Patterns of speciation in a parapatric pair of Saturnia moths as revealed by target capture.

The focus of this study has been to understand the evolutionary relationships and taxonomy of a widely distributed parapatric species pair of wild silk moths in Europe: Saturnia pavonia and Saturnia pavoniella (Lepidoptera: Saturniidae). To address species delimitation in these parapatric taxa, target enrichment and mtDNA sequencing was employed alongside phylogenetic, admixture, introgression, and species delimitation analyses. The dataset included individuals from both species close to and farther away from the contact zone as well as two hybrids generated in the lab. Nuclear markers strongly supported both S. pavonia and S. pavoniella as two distinct species, with hybrids forming a sister group to S. pavoniella. However, the Maximum Likelihood (ML) tree generated from mtDNA sequencing data presented a different picture, showing both taxa to be phylogenetically intermixed. This inconsistency is likely attributable to mitonuclear discordance, which can arise from biological factors (e.g., introgressive hybridization and/or incomplete lineage sorting). Our analyses indicate that past introgressions have taken place, but that there is no evidence to suggest an ongoing admixture between the two species, demonstrating that the taxa have reached full postzygotic reproductive isolation and hence represent two distinct biological species. Finally, we discuss our results from an evolutionary point of view taking into consideration the past climatic oscillations that have likely shaped the present dynamics between the two species. Overall, our study demonstrates the effectiveness of the target enrichment approach in resolving shallow phylogenetic relationships under complex evolutionary circumstances and that this approach is useful in establishing robust and well-informed taxonomic delimitations involving parapatric taxa.

Phylogeny of gracillariid leaf-mining moths: evolution of larval behaviour inferred from phylogenomic and Sanger data.

Gracillariidae is the most taxonomically diverse cosmopolitan leaf-mining moth family, consisting of nearly 2000 named species in 105 described genera, classified into eight extant subfamilies. The majority of gracillariid species are internal plant feeders as larvae, creating mines and galls in plant tissue. Despite their diversity and ecological adaptations, their phylogenetic relationships, especially among subfamilies, remain uncertain. Genomic data (83 taxa, 589 loci) were integrated with Sanger data (130 taxa, 22 loci), to reconstruct a phylogeny of Gracillariidae. Based on analyses of both datasets combined and analyzed separately, monophyly of Gracillariidae and all its subfamilies, monophyly of the clade "LAMPO" (subfamilies: Lithocolletinae, Acrocercopinae, Marmarinae, Phyllocnistinae, and Oecophyllembiinae) and relationships of its subclade "AMO" (subfamilies: Acrocercopinae, Marmarinae, and Oecophyllembiinae) were strongly supported. A sister-group relationship of Ornixolinae to the remainder of the family, and a monophyletic leaf roller lineage (Callicercops Vári + Parornichinae) + Gracillariinae, as sister to the "LAMPO" clade were supported by the most likely tree. Dating analyses indicate a mid-Cretaceous (105.3 Ma) origin of the family, followed by a rapid diversification into the nine subfamilies predating the Cretaceous-Palaeogene extinction. We hypothesize that advanced larval behaviours, such as making keeled or tentiform blotch mines, rolling leaves and galling, allowed gracillariids to better avoid larval parasitoids allowing them to further diversify. Finally, we stabilize the classification by formally re-establishing the subfamily ranks of Marmarinae stat.rev., Oecophyllembiinae stat.rev. and Parornichinae stat.rev., and erect a new subfamily, Callicercopinae Li, Ohshima and Kawahara to accommodate the enigmatic genus Callicercops.

DNA barcodes reveal deeply neglected diversity and numerous invasions of micromoths in Madagascar 1.

Madagascar is a prime evolutionary hotspot globally, but its unique biodiversity is under threat, essentially from anthropogenic disturbance. There is a race against time to describe and protect the Madagascan endangered biota. Here we present a first molecular characterization of the micromoth fauna of Madagascar. We collected 1572 micromoths mainly using light traps in both natural and anthropogenically disturbed habitats in 24 localities across eastern and northwest Madagascar. We also collected 1384 specimens using a Malaise trap in a primary rain forest at Andasibe, eastern Madagascar. In total, we DNA barcoded 2956 specimens belonging to 1537 Barcode Index Numbers (BINs), 88.4% of which are new to BOLD. Only 1.7% of new BINs were assigned to species. Of 47 different families found, Dryadaulidae, Bucculatricidae, Bedelliidae, Batrachedridae, and Blastobasidae are newly reported for Madagascar and the recently recognized Tonzidae is confirmed. For test faunas of Canada and Australia, 98.9%-99.4% of Macroheterocera BINs exhibited the molecular synapomorphy of a phenylalanine in the 177th complete DNA barcode codon. Non-macroheteroceran BINs could thus be sifted out efficiently in the Malaise sample. The Madagascar micromoth fauna shows highest affinity with the Afrotropics (146 BINs also occur in the African continent). We found 22 recognised pests or invasive species, mostly occurring in disturbed habitats. Malaise trap samples show high temporal turnover and alpha diversity with as many as 507 BINs collected; of these, astonishingly, 499 (98.4%) were novel to BOLD and 292 (57.6%) were singletons. Our results provide a baseline for future surveys across the island.

Characterization and comparison of poorly known moth communities through DNA barcoding in two Afrotropical environments in Gabon 1.

Biodiversity research in tropical ecosystems-popularized as the most biodiverse habitats on Earth-often neglects invertebrates, yet invertebrates represent the bulk of local species richness. Insect communities in particular remain strongly impeded by both Linnaean and Wallacean shortfalls, and identifying species often remains a formidable challenge inhibiting the use of these organisms as indicators for ecological and conservation studies. Here we use DNA barcoding as an alternative to the traditional taxonomic approach for characterizing and comparing the diversity of moth communities in two different ecosystems in Gabon. Though sampling remains very incomplete, as evidenced by the high proportion (59%) of species represented by singletons, our results reveal an outstanding diversity. With about 3500 specimens sequenced and representing 1385 BINs (Barcode Index Numbers, used as a proxy to species) in 23 families, the diversity of moths in the two sites sampled is higher than the current number of species listed for the entire country, highlighting the huge gap in biodiversity knowledge for this country. Both seasonal and spatial turnovers are strikingly high (18.3% of BINs shared between seasons, and 13.3% between sites) and draw attention to the need to account for these when running regional surveys. Our results also highlight the richness and singularity of savannah environments and emphasize the status of Central African ecosystems as hotspots of biodiversity.

Species-Level Para- and Polyphyly in DNA Barcode Gene Trees: Strong Operational Bias in European Lepidoptera.

The proliferation of DNA data is revolutionizing all fields of systematic research. DNA barcode sequences, now available for millions of specimens and several hundred thousand species, are increasingly used in algorithmic species delimitations. This is complicated by occasional incongruences between species and gene genealogies, as indicated by situations where conspecific individuals do not form a monophyletic cluster in a gene tree. In two previous reviews, non-monophyly has been reported as being common in mitochondrial DNA gene trees. We developed a novel web service "Monophylizer" to detect non-monophyly in phylogenetic trees and used it to ascertain the incidence of species non-monophyly in COI (a.k.a. cox1) barcode sequence data from 4977 species and 41,583 specimens of European Lepidoptera, the largest data set of DNA barcodes analyzed from this regard. Particular attention was paid to accurate species identification to ensure data integrity. We investigated the effects of tree-building method, sampling effort, and other methodological issues, all of which can influence estimates of non-monophyly. We found a 12% incidence of non-monophyly, a value significantly lower than that observed in previous studies. Neighbor joining (NJ) and maximum likelihood (ML) methods yielded almost equal numbers of non-monophyletic species, but 24.1% of these cases of non-monophyly were only found by one of these methods. Non-monophyletic species tend to show either low genetic distances to their nearest neighbors or exceptionally high levels of intraspecific variability. Cases of polyphyly in COI trees arising as a result of deep intraspecific divergence are negligible, as the detected cases reflected misidentifications or methodological errors. Taking into consideration variation in sampling effort, we estimate that the true incidence of non-monophyly is ∼23%, but with operational factors still being included. Within the operational factors, we separately assessed the frequency of taxonomic limitations (presence of overlooked cryptic and oversplit species) and identification uncertainties. We observed that operational factors are potentially present in more than half (58.6%) of the detected cases of non-monophyly. Furthermore, we observed that in about 20% of non-monophyletic species and entangled species, the lineages involved are either allopatric or parapatric-conditions where species delimitation is inherently subjective and particularly dependent on the species concept that has been adopted. These observations suggest that species-level non-monophyly in COI gene trees is less common than previously supposed, with many cases reflecting misidentifications, the subjectivity of species delimitation or other operational factors.

Phylogeography of the Spanish Moon Moth Graellsia isabellae (Lepidoptera, Saturniidae).

BACKGROUND: Geographic and demographic factors as well as specialisation to a new host-plant may lead to host-associated differentiation in plant-feeding insects. We explored the phylogeography of a protected moth, Graellsia isabellae, and its two recognised host-plant species (Pinus sylvestris and P. nigra) in order to seek for any concordance useful to disentangle the evolutionary history of this iconic lepidopteran. RESULTS: DNA variation in one mitochondrial marker and nine nuclear microsatellite loci revealed a strong phylogeographic pattern across 28 populations of G. isabellae studied in Spain and France comprising six groups mostly distributed along different mountain ranges. Reanalysis of a previously published chloroplast microsatellite dataset revealed a three and two-group structure for Spanish P. sylvestris and P. nigra, respectively. Overall, the population groupings of this protected moth did not match the ones of P. sylvestris and P. nigra. CONCLUSIONS: There was no evidence of host-associated differentiation between populations using P. sylvestris and the ones inhabiting P. nigra. The two major mitochondrial clades of G. isabellae likely diverged before the Last Glacial Maximum and geographically separated the species into a "southern" (Central and Southern Iberian clusters) and a "northern" lineage (Eastern Iberian, Pyrenean and French Alpine clusters). The Eastern Iberian System, where this insect uses both host-plants, harboured the highest level of genetic diversity. Such a group independently colonised the West and East parts of the Pyrenees. Our results point to a native origin for the French populations occurring in the Alps, genetically related to the Eastern Iberian and Pyrenean sites. The Central Iberian group derived from Southern Iberian ancestors. Secondary contacts were inferred between the Southern/Central Iberian populations and Eastern Iberian cluster as well as between the two Pyrenean ones. The mito-nuclear discordance observed with regard to the Eastern Iberian cluster is congruent with a secondary contact after the evolution of mito-nuclear incompatibilities in geographically isolated areas.

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.].

Shared but overlooked: 30 species of Holarctic Microlepidoptera revealed by DNA barcodes and morphology.

This study reports 30 species of Lepidoptera previously known from either the Palearctic or the Nearctic that are newly recorded as Holarctic. For 28 of these species, their intercontinental distributions were initially detected through DNA barcode analysis and subsequently confirmed by morphological examination; two Palearctic species were first detected in North America through morphology and then barcoded. When possible, the origin and status of each species (introduced, overlooked Holarctic species, or unknowingly re-described) is discussed, and its morphology is diagnosed and illustrated. The species involved include Tineidae: Scardia amurensis Zagulajev, Triaxomera parasitella (Hübner), Nemapogon cloacella (Haworth), Elatobia montelliella (Schantz), Tinea svenssoni Opheim; Gracillariidae: Caloptilia suberinella (Tengström), Parornix betulae (Stainton); Phyllonorycter maestingella (Müller); Yponomeutidae: Paraswammerdamia albicapitella (Scharfenberg), P. conspersella (Tengström); Plutellidae: Plutella hyperboreella Strand; Lyonetiidae: Lyonetia pulverulentella Zeller; Autostichidae: Oegoconia deauratella (Herrich-Schäffer), O. novimundi (Busck); Blastobasidae: Blastobasis glandulella (Riley), B. maroccanella (Amsel), B. tarda Meyrick; Depressariidae: Agonopterix conterminella (Zeller), Depressaria depressana (F.); Coleophoridae: Coleophora atriplicis Meyrick, C. glitzella Hofmann, C. granulatella Zeller, C. texanella Chambers, C. vitisella Gregson; Scythrididae: Scythris sinensis (Felder & Rogenhofer); Gelechiidae: Altenia perspersella (Wocke), Gnorimoschema jalavai Povolný, Scrobipalpa acuminatella (Sircom), Sophronia gelidella Nordman; Choreutidae: Anthophila fabriciana (L.); and Tortricidae: Phiaris bipunctana (F.). These cases of previously unrecognized faunal overlap have led to their redescription in several instances. Five new synonyms are proposed: Blastobasis glandulella (Riley, 1871) = B. huemeri Sinev, 1993, syn. nov.; B. tarda Meyrick, 1902 = Neoblastobasis ligurica Nel & Varenne, 2004, syn. nov.; Coleophora atriplicis Meyrick, 1928 = C. cervinella McDunnough, 1946, syn. nov.; C. texanella Chambers, 1878 = C. coxi Baldizzone & van der Wolf, 2007, syn. nov., and = C. vagans Walsingham, 1907, syn. nov. Lectotypes are designated for Blastobasis tarda Meyrick and Coleophora texanella Chambers. Type specimens were examined where pertinent to establish new synonymies. We identify 12 previously overlooked cases of species introductions, highlighting the power of DNA barcoding as a tool for biosurveillance.

Persisting roadblocks in arthropod monitoring using non-destructive metabarcoding from collection media of passive traps.

Background: Broad-scale monitoring of arthropods is often carried out with passive traps (e.g., Malaise traps) that can collect thousands of specimens per sample. The identification of individual specimens requires time and taxonomic expertise, limiting the geographical and temporal scale of research and monitoring studies. DNA metabarcoding of bulk-sample homogenates has been found to be faster, efficient and reliable, but the destruction of samples prevents a posteriori validation of species occurrences and relative abundances. Non-destructive metabarcoding of DNA extracted from collection medium has been applied in a limited number of studies, but further tests of efficiency are required with different trap types and collection media to assess the consistency of the method. Methods: We quantified the detection rate of arthropod species when applying non-destructive DNA metabarcoding with a short (127-bp) fragment of mitochondrial COI on two combinations of passive traps and collection media: (1) water with monopropylene glycol (H2O-MPG) used in window-flight traps (WFT, 53 in total); (2) ethanol with monopropylene glycol (EtOH-MPG) used in Malaise traps (MT, 27 in total). We then compared our results with those obtained for the same samples using morphological identification (for WFTs) or destructive metabarcoding of bulk homogenate (for MTs). This comparison was applied as part of a larger study of arthropod species richness in silver fir (Abies alba Mill., 1759) stands across a range of climate-induced tree dieback levels and forest management strategies. Results: Of the 53 H2O-MPG samples from WFTs, 16 produced no metabarcoding results, while the remaining 37 samples yielded 77 arthropod MOTUs in total, of which none matched any of the 343 beetle species morphologically identified from the same traps. Metabarcoding of 26 EtOH-MPG samples from MTs detected more arthropod MOTUs (233) than destructive metabarcoding of homogenate (146 MOTUs, 8 orders), of which 71 were shared MOTUs, though MOTU richness per trap was similar between treatments. While we acknowledge the failure of metabarcoding from WFT-derived collection medium (H2O-MPG), the treatment of EtOH-based Malaise trapping medium remains promising. We conclude however that DNA metabarcoding from collection medium still requires further methodological developments and cannot replace homogenate metabarcoding as an approach for arthropod monitoring. It can be used nonetheless as a complementary treatment when enhancing the detection of soft-bodied arthropods like spiders and Diptera.

The global phylogeny of the subfamily Sycoryctinae (Pteromalidae): parasites of an obligate mutualism.

The inflorescences of fig trees (Ficus, Moraceae) host well-defined, host plant specific wasp communities that lend themselves to tests of hypotheses on insect diversification. We provide the first estimate of the global molecular phylogeny for the Sycoryctinae - a large subfamily of fig wasps consisting mainly of parasitoids of fig-pollinating wasps. We find strong support for a large Old World clade that contains eight of the eleven genera, in the tribes Sycoryctini and Philotrypesini. The sister taxon is tribe Apocryptini, comprising the genera Apocrypta and Bouceka. Finally, a new tribe, Critogastrini, is raised for the genus Critogaster, sister to all other sycoryctines. At the genus level, we found a general pattern of strong host conservatism, in which closely related wasps associate with closely related figs. Despite this, there is also evidence for multiple host shifts between more distantly related figs in some wasp genera (especially Philotrypesis). We estimate Sycoryctinae to have originated 49-64 Ma, after the initial co-radiation of the host figs and pollinators. Further, conservative assumptions in our analyses probably overestimate the age of the sycoryctines. Together, patterns of host association, evidence for a mix of host constraints and host shifting, and molecular dating suggest that sycoryctine parasites radiated through delayed phylogenetic tracking of their hosts. This contributes to the growing body of literature suggesting that coevolving parasites often radiate after their hosts.

Phylogenetic, functional and taxonomic responses of wild bee communities along urbanisation gradients.

Increasing urbanisation is one of the primary drivers of land-use change that threaten biodiversity. Wild bee communities have been reported with contrasting responses to urbanisation, with varying effects on abundance and taxonomical diversity. The suite of functional traits exhibited by wild bee species might determine their persistence in urban areas. Urbanisation thus can impose an environmental filter with potential consequences on the functional and phylogenetical diversity of wild bee communities. Here, we sampled 2944 wild bee specimens from 156 species in 29 sites located along an urbanisation gradient using a replicated design in three mid-sized cities in the Loire valley (France). We show that urban landscape cover has a negative effect on overall species richness and taxonomical diversity indices, while total abundance remains constant. Species loss was taxon dependent, mainly driven by Andrenidae and Halictidae. Only a few species, especially of the genus Lasioglossum, were positively affected by the urban landscape cover. Urban and peri-urban areas differed in their composition of bee assemblages. Species turnover was the main component of beta diversity, driving community dissimilarities through the urban gradient. Urbanisation favours bees with small body sizes, social structure and extended flight periods but did not affect the phylogenetic or the functional diversity of communities. Our findings have implications for understanding the factors involved in the environmental filter exerted through the urban gradient on bee communities helping to implement conservation measures and managing urban spaces for bees.

Tracing the invasion of a leaf-mining moth in the Palearctic through DNA barcoding of historical herbaria.

The lime leaf-miner, Phyllonorycter issikii is an invasive micromoth with an unusually higher number of haplotypes in the invaded area (Europe, Western Siberia) compared to its putative native region (East Asia). The origin of the genetic diversity in the neocolonized region remains unclear. We surveyed over 15 thousand herbarium specimens of lime trees (Tilia spp.) collected across the Palearctic over a period of 252 years (1764-2016) looking for preserved larvae within the archival leaf mines. We found 203 herbarium specimens with leaf mines of Ph. issikii collected in East Asia, one of them dating back to 1830, i.e. 133 years before the description of the species. In contrast, only 22 herbarium specimens collected in the West Palearctic in the last three decades (1987-2015) carried leaf mines. DNA barcoding of archival specimens revealed 32 haplotypes out of which 23 were novel (not known from modern populations) and found exclusively in East Asia. Six haplotypes are shared between both native and invaded areas and only two were responsible for the recent invasion of the Western Palearctic. The remarkable number of newly discovered haplotypes in archival populations supports East Asia as the native region and the source area of invasion.

Climate-induced forest dieback drives compositional changes in insect communities that are more pronounced for rare species.

Species richness, abundance and biomass of insects have recently undergone marked declines in Europe. We metabarcoded 211 Malaise-trap samples to investigate whether drought-induced forest dieback and subsequent salvage logging had an impact on ca. 3000 species of flying insects in silver fir Pyrenean forests. While forest dieback had no measurable impact on species richness, there were significant changes in community composition that were consistent with those observed during natural forest succession. Importantly, most observed changes were driven by rare species. Variation was explained primarily by canopy openness at the local scale, and the tree-related microhabitat diversity and deadwood amount at landscape scales. The levels of salvage logging in our study did not explain compositional changes. We conclude that forest dieback drives changes in species assemblages that mimic natural forest succession, and markedly increases the risk of catastrophic loss of rare species through homogenization of environmental conditions.

A large-scale dataset reveals taxonomic and functional specificities of wild bee communities in urban habitats of Western Europe.

Wild bees are declining, mainly due to the expansion of urban habitats that have led to land-use changes. Effects of urbanization on wild bee communities are still unclear, as shown by contrasting reports on their species and functional diversities in urban habitats. To address this current controversy, we built a large dataset, merging 16 surveys carried out in 3 countries of Western Europe during the past decades, and tested whether urbanization influences local wild bee taxonomic and functional community composition. These surveys encompassed a range of urbanization levels, that were quantified using two complementary metrics: the proportion of impervious surfaces and the human population density. Urban expansion, when measured as a proportion of impervious surfaces, but not as human population density, was significantly and negatively correlated with wild bee community species richness. Taxonomic dissimilarity of the bee community was independent of both urbanization metrics. However, occurrence rates of functional traits revealed significant differences between lightly and highly urbanized communities, for both urbanization metrics. With higher human population density, probabilities of occurrence of above-ground nesters, generalist and small species increased. With higher soil sealing, probabilities of occurrence of above-ground nesters, generalists and social bees increased as well. Overall, these results, based on a large European dataset, suggest that urbanization can have negative impacts on wild bee diversity. They further identify some traits favored in urban environments, showing that several wild bee species can thrive in cities.

Increased gene sampling strengthens support for higher-level groups within leaf-mining moths and relatives (Lepidoptera: Gracillariidae).

BACKGROUND: Researchers conducting molecular phylogenetic studies are frequently faced with the decision of what to do when weak branch support is obtained for key nodes of importance. As one solution, the researcher may choose to sequence additional orthologous genes of appropriate evolutionary rate for the taxa in the study. However, generating large, complete data matrices can become increasingly difficult as the number of characters increases. A few empirical studies have shown that augmenting genes even for a subset of taxa can improve branch support. However, because each study differs in the number of characters and taxa, there is still a need for additional studies that examine whether incomplete sampling designs are likely to aid at increasing deep node resolution. We target Gracillariidae, a Cretaceous-age (~100 Ma) group of leaf-mining moths to test whether the strategy of adding genes for a subset of taxa can improve branch support for deep nodes. We initially sequenced ten genes (8,418 bp) for 57 taxa that represent the major lineages of Gracillariidae plus outgroups. After finding that many deep divergences remained weakly supported, we sequenced eleven additional genes (6,375 bp) for a 27-taxon subset. We then compared results from different data sets to assess whether one sampling design can be favored over another. The concatenated data set comprising all genes and all taxa and three other data sets of different taxon and gene sub-sampling design were analyzed with maximum likelihood. Each data set was subject to five different models and partitioning schemes of non-synonymous and synonymous changes. Statistical significance of non-monophyly was examined with the Approximately Unbiased (AU) test. RESULTS: Partial augmentation of genes led to high support for deep divergences, especially when non-synonymous changes were analyzed alone. Increasing the number of taxa without an increase in number of characters led to lower bootstrap support; increasing the number of characters without increasing the number of taxa generally increased bootstrap support. More than three-quarters of nodes were supported with bootstrap values greater than 80% when all taxa and genes were combined. Gracillariidae, Lithocolletinae + Leucanthiza, and Acrocercops and Parectopa groups were strongly supported in nearly every analysis. Gracillaria group was well supported in some analyses, but less so in others. We find strong evidence for the exclusion of Douglasiidae from Gracillarioidea sensu Davis and Robinson (1998). Our results strongly support the monophyly of a G.B.R.Y. clade, a group comprised of Gracillariidae + Bucculatricidae + Roeslerstammiidae + Yponomeutidae, when analyzed with non-synonymous changes only, but this group was frequently split when synonymous and non-synonymous substitutions were analyzed together. CONCLUSIONS: 1) Partially or fully augmenting a data set with more characters increased bootstrap support for particular deep nodes, and this increase was dramatic when non-synonymous changes were analyzed alone. Thus, the addition of sites that have low levels of saturation and compositional heterogeneity can greatly improve results. 2) Gracillarioidea, as defined by Davis and Robinson (1998), clearly do not include Douglasiidae, and changes to current classification will be required. 3) Gracillariidae were monophyletic in all analyses conducted, and nearly all species can be placed into one of six strongly supported clades though relationships among these remain unclear. 4) The difficulty in determining the phylogenetic placement of Bucculatricidae is probably attributable to compositional heterogeneity at the third codon position. From our tests for compositional heterogeneity and strong bootstrap values obtained when synonymous changes are excluded, we tentatively conclude that Bucculatricidae is closely related to Gracillariidae + Roeslerstammiidae + Yponomeutidae.

Molecular analysis of parasitoid linkages (MAPL): gut contents of adult parasitoid wasps reveal larval host.

Metamorphosing insects often have complex and poorly known life histories. In particular, what they feed on during their larval stages remains unknown for the vast majority of species, and its documentation only results from difficult and time-intensive field observations, rearing or dissections. Through the application of a DNA analysis of gut contents in adult parasitoid wasps, we were able to selectively sequence a diagnostic DNA marker that permitted the identification of the host used by these wasps during their larval stages. By reproducing these results in species with different life histories, we excluded other potential sources of host DNA, confirming that after ingestion by the parasitoid larva the host DNA can persist through metamorphosis in the abdominal contents of the adult wasp. Our discovery considerably extends the applicability of molecular analysis of gut contents by enabling the documentation of food used by insects during their larval stages and thus increasing the accuracy and precision of food web studies. The 24% success rate of our approach is surprisingly high considering the challenging context for host DNA preservation, and we discuss the factors possibly affecting this rate. We propose molecular analysis of parasitoid linkages (MAPL) as a new method to document host-parasitoid associations at a faster pace and with unrivalled precision. Because of the key regulatory role of parasitoid wasps in ecosystems, which makes them the most commonly used biological control agents, MAPL will have immediate applications in both basic and applied biological sciences.

A DNA barcode-based survey of wild urban bees in the Loire Valley, France.

The current decline of wild bees puts important ecosystem services such as pollination at risk. Both inventory and monitoring programs are needed to understand the causes of wild bee decline. Effective insect monitoring relies on both mass-trapping methods coupled with rapid and accurate identifications. Identifying wild bees using only morphology can be challenging, in particular, specimens from mass-trapped samples which are often in poor condition. We generated DNA barcodes for 2931 specimens representing 157 species (156 named and one unnamed species) and 28 genera. Automated cluster delineation reveals 172 BINs (Barcodes Index Numbers). A total of 36 species (22.93%) were found in highly urbanized areas. The majority of specimens, representing 96.17% of the species barcoded form reciprocally exclusive groups, allowing their unambiguous identification. This includes several closely related species notoriously difficult to identify. A total of 137 species (87.26%) show a "one-to-one" match between a named species and the BIN assignment. Fourteen species (8.92%) show deep conspecific lineages with no apparent morphological differentiation. Only two species pairs shared the same BIN making their identification with DNA barcodes alone uncertain. Therefore, our DNA barcoding reference library allows reliable identification by non-experts for the vast majority of wild bee species in the Loire Valley.

DNA barcoding for bio-surveillance of emerging pests and species identification in Afrotropical Prioninae (Coleoptera, Cerambycidae).

DNA barcoding has been succesfully used for bio-surveillance of forest and agricultural pests in temperate areas, but has few applications in the tropics and particulary in Africa. Cacosceles newmannii (Coleoptera: Cerambycidae) is a Prioninae species that is locally causing extensive damage in commercially-grown sugarcane in the KwaZulu-Natal Province in South Africa. Due to the risk of spread of this species to the rest of southern Africa and to other sugarcane growing regions, clear and easy identification of this pest is critical for monitoring and for phytosanitary services. The genus Cacosceles Newman, 1838 includes four species, most being very similar in morphology. The damaging stage of the species is the larva, which is inherently difficult to distinguish morphologically from other Cerambycidae species. A tool for rapid and reliable identification of this species was needed by plant protection and quarantine agencies to monitor its potential abundance and spread. Here, we provide newly-generated barcodes for C. newmannii that can be used to reliably identify any life stage, even by non-trained taxonomists. In addition, we compiled a curated DNA barcoding reference library for 70 specimens of 20 named species of Afrotropical Prioninae to evaluate DNA barcoding as a valid tool to identify them. We also assessed the level of deeply conspecific mitochondrial lineages. Sequences were assigned to 42 different Barcode Index Numbers (BINs), 28 of which were new to BOLD. Out of the 20 named species barcoded, 11 (52.4%) had their own unique Barcode Index Number (BIN). Eight species (38.1%) showed multiple BINs with no morphological differentiation. Amongst them, C. newmannii showed two highly divergent genetic clusters which co-occur sympatrically, but further investigation is required to test whether they could represent new cryptic species.

Identification of the sex pheromone of a protected species, the Spanish moon moth Graellsia isabellae.

Sex attractant pheromones are highly sensitive and selective tools for detecting and monitoring populations of insects, yet there has been only one reported case of pheromones being used to monitor protected species. Here, we report the identification and synthesis of the sex pheromone of a protected European moth species, Graellsia isabellae (Lepidoptera: Saturniidae), as the single component, (4E,6E,11Z)-hexadecatrienal. In preliminary field trials, lures loaded with this compound attracted male moths from populations of this species at a number of widely separated field sites in France, Switzerland, and Spain, clearly demonstrating the utility of pheromones in sampling potentially endangered insect species.

Social parasitism by male-producing reproductive workers in a eusocial insect.

The evolution of extreme cooperation, as found in eusocial insects (those with a worker caste), is potentially undermined by selfish reproduction among group members. In some eusocial Hymenoptera (ants, bees and wasps), workers can produce male offspring from unfertilized eggs. Kin selection theory predicts levels of worker reproduction as a function of the relatedness structure of the workers' natal colony and the colony-level costs of worker reproduction. However, the theory has been only partially successful in explaining levels of worker reproduction. Here we show that workers of a eusocial bumble bee (Bombus terrestris) enter unrelated, conspecific colonies in which they then produce adult male offspring, and that such socially parasitic workers reproduce earlier and are significantly more reproductive and aggressive than resident workers that reproduce within their own colonies. Explaining levels of worker reproduction, and hence the potential of worker selfishness to undermine the evolution of cooperation, will therefore require more than simply a consideration of the kin-selected interests of resident workers. It will also require knowledge of the full set of reproductive options available to workers, including intraspecific social parasitism.