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We critically re-examine 17 records of fossils currently assigned to the lepidopteran superfamily Bombycoidea, which includes the silk moths, emperor moths and hawk moths. These records include subfossils, compression and impression fossils, permineralizations and ichnofossils. We assess whether observable morphological features warrant their confident assignment to the superfamily. None of the examined fossils displays characters that allow unequivocal identification as Sphingidae, but three fossils and a subfossil ( Mioclanis shanwangiana Zhang, Sun and Zhang, 1994, two fossil larvae, and a proboscis in asphaltum) have combinations of diagnostic features that support placement in the family. The identification of a fossil pupa as Bunaeini (Saturniidae) is well supported. The other fossils that we evaluate lack definitive bombycoid and, in several cases, even lepidopteran characters. Some of these dubious fossils have been used as calibration points in earlier studies casting doubt on the resulting age estimates. All fossil specimens reliably assigned to Bombycoidea are relatively young, the earliest fossil evidence of the superfamily dating to the middle Miocene., Competing Interests: The authors declare that they have no competing interests., (© 2023 Heikkilä et al.)

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 (H 2 O-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 H 2 O-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 (H 2 O-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., Competing Interests: The authors declare there are no competing interests., (©2023 Sire et al.)

The genus Lonomia Walker, 1855 (Lepidoptera: Saturniidae) is of particular interest to the medical community, since the scoli of these caterpillars harbor a venom that induces hemorrhaging in humans. In Colombia, deadly encounters with Lonomia achelous (Cramer, 1777), have been reported since 2000. There is little information on the main biological and ecological aspects of this genus to help better understand and develop prevention strategies. This study aimed to describe morphological and biological aspects (especially of immature stages) of four recently reported species of Lonomia in Colombia that pose a risk to humans. We collected caterpillars and adults from five localities and reared them under laboratory conditions. Specimens were identified using DNA barcoding and dissection of adult male genitalia. We provided the first description, to our knowledge, of part of the life cycles of Lonomia casanarensis Brechlin, 2017 and Lonomia orientoandensis Brechlin & Meister, 2011 and the complete life cycles of Lonomia columbiana Lemaire, 1972 and Lonomia orientocordillera Brechlin, Käch & Meister, 2013. We also present the first records of the parasitoids of L. orientocordillera, and L. casanarensis and new host plants. This information will guide not only their morphological recognition and the identification of their parasitoids and hosts, but also will guide rearing methods of these and other Lonomia species in new studies to prevent incidents with humans and create specific antivenoms., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Toro-Vargas et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Caterpillars of the Neotropical genus Lonomia (Lepidoptera: Saturniidae) are responsible for some fatal envenomation of humans in South America inducing hemostatic disturbances in patients upon skin contact with the caterpillars' spines. Currently, only two species have been reported to cause hemorrhagic syndromes in humans: Lonomia achelous and Lonomia obliqua. However, species identifications have remained largely unchallenged despite improved knowledge of venom diversity and growing evidence that the taxonomy used over past decades misrepresents and underestimates species diversity. Here, we revisit the taxonomic diversity and distribution of Lonomia species using the most extensive dataset assembled to date, combining DNA barcodes, morphological comparisons, and geographical information. Considering new evidence for seven undescribed species as well as three newly proposed nomenclatural changes, our integrative approach leads to the recognition of 60 species, of which seven are known or strongly suspected to cause severe envenomation in humans. From a newly compiled synthesis of epidemiological data, we also examine the consequences of our results for understanding Lonomia envenomation risks and call for further investigations of other species' venom activities. This is required and necessary to improve alertness in areas at risk, and to define adequate treatment strategies for envenomed patients, including performing species identification and assessing the efficacy of anti-Lonomia serums against a broader diversity of species., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 González et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

One of the key objectives in biological research is understanding how evolutionary processes have produced Earth's diversity. A critical step toward revealing these processes is an investigation of evolutionary tradeoffs-that is, the opposing pressures of multiple selective forces. For millennia, nocturnal moths have had to balance successful flight, as they search for mates or host plants, with evading bat predators. However, the potential for evolutionary trade-offs between wing shape and body size are poorly understood. In this study, we used phylogenomics and geometric morphometrics to examine the evolution of wing shape in the wild silkmoth subfamily Arsenurinae (Saturniidae) and evaluate potential evolutionary relationships between body size and wing shape. The phylogeny was inferred based on 782 loci from target capture data of 42 arsenurine species representing all 10 recognized genera. After detecting in our data one of the most vexing problems in phylogenetic inference-a region of a tree that possesses short branches and no "support" for relationships (i.e., a polytomy), we looked for hidden phylogenomic signal (i.e., inspecting differing phylogenetic inferences, alternative support values, quartets, and phylogenetic networks) to better illuminate the most probable generic relationships within the subfamily. We found there are putative evolutionary trade-offs between wing shape, body size, and the interaction of fore- and hindwing (HW) shape. Namely, body size tends to decrease with increasing HW length but increases as forewing (FW) shape becomes more complex. Additionally, the type of HW (i.e., tail or no tail) a lineage possesses has a significant effect on the complexity of FW shape. We outline possible selective forces driving the complex HW shapes that make Arsenurinae, and silkmoths as a whole, so charismatic. [Anchored hybrid enrichment; Arsenurinae; geometric morphometrics; Lepidoptera; phylogenomics; Saturniidae.]., (© The Author(s) 2021. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The regions of the Andes and Caribbean-Mesoamerica are both hypothesized to be the cradle for many Neotropical lineages, but few studies have fully investigated the dynamics and interactions between Neotropical bioregions. The New World hawkmoth genus Xylophanes is the most taxonomically diverse genus in the Sphingidae, with the highest endemism and richness in the Andes and Caribbean-Mesoamerica. We integrated phylogenomic and DNA barcode data and generated the first time-calibrated tree for this genus, covering 93.8% of the species diversity. We used event-based likelihood ancestral area estimation and biogeographic stochastic mapping to examine the speciation and dispersal dynamics of Xylophanes across bioregions. We also used trait-dependent diversification models to compare speciation and extinction rates of lineages associated with different bioregions. Our results indicate that Xylophanes originated in Caribbean-Mesoamerica in the Late Miocene, and immediately diverged into five major clades. The current species diversity and distribution of Xylophanes can be explained by two consecutive phases. In the first phase, the highest Xylophanes speciation and emigration rates occurred in the Caribbean-Mesoamerica, and the highest immigration rates occurred in the Andes, whereas in the second phase the highest immigration rates were found in Amazonia, and the Andes had the highest speciation and emigration rates.

Based on the review of literature and biological collections, information retrieved from public online databases, and from fieldwork conducted between 2015 and 2020, we provide more than 3500 occurrence records and an updated checklist of Colombian Saturniidae, annotated with distribution data for all species. In the first checklist of Colombian saturniids published two decades ago, a total of 184 species were cited; in the current update the number has risen to 653 species/subspecies classified in 55 genera, representing all six recognized Neotropical subfamilies. The Andean and Pacific regions are the richest, accounting for about three quarters of all species listed herein. We call attention to the fact that for most speciose saturniid genera in the Neotropics, there remains a significant need for further taxonomic and systematic research with objective and integrative approaches. Likewise, it is necessary to continue the study of Saturniidae moths in Colombia, strategically increasing the sampling efforts in specific under-sampled natural regions to further document the diversity of this family in the country.

The Saturniidae is one of the most emblematic families of moths, comprising nearly 3000 species distributed globally. In this study, DNA barcode analysis and comparative morphology were combined to describe three new species within the genus Automeris , which is the most diverse genus in the family. Automeris llaneros Decaëns, Rougerie & Bonilla, sp. nov. , Automeris mineros Decaëns, Rougerie & Bonilla, sp. nov. , and Automeris belemensis Decaëns, Rougerie & Bénéluz, sp. nov. are described from the Colombian Orinoco watershed, the Colombian Eastern Cordillera, and the area of endemism of Belém in the Brazilian Amazonia, respectively. They all belong to the Automeris bilinea (Walker, 1855) species subgroup, which comprises a number of species that are sometimes difficult to distinguish from each other using morphology alone. Here, the description of these three new species is based on significant differences from their closest relatives, either in terms of wing patterns, genitalia, DNA barcodes or a combination of these features., (Thibaud Decaëns, Frédéric Bénéluz, Liliana Ballesteros-Mejia, Diego Bonilla, Rodolphe Rougerie.)

Gamelia bennetti sp. nov. is described from Trinidad, Trinidad and Tobago, West Indies, and compared with members of the Gamelia abas species group: G. abas (Cramer, [1775]), G. berliozi Lemaire, 1967, G. lichyi Lemaire, 1973, G. rubriluna (Walker, 1862) and G. septentrionalis (Bouvier, 1936). A photographic record suggests G. bennetti sp. nov. may also occur in Tobago.

Background: Herbivorous insects represent a major fraction of global biodiversity and the relationships they have established with their food plants range from strict specialists to broad generalists. Our knowledge of these relationships is of primary importance to basic (e.g. the study of insect ecology and evolution) and applied biology (e.g. monitoring of pest or invasive species) and yet remains very fragmentary and understudied. In Lepidoptera, caterpillars of families Saturniidae and Sphingidae are rather well known and considered to have adopted contrasting preferences in their use of food plants. The former are regarded as being rather generalist feeders, whereas the latter are more specialist., New Information: To assemble and synthesise the vast amount of existing data on food plants of Lepidoptera families Saturniidae and Sphingidae, we combined three major existing databases to produce a dataset collating more than 26,000 records for 1256 species (25% of all species) in 121 (67%) and 167 (81%) genera of Saturniidae and Sphingidae, respectively. This dataset is used here to document the level of polyphagy of each of these genera using summary statistics, as well as the calculation of a polyphagy score derived from the analysis of Phylogenetic Diversity of the food plants used by the species in each genus., (Liliana Ballesteros Mejia, Pierre Arnal, Winnie Hallwachs, Jean Haxaire, Daniel Janzen, Ian J. Kitching, Rodolphe Rougerie.)

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.

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.

Assessing how much management of agricultural landscapes, in addition to protected areas, can offset biodiversity erosion in the tropics is a central issue for conservation that still requires cross-taxonomic and landscape-scale studies. We measured the effects of Amazonia deforestation and subsequent land-use intensification in 6 agricultural areas (landscape scale), where we sampled plants and 4 animal groups (birds, earthworms, fruit flies, and moths). We assessed land-use intensification with a synthetic index based on landscape metrics (total area and relative percentages of land uses, edge density, mean patch density and diversity, and fractal structures at 5 dates from 1990 to 2007). Species richness decreased consistently as agricultural intensification increased despite slight differences in the responses of sampled groups. Globally, in moderately deforested landscapes species richness was relatively stable, and there was a clear threshold in biodiversity loss midway along the intensification gradient, mainly linked to a drop in forest cover and quality. Our results suggest anthropogenic landscapes with high-quality forest covering >40 % of the surface area may prevent biodiversity loss in Amazonia., (© 2018 Society for Conservation Biology.)

Background: Bombycoidea is an ecologically diverse and speciose superfamily of Lepidoptera. The superfamily includes many model organisms, but the taxonomy and classification of the superfamily has remained largely in disarray. Here we present a global checklist of Bombycoidea. Following Zwick (2008) and Zwick et al. (2011), ten families are recognized: Anthelidae, Apatelodidae, Bombycidae, Brahmaeidae, Carthaeidae, Endromidae, Eupterotidae, Phiditiidae, Saturniidae and Sphingidae. The former families Lemoniidae and Mirinidae are included within Brahmaeidae and Endromidae respectively. The former bombycid subfamilies Oberthueriinae and Prismostictinae are also treated as synonyms of Endromidae, and the former bombycine subfamilies Apatelodinae and Phitditiinae are treated as families., New Information: This checklist represents the first effort to synthesize the current taxonomic treatment of the entire superfamily. It includes 12,159 names and references to their authors, and it accounts for the recent burst in species and subspecies descriptions within family Saturniidae (ca. 1,500 within the past 10 years) and to a lesser extent in Sphingidae (ca. 250 species over the same period). The changes to the higher classification of Saturniidae proposed by Nässig et al. (2015) are rejected as premature and unnecessary. The new tribes, subtribes and genera described by Cooper (2002) are here treated as junior synonyms. We also present a new higher classification of Sphingidae, based on Kawahara et al. (2009), Barber and Kawahara (2013) and a more recent phylogenomic study by Breinholt et al. (2017), as well as a reviewed genus and species level classification, as documented by Kitching (2018).

We have little knowledge of the response of invertebrate assemblages to climate change in tropical ecosystems, and few studies have compiled long-term data on invertebrates from tropical rainforests. We provide an updated list of the 72 species of Saturniidae moths collected on Barro Colorado Island (BCI), Panama, during the period 1958-2016. This list will serve as baseline data for assessing long-term changes of saturniids on BCI in the future, as 81% of the species can be identified by their unique DNA Barcode Index Number, including four cryptic species not yet formally described. A local species pool of 60 + species breeding on BCI appears plausible, but more cryptic species may be discovered in the future. We use monitoring data obtained by light trapping to analyze recent population trends on BCI for saturniid species that were relatively common during 2009-2016, a period representing >30 saturniid generations. The abundances of 11 species, of 14 tested, could be fitted to significant time-series models. While the direction of change in abundance was uncertain for most species, two species showed a significant increase over time, and forecast models also suggested continuing increases for most species during 2017-2018, as compared to the 2009 base year. Peaks in saturniid abundance were most conspicuous during El Niño and La Niña years. In addition to a species-specific approach, we propose a reproducible functional classification based on five functional traits to analyze the responses of species sharing similar functional attributes in a fluctuating climate. Our results suggest that the abundances of larger body-size species with good dispersal abilities may increase concomitantly with rising air temperature in the future, because short-lived adults may allocate less time to increasing body temperature for flight, leaving more time available for searching for mating partners or suitable oviposition sites.

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., (© The Author(s) 2016. Published by Oxford University Press, on behalf of the Society of Systematic Biologists.)

The morphological species delimitations (i.e. morphospecies) have long been the best way to avoid the taxonomic impediment and compare insect taxa biodiversity in highly diverse tropical and subtropical regions. The development of DNA barcoding, however, has shown great potential to replace (or at least complement) the morphospecies approach, with the advantage of relying on automated methods implemented in computer programs or even online rather than in often subjective morphological features. We sampled moths extensively for two years using light traps in a patch of the highly endangered Atlantic Forest of Brazil to produce a nearly complete census of arctiines (Noctuoidea: Erebidae), whose species richness was compared using different morphological and molecular approaches (DNA barcoding). A total of 1,075 barcode sequences of 286 morphospecies were analyzed. Based on the clustering method Barcode Index Number (BIN) we found a taxonomic bias of approximately 30% in our initial morphological assessment. However, a morphological reassessment revealed that the correspondence between morphospecies and molecular operational taxonomic units (MOTUs) can be up to 94% if differences in genitalia morphology are evaluated in individuals of different MOTUs originated from the same morphospecies (putative cases of cryptic species), and by recording if individuals of different genders in different morphospecies merge together in the same MOTU (putative cases of sexual dimorphism). The results of two other clustering methods (i.e. Automatic Barcode Gap Discovery and 2% threshold) were very similar to those of the BIN approach. Using empirical data we have shown that DNA barcoding performed substantially better than the morphospecies approach, based on superficial morphology, to delimit species of a highly diverse moth taxon, and thus should be used in species inventories.

Background: Comprehensive biotic surveys, or 'all taxon biodiversity inventories' (ATBI), have traditionally been limited in scale or scope due to the complications surrounding specimen sorting and species identification. To circumvent these issues, several ATBI projects have successfully integrated DNA barcoding into their identification procedures and witnessed acceleration in their surveys and subsequent increase in project scope and scale. The Biodiversity Institute of Ontario partnered with the rare Charitable Research Reserve and delegates of the 6th International Barcode of Life Conference to complete its own rapid, barcode-assisted ATBI of an established land trust in Cambridge, Ontario, Canada., New Information: The existing species inventory for the rare Charitable Research Reserve was rapidly expanded by integrating a DNA barcoding workflow with two surveying strategies - a comprehensive sampling scheme over four months, followed by a one-day bioblitz involving international taxonomic experts. The two surveys resulted in 25,287 and 3,502 specimens barcoded, respectively, as well as 127 human observations. This barcoded material, all vouchered at the Biodiversity Institute of Ontario collection, covers 14 phyla, 29 classes, 117 orders, and 531 families of animals, plants, fungi, and lichens. Overall, the ATBI documented 1,102 new species records for the nature reserve, expanding the existing long-term inventory by 49%. In addition, 2,793 distinct Barcode Index Numbers (BINs) were assigned to genus or higher level taxonomy, and represent additional species that will be added once their taxonomy is resolved. For the 3,502 specimens, the collection, sequence analysis, taxonomic assignment, data release and manuscript submission by 100+ co-authors all occurred in less than one week. This demonstrates the speed at which barcode-assisted inventories can be completed and the utility that barcoding provides in minimizing and guiding valuable taxonomic specialist time. The final product is more than a comprehensive biotic inventory - it is also a rich dataset of fine-scale occurrence and sequence data, all archived and cross-linked in the major biodiversity data repositories. This model of rapid generation and dissemination of essential biodiversity data could be followed to conduct regional assessments of biodiversity status and change, and potentially be employed for evaluating progress towards the Aichi Targets of the Strategic Plan for Biodiversity 2011-2020.

Saproxylic beetles - associated with dead wood or with other insects, fungi and microorganisms that decompose it - play a major role in forest nutrient cycling. They are important ecosystem service providers and are used as key bio-indicators of old-growth forests. In France alone, where the present study took place, there are about 2500 species distributed within 71 families. This high diversity represents a major challenge for specimen sorting and identification. The PASSIFOR project aims at developing a DNA metabarcoding approach to facilitate and enhance the monitoring of saproxylic beetles as indicators in ecological studies. As a first step toward that goal we assembled a library of DNA barcodes using the standard genetic marker for animals, i.e. a portion of the COI mitochondrial gene. In the present contribution, we release a library including 656 records representing 410 species in 40 different families. Species were identified by expert taxonomists, and each record is linked to a voucher specimen to enable future morphological examination. We also highlight and briefly discuss cases of low interspecific divergences, as well as cases of high intraspecific divergences that might represent cases of overlooked or cryptic diversity.

Europe has one of the best-known Lepidopteran faunas in the world, yet many species are still being discovered, especially in groups of small moths. Here we describe a new gracillariid species from the south-eastern Alps, Callistobasistrigella Huemer, Deutsch & Triberti, sp. n. It shows differences from its sister species Callistocoffeella in morphology, the barcode region of the cytochrome c oxidase I gene and the nuclear gene histone H3. Both Callistobasistrigella and Callistocoffeella can co-occur in sympatry without evidence of admixture. Two Callistobasistrigella specimens show evidence of introgression. We highlight the importance of an integrative approach to delimit species, combining morphological and ecological data with mitochondrial and nuclear sequence data. Furthermore, in connection with this study, Ornixblandella Müller-Rutz, 1920, syn. n. is synonymized with Callistocoffeella (Zetterstedt, 1839).

Main Objective: We examine the extent of taxonomic and biogeographical uncertainty in a well-studied group of Australian Lepidoptera, the hawkmoths (Sphingidae)., Methods: We analysed the diversity of Australian sphingids through the comparative analysis of their DNA barcodes, supplemented by morphological re-examinations and sequence information from a nuclear marker in selected cases. The results from the analysis of Australian sphingids were placed in a broader context by including conspecifics and closely related taxa from outside Australia to test taxonomic boundaries., Results: Our results led to the discovery of six new species in Australia, one case of erroneously synonymized species, and three cases of synonymy. As a result, we establish the occurrence of 75 species of hawkmoths on the continent. The analysis of records from outside Australia also challenges the validity of current taxonomic boundaries in as many as 18 species, including Agrius convolvuli (Linnaeus, 1758), a common species that has gained adoption as a model system. Our work has revealed a higher level of endemism than previously recognized. Most (90%) Australian sphingids are endemic to the continent (45%) or to Australia, the Pacific Islands and the Papuan and Wallacean regions (45%). Only seven species (10%) have ranges that extend beyond this major biogeographical boundary toward SE Asia and other regions of the Old World., Main Conclusions: This study has established that overlooked cryptic diversity and inaccurate species delineation produced significant misconceptions concerning diversity and distribution patterns in a group of insects that is considered well known taxonomically. Because DNA barcoding represents a straightforward way to test taxonomic boundaries, its implementation can improve the accuracy of primary diversity data in biogeography and conservation studies.

Westindia Vincent, a new genus, is proposed for W. haxairei Vincent, a new species of Neotropical tiger-moth described from Dominican Republic. Habitus, male and female genitalia are described and figured. The systematic position of the new genus within Arctiinae is discussed in light of a comparative morphology and a molecular phylogeny derived from the DNA barcode fragment of the mitochondrial COI gene and of the D2 region of the 28S rDNA gene.

Background: The geometrid moths of Europe are one of the best investigated insect groups in traditional taxonomy making them an ideal model group to test the accuracy of the Barcode Index Number (BIN) system of BOLD (Barcode of Life Datasystems), a method that supports automated, rapid species delineation and identification., Methodology/principal Findings: This study provides a DNA barcode library for 219 of the 249 European geometrid moth species (88%) in five selected subfamilies. The data set includes COI sequences for 2130 specimens. Most species (93%) were found to possess diagnostic barcode sequences at the European level while only three species pairs (3%) were genetically indistinguishable in areas of sympatry. As a consequence, 97% of the European species we examined were unequivocally discriminated by barcodes within their natural areas of distribution. We found a 1:1 correspondence between BINs and traditionally recognized species for 67% of these species. Another 17% of the species (15 pairs, three triads) shared BINs, while specimens from the remaining species (18%) were divided among two or more BINs. Five of these species are mixtures, both sharing and splitting BINs. For 82% of the species with two or more BINs, the genetic splits involved allopatric populations, many of which have previously been hypothesized to represent distinct species or subspecies., Conclusions/significance: This study confirms the effectiveness of DNA barcoding as a tool for species identification and illustrates the potential of the BIN system to characterize formal genetic units independently of an existing classification. This suggests the system can be used to efficiently assess the biodiversity of large, poorly known assemblages of organisms. For the moths examined in this study, cases of discordance between traditionally recognized species and BINs arose from several causes including overlooked species, synonymy, and cases where DNA barcodes revealed regional variation of uncertain taxonomic significance.

Classical biological control is often advocated as a tool for managing invasive species. However, accurate evaluations of parasitoid species complexes and assessment of host specificity are impeded by the lack of morphological variation. Here, we study the possibility of host races/species within the eulophid wasp Pediobius saulius, a pupal generalist parasitoid that parasitize the highly invasive horse-chestnut leaf-mining moth Cameraria ohridella. We analysed the population genetic structure, host associations and phylogeographic patterns of P. saulius in Europe using the COI mitochondrial gene. This marker strongly supports a division into at least five highly differentiated parasitoid complexes, within two of which clades with differing degrees of host specialization were found: a Balkan clade that mainly (but not only) attacks C. ohridella and a more generalist European group that attacks many hosts, including C. ohridella. The divergence in COI (up to 7.6%) suggests the existence of cryptic species, although this is neither confirmed by nuclear divergence nor morphology. We do not find evidence of host tracking. The higher parasitism rates observed in the Balkans and the scarcity of the Balkan-Cameraria haplotypes out of the Balkans open the possibility of using these Balkan haplotypes as biological control agents of C. ohridella elsewhere in Europe.

The wild silkmoth genus Sinobirma Bryk, 1944 is a poorly known monotypic taxon from the eastern end of the Himalaya Range. It was convincingly proposed to be closely related to some members of an exclusively Afro-tropical group of Saturniidae, but its biogeographical and evolutionary history remains enigmatic. After examining recently collected material from Tibet, northern India, and northeastern Myanmar, we realized that this unique species, S. malaisei Bryk, 1944 only known so far from a few specimens and from a very restricted area near the border between north-eastern Myanmar and the Yunnan province of China, may in fact belong to a group of closely related cryptic species. In this work, we combined morphological comparative study, DNA barcoding, and the sequences of a nuclear marker (D2 expansion segment of the 28S rRNA gene) to unequivocally delimit three distinct species in the genus Sinobirma, of which two are described as new to science: S. myanmarensis sp. n. and S. bouyeri sp. n. An informative DNA barcode sequence was obtained from the female holotype of S. malaisei--collected in 1934--ensuring the proper assignation of this name to the newly collected and studied specimens. Our findings represent another example of the potential of coupling traditional taxonomy and DNA barcoding for revealing and solving difficult cases of cryptic diversity. This approach is now being generalized to the world fauna of Saturniidae, with the participation of most of the taxonomists studying these moths.

Wolbachia is a genus of bacterial endosymbionts that impacts the breeding systems of their hosts. Wolbachia can confuse the patterns of mitochondrial variation, including DNA barcodes, because it influences the pathways through which mitochondria are inherited. We examined the extent to which these endosymbionts are detected in routine DNA barcoding, assessed their impact upon the insect sequence divergence and identification accuracy, and considered the variation present in Wolbachia COI. Using both standard PCR assays (Wolbachia surface coding protein--wsp), and bacterial COI fragments we found evidence of Wolbachia in insect total genomic extracts created for DNA barcoding library construction. When >2 million insect COI trace files were examined on the Barcode of Life Datasystem (BOLD) Wolbachia COI was present in 0.16% of the cases. It is possible to generate Wolbachia COI using standard insect primers; however, that amplicon was never confused with the COI of the host. Wolbachia alleles recovered were predominantly Supergroup A and were broadly distributed geographically and phylogenetically. We conclude that the presence of the Wolbachia DNA in total genomic extracts made from insects is unlikely to compromise the accuracy of the DNA barcode library; in fact, the ability to query this DNA library (the database and the extracts) for endosymbionts is one of the ancillary benefits of such a large scale endeavor--which we provide several examples. It is our conclusion that regular assays for Wolbachia presence and type can, and should, be adopted by large scale insect barcoding initiatives. While COI is one of the five multi-locus sequence typing (MLST) genes used for categorizing Wolbachia, there is limited overlap with the eukaryotic DNA barcode region.

Background: When a specimen belongs to a species not yet represented in DNA barcode reference libraries there is disagreement over the effectiveness of using sequence comparisons to assign the query accurately to a higher taxon. Library completeness and the assignment criteria used have been proposed as critical factors affecting the accuracy of such assignments but have not been thoroughly investigated. We explored the accuracy of assignments to genus, tribe and subfamily in the Sphingidae, using the almost complete global DNA barcode reference library (1095 species) available for this family. Costa Rican sphingids (118 species), a well-documented, diverse subset of the family, with each of the tribes and subfamilies represented were used as queries. We simulated libraries with different levels of completeness (10-100% of the available species), and recorded assignments (positive or ambiguous) and their accuracy (true or false) under six criteria., Results: A liberal tree-based criterion assigned 83% of queries accurately to genus, 74% to tribe and 90% to subfamily, compared to a strict tree-based criterion, which assigned 75% of queries accurately to genus, 66% to tribe and 84% to subfamily, with a library containing 100% of available species (but excluding the species of the query). The greater number of true positives delivered by more relaxed criteria was negatively balanced by the occurrence of more false positives. This effect was most sharply observed with libraries of the lowest completeness where, for example at the genus level, 32% of assignments were false positives with the liberal criterion versus < 1% when using the strict. We observed little difference (< 8% using the liberal criterion) however, in the overall accuracy of the assignments between the lowest and highest levels of library completeness at the tribe and subfamily level., Conclusions: Our results suggest that when using a strict tree-based criterion for higher taxon assignment with DNA barcodes, the likelihood of assigning a query a genus name incorrectly is very low, if a genus name is provided it has a high likelihood of being accurate, and if no genus match is available the query can nevertheless be assigned to a subfamily with high accuracy regardless of library completeness. DNA barcoding often correctly assigned sphingid moths to higher taxa when species matches were unavailable, suggesting that barcode reference libraries can be useful for higher taxon assignments long before they achieve complete species coverage.

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., (© 2010 Blackwell Publishing Ltd.)

The widely studied and invasive earthworm, Lumbricus terrestris L., 1758 has been the subject of nomenclatural debate for many years. However these disputes were not based on suspicions of heterogeneity, but rather on the descriptions and nomenclatural acts associated with the species name. Large numbers of DNA barcode sequences of the cytochrome oxidase I obtained for nominal L. terrestris and six congeneric species reveal that there are two distinct lineages within nominal L. terrestris. One of those lineages contains the Swedish population from which the name-bearing specimen of L. terrestris was obtained. The other contains the population from which the syntype series of Enterion herculeum Savigny, 1826 was collected. In both cases modern and old representatives yielded barcode sequences allowing us to clearly establish that these are two distinct species, as different from one another as any other pair of congeners in our data set. The two are morphologically indistinguishable, except by overlapping size-related characters. We have designated a new neotype for L. terrestris. The newly designated neotype and a syntype of L. herculeus yielded DNA adequate for sequencing part of the cytochrome oxidase I gene (COI). The sequence data make possible the objective determination of the identities of earthworms morphologically identical to L. terrestris and L. herculeus, regardless of body size and segment number. Past work on nominal L. terrestris could have been on either or both species, although L. herculeus has yet to be found outside of Europe.

Here, we describe a simple method adapted for high-throughput protocols allowing voucher specimen recovery for Collembola and by extension for other soft-bodied small arthropods. A standard extraction protocol was tested to examine the effects of lysis duration (1, 2, 4, 12 h) on DNA concentration, amplification success and specimen condition. Good quality DNA was obtained after 1 h of lysis, while voucher condition was fine for up to 12 h. The lysis step substantially shortens the clearing process necessary for morphological examination., (© 2010 Blackwell Publishing Ltd.)

Inventory of the caterpillars, their food plants and parasitoids began in 1978 for today's Area de Conservacion Guanacaste (ACG), in northwestern Costa Rica. This complex mosaic of 120 000 ha of conserved and regenerating dry, cloud and rain forest over 0-2000 m elevation contains at least 10 000 species of non-leaf-mining caterpillars used by more than 5000 species of parasitoids. Several hundred thousand specimens of ACG-reared adult Lepidoptera and parasitoids have been intensively and extensively studied morphologically by many taxonomists, including most of the co-authors. DNA barcoding - the use of a standardized short mitochondrial DNA sequence to identify specimens and flush out undisclosed species - was added to the taxonomic identification process in 2003. Barcoding has been found to be extremely accurate during the identification of about 100 000 specimens of about 3500 morphologically defined species of adult moths, butterflies, tachinid flies, and parasitoid wasps. Less than 1% of the species have such similar barcodes that a molecularly based taxonomic identification is impossible. No specimen with a full barcode was misidentified when its barcode was compared with the barcode library. Also as expected from early trials, barcoding a series from all morphologically defined species, and correlating the morphological, ecological and barcode traits, has revealed many hundreds of overlooked presumptive species. Many but not all of these cryptic species can now be distinguished by subtle morphological and/or ecological traits previously ascribed to 'variation' or thought to be insignificant for species-level recognition. Adding DNA barcoding to the inventory has substantially improved the quality and depth of the inventory, and greatly multiplied the number of situations requiring further taxonomic work for resolution., (© 2009 Blackwell Publishing Ltd and Crown in the right of Canada.)

The current classification of African representatives of the family Saturniidae is largely unsatisfactory and needs to be revised following a proper and rigorous phylogenetic analysis. In this regard, the use of characters of preimaginal stages in phylogeny reconstruction was already emphasized in the literature; however, few details are known about the morphology of the eggs, larvae, and pupae of these moths. To fill this gap and to provide a preliminary basis for further phylogenetic studies, the early stages of Bunaeopsis licharbas, a member of the tribe Bunaeini within the Saturniinae, are described. Egg chorionic ultrastructure, chaetotaxy of the larva in every instar, and morphology of the pupa are described and illustrated. A total of 22 previously overlooked characters was found and their potential phylogenetic significance is discussed in the light of a comparative study which includes a large set of representatives of all five tribes in the subfamily. Until further phylogenetic analyses, some characters are considered to be potential synapomorphies supporting, in particular, the monophyly of the tribe Bunaeini, the close relationship between this tribe and part of the Urotini, as well as the existence of an Afrotropical lineage within the Saturniinae uniting the tribes Bunaeini, Urotini, and Micragonini. In a group of moths whose preimaginal instars are among the best known within the Lepidoptera, the unexpected, and relatively high number of potentially informative characters found by the present study stresses the value of minute and exhaustive descriptive studies to make use of morphological characters available for phylogenetic studies., (Copyright (c) 2007 Wiley-Liss, Inc.)