Metabarcoding is a powerful tool for biodiversity comparisons, where standard-size DNA barcodes (> 500 bases) offer better taxonomic resolution than shorter ones. Still, the choice of sequencing platforms and bioinformatics pipelines may strongly affect inferred diversity due to various technical biases. We assessed the relative performance of Illumina MiSeq i100 (2 × 500 paired-end), PacBio Revio and Oxford Nanopore MinION sequencing and bioinformatics pipelines, using full-length ITS amplicon sequencing datasets from a 103-species mock community and 45 composite soil samples. Despite numerous low-quality reads, PacBio yielded the lowest overall error rate and highest number of taxa. Illumina revealed the highest proportion of chimeric and index-switched reads, along with a strong bias towards shorter amplicons. MinION data analysed using PRONAME and Minovar-a bioinformatics pipeline presented here-had the largest proportion of low-quality data, and rare taxa were lost during data filtering and read polishing steps. Although Minovar enabled amplicon sequence variant (ASV) level precision for common taxa, we recommend clustering ASVs into OTUs. For PacBio, standard filtering approaches outperformed the ASV approach because they retained rare taxa. For Illumina, a stringent ASV approach or removal of rare OTUs would limit artefacts. Across all platforms, excess PCR cycles promoted chimeric and low-quality reads and lost quantitativity in biodiversity assessments. With moderate differences in effect sizes, all analytical approaches supported the conclusion that sampling design determines how we see soil biodiversity responses to land use. For biodiversity surveys based on the full-length ITS metabarcoding, we recommend using PacBio sequencing with standard, non-ASV pipelines.

DNA barcoding, the use of a standard DNA fragment for species identification, has emerged as a major field of biodiversity research. The effectiveness of this approach rests on the premise that much less variation exists within species than between them. While exceptions occur, this has been demonstrated in many animal taxa where the COI gene is effective in species discrimination. Sawflies are an exception to this pattern because DNA barcodes often fail to distinguish congeneric species. Using high-throughput single-molecule DNA sequencing to recover COI sequences from thousands of sawflies, we found that single individuals often possess multiple, seemingly functional, full-length DNA barcodes (i.e., unrecognizable as nuclear pseudogenes)-a phenomenon not documented at similar prevalence in any animal taxon. While the evolutionary causes of multiple variants require further investigation, our observation is remarkable as it violates the one-barcode-one-specimen assumption. The presence of multiple variants of barcodes within individuals does not jeopardize the concept, but it introduces a complexity for species inventories based on metabarcoding. They will overestimate the species count when barcode-based operational species units are used as species proxies. Similarly, DNA barcode reference libraries must consider how best to deal with the high frequency of multiple intra-individual variants.

The sawfly fauna of Greenland is reviewed, based on specimens collected in 2025 and a re-examination of material in museum collections. Ametastegia pallipes (Spinola, 1808) and Euura vaga (Fabricius, 1781) are recorded from Greenland for the first time. Pristiphora laricis (Hartig, 1837) is confirmed to be well established in a restricted area of South Greenland. Euura obscuripes (Holmgren, 1883) is the valid name for the nominal taxa described from Greenland as Nematus borealis Marlatt, 1892 (new synonym) and Amauronematus groenlandicus Malaise, 1933 (new synonym). Greenlandic specimens of Euura cornuta (Lindqvist, 1962) have formerly been misidentified as Amauronematus viduatus (Zetterstedt, 1838) or A. nitidipleuris Malaise, 1931. Now including a total of only nine recorded species, the sawfly fauna is extremely species-poor, thus fitting the general pattern exhibited by most groups of insects in Greenland. Genetic sequencing and existing distribution data indicate that most species are likely to have reached Greenland from the West Palaearctic. The possibility that some of them may have been accidentally introduced by humans is discussed.

New data on the distribution of 11 Symphyta species phyllophagous on birch trees (Betula L.) are presented. Dineura virididorsata (Retzius,1783), Heterarthrus nemoratus (Fall & eacute;n, 1808), Nematinus caledonicus (Cameron, 1882), Pamphilius varius (Audinet-Serville,1823), and Pristiphora alpestris (Konow, 1903) are reported from Slovakia for the first time.The little-known Allantus cingillum (Klug,1814), Euura ampla (Konow, 1895), E. leionota (Benson, 1933), Profenusa thomsoni (Konow, 1886), Trichiosoma lucorum (Linnaeus, 1758), and T. vitellina (Linnaeus, 1761) were found in Slovakia, either after many decades or for the second time.

DNA barcoding, the use of standard segments of DNA to assign specimens to a species, has emerged as a major field of biodiversity research over the last 20 years. Large-scale global initiatives are building DNA barcode reference libraries for animals, fungi, and plants, while pipelines are being developed for metabarcoding-based biomonitoring. The effectiveness of these approaches rests on the premise that much less variation exists within species than between them. While exceptions occur, this principle has been demonstrated to apply in the many animal taxa where the barcode region of the COI gene is effective in species discrimination. Sawflies are an exception to this general pattern because DNA barcodes often fail to distinguish congeneric species, an observation which prompted us to search for an explanation. Using high-throughput single-molecule DNA sequencing to recover COI sequences from thousands of sawflies, we found that single individuals often possess multiple, seemingly functional, full-length DNA barcodes – a phenomenon not documented at similar prevalence in any animal taxon. While the evolutionary causes of multiple variants require further investigation, our observation is remarkable as it violates the one-barcode-one-specimen assumption. The presence of multiple variants of barcodes within individuals does not jeopardize the concept, but its occurrence does introduce a complexity for species inventories based on metabarcoding. They will overestimate the species count when barcode-based operational species units are used as species proxies. Similarly, reference libraries must consider how best to deal with the high frequency of multiple variants in sawflies and any other groups of organisms. Significance Statement DNA barcoding is revolutionizing biodiversity science by enabling the accurate identification of organisms, accelerating taxonomic workflows, and permitting DNA-based biomonitoring. The DNA barcode region for the animal kingdom, mitochondrial COI, is highly effective in discriminating species in almost all studied animal groups. However, the use DNA barcoding is sometimes complicated by the presence of nuclear pseudogenes (NUMTs) or by variants of the mitogenome itself (heteroplasmy) within individuals. By using high-throughput sequencing (HTS) to analyze thou-sands of specimens, we demonstrate that multiple, seemingly functional, full-length variants of the COI barcode region are frequent in North European sawflies. Since these variants are sometimes very divergent, it is important to consider the impact of this within-individual variability on studies based on DNA barcodes. ### Competing Interest Statement The authors have declared no competing interest.

Excluding 102 species of the previously revised West Palaearctic species groups of Euura Newman, 1837 (gall-making groups, 84 species; amentorum group, 8 species; bergmanni group, 8 species; and oligospila group, 2 species), we treat here the remaining 164 West Palaearctic species of the genus. 145 new synonymies are proposed (one in Platycampus, one in Nematus, two in Pristiphora, and the rest in Euura: see Table 4 for details). 153 lectotypes are designated (see Table 4). Four nominal species are reinstated (valid name in square brackets): Pteronidea fuscarima Benson, 1933 [E. fuscarima (Benson, 1933) comb. nov.], P. fuscodorsata Lindqvist, 1949 [Euura fuscodorsata (Lindqvist, 1949) comb. nov.], Pachynematus perkioemaekii Lindqvist, 1960 [E. perkioemaekii (Lindqvist, 1960) comb. nov.], and Pontania poppii Konow, 1904 [Euura poppii (Konow, 1904) comb. nov.]. Euura tiliae (Zinovjev, 1998) comb. nov. and E. wuyishanica (Wei, 2003) comb. nov. are transferred from Nematus. Euura memoriakaszabi (Haris, 2002) comb. nov. is transferred from Pristiphora. Five new species are described: Euura halo Prous, Liston & Mutanen sp. nov., E. histriato Prous, Liston & Mutanen sp. nov., E. minivittata Prous & Mutanen sp. nov., E. polepso Prous & Mutanen sp. nov., and E. serela Prous & Mutanen sp. nov. Euura telos Liston & Prous nom. nov. (an East Palaearctic species) is proposed for Amauronematus terminalis Malaise, 1931, a secondary homonym of Pontania terminalis Marlatt, 1896 [Euura terminalis (Marlatt, 1896)]. Three treated putative species based on single males (E. bergmanni and E. clitellata group) and a female (E. bipartita group) remain unidentified pending further research. An identification key is provided to separate the genus Euura from the other similar genera. Host plants are now known for 80% (132) of the treated species (88% for all West Palaearctic Euura). Genetic data (at least mitochondrial COI and nuclear NaK and POL2) are reported for 91% (151) of the treated species. The genetic data were obtained with Sanger and Nanopore sequencing. In numerous cases, identification of one sex of a species remains difficult using morphological characters but is clear when using genetic data. In a few cases, however, identification based on morphology is reliable, while support from available genetic data is weak. Often, large morphological and genetic variability makes species delimitation ambiguous. Within-species genetic diversity, as estimated from diploid females (i.e., within-individual genetic diversity, which is an underestimate of within-species diversity), is large in Euura, on average with 0.3% divergence between the haplotypes (max 1.4%), while between-species divergence for a given species group is often only slightly higher (on average varies between 0.5–2.1%). Strong mito-nuclear discordance is observed within most species groups, but in some cases even between species groups. Over 50% of the species cannot be reliably identified based on mitochondrial COI barcodes. While nuclear DNA is significantly more congruent with morphology, identification of about 15% of the species can be ambiguous due to large genetic variability. Remarkably, two or more apparently functional COI variants are frequently observed within the same individual, with variants diverging by up to 9.6% in Euura lappo (for the 658 bp barcoding region).

Phylogenomic approaches have recently helped elucidate various insect relationships, but large-scale comprehensive analyses on relationships within sawflies and woodwasps are still lacking. Here, we infer the relationships and long-term biogeographic history of these hymenopteran groups using a large dataset of 354 UCE loci collected from 385 species that represent all major lineages. Early Hymenoptera started diversifying during the Early Triassic ∼249 Ma and spread all over the ancient supercontinent Pangaea. We recovered Xyeloidea as a monophyletic sister group to other Hymenoptera and Pamphilioidea as sister to Unicalcarida. Within the diverse family Tenthredinidae, our taxonomically and geographically expanded taxon sampling highlights the non-monophyly of several traditionally defined subfamilies. In addition, the recent removal of Athalia and related genera from the Tenthredinidae into the separate family Athaliidae is supported. The deep historical biogeography of the group is characterised by independent dispersals and re-colonisations between the northern (Laurasia) and southern (Gondwana) palaeocontinents. The breakup of these landmasses led to ancient vicariance in several Gondwanan lineages, while interchange across the Northern Hemisphere has continued until the Recent. The little-studied African sawfly fauna is likewise a diverse mixture of groups with varying routes of colonization. Our results reveal interesting parallels in the evolution and biogeography of early hymenopterans and other ancient insect groups.

Molecular identification of micro- and macroorganisms based on nuclear markers has revolutionized our understanding of their taxonomy, phylogeny and ecology. Today, research on the diversity of eukaryotes in global ecosystems heavily relies on nuclear ribosomal RNA (rRNA) markers. Here, we present the research community-curated reference database EUKARYOME for nuclear ribosomal 18S rRNA, internal transcribed spacer (ITS) and 28S rRNA markers for all eukaryotes, including metazoans (animals), protists, fungi and plants. It is particularly useful for the identification of arbuscular mycorrhizal fungi as it bridges the four commonly used molecular markers-ITS1, ITS2, 18S V4-V5 and 28S D1-D2 subregions. The key benefits of this database over other annotated reference sequence databases are that it is not restricted to certain taxonomic groups and it includes all rRNA markers. EUKARYOME also offers a number of reference long-read sequences that are derived from (meta)genomic and (meta)barcoding-a unique feature that can be used for taxonomic identification and chimera control of third-generation, long-read, high-throughput sequencing data. Taxonomic assignments of rRNA genes in the database are verified based on phylogenetic approaches. The reference datasets are available in multiple formats from the project homepage, http://www.eukaryome.org.

We sequenced and assembled mitochondrial genomes of three tenthredinid sawflies (Euura poecilonota, E. striata, and Dolerus timidus) using Oxford Nanopore Technologies' MinION. The Canu assembler produced circular assemblies (23,000-40,000 bp). Still, errors were found in the highly repetitive non-coding control region because of the fragmented DNA which led to no reads spanning the complete control region, preventing its reliable assembly. Based on the non-repetitive coding region's sequencing coverage, we estimate the lengths of mitochondrial genomes of E. poecilonota, D. timidus, and E. striata to be about 30,000 bp, 31,000 bp, and 37,000 bp and control region to be 15,000 bp, 16,000 bp, and 22,000 bp respectively. All standard bilaterian mitochondrial genes are in the same order and orientation, except trnQ, which is on the minus strand in Euura and the plus strand in Dolerus. Using published tenthredinid genome data, we show that control region lengths are often underestimated.

The Euura amentorum species group is Holarctic, and in Europe it is most species-rich in the North. Their larvae develop entirely within the female catkins of Salix species: some species bore in the central stalk, whereas others live outside this and feed mainly on the developing seeds. Eight Palaearctic species are treated here as valid, and a key to these is provided. Males of five species are known. Two new species are described from northern Europe: Euura pohjola sp. n. and E. ursaminor sp. n. First records of E. itelmena (Malaise, 1931) from the West Palaearctic are presented. We propose seven new synonymies: Pontopristia montana Lindqvist, 1961 (junior secondary homonym in Euura) with Euura freyja (Liston, Taeger & Blank, 2009); Pontopristia brevilabris Malaise, 1921, Amauronematus fennicus Lindqvist, 1944, Pontopristia boreoalpina Lindqvist, 1961, Pontopristia punctulata Lindqvist, 1961, and Amauronematus pyrenaeus Lacourt, 1995 with Euura microphyes (Frster, 1854); and Pteronidea holmgreni Lindqvist, 1968 with Nematus umbratus Thomson, 1871. Lectotypes are designated for: Amauronematus fennicus Lindqvist, 1944, Nematus amentorum Frster, 1854, Nematus suavis Ruthe, 1859, Pontopristia brevilabris Malaise, 1921, Pontopristia itelmena Malaise, 1931, Pontopristia kamtchatica Malaise, 1931, Pontopristia lapponica Malaise, 1921, Pontopristia latiserra Malaise, 1921, Pontopristia romani Malaise, 1921, and Pristiphora amentorum var. nigripleuris Enslin, 1916. Many new host plant associations are recorded.