sharing and diversification in the fig–wasp mutualism: insights from Ficus natalensis, and its two wasp pollinators (South Africa)

Combining biogeographic, ecological, morphological, molecular and chemical data, we document departure from strict specialization in the fig-pollinating wasp mutualism. We show that the pollinating wasps Elisabethiella stuckenbergi and Elisabethiella socotrensis form a species complex of five lineages in East and Southern Africa. Up to two morphologically distinct lineages were found to co-occur locally in the southern African region. Wasps belonging to a single lineage were frequently the main regional pollinators of several Ficus species. In South Africa, two sister lineages, E. stuckenbergi and E. socotrensis, pollinate Ficus natalensis but only E. stuckenbergi also regularly pollinates Ficus burkei. The two wasp species co-occur in individual trees of F. natalensis throughout KwaZulu-Natal. Floral volatile blends emitted by F. natalensis in KwaZulu-Natal were similar to those emitted by F. burkei and different from those produced by other African Ficus species. The fig odour similarity suggests evolutionary convergence to attract particular wasp species. The observed pattern may result from selection for pollinator sharing among Ficus species. Such a process, with one wasp species regionally pollinating several hosts, but several wasp species pollinating a given Ficus species across its geographical range could play an important role in the evolutionary dynamics of the Ficus-pollinating wasp association.

Testing the Emergence of New Caledonia: Fig Wasp Mutualism as a Case Study and a Review of Evidence

While geologists suggest that New Caledonian main island (Grande Terre) was submerged until ca 37 Ma, biologists are struck by the presence of supposedly Gondwanan groups on the island. Among these groups are the Oreosycea fig trees (Ficus, Moraceae) and their Dolichoris pollinators (Hymenoptera, Agaonidae). These partners are distributed in the Paleotropics and Australasia, suggesting that their presence on New Caledonia could result from Gondwanan vicariance. To test this hypothesis, we obtained mitochondrial and nuclear markers (5.3 kb) from 28 species of Dolichoris, used all available sequences for Oreosycea, and conducted phylogenetic and dating analyses with several calibration strategies. All our analyses ruled out a vicariance scenario suggesting instead that New Caledonian colonization by Dolichoris and Oreosycea involved dispersal across islands from Sundaland ca 45.9-32.0 Ma. Our results show that successful long-distance dispersal of obligate mutualists may happen further suggesting that presence of intimate mutualisms on isolated islands should not be used as a priori evidence for vicariance. Comparing our results to a review of all the published age estimates for New Caledonian plant and animal taxa, we showed that support for a vicariant origin of the island biota is still lacking. Finally, as demonstrating a causal relationship between geology and biology requires independent evidence, we argue that a priori assumptions about vicariance or dispersal should not be used to constrain chronograms. This circular reasoning could lead to under or overestimation of age estimates.

Is ecological speciation a major trend in aphids? Insights from a molecular phylogeny of the conifer-feeding genus Cinara

IntroductionIn the past decade ecological speciation has been recognized as having an important role in the diversification of plant-feeding insects. Aphids are host-specialised phytophagous insects that mate on their host plants and, as such, they are prone to experience reproductive isolation linked with host plant association that could ultimately lead to species formation. The generality of such a scenario remains to be tested through macroevolutionary studies. To explore the prevalence of host-driven speciation in the diversification of the aphid genus Cinara and to investigate alternative modes of speciation, we reconstructed a phylogeny of this genus based on mitochondrial, nuclear and Buchnera aphidicola DNA sequence fragments and applied a DNA-based method of species delimitation. Using a recent software (PhyloType), we explored evolutionary transitions in host-plant genera, feeding sites and geographic distributions in the diversification of Cinara and investigated how transitions in these characters have accompanied speciation events.ResultsThe diversification of Cinara has been constrained by host fidelity to conifer genera sometimes followed by sequential colonization onto different host species and by feeding-site specialisation. Nevertheless, our analyses suggest that, at the most, only half of the speciation events were accompanied by ecological niche shifts. The contribution of geographical isolation in the speciation process is clearly apparent in the occurrence of species from two continents in the same clades in relatively terminal positions in our phylogeny. Furthermore, in agreement with predictions from scenarios in which geographic isolation accounts for speciation events, geographic overlap between species increased significantly with time elapsed since their separation.ConclusionsThe history of Cinara offers a different perspective on the mode of speciation of aphids than that provided by classic models such as the pea aphid. In this genus of aphids, the role of climate and landscape history has probably been as important as host-plant specialisation in having shaped present-day diversity.

An integrative approach to species discrimination in the Eupelmus urozonus complex (Hymenoptera, Eupelmidae), with the description of 11 new species from the Western Palaearctic

The systematics of the European species of Eupelmus (Eupelmus) Dalman (Hymenoptera: Eupelmidae) belonging to the ‘urozonus‐complex’ is elucidated through combined molecular and morphological characterization. One mitochondrial gene fragment (Cytochrome oxidase I) and one nuclear protein‐coding gene fragment (Wingless) were sequenced and the results compared with those of a detailed morphological study of the specimens from an extensive sampling. Knowledge of the biodiversity of Eupelmus in the Western Palearctic Region is significantly improved through the separation and description of 11 new species: E. (Eupelmus) confusus Al khatib sp.n., E. gemellus Al khatib sp.n., E. janstai Delvare & Gibson sp.n., E. longicalvus Al khatib & Fusu sp.n., E. minozonus Delvare sp.n., E. opacus Delvare sp.n., E. pistaciae Al khatib sp.n., E. priotoni Delvare sp.n., E. purpuricollis Fusu & Al khatib sp.n., E. simizonus Al khatib sp.n. and E. tremulae Delvare sp.n. Illustrated keys to females and, when known, males of these new 11 species plus the other already described species considered to belong to the ‘urozonus‐complex’ (E. acinellus Askew, E. annulatus Nees, E. azureus Ratzeburg, E. cerris Förster, E. fulvipes Förster, E. kiefferi De Stefani, E. martellii Masi, E. stenozonus Askew, E. tibicinis Bouček and E. urozonus Dalman) are provided and all the species are described based on morphology. Eupelmus kiefferi is removed from synonymy under E. urozonus and E. azureus is recognized as the valid senior synonym of Pteromalus cordairii Ratzeburg, and Eupelmus spongipartus Förster, syn.n. The discrimination of the species included in this complex is particularly relevant because some are potential biological control agents and have been confused in the past.