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

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

Genomic evidence for contrasting patterns of host-associated genetic differentiation across shared host-plant species in leaf- and bud-galling sawflies.

Resource specialization and ecological speciation arising through host-associated genetic differentiation (HAD) are frequently invoked as an explanation for the high diversity of plant-feeding insects and other organisms with a parasitic lifestyle. While genetic studies have demonstrated numerous examples of HAD in insect herbivores, the rarity of comparative studies means that we still lack an understanding of how deterministic HAD is, and whether patterns of host shifts can be predicted over evolutionary timescales. We applied genome-wide single nucleotide polymorphism and mitochondrial DNA sequence data obtained through genome resequencing to define species limits and to compare host-plant use in population samples of leaf- and bud-galling sawflies (Hymenoptera: Tenthredinidae: Nematinae) collected from seven shared willow (Salicaceae: Salix) host species. To infer the repeatability of long-term cophylogenetic patterns, we also contrasted the phylogenies of the two galler groups with each other as well as with the phylogeny of their Salix hosts estimated based on RADseq data. We found clear evidence for host specialization and HAD in both of the focal galler groups, but also that leaf gallers are more specialized to single host species compared with most bud gallers. In contrast to bud gallers, leaf gallers also exhibited statistically significant cophylogenetic signal with their Salix hosts. The observed discordant patterns of resource specialization and host shifts in two related galler groups that have radiated in parallel across a shared resource base indicate a lack of evolutionary repeatability in the focal system, and suggest that short- and long-term host use and ecological diversification in plant-feeding insects are dominated by stochasticity and/or lineage-specific effects.

Diversification linked to larval host plant in the butterfly Eumedonia eumedon.

It is widely accepted that the relationship between phytophagous insects and their host plants influences insect diversification. However, studies addressed at documenting host-associated genetic differentiation (HAD) and the mechanisms that drive reproductive isolation in host-associated lineages (or host races) are still scarce relative to insect diversity. To uncover further evidence on the HAD processes in Lepidoptera, we investigated the genetic structure of the geranium argus butterfly (Eumedonia eumedon) and tested for isolation by ecology (IBE) vs. isolation by distance (IBD). Genomic data revealed an array of host races (three of them in the same mountain range, the Cantabrian Mountains, northern Iberia) at apparently distinct levels of reproductive isolation. We found a pattern of IBE mediated by HAD at both local and European scales, in which genetic differentiation between populations and individuals correlated significantly with the taxonomic relatedness of the host plants. IBD was significant only when considered at the wider European scale. We hypothesize that, locally, HAD between Geranium-feeding populations was caused (at least partially) by allochrony, that is via adaptation of adult flight time to the flowering period of each host plant species. Nevertheless, the potential reproductive isolation between populations using Erodium and populations using Geranium cannot be explained by allochrony or IBD, and other mechanisms are expected to be at play.

Hydric effects on thermal tolerances influence climate vulnerability in a high-latitude beetle.

Species' thermal tolerances are used to estimate climate vulnerability, but few studies consider the role of the hydric environment in shaping thermal tolerances. As environments become hotter and drier, organisms often respond by limiting water loss to lower the risk of desiccation; however, reducing water loss may produce trade-offs that lower thermal tolerances if respiration becomes inhibited. Here, we measured the sensitivity of water loss rate and critical thermal maximum (CTmax ) to precipitation in nature and laboratory experiments that exposed click beetles (Coleoptera: Elateridae) to acute- and long-term humidity treatments. We also took advantage of their unique clicking behavior to characterize subcritical thermal tolerances. We found higher water loss rates in the dry acclimation treatment compared to the humid, and water loss rates were 3.2-fold higher for individuals that had experienced a recent precipitation event compared to individuals that had not. Acute humidity treatments did not affect CTmax , but precipitation indirectly affected CTmax through its effect on water loss rates. Contrary to our prediction, we found that CTmax was negatively associated with water loss rate, such that individuals with high water loss rate exhibited a lower CTmax . We then incorporated the observed variation of CTmax into a mechanistic niche model that coupled leaf and click beetle temperatures to predict climate vulnerability. The simulations indicated that indices of climate vulnerability can be sensitive to the effects of water loss physiology on thermal tolerances; moreover, exposure to temperatures above subcritical thermal thresholds is expected to increase by as much as 3.3-fold under future warming scenarios. The correlation between water loss rate and CTmax identifies the need to study thermal tolerances from a "whole-organism" perspective that considers relationships between physiological traits, and the population-level variation in CTmax driven by water loss rate complicates using this metric as a straightforward proxy of climate vulnerability.

Molecular phylogeny, divergence time, biogeography and trends in host plant usage in the agriculturally important tortricid tribe Grapholitini (Lepidoptera: Tortricidae: Olethreutinae).

The leaf-roller moth tribe Grapholitini comprises about 1200 described species and contains numerous notorious pests of fruits and seeds. The phylogeny of the tribe has been little studied using contemporary methods, and the monophyly of several genera remains questionable. In order to provide a more robust phylogenetic framework for the group, we conducted a multiple-gene phylogenetic analysis of 104 species representing 27 genera of Grapholitini and 29 outgroup species. Divergence time, ancestral area, and host plant usage were also inferred to explore evolutionary trends in the tribe. Our analyses indicate that Larisa and Corticivora, traditionally assigned to Grapholitini, are best excluded from the tribe. After removal of these two genera, the tribe is found to be monophyletic, represented by two major lineages-a Dichrorampha clade and a Cydia clade, the latter of which can be divided into seven generic groups. The genus Grapholita was found to be polyphyletic, comprising three different clades, and we propose three genera to accommodate these groups: Grapholita (sensu stricto), Aspila (formerly a subgenus of Grapholita) and Ephippiphora (formerly considered a synonym of Grapholita). We summarize each generic group, including related genera not included in our analysis, providing morphological, pheromone and food plant characters that support particular branches within the molecular hypotheses. Biogeographical analyses indicate that Grapholitini probably originated in the Nearctic, Afrotropical and Neotropical regions in the Lutetian of the middle Eocene (ca. 44.3 Ma). Our results also indicate that most groups in Grapholitini originated from Fabaceae-feeding monophagous or oligophagous ancestors, and that host plant shifts probably promoted species diversification within the tribe.

Linking large-scale genetic structure of three Argynnini butterfly species to geography and environment.

Understanding which factors and processes are associated with genetic differentiation within and among species remains a major goal in evolutionary biology. To explore differences and similarities in genetic structure and its association with geographical and climatic factors in sympatric sister species, we conducted a large-scale (>32° latitude and >36° longitude) comparative phylogeographical study on three Argynnini butterfly species (Speyeria aglaja, Fabriciana adippe and F. niobe) that have similar life histories, but differ in ecological generalism and dispersal abilities. Analyses of nuclear (ddRAD-sequencing derived SNP markers) and mitochondrial (COI sequences) data revealed differences between species in genetic structure and how genetic differentiation was associated with climatic factors (temperature, solar radiation, precipitation, wind speed). Geographical proximity accounted for much of the variation in nuclear and mitochondrial structure and evolutionary relationships in F. adippe and F. niobe, but only explained the pattern observed in the nuclear data in S. aglaja, for which mitonuclear discordance was documented. In all species, Iberian and Balkan individuals formed genetic clusters, suggesting isolation in glacial refugia and limited postglacial expansion. Solar radiation and precipitation were associated with the genetic structure on a regional scale in all species, but the specific combinations of environmental and geographical factors linked to variation within species were unique, pointing to species-specific responses to common environments. Our findings show that the species share similar colonization histories, and that the same ecological factors, such as niche breadth and dispersal capacity, covary with genetic differentiation within these species to some extent, thereby highlighting the importance of comparative phylogeographical studies in sympatric sister species.

Dynamics of a host-parasitoid interaction clarified by modelling and DNA sequencing.

It has been hypothesised that the 2-year oscillations in abundance of Xestia moths are mediated by interactions with 1-year Ophion parasitoid wasps. We tested this hypothesis by modelling a 35-year time series of Xestia and Ophion from Northern Finland. Additionally, we used DNA barcoding to ascertain the species diversity of Ophion and targeted amplicon sequencing of their gut contents to confirm their larval hosts. Modelling of the time-series data strongly supported the hypothesised host-parasitoid dynamics and that periodic occurrence of Xestia moths is mediated by Ophion. DNA barcodes revealed that Ophion included five species rather than just one while targeted amplicon sequencing verified that Ophion does parasitise Xestia. At least one Ophion species employs 1-year Syngrapha interrogationis as an alternate host, but it did not detectably affect Xestia-Ophion dynamics. We also demonstrate the previously unrecognised complexity of this system due to cryptic parasitoid diversity.

Rapid colour shift by reproductive character displacement in Cupido butterflies.

Reproductive character displacement occurs when competition for successful breeding imposes a divergent selection on the interacting species, causing a divergence of reproductive traits. Here, we show that a disputed butterfly taxon is actually a case of male wing colour shift, apparently produced by reproductive character displacement. Using double digest restriction-site associated DNA sequencing and mitochondrial DNA sequencing we studied four butterfly taxa of the subgenus Cupido (Lepidoptera: Lycaenidae): Cupido minimus and the taxon carswelli, both characterized by brown males and females, plus C. lorquinii and C. osiris, both with blue males and brown females. Unexpectedly, taxa carswelli and C. lorquinii were close to indistinguishable based on our genomic and mitochondrial data, despite displaying strikingly different male coloration. In addition, we report and analysed a brown male within the C. lorquinii range, which demonstrates that the brown morph occurs at very low frequency in C. lorquinii. Such evidence strongly suggests that carswelli is conspecific with C. lorquinii and represents populations with a fixed male brown colour morph. Considering that these brown populations occur in sympatry with or very close to the blue C. osiris, and that the blue C. lorquinii populations never do, we propose that the taxon carswelli could have lost the blue colour due to reproductive character displacement with C. osiris. Since male colour is important for conspecific recognition during courtship, we hypothesize that the observed colour shift may eventually trigger incipient speciation between blue and brown populations. Male colour seems to be an evolutionarily labile character in the Polyommatinae, and the mechanism described here might be at work in the wide diversification of this subfamily of butterflies.

Host dispersal shapes the population structure of a tick-borne bacterial pathogen.

Birds are hosts for several zoonotic pathogens. Because of their high mobility, especially of longdistance migrants, birds can disperse these pathogens, affecting their distribution and phylogeography. We focused on Borrelia burgdorferi sensu lato, which includes the causative agents of Lyme borreliosis, as an example for tick-borne pathogens, to address the role of birds as propagation hosts of zoonotic agents at a large geographical scale. We collected ticks from passerine birds in 11 European countries. B. burgdorferi s.l. prevalence in Ixodes spp. was 37% and increased with latitude. The fieldfare Turdus pilaris and the blackbird T. merula carried ticks with the highest Borrelia prevalence (92 and 58%, respectively), whereas robin Erithacus rubecula ticks were the least infected (3.8%). Borrelia garinii was the most prevalent genospecies (61%), followed by B. valaisiana (24%), B. afzelii (9%), B. turdi (5%) and B. lusitaniae (0.5%). A novel Borrelia genospecies "Candidatus Borrelia aligera" was also detected. Multilocus sequence typing (MLST) analysis of B. garinii isolates together with the global collection of B. garinii genotypes obtained from the Borrelia MLST public database revealed that: (a) there was little overlap among genotypes from different continents, (b) there was no geographical structuring within Europe, and (c) there was no evident association pattern detectable among B. garinii genotypes from ticks feeding on birds, questing ticks or human isolates. These findings strengthen the hypothesis that the population structure and evolutionary biology of tick-borne pathogens are shaped by their host associations and the movement patterns of these hosts.

Cross-contamination and strong mitonuclear discordance in Empria sawflies (Hymenoptera, Tenthredinidae) in the light of phylogenomic data.

In several sawfly taxa strong mitonuclear discordance has been observed, with nuclear genes supporting species assignments based on morphology, whereas the barcode region of the mitochondrial COI gene suggests different relationships. As previous studies were based on only a few nuclear genes, the causes and the degree of mitonuclear discordance remain ambiguous. Here, we obtained genomic-scale ddRAD data together with Sanger sequences of mitochondrial COI and two to three nuclear protein coding genes to investigate species limits and mitonuclear discordance in two closely related species groups of the sawfly genus Empria. As found previously based on nuclear ITS and mitochondrial COI sequences, species are in most cases supported as monophyletic based on new nuclear data reported here, but not based on mitochondrial COI. This mitonuclear discordance can be explained by occasional mitochondrial introgression with little or no nuclear gene flow, a pattern that might be common in haplodiploid taxa with slowly evolving mitochondrial genomes. Some species in the E. immersa group are not recovered as monophyletic according to either mitochondrial or nuclear data, but this could partly be because of unresolved taxonomy. Preliminary analyses of ddRAD data did not recover monophyly of E. japonica within the E. longicornis group (three Sanger sequenced nuclear genes strongly supported monophyly), but closer examination of the data and additional Sanger sequencing suggested that both specimens were substantially (possibly 10-20% of recovered loci) cross-contaminated. A reason could be specimen identification tag jumps during sequencing library preparation that in previous studies have been shown to affect up to 2.5% of the sequenced reads. We provide an R script to examine patterns of identical loci among the specimens and estimate that the cross-contamination rate is not unusually high for our ddRAD dataset as a whole (based on counting of identical sequences in the immersa and longicornis groups, which are well separated from each other and probably do not hybridise). The high rate of cross-contamination for both E. japonica specimens might be explained by the small number of recovered loci (~1000) compared to most other specimens (>10 000 in some cases) because of poor sequencing results. We caution against drawing unexpected biological conclusions when closely related specimens are pooled before sequencing and tagged only at one end of the molecule or at both ends using a unique combination of limited number of tags (less than the number of specimens).

Climate change-driven elevational changes among boreal nocturnal moths.

Climate change has shifted geographical ranges of species northwards or to higher altitudes on elevational gradients. These changes have been associated with increases in ambient temperatures. For ectotherms in seasonal environments, however, life history theory relies largely on the length of summer, which varies somewhat independently of ambient temperature per se. Extension of summer reduces seasonal time constraints and enables species to establish in new areas as a result of over-wintering stage reaching in due time. The reduction of time constraints is also predicted to prolong organisms' breeding season when reproductive potential is under selection. We studied temporal change in the summer length and its effect on species' performance by combining long-term data on the occurrence and abundance of nocturnal moths with weather conditions in a boreal location at Värriötunturi fell in NE Finland. We found that summers have lengthened on average 5 days per decade from the late 1970s, profoundly due to increasing delays in the onset of winters. Moth abundance increased with increasing season length a year before. Most of the species occurrences expanded upwards in elevation. Moth communities in low elevation pine heath forest and middle elevation mountain birch forest have become inseparable. Yet, the flight periods have remained unchanged, probably due to unpredictable variation in proximate conditions (weather) that hinders life histories from selection. We conclude that climate change-driven changes in the season length have potential to affect species' ranges and affect the structure of insect assemblages, which may contribute to alteration of ecosystem-level processes.

The conundrum of species delimitation: a genomic perspective on a mitogenetically super-variable butterfly.

The Palaearctic butterfly Melitaea didyma stands out as one of the most striking cases of intraspecific genetic differentiation detected in Lepidoptera: 11 partially sympatric mitochondrial lineages have been reported, displaying levels of divergence of up to 7.4%. To better understand the evolutionary processes underlying the diversity observed in mtDNA, we compared mtDNA and genome-wide SNP data using double-digest restriction site-associated DNA sequencing (ddRADseq) results from 93 specimens of M. didyma ranging from Morocco to eastern Kazakhstan. We found that, between ddRADseq and mtDNA results, there is a match only in populations that probably remained allopatric for long periods of time. Other mtDNA lineages may have resulted from introgression events and were probably affected by Wolbachia infection. The five main ddRADseq clades supported by STRUCTURE were parapatric or allopatric and showed high pairwise FST values, but some were also estimated to display various levels of gene flow. Melitaea didyma represents one of the first cases of deep mtDNA splits among European butterflies assessed by a genome-wide DNA analysis and reveals that the interpretation of patterns remains challenging even when a high amount of genomic data is available. These findings actualize the ongoing debate of species delimitation in allopatry, an issue probably of relevance to a significant proportion of global biodiversity.

Information Dropout Patterns in Restriction Site Associated DNA Phylogenomics and a Comparison with Multilocus Sanger Data in a Species-Rich Moth Genus.

A rapid shift from traditional Sanger sequencing-based molecular methods to the phylogenomic approach with large numbers of loci is underway. Among phylogenomic methods, restriction site associated DNA (RAD) sequencing approaches have gained much attention as they enable rapid generation of up to thousands of loci randomly scattered across the genome and are suitable for nonmodel species. RAD data sets however suffer from large amounts of missing data and rapid locus dropout along with decreasing relatedness among taxa. The relationship between locus dropout and the amount of phylogenetic information retained in the data has remained largely uninvestigated. Similarly, phylogenetic hypotheses based on RAD have rarely been compared with phylogenetic hypotheses based on multilocus Sanger sequencing, even less so using exactly the same species and specimens. We compared the Sanger-based phylogenetic hypothesis (8 loci; 6172 bp) of 32 species of the diverse moth genus Eupithecia (Lepidoptera, Geometridae) to that based on double-digest RAD sequencing (3256 loci; 726,658 bp). We observed that topologies were largely congruent, with some notable exceptions that we discuss. The locus dropout effect was strong. We demonstrate that number of loci is not a precise measure of phylogenetic information since the number of single-nucleotide polymorphisms (SNPs) may remain low at very shallow phylogenetic levels despite large numbers of loci. As we hypothesize, the number of SNPs and parsimony informative SNPs (PIS) is low at shallow phylogenetic levels, peaks at intermediate levels and, thereafter, declines again at the deepest levels as a result of decay of available loci. Similarly, we demonstrate with empirical data that the locus dropout affects the type of loci retained, the loci found in many species tending to show lower interspecific distances than those shared among fewer species. We also examine the effects of the numbers of loci, SNPs, and PIS on nodal bootstrap support, but could not demonstrate with our data our expectation of a positive correlation between them. We conclude that RAD methods provide a powerful tool for phylogenomics at an intermediate phylogenetic level as indicated by its broad congruence with an eight-gene Sanger data set in a genus of moths. When assessing the quality of the data for phylogenetic inference, the focus should be on the distribution and number of SNPs and PIS rather than on loci.

Mitonuclear discordance in wolf spiders: Genomic evidence for species integrity and introgression.

Systematists and taxonomists have benefited greatly from the emergence of molecular methods. Species identification has become straightforward through DNA barcoding and the rapid build-up of massive DNA barcode reference libraries. In animals, mitonuclear discordance can significantly complicate the process of species identification and delimitation. The causes of mitonuclear discordance are either biological (e.g., introgression, incomplete lineage sorting, horizontal gene transfer androgenesis) or induced by operational factors (e.g., human error with specimen misidentification or incorrect species delimitation). Moreover, endosymbionts may play an important role in promoting fixation of mitochondrial genomes. Here, we study the mitonuclear discordance of wolf spiders species (Lycosidae) (independent cases from Alopecosa aculeata and Pardosa pullata groups) that share identical COI DNA barcodes. We approached the case utilizing double-digest restriction site-associated DNA sequencing (ddRADseq) to obtain and analyse genomic-scale data. Our results suggest that the observed cases of mitonuclear discordance are not due to operational reasons but result from biological processes. Further analysis indicated introgression and that incomplete lineage sorting is unlikely to have been responsible for the observed discrepancy. Additional survey of endosymbionts provided ideas on further research and their role in shaping mitochondrial DNA distribution patterns. Thus, ddRADseq grants an efficient way to study the taxonomy of problematic groups with insight into underlying evolutionary processes.

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

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

Elusive ditrysian phylogeny: an account of combining systematized morphology with molecular data (Lepidoptera).

BACKGROUND: Ditrysia comprise close to 99 % of all butterflies and moths. The evolutionary relationships among the ditrysian superfamilies have received considerable attention in phylogenetic studies based on DNA and transcriptomic data, but the deepest divergences remain for large parts unresolved or contradictory. To obtain complementary insight into the evolutionary history of the clade, and to test previous hypotheses on the subdivision of Ditrysia based on morphology, we examine the morphology of larvae, pupae and adult males and females of 318 taxa representing nearly all ditrysian superfamilies and families. We present the most comprehensive morphological dataset on Ditrysia to date, consisting of over 500 morphological characters. The data are analyzed alone and combined with sequence data (one mitochondrial and seven nuclear protein-coding gene regions, sequenced from 422 taxa). The full dataset consists of 473 exemplar species. Analyses are performed using maximum likelihood methods, and parsimony methods for the morphological dataset. We explore whether combining morphological data and DNA-data can stabilize taxa that are unstable in phylogenetic studies based on genetic data only. RESULTS: Morphological characters are found phylogenetically informative in resolving apical nodes (superfamilies and families), but characters serving as evidence of relatedness of larger assemblages are few. Results include the recovery of a monophyletic Tineoidea, Sesioidea and Cossoidea, and a stable position for some unstable taxa (e.g. Epipyropidae, Cyclotornidae, Urodoidea + Schreckensteinioidea). Several such taxa, however, remain unstable even though morphological characters indicate a position in the tree (e.g. Immidae). Evidence supporting affinities between clades are suggested, e.g. a novel larval synapomorphy for Tineidae. We also propose the synonymy of Tineodidae with Alucitidae, syn. nov. CONCLUSIONS: The large morphological dataset provides information on the diversity and distribution of morphological traits in Ditrysia, and can be used in future research on the evolution of these traits, in identification keys and in identification of fossil Lepidoptera. The "backbone" of the phylogeny for Ditrysia remains largely unresolved. As previously proposed as an explanation for the scarcity of molecular signal in resolving the deeper nodes, this may be due to the rapid radiation of Ditrysia in the Cretaceous.

Morphology reinforces proposed molecular phylogenetic affinities: a revised classification for Gelechioidea (Lepidoptera).

Gelechioidea are one of the most species rich and least studied superfamilies of Lepidoptera. We examine the interrelationships within the superfamily using the densest taxon sampling to date, combined with the most extensive ever morphological and molecular character data. We perform partitioned and combined analyses using maximum likelihood, Bayesian and parsimony approaches. The combined dataset consists of 155 exemplar species of Gelechioidea, representing nearly all subfamilies recognized in recent classifications. Parsimony analyses are performed with a dataset including 28 additional terminal taxa with only morphological data available. We use eight genes with a total of 6127 bp, and morphological data with 253 characters derived from larval, pupal, and adult morphology. The analyses of combined data yield more resolved trees and significantly better-supported groupings than either dataset when analysed alone. The recurrent monophyletic groupings in all our model-based analyses support a revision of the family classification. Deeper relationships vary between analyses and data partitions, leaving them ambiguous. The place of the root remains a challenge for future research. We propose a revised classification and suggest the division of Gelechioidea into 16 families. We redefine Depressariidae Meyrick, 1883 for a monophylum that includes Acriinae, Aeolanthinae, Cryptolechiinae, Depressariinae, Ethmiinae, Hypercalliinae, Hypertrophinae, Peleopodinae, Oditinae, Stenomatinae, Carcina, and a diversity of predominantly New World taxa previously excluded from Lypusidae (Amphisbatidae s. authors) but left without family position. A monophyletic Oecophoridae s. s., including Deuterogoniinae and Pleurotinae, is obtained for the first time with significant support. Elachistidae s. l. is found to be polyphyletic, and Elachistidae is restricted to comprise Agonoxeninae, Elachistinae, and Parametriotinae. Batrachedridae are polyphyletic, with several genera pending further study. Apart from the core Batrachedra, the taxa previously included in this family are grouped in an expanded Pterolonchidae, together with Coelopoetinae and Syringopainae. Lypusidae s. s. and Chimabachidae form a monophylum; Chimabachinae is united with Lypusidae as a subfamily, stat. n. Our results contradict the subfamily classifications of several families, notably Lecithoceridae and Autostichidae, but due to insufficient sampling of taxa we refrain from comprehensive taxonomic conclusions on the subfamily level, and encourage focused studies to resolve these groups.

Diet shift induced rapid evolution of size and function in a predatory bird.

A predator's body size correlates with its prey size. Change in the diet may call for changes in the hunting mode and traits determining hunting success. We explored long-term trends in sternum size and shape in the northern goshawk by applying geometric morphometrics. Tetraonids, the primary prey of the goshawk, have decreased and been replaced by smaller birds in the diet. We expected that the size of the goshawk has decreased accordingly more in males than females based on earlier observations of outer morphology. We also expected changes in sternum shape as a function of changes in hunting mode. Size of both sexes has decreased during the preceding decades (1962-2008), seemingly reflecting a shift in prey size and hunting mode. Female goshawks hunting also mammalian prey tend to have a pronouncedly "Buteo-type" sternum compared to males preying upon birds. Interestingly, the shrinkage of body size resulted in an increasingly "Buteo-type" sternum in both sexes. In addition, the sternum shape in birds that died accidentally (i.e., fit individuals) was more Buteo-type than in starved ones, hinting that selection was towards a Buteo-type sternum shape. We conclude that these observed patterns are likely due to directional selection driven by changes in the diet towards smaller and more agile prey. On the other hand, global warming is predicted to also cause a decrease in size, thus these two scenarios are inseparable. Because of difficulties in studying fitness-related phenotypic changes of large raptors in the field, time series of museum exemplars collected over a wide geographical area may give answers to this conundrum.

ddRAD Sequencing and DNA Barcoding.

Double-digest restriction site-associated DNA sequencing is a library preparation protocol that enables capturing variable sites across the genome including single-nucleotide polymorphisms (SNPs). These SNPs can be utilized to gain evolutionary insights into patterns observed in DNA barcodes, to infer population structure and phylogenies, to detect gene flow and introgression, and to perform species delimitation analyses. The protocol includes chemically shearing genomic DNA with restriction enzymes, unique tagging, size selection, and amplification of the resulting DNA fragments. Here we provide a detailed description of each step of the protocol, as well as information on essential equipment and common issues encountered during laboratory work.

Global arthropod beta-diversity is spatially and temporally structured by latitude.

Global biodiversity gradients are generally expected to reflect greater species replacement closer to the equator. However, empirical validation of global biodiversity gradients largely relies on vertebrates, plants, and other less diverse taxa. Here we assess the temporal and spatial dynamics of global arthropod biodiversity dynamics using a beta-diversity framework. Sampling includes 129 sampling sites whereby malaise traps are deployed to monitor temporal changes in arthropod communities. Overall, we encountered more than 150,000 unique barcode index numbers (BINs) (i.e. species proxies). We assess between site differences in community diversity using beta-diversity and the partitioned components of species replacement and richness difference. Global total beta-diversity (dissimilarity) increases with decreasing latitude, greater spatial distance and greater temporal distance. Species replacement and richness difference patterns vary across biogeographic regions. Our findings support long-standing, general expectations of global biodiversity patterns. However, we also show that the underlying processes driving patterns may be regionally linked.