Historical museum samples reveal signals of selection and drift in response to changing insecticide use in an agricultural pest moth.

In response to environmental and human-imposed selective pressures, agroecosystem pests frequently undergo rapid evolution, with some species having a remarkable capacity to rapidly develop pesticide resistance. Temporal sampling of genomic data can comprehensively capture such adaptive changes over time, for example, by elucidating allele frequency shifts in pesticide resistance loci in response to different pesticides. Here, we leveraged museum specimens spanning over a century of collections to generate temporal contrasts between pre- and post-insecticide populations of an agricultural pest moth, Helicoverpa armigera. We used targeted exon sequencing of 254 samples collected across Australia from the pre-1950s (prior to insecticide introduction) to the 1990s, encompassing decades of changing insecticide use. Our sequencing approach focused on genes that are known to be involved in insecticide resistance, environmental sensation, and stress tolerance. We found an overall lack of spatial and temporal population structure change across Australia. In some decades (e.g., 1960s and 1970s), we found a moderate reduction of genetic diversity, implying stochasticity in evolutionary trajectories due to genetic drift. Temporal genome scans showed extensive evidence of selection following insecticide use, although the majority of selected variants were low impact, and alternating trajectories of allele frequency change were suggestive of potential antagonistic pleiotropy. Our results provide new insights into recent evolutionary responses in an agricultural pest and show how temporal contrasts using museum specimens can improve mechanistic understanding of rapid evolution.

Large-scale genomic data reveal the phylogeny and evolution of owlet moths (Noctuoidea).

The owlet moths (Noctuoidea; ~43-45K described species) are one of the most ecologically diverse and speciose superfamilies of animals. Moreover, they comprise some of the world's most notorious pests of agriculture and forestry. Despite their contributions to terrestrial biodiversity and impacts on ecosystems and economies, the evolutionary history of Noctuoidea remains unclear because the superfamily lacks a statistically robust phylogenetic and temporal framework. We reconstructed the phylogeny of Noctuoidea using data from 1234 genes (946.4 kb nucleotides) obtained from the genome and transcriptome sequences of 76 species. The relationships among the six families of Noctuoidea were well resolved and consistently recovered based on both concatenation and gene coalescence approaches, supporting the following relationships: Oenosandridae + (Notodontidae + (Erebidae + (Nolidae + (Euteliidae + Noctuidae)))). A Yule tree prior with three unlinked molecular clocks was identified as the preferred BEAST analysis using marginal-likelihood estimations. The crown age of Noctuoidea was estimated at 74.5 Ma, with most families originating before the end of the Paleogene (23 Ma). Our study provides the first statistically robust phylogenetic and temporal framework for Noctuoidea, including all families of owlet moths, based on large-scale genomic data.

Museomics unveil systematics, diversity and evolution of Australian cycad-pollinating weevils.

Weevils have been shown to play significant roles in the obligate pollination of Australian cycads. In this study, we apply museomics to produce a first molecular phylogeny estimate of the Australian cycad weevils, allowing an assessment of their monophyly, placement and relationships. Divergence dating suggests that the Australian cycad weevils originated from the Late Oligocene to the Middle Miocene and that the main radiation of the cycad-pollinating groups occurred from the Middle to the Late Miocene, which is congruent with the diversification of the Australian cycads, thus refuting any notion of an ancient ciophilous system in Australia. Taxonomic studies reveal the existence of 19 Australian cycad weevil species and that their associations with their hosts are mostly non-species-specific. Co-speciation analysis shows no extensive co-speciation events having occurred in the ciophilous system of Australian cycads. The distribution pattern suggests that geographical factors, rather than diversifying coevolution, constitute the overriding process shaping the Australian cycad weevil diversity. The synchronous radiation of cycads and weevil pollinators is suggested to be a result of the post-Oligocene diversification common in Australian organisms.

Pollen DNA metabarcoding reveals cryptic diversity and high spatial turnover in alpine plant-pollinator networks.

Alpine plant-pollinator communities play an important role in the functioning of alpine ecosystems, which are highly threatened by climate change. However, we still have a poor understanding of how environmental factors and spatiotemporal variability shape these communities. Here, we investigate what drives structure and beta diversity in a plant-pollinator metacommunity from the Australian alpine region using two approaches: pollen DNA metabarcoding (MB) and observations. Individual pollinators often carry pollen from multiple plant species, and therefore we expected MB to reveal a more diverse and complex network structure. We used two gene regions (ITS2 and trnL) to identify plant species present in the pollen loads of 154 insect pollinator specimens from three alpine habitats and construct MB networks, and compared them to networks based on observations alone. We compared species and interaction turnover across space for both types of networks, and evaluated their differences for plant phylogenetic diversity and beta diversity. We found significant structural differences between the two types of networks; notably, MB networks were much less specialized but more diverse than observation networks, with MB detecting many cryptic plant species. Both approaches revealed that alpine pollination networks are very generalized, but we estimated a high spatial turnover of plant species (0.79) and interaction rewiring (0.6) as well as high plant phylogenetic diversity (0.68) driven by habitat differences based on the larger diversity of plant species and species interactions detected with MB. Overall, our findings show that habitat and microclimatic heterogeneity drives diversity and fine-scale spatial turnover of alpine plant-pollinator networks.

Phylogenomics resolves timing and patterns in the evolution of Australasian Cerambycinae (Coleoptera: Cerambycidae), and reveals new insights into the subfamily-level classification and historical biogeography of longhorn beetles.

Cerambycinae is the second-largest subfamily of longhorn beetles in the Southern Hemisphere. The phylogeny of Cerambycinae is poorly known, resulting in a highly artificial tribal-level classification and a largely speculative evolutionary history. We reconstructed the phylogenetic relationships of Cerambycinae at the generic level using anchored hybrid enrichment data from hundreds of nuclear genes, with a primary focus on the extraordinarily diverse faunas of Australia and New Zealand. We also estimated divergence times by incorporating fossil calibrations in our analyses. We identified two main clades within Cerambycinae, which can also be separated morphologically by a distinct type of antennal foramen. We recovered a Late Jurassic origin of crown Cerambycinae. Dorcasominae, which was newly found to have representatives in Australia, was notably derived from within Cerambycinae. We recovered two independent origins of Australian Cerambycinae: one clade originated in the Early Cretaceous and is likely endemic to the Southern Hemisphere, while the other clade appears to have immigrated to Australia, perhaps from the Northern Hemisphere. Within the Australian lineages were multiple independent origins of New Zealand taxa, all of which are relative host-plant generalists. Tribal relationships and assignments are discussed, and based on our results, the following major nomenclatural acts were made: Dorcasominae Lacordaire, 1868 is downgraded to a tribe Dorcasomini of Cerambycinae Latreille, 1804; Neostenini Lacordaire, 1868 syn. nov. is treated as a junior synonym of Uracanthini Blanchard, 1851.

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.

Phylogenomics reveals the evolutionary timing and pattern of butterflies and moths.

Butterflies and moths (Lepidoptera) are one of the major superradiations of insects, comprising nearly 160,000 described extant species. As herbivores, pollinators, and prey, Lepidoptera play a fundamental role in almost every terrestrial ecosystem. Lepidoptera are also indicators of environmental change and serve as models for research on mimicry and genetics. They have been central to the development of coevolutionary hypotheses, such as butterflies with flowering plants and moths' evolutionary arms race with echolocating bats. However, these hypotheses have not been rigorously tested, because a robust lepidopteran phylogeny and timing of evolutionary novelties are lacking. To address these issues, we inferred a comprehensive phylogeny of Lepidoptera, using the largest dataset assembled for the order (2,098 orthologous protein-coding genes from transcriptomes of 186 species, representing nearly all superfamilies), and dated it with carefully evaluated synapomorphy-based fossils. The oldest members of the Lepidoptera crown group appeared in the Late Carboniferous (∼300 Ma) and fed on nonvascular land plants. Lepidoptera evolved the tube-like proboscis in the Middle Triassic (∼241 Ma), which allowed them to acquire nectar from flowering plants. This morphological innovation, along with other traits, likely promoted the extraordinary diversification of superfamily-level lepidopteran crown groups. The ancestor of butterflies was likely nocturnal, and our results indicate that butterflies became day-flying in the Late Cretaceous (∼98 Ma). Moth hearing organs arose multiple times before the evolutionary arms race between moths and bats, perhaps initially detecting a wide range of sound frequencies before being co-opted to specifically detect bat sonar. Our study provides an essential framework for future comparative studies on butterfly and moth evolution.

Horizontally acquired antibacterial genes associated with adaptive radiation of ladybird beetles.

BACKGROUND: Horizontal gene transfer (HGT) has been documented in many herbivorous insects, conferring the ability to digest plant material and promoting their remarkable ecological diversification. Previous reports suggest HGT of antibacterial enzymes may have contributed to the insect immune response and limit bacterial growth. Carnivorous insects also display many evolutionary successful lineages, but in contrast to the plant feeders, the potential role of HGTs has been less well-studied. RESULTS: Using genomic and transcriptomic data from 38 species of ladybird beetles, we identified a set of bacterial cell wall hydrolase (cwh) genes acquired by this group of beetles. Infection with Bacillus subtilis led to upregulated expression of these ladybird cwh genes, and their recombinantly produced proteins limited bacterial proliferation. Moreover, RNAi-mediated cwh knockdown led to downregulation of other antibacterial genes, indicating a role in antibacterial immune defense. cwh genes are rare in eukaryotes, but have been maintained in all tested Coccinellinae species, suggesting that this putative immune-related HGT event played a role in the evolution of this speciose subfamily of predominant predatory ladybirds. CONCLUSION: Our work demonstrates that, in a manner analogous to HGT-facilitated plant feeding, enhanced immunity through HGT might have played a key role in the prey adaptation and niche expansion that promoted the diversification of carnivorous beetle lineages. We believe that this represents the first example of immune-related HGT in carnivorous insects with an association with a subsequent successful species radiation.

Phylogenomics reveals accelerated late Cretaceous diversification of bee flies (Diptera: Bombyliidae).

Bombyliidae is a very species-rich and widespread family of parasitoid flies with more than 250 genera classified into 17 extant subfamilies. However, little is known about their evolutionary history or how their present-day diversity was shaped. Transcriptomes of 15 species and anchored hybrid enrichment (AHE) sequence captures of 86 species, representing 94 bee fly species and 14 subfamilies, were used to reconstruct the phylogeny of Bombyliidae. We integrated data from transcriptomes across each of the main lineages in our AHE tree to build a data set with more genes (550 loci versus 216 loci) and higher support levels. Our overall results show strong congruence with the current classification of the family, with 11 out of 14 included subfamilies recovered as monophyletic. Heterotropinae and Mythicomyiinae are successive sister groups to the remainder of the family. We examined the evolution of key morphological characters through our phylogenetic hypotheses and show that neither the "sand chamber subfamilies" nor the "Tomophthalmae" are monophyletic in our phylogenomic analyses. Based on our results, we reinstate two tribes at the subfamily level (Phthiriinae stat. rev. and Ecliminae stat. rev.) and we include the genus Sericosoma Macquart (previously incertae sedis) in the subfamily Oniromyiinae, bringing the total number of bee fly subfamilies to 19. Our dating analyses indicate a Jurassic origin of the family (165-194 Ma), with the sand chamber evolving early in bee fly evolution, in the late Jurassic or mid-Cretaceous (100-165 Ma). We hypothesize that the angiosperm radiation and the hothouse climate established during the late Cretaceous accelerated the diversification of bee flies, by providing an expanded range of resources for the parasitoid larvae and nectarivorous adults.

Hybridization and gene flow in the mega-pest lineage of moth, Helicoverpa.

Within the mega-pest lineage of heliothine moths are a number of polyphagous, highly mobile species for which the exchange of adaptive traits through hybridization would affect their properties as pests. The recent invasion of South America by one of the most significant agricultural pests, Helicoverpa armigera, raises concerns for the formation of novel combinations of adaptive genes following hybridization with the closely related Helicoverpa zea To investigate the propensity for hybridization within the genus Helicoverpa, we carried out whole-genome resequencing of samples from six species, focusing in particular upon H. armigera population structure and its relationship with H. zea We show that both H. armigera subspecies have greater genetic diversity and effective population sizes than do the other species. We find no signals for gene flow among the six species, other than between H. armigera and H. zea, with nine Brazilian individuals proving to be hybrids of those two species. Eight had largely H. armigera genomes with some introgressed DNA from H. zea scattered throughout. The ninth resembled an F1 hybrid but with stretches of homozygosity for each parental species that reflect previous hybridization. Regions homozygous for H. armigera-derived DNA in this individual included one containing a gustatory receptor and esterase genes previously associated with host range, while another encoded a cytochrome P450 that confers insecticide resistance. Our data point toward the emergence of novel hybrid ecotypes and highlight the importance of monitoring H. armigera genotypes as they spread through the Americas.

Molecular phylogeny reveals food plasticity in the evolution of true ladybird beetles (Coleoptera: Coccinellidae: Coccinellini).

BACKGROUND: The tribe Coccinellini is a group of relatively large ladybird beetles that exhibits remarkable morphological and biological diversity. Many species are aphidophagous, feeding as larvae and adults on aphids, but some species also feed on other hemipterous insects (i.e., heteropterans, psyllids, whiteflies), beetle and moth larvae, pollen, fungal spores, and even plant tissue. Several species are biological control agents or widespread invasive species (e.g., Harmonia axyridis (Pallas)). Despite the ecological importance of this tribe, relatively little is known about the phylogenetic relationships within it. The generic concepts within the tribe Coccinellini are unstable and do not reflect a natural classification, being largely based on regional revisions. This impedes the phylogenetic study of important traits of Coccinellidae at a global scale (e.g. the evolution of food preferences and biogeography). RESULTS: We present the most comprehensive phylogenetic analysis of Coccinellini to date, based on three nuclear and one mitochondrial gene sequences of 38 taxa, which represent all major Coccinellini lineages. The phylogenetic reconstruction supports the monophyly of Coccinellini and its sister group relationship to Chilocorini. Within Coccinellini, three major clades were recovered that do not correspond to any previously recognised divisions, questioning the traditional differentiation between Halyziini, Discotomini, Tytthaspidini, and Singhikaliini. Ancestral state reconstructions of food preferences and morphological characters support the idea of aphidophagy being the ancestral state in Coccinellini. This indicates a transition from putative obligate scale feeders, as seen in the closely related Chilocorini, to more agile general predators. CONCLUSIONS: Our results suggest that the classification of Coccinellini has been misled by convergence in morphological traits. The evolutionary history of Coccinellini has been very dynamic in respect to changes in host preferences, involving multiple independent host switches from different insect orders to fungal spores and plants tissues. General predation on ephemeral aphids might have created an opportunity to easily adapt to mixed or specialised diets (e.g. obligate mycophagy, herbivory, predation on various hemipteroids or larvae of leaf beetles (Chrysomelidae)). The generally long-lived adults of Coccinellini can consume pollen and floral nectars, thereby surviving periods of low prey frequency. This capacity might have played a central role in the diversification history of Coccinellini.

Diversification shifts in leafroller moths linked to continental colonization and the rise of angiosperms.

Tectonic dynamics and niche availability play intertwined roles in determining patterns of diversification. Such drivers explain the current distribution of many clades, whereas events such as the rise of angiosperms can have more specific impacts, such as on the diversification rates of herbivores. The Tortricidae, a diverse group of phytophagous moths, are ideal for testing the effects of these determinants on the diversification of herbivorous clades. To estimate ancestral areas and diversification patterns in Tortricidae, a complete tribal-level dated tree was inferred using molecular markers (one mitochondrial and five nuclear) and calibrated using fossil constraints. We found that Tortricidae diverged from their sister group c. 120 Myr ago (Ma) and diversified c. 97 Ma, a timeframe synchronous with the rise of angiosperms in the Early-mid Cretaceous. Ancestral areas analysis, based on updated Wallace's biogeographical regions, supports the hypothesis of a Gondwanan origin of Tortricidae in the South American plate. We also detected an increase in speciation rate that coincided with the peak of angiosperm diversification in the Cretaceous. This in turn probably was further heightened by continental colonization of the Palaeotropics when angiosperms became dominant by the end of the Late Cretaceous.

Arthropod relationships revealed by phylogenomic analysis of nuclear protein-coding sequences.

The remarkable antiquity, diversity and ecological significance of arthropods have inspired numerous attempts to resolve their deep phylogenetic history, but the results of two decades of intensive molecular phylogenetics have been mixed. The discovery that terrestrial insects (Hexapoda) are more closely related to aquatic Crustacea than to the terrestrial centipedes and millipedes (Myriapoda) was an early, if exceptional, success. More typically, analyses based on limited samples of taxa and genes have generated results that are inconsistent, weakly supported and highly sensitive to analytical conditions. Here we present strongly supported results from likelihood, Bayesian and parsimony analyses of over 41 kilobases of aligned DNA sequence from 62 single-copy nuclear protein-coding genes from 75 arthropod species. These species represent every major arthropod lineage, plus five species of tardigrades and onychophorans as outgroups. Our results strongly support Pancrustacea (Hexapoda plus Crustacea) but also strongly favour the traditional morphology-based Mandibulata (Myriapoda plus Pancrustacea) over the molecule-based Paradoxopoda (Myriapoda plus Chelicerata). In addition to Hexapoda, Pancrustacea includes three major extant lineages of 'crustaceans', each spanning a significant range of morphological disparity. These are Oligostraca (ostracods, mystacocarids, branchiurans and pentastomids), Vericrustacea (malacostracans, thecostracans, copepods and branchiopods) and Xenocarida (cephalocarids and remipedes). Finally, within Pancrustacea we identify Xenocarida as the long-sought sister group to the Hexapoda, a result confirming that 'crustaceans' are not monophyletic. These results provide a statistically well-supported phylogenetic framework for the largest animal phylum and represent a step towards ending the often-heated, century-long debate on arthropod relationships.

Biodiversity into your hands - A call for a virtual global natural history 'metacollection'.

BACKGROUND: Many scientific disciplines rely on correct taxon delineations and identifications. So does a great part of the general public as well as decision makers. Researchers, students and enthusiastic amateurs often feel frustrated because information about species remains scattered, difficult to access, or difficult to decipher. Together, this affects almost anyone who wishes to identify species or verify identifications. Many remedies have been proposed, but we argue that the role of natural history collections remains insufficiently appreciated. We suggest using state-of-the-art mass imaging technology and to join forces to create a global natural history metacollection on the internet, providing access to the morphology of tens of millions of specimens and making them available for automated digital image analysis. DISCUSSION: Robotic high-resolution imaging technology and fast (high performance) computer-based image stitching make it now feasible to digitize entire collection drawers typically used for arthropod collections, or trays or containers used for other objects. Resolutions of 500 megapixels and much higher are already utilized to capture the contents of 40x50 cm collection drawers, providing amazing detail of specimens. Flanked by metadata entry, this helps to create access to tens of thousands of specimens in days. By setting priorities and combining the holdings of the most comprehensive collections for certain taxa, drawer digitizing offers the unique opportunity to create a global, virtual metacollection.The taxonomic and geographic coverage of such a collection could never be achieved by a single institution or individual. We argue that by joining forces, many new impulses will emerge for systematic biology, related fields and understanding of biodiversity in general.Digitizing drawers containing unidentified, little-curated specimens is a contribution towards the beginning of a new era of online curation. It also will help taxonomists and curators to discover and process the millions of "gems" of undescribed species hidden in museum accessions. SUMMARY: Our proposal suggests creating virtual, high-resolution image resources that will, for the first time in history, provide access for expert scientists as well as students and the general public to the enormous wealth of the world's natural history collections. We foresee that this will contribute to a better understanding, appreciation and increased use of biodiversity resources and the natural history collections serving this cause.

Revision of the African Neoperla Needham, 1905 (Plecoptera: Perlidae: Perlinae) based on morphological and molecular data.

Adults of the African species of the genus Neoperla Needham, 1905 (Plecoptera: Perlidae: Perlinae) are revised, and 82 valid species are recognised. Of the original 29 named species, 14 valid ones are redescribed from types, nine lectotypes are designated, and nine new synonymies are proposed. Sixty-two new species are named, and several additional new species only known from material insufficient for formal description are listed. There are one unavailable nomen nudum and three doubtful names. Diagnostic morphological traits of the recognised species are described and illustrated in detail where available of males, females, and eggs. Dichotomous keys to all species are provided. Mitochondrial DNA-data of 71 species-group taxa are available, which permits reliable association of sexes and minimises the risk of synonymies between species known from only one sex. Importantly, this includes DNA sequences from 50 holotypes. The DNA-data will also permit future monitoring with eDNA sequencing and identification of nymphs, which are probably important but so far unidentifiable bioindicators in streams. The genus Neoperla occurs largely in the northern hemisphere and in Africa, where it is restricted to the Ethiopian region south of the Sahara but unknown from Madagascar. A single specimen not clearly distinct from a widespread mainland species was taken on Comoro Island. All African species are endemic, but one African species group has a few outlying members in Asia. The Asian N. montivaga-group is not known from Africa. The following new species are described and named: N. aethiopica n. sp., N. amoena n. sp., N. angolana n. sp., N. bareensis n. sp., N. bella n. sp., N. benti n. sp., N. beta n. sp., N. bipolaris n. sp., N. biserrata n. sp., N. brachyphallus n. sp., N. caeleps n. sp., N. cataractae n. sp., N. claviger n. sp., N. coffea n. sp., N. costata n. sp., N. crenulata n. sp., N. crustata n. sp., N. decorata n. sp., N. dianae n. sp., N. dolium n. sp., N. dundoana n. sp., N. duodeviginti n. sp., N. erinaceus n. sp., N. excavata n. sp., N. filamentosa n. sp., N. funiculata n. sp., N. gibbosa n. sp., N. gordius n. sp., N. heideae n. sp., N. ivanloebli n. sp., N. juxtadidita n. sp., N. kalengonis n. sp., N. larvata n. sp., N. lineata n. sp., N. luhohonis n. sp., N. massevensis n. sp., N. multiserrata n. sp., N. muyukae n. sp., N. nichollsi n. sp., N. occulta n. sp., N. orthonema n. sp., N. pallidogigas n. sp., N. panafricana n. sp., N. pickeri n. sp., N. pilulifera n. sp., N. pirus n. sp., N. planidorsum n. sp., N. plicata n. sp., N. proxima n. sp., N. pusilla n. sp., N. rostrata n. sp., N. sambarua n. sp., N. sassandrae n. sp., N. schuelei n. sp., N. serrula n. sp., N. simplex n. sp., N. socia n. sp., N. sorella n. sp., N. spaghetti n. sp., N. spectabilis n. sp., N. spironema n. sp., N. tangana n. sp., N. tansanica n. sp., N. usambara n. sp., and N. vicina n. sp..

Critical re-examination of known purported fossil Bombycoidea (Lepidoptera).

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.

Can deliberately incomplete gene sample augmentation improve a phylogeny estimate for the advanced moths and butterflies (Hexapoda: Lepidoptera)?

This paper addresses the question of whether one can economically improve the robustness of a molecular phylogeny estimate by increasing gene sampling in only a subset of taxa, without having the analysis invalidated by artifacts arising from large blocks of missing data. Our case study stems from an ongoing effort to resolve poorly understood deeper relationships in the large clade Ditrysia ( > 150,000 species) of the insect order Lepidoptera (butterflies and moths). Seeking to remedy the overall weak support for deeper divergences in an initial study based on five nuclear genes (6.6 kb) in 123 exemplars, we nearly tripled the total gene sample (to 26 genes, 18.4 kb) but only in a third (41) of the taxa. The resulting partially augmented data matrix (45% intentionally missing data) consistently increased bootstrap support for groupings previously identified in the five-gene (nearly) complete matrix, while introducing no contradictory groupings of the kind that missing data have been predicted to produce. Our results add to growing evidence that data sets differing substantially in gene and taxon sampling can often be safely and profitably combined. The strongest overall support for nodes above the family level came from including all nucleotide changes, while partitioning sites into sets undergoing mostly nonsynonymous versus mostly synonymous change. In contrast, support for the deepest node for which any persuasive molecular evidence has yet emerged (78-85% bootstrap) was weak or nonexistent unless synonymous change was entirely excluded, a result plausibly attributed to compositional heterogeneity. This node (Gelechioidea + Apoditrysia), tentatively proposed by previous authors on the basis of four morphological synapomorphies, is the first major subset of ditrysian superfamilies to receive strong statistical support in any phylogenetic study. A "more-genes-only" data set (41 taxa×26 genes) also gave strong signal for a second deep grouping (Macrolepidoptera) that was obscured, but not strongly contradicted, in more taxon-rich analyses.

Development and evaluation of a custom bait design based on 469 single-copy protein-coding genes for exon capture of isopods (Philosciidae: Haloniscus).

Transcriptome-based exon capture approaches, along with next-generation sequencing, are allowing for the rapid and cost-effective production of extensive and informative phylogenomic datasets from non-model organisms for phylogenetics and population genetics research. These approaches generally employ a reference genome to infer the intron-exon structure of targeted loci and preferentially select longer exons. However, in the absence of an existing and well-annotated genome, we applied this exon capture method directly, without initially identifying intron-exon boundaries for bait design, to a group of highly diverse Haloniscus (Philosciidae), paraplatyarthrid and armadillid isopods, and examined the performance of our methods and bait design for phylogenetic inference. Here, we identified an isopod-specific set of single-copy protein-coding loci, and a custom bait design to capture targeted regions from 469 genes, and analysed the resulting sequence data with a mapping approach and newly-created post-processing scripts. We effectively recovered a large and informative dataset comprising both short (<100 bp) and longer (>300 bp) exons, with high uniformity in sequencing depth. We were also able to successfully capture exon data from up to 16-year-old museum specimens along with more distantly related outgroup taxa, and efficiently pool multiple samples prior to capture. Our well-resolved phylogenies highlight the overall utility of this methodological approach and custom bait design, which offer enormous potential for application to future isopod, as well as broader crustacean, molecular studies.

Toward reconstructing the evolution of advanced moths and butterflies (Lepidoptera: Ditrysia): an initial molecular study.

BACKGROUND: In the mega-diverse insect order Lepidoptera (butterflies and moths; 165,000 described species), deeper relationships are little understood within the clade Ditrysia, to which 98% of the species belong. To begin addressing this problem, we tested the ability of five protein-coding nuclear genes (6.7 kb total), and character subsets therein, to resolve relationships among 123 species representing 27 (of 33) superfamilies and 55 (of 100) families of Ditrysia under maximum likelihood analysis. RESULTS: Our trees show broad concordance with previous morphological hypotheses of ditrysian phylogeny, although most relationships among superfamilies are weakly supported. There are also notable surprises, such as a consistently closer relationship of Pyraloidea than of butterflies to most Macrolepidoptera. Monophyly is significantly rejected by one or more character sets for the putative clades Macrolepidoptera as currently defined (P < 0.05) and Macrolepidoptera excluding Noctuoidea and Bombycoidea sensu lato (P < or = 0.005), and nearly so for the superfamily Drepanoidea as currently defined (P < 0.08). Superfamilies are typically recovered or nearly so, but usually without strong support. Relationships within superfamilies and families, however, are often robustly resolved. We provide some of the first strong molecular evidence on deeper splits within Pyraloidea, Tortricoidea, Geometroidea, Noctuoidea and others.Separate analyses of mostly synonymous versus non-synonymous character sets revealed notable differences (though not strong conflict), including a marked influence of compositional heterogeneity on apparent signal in the third codon position (nt3). As available model partitioning methods cannot correct for this variation, we assessed overall phylogeny resolution through separate examination of trees from each character set. Exploration of "tree space" with GARLI, using grid computing, showed that hundreds of searches are typically needed to find the best-feasible phylogeny estimate for these data. CONCLUSION: Our results (a) corroborate the broad outlines of the current working phylogenetic hypothesis for Ditrysia, (b) demonstrate that some prominent features of that hypothesis, including the position of the butterflies, need revision, and (c) resolve the majority of family and subfamily relationships within superfamilies as thus far sampled. Much further gene and taxon sampling will be needed, however, to strongly resolve individual deeper nodes.

Resolving arthropod phylogeny: exploring phylogenetic signal within 41 kb of protein-coding nuclear gene sequence.

This study attempts to resolve relationships among and within the four basal arthropod lineages (Pancrustacea, Myriapoda, Euchelicerata, Pycnogonida) and to assess the widespread expectation that remaining phylogenetic problems will yield to increasing amounts of sequence data. Sixty-eight regions of 62 protein-coding nuclear genes (approximately 41 kilobases (kb)/taxon) were sequenced for 12 taxonomically diverse arthropod taxa and a tardigrade outgroup. Parsimony, likelihood, and Bayesian analyses of total nucleotide data generally strongly supported the monophyly of each of the basal lineages represented by more than one species. Other relationships within the Arthropoda were also supported, with support levels depending on method of analysis and inclusion/exclusion of synonymous changes. Removing third codon positions, where the assumption of base compositional homogeneity was rejected, altered the results. Removing the final class of synonymous mutations--first codon positions encoding leucine and arginine, which were also compositionally heterogeneous--yielded a data set that was consistent with a hypothesis of base compositional homogeneity. Furthermore, under such a data-exclusion regime, all 68 gene regions individually were consistent with base compositional homogeneity. Restricting likelihood analyses to nonsynonymous change recovered trees with strong support for the basal lineages but not for other groups that were variably supported with more inclusive data sets. In a further effort to increase phylogenetic signal, three types of data exploration were undertaken. (1) Individual genes were ranked by their average rate of nonsynonymous change, and three rate categories were assigned--fast, intermediate, and slow. Then, bootstrap analysis of each gene was performed separately to see which taxonomic groups received strong support. Five taxonomic groups were strongly supported independently by two or more genes, and these genes mostly belonged to the slow or intermediate categories, whereas groups supported only by a single gene region tended to be from genes of the fast category, arguing that fast genes provide a less consistent signal. (2) A sensitivity analysis was performed in which increasing numbers of genes were excluded, beginning with the fastest. The number of strongly supported nodes increased up to a point and then decreased slightly. Recovery of Hexapoda required removal of fast genes. Support for Mandibulata (Pancrustacea + Myriapoda) also increased, at times to "strong" levels, with removal of the fastest genes. (3) Concordance selection was evaluated by clustering genes according to their ability to recover Pancrustacea, Euchelicerata, or Myriapoda and analyzing the three clusters separately. All clusters of genes recovered the three concordance clades but were at times inconsistent in the relationships recovered among and within these clades, a result that indicates that the a priori concordance criteria may bias phylogenetic signal in unexpected ways. In a further attempt to increase support of taxonomic relationships, sequence data from 49 additional taxa for three slow genes (i.e., EF-1 alpha, EF-2, and Pol II) were combined with the various 13-taxon data sets. The 62-taxon analyses supported the results of the 13-taxon analyses and provided increased support for additional pancrustacean clades found in an earlier analysis including only EF-1 alpha, EF-2, and Pol II.