Museum collections around the world contain billions of specimens, including rare and extinct species. If their genetic information could be retrieved at a large scale, this would dramatically increase our knowledge of genetic and taxonomic diversity information, and support evolutionary, ecological and systematic studies. We here present a target enrichment kit for 2953 loci in 1753 orthologous nuclear genes + the barcoding region of cytochrome C oxidase 1, for Lepidoptera and demonstrate its utility to obtain a large number of nuclear loci from dry, pinned museum material collected from 1892 to 2017. We sequenced enriched libraries of 37 museum specimens across the order Lepidoptera, many from higher taxa not yet included in high‐throughput molecular studies, showing that our kit can be used to generate comparable data across the order, and provides resolution both for shallower and deeper nodes. The filtered datasets (172 taxa, 234 464 amino acid positions and corresponding nucleotides from 1835 CDS regions) were used to infer a phylogeny of Lepidoptera, which is largely congruent in topology to recent phylogenomic studies, but with the addition of some key taxa. We furthermore present our TEnriAn (Target Enrichment Analysis) workflow for processing and combining target enrichment, transcriptomic and genomic data.

Environmental DNA studies targeting multiple taxa using metabarcoding provide remarkable insights into levels of species diversity in any habitat. The main drawbacks are the presence of primer bias and difficulty in identifying rare species. We tested a DNA sequence‐capture method in parallel with the metabarcoding approach to reveal possible advantages of one method over the other. Both approaches were performed using the same eDNA samples and the same 18S and COI regions, followed by high throughput sequencing. Metabarcoded eDNA libraries were PCR amplified with one primer pair from 18S and COI genes. DNA sequence‐capture libraries were enriched with 3,639 baits targeting the same gene regions. We tested amplicon sequence variants (ASVs) and operational taxonomic units (OTUs) in silico approaches for both markers and methods, using for this purpose the metabarcoding data set. ASVs methods uncovered more species for the COI gene, whereas the opposite occurred for the 18S gene, suggesting that clustering reads into OTUs could bias diversity richness especially using 18S with relaxed thresholds. Additionally, metabarcoding and DNA sequence‐capture recovered 80%–90% of the control sample species. DNA sequence‐capture was 8x more expensive, nonetheless it identified 1.5x more species for COI and 13x more genera for 18S than metabarcoding. Both approaches offer reliable results, sharing ca. 40% species and 72% families and retrieve more taxa when nuclear and mitochondrial markers are combined. eDNA metabarcoding is quite well established and low‐cost, whereas DNA‐sequence capture for biodiversity assessment is still in its infancy, is more time‐consuming but provides more taxonomic assignments.

Species are the fundamental units of life and evolution. Their recognition is essential for science and society. Molecular methods have been increasingly used for the identification of animal species, despite several challenges. Here, we explore with genomic data from nine animal lineages a set of nuclear markers, namely metazoan‐level universal single‐copy orthologs (metazoan USCOs), for their use in species delimitation. Our data sets include arthropods and vertebrates. We use various data assembly strategies and use coalescent‐based species inference as well as population admixture analyses and phenetic methods. We demonstrate that metazoan USCOs distinguish well closely related morphospecies and consistently outperform classical mitochondrial DNA barcoding in discriminating closely related species in different animal taxa, as judged by comparison with morphospecies delimitations. USCOs overcome the general shortcomings of mitochondrial DNA barcodes, and due to standardization across Metazoa, also those of other approaches. They accurately assign samples not only to lower but also to higher taxonomic levels. Metazoan USCOs provide a powerful and unifying framework for DNA‐based species delimitation and taxonomy in animals and their employment could result in a more efficient use of research data and resources.

Cuckoo wasps (Hymenoptera: Chrysididae) are a species-rich family of obligate brood parasites (i.e. parasitoids and kleptoparasites) whose hosts range from sawflies, wasps and bees, to walking sticks and moths. Their brood parasitic lifestyle has led to the evolution of fascinating adaptations, including chemical mimicry of host odours by some species. Long-term nomenclatural stability of the higher taxonomic units (e.g. genera, tribes, and subfamilies) in this family and a thorough understanding of the family's evolutionary history critically depend on a robust phylogeny of cuckoo wasps. Here we present the results from phylogenetically analysing ten nuclear-encoded genes and one mitochondrial gene, all protein-coding, in a total of 186 different species of cuckoo wasps representing most major cuckoo wasp lineages. The compiled data matrix comprised 4946 coding nucleotide sites and was phylogenetically analysed using classical maximum-likelihood and Bayesian inference methods. The results of our phylogenetic analyses are mostly consistent with earlier ideas on the phylogenetic relationships of the cuckoo wasps' subfamilies and tribes, but cast doubts on the hitherto hypothesized phylogenetic position of the subfamily Amiseginae. However, the molecular data are not fully conclusive in this respect due to low branch support values at deep nodes. In contrast, our phylogenetic estimates clearly indicate that the current systematics of cuckoo wasps at the genus level is artificial. Several of the Correspondence: Oliver Niehuis, Phylogeny and host associations of cuckoo wasps 323 currently recognized genera are para-or polyphyletic (e.g. Cephaloparnops, Chrysis, Chrysura, Euchroeus, Hedychridium, Praestochrysis, Pseudochrysis, Spintharina, and Spinolia). At the same time, our data support the validity of the genus Colpopyga, previously synonymized with Hedychridium. We discuss possible solutions for how to resolve the current shortcomings in the systematics of cuckoo wasp genera and decided to grant Prospinolia the status of a valid genus (Prospinolia stat.n.) and transferring Spinolia theresae [du Buysson 1900] from Spinolia to Prospinolia (Prospinolia theresae stat.restit.). We discuss the implications of our phylogenetic inferences for understanding the evolution of host associations in this group. The results of our study not only shed new light on the evolutionary history of cuckoo wasps, but also set the basis for future phylogenomic investigations on this captivating group of wasps by guiding taxonomic sampling efforts and the design of probes for target DNA enrichment approaches.

Species are the fundamental units of life and their recognition is essential for science and society. DNA barcoding, the use of a single and often mitochondrial gene, has been increasingly employed as a universal approach for the identification of animal species. However, this approach faces several challenges. Here, we demonstrate with empirical data from a number of metazoan animal lineages that multiple nuclear-encoded markers, so called universal single-copy orthologs (USCOs) performs much better than the single barcode gene to discriminate closely related species. Overcoming the general shortcomings of mitochondrial DNA barcodes, USCOs also accurately assign samples to higher taxonomic levels. These loci thus provide a powerful and unifying framework for species delimitation which considerably improves the DNA-based inference of animal species.