Nematinae, Thomson, 1871

Prous, Marko, Liston, Andrew, Monckton, Spencer K., Kramp, Katja, Vårdal, Hege, Vikberg, Veli, Heibo, Erik & Mutanen, Marko, 2025, West Palaearctic species of Euura Newman, 1837 (Hymenoptera, Tenthredinidae), European Journal of Taxonomy 977, pp. 1-377 : 11-19

Monophyly and delimitation of Nematinae

Based on ten genes (COI and 9 nuclear genes), the subfamily Nematinae is strongly supported as monophyletic ( Fig. 1 ), as found in previous molecular phylogenetic analyses with adequate taxon and gene sampling ( Prous et al. 2014; Malm & Nyman 2015; Vilhelmsen et al. 2024; Wutke et al. 2024). Support for monophyly of Nematinae based on morphology is ambiguous( Vilhelmsen 2015). Unusually, some genera of Nematinae ( Analcellicampa , Caulocampus , Cladius , Hoplocampa , Monocellicampa , and Susana ) are classified under a separate subfamily Hoplocampinae Rohwer, 1911 in NCBI GenBank. This arrangement of genera ( Hoplocampinae ) finds no support in any phylogenetic analyses ( Prous et al. 2014; Malm & Nyman 2015; Vilhelmsen 2015; Fig. 1 ), except perhaps a classification proposal by Wei & Nie (1998), which is not widely accepted ( Taeger et al. 2010). The genera of Hoplocampinae are strongly supported as part of Nematinae radiation, but the exact relationships between many of these and the other genera of Nematinae are poorly supported ( Prous et al. 2014; Malm & Nyman 2015; Niu et al. 2021; Fig. 1 ). The best supported clades within the Nematinae are Dineurini Ashmead, 1898 ( Prous et al. 2014) and Nematini ( Figs 1–2 ), the latter containing the majority of species of Nematinae (more than 75%), including the genus Euura .

Phylogeny and groups of Euura

The core of the genus Euura (i.e., without the Euura epilosa group) is strongly supported as monophyletic based on phylogenetic analyses of ten genes ( Fig. 2 ). The Euura epilosa group (sampled species E. brachycera and E. epilosa ) is weakly or moderately supported as the sister group of the core of Euura ( Fig. 2 ). The Nematus wahlbergi group is found as the sister group of Euura with moderate support, rendering Nematus paraphyletic ( Fig. 2 ). Nematus together with E. brachycera (DEI-GISHym86097 as the only representative of the group which was identified as Nematus spec. ) formed a weakly supported sister clade of core Euura in a previous analysis based on a smaller dataset ( Prous et al. 2019). Euura brachycera ( Lindqvist, 1975) ( Euura brevicera Taeger & Blank, 2014 is an unnecessary replacement name) was transferred to Euura by Prous et al. (2014), although no genetic data was available for this or other related species at the time. Even though the Euura epilosa group is not genetically strongly supported as a member of Euura , we retain it in this genus because of its similar morphology [see under Euura epilosa ( Lindqvist, 1970) ]. To reconstruct internal phylogeny of Euura , we excluded mitochondrial COI, because of strong conflict with nuclear genes: in some cases even above group level (see under Mitonuclear discordance). For most species we included 1– 2 specimens having the greatest amount of sequence data, but more specimens in some cases because of large genetic variability ( Fig. 3 ). At the base of core Euura (excluding the Euura epilosa group) 15 clades are well-supported, but relationships among them are poorly resolved ( Fig. 3 ). In combination with morphology, we have defined 37 species groups, 7 of which are gall-makers ( Liston et al. 2017), and 12 species not assigned to groups. The largest well-supported clade (clade A) includes roughly 30% of species of Euura , containing most of the former “ Amauronematus ” (including former “ Pontopristia ”, but excluding “ Brachycoluma ”), i.e., the punicea group sensu lato (s. lat.), and flavescens , scutellata , miliaris , melanocephalus , vicina , caeruleocarpus groups, and some species not assigned to groups ( imperfecta , salicis , distinguenda , anthracina , hyperborea ) ( Fig. 3 ). Within this clade, the punicea group s. lat. is strongly supported, containing the former “ Amauronematus ” groups: aegra , amentorum , histrio , humeralis , longiserra (not monophyletic based on nuclear data), opacipleuris , punicea sensu stricto (s. str.), toeniata , tunicata , variator , vittata , and E. brunnea not assigned to a group. Clade A also includes a well-supported clade comprising the miliaris , scutellata , and flavescens groups, where the last two may be sisters. The second largest well-supported clade comprises the gallmakers, followed by three other well or moderately supported clades containing two groups each: miltonota + viduata , myosotidis + bipartita , and pallescens + ribesii ( Fig. 3 ). A neighbour-joining distance tree including specimens with minimum of 1529 bp of nuclear data (1381 specimens) is included as supplementary material.

Within-species genetic diversity in Euura

Without any knowledge of species boundaries, it is possible to get information about within-species genetic diversity based on haplotype divergence of nuclear genes of single individuals. As Hymenoptera are haplodiploid (males are haploid, females diploid), this assessment is normally only possible for female individuals (although rare diploid males exist). Averaging across individual females with at least 1529 bp of nuclear data, the average divergence between the haplotypes in Euura is 0.32% (max 1.41%). When considering all individuals of each species as delimited here, the average within-species divergence is 0.23%. This is counter-intuitive, as within-individual haplotype divergence should underestimate within-species divergence; however, this result is because we coded heterozygous positions using IUPAC ambiguity symbols, which are treated as missing data in the calculations. When only males are considered (i.e., hemizygous, therefore no ambiguous positions), average within-species divergence is 0.42%, which is larger than estimated from within-individual female haplotypes. Consistent with morphological observations, the between-species genetic divergence within species groups is often only slightly larger (on average varies between 0.5–2.1%) than within-species divergence, highlighting the difficulty of delimiting species in Euura .

Mitonuclear discordance

As in Pristiphora ( Prous et al. 2017) , in Euura there is a strong conflict between mitochondrial COI on one hand, and nuclear and morphological data on the other hand when delimiting species – or even groups above species level in some cases (see supplementary distance COI and nuclear trees). Currently, over 50% of the species in Euura cannot be reliably identified using COI sequences. However, the precision of identification could probably be increased by comparing exact COI variants (i.e., differing by at least one nucleotide), which often correlate with species boundaries. At present, due to the large diversity of COI variants, the correspondence between exact variants and species has not yet been established in most cases. Many additional COI variants (hundreds, if not thousands) can be expected in Finland alone, where more than 4500 Euura specimens have been barcoded. Even ignoring this challenge, different species frequently have identical sequences: based on current data (>1300 specimens for which nuclear genes are also available) this is the case for 47 species using just the standard barcode region, or 42 species using the full 1078 bp. This is more than 20% of the species, not counting gall-makers. Identification success using COI in Euura may be higher in southern and central Europe because of lower species richness and lower within-species genetic diversity.

Notably, in about 15% of species, genetic diversity is sufficiently high that species identification based on 1–2 nuclear genes can also be ambiguous. The success rate increases when more nuclear genes are included.

Remarkably, in some cases there is strong disagreement between mitochondrial COI and nuclear genes for delineating groups of species. The most remarkable is the non-monophyly of the E. amentorum group based on COI, contrasting with strong support based on nuclear genes ( Liston et al. 2023). Another example, albeit less extreme, is the clade comprising the E. histrio , E. variator , and E. opacipleuris groups, wherein groups can be delimited well by morphology and nuclear genes, but COI sequences of the same species can in several cases appear to belong to different groups. There are a few other such examples throughout Euura (see supplementary distance COI and nuclear trees of the specimens analysed here).

Intraindividual mitochondrial variants in Nematinae

As already reported ( Prous et al. 2021; Liston et al. 2023), multiple intraindividual COI variants have been detected in many Nematinae using Nanopore sequencing. The vast majority of these variants appear to be functional, because they lack stop codons or frame shifting indels (insertions or deletions). This would suggest heteroplasmy, rather than the presence of nuclear-encoded mitochondrial pseudogenes (NUMTs). Excluding the detectable NUMTs, about 25% of about 1000 specimens still have more than one apparently functional variant (1078–1087 bp amplicons). It is possible that some of the detected intraindividual variants are PCR chimeras (if there is mixture of different sequences, DNA polymerase sometimes switches the template molecule during PCR), but this seems to be a minor issue. This is indicated by the fact that for nuclear genes of males (which are haploid) almost always only a single variant is detected (contrasting with frequent multiple COI variant detection) and for nuclear genes of females (diploid) usually two variants are detected (sometimes one, almost never more than two), again contrasting with frequent detection of more than two variants for COI. We did notice possible PCR chimeras among single Nanopore reads and sometimes detected such consensus variant sequences, but these have usually been of sufficiently low frequency that the variants are not detected or the variants have been filtered out because of low read counts. The frequent presence of multiple COI variants within the same individual has also been evident in the results of Sanger sequencing ( Prous et al. 2021), although this method does not allow determination of the number and exact sequences of the variants, unlike Nanopore sequencing as employed here. Interestingly, the presence of heteroplasmy seems to correlate well with mito-nuclear discordance: the groups where species boundaries are more reliably defined by nuclear than mitochondrial DNA tend also to have a higher incidence of heteroplasmy. If the mis-match between mitochondrial and nuclear sequences in terms of species boundaries is mainly due to occasional hybridisation between species (a pattern expected in theory for haplodiploid species and at least partly supported by empirical studies; Linnen & Farrell 2007; Patten et al. 2015; Prous et al. 2020), then this could also explain the extensive occurrence of heteroplasmy, which has been suggested to be more likely when heterospecific hybridization has occurred ( Ladoukakis & Zouros 2017; Mastrantonio et al. 2019). A neighbour-joining distance tree including specimens with a minimum of 621 bp of COI sequence and the detected variants (1905 specimens, 2254 sequences) is included in the supplementary material.

Possible diploid males and tri- or tetraploid females

In a few instances, sequencing of multiple nuclear genes consistently indicated two haplotypes for males and three or even four haplotypes in Euura vaga females. Diploid males are known ( Naito & Suzuki 1991; Harper et al. 2016) and can be found in nature ( Cook et al. 2013). They are usually sterile, less viable, or both, and are produced (instead of females) when there is no heterozygosity at the complementary sex determination (CSD) locus ( Naito & Suzuki 1991; Harper et al. 2016). Although polyploidy is apparently rare among parthenogenetic haplodiploids ( van der Kooi et al. 2017), the largely parthenogenetic European E. vaga might be an additional example.

Host plants

Host plants are known for 88% of the West Palaearctic species of Euura , most of which feed on Salix L. (73%). Other utilized hosts are Betula L. (7% of the species), Polygonoideae Eaton ( Polygonaceae ) (3%), Poales E.Small (3%), Ribes L. (3%), Faboideae Rudd ( Fabaceae ) (2%), Populus L. (2%), Picea A.Dietr. (2%), Larix Mill. (1%), and Vaccinium L. (1%). Alnus Mill. , Aruncus L., Comarum L., Fagus L., Rhododendron L., and Spiraea L. are hosts for only one (0.4%) species each in the West Palaearctic.

To identify host plants of swept larvae, we amplified the ITS1-5.8S-ITS2 region with plant-specific primers. Two Nanopore runs of 96 specimens containing mostly larvae (half of thorax cut from left or right side) and one run of 96 specimens containing mostly adults (legs) were done.

The larval dataset contained about 49 000 demultiplexed and quality-filtered plant ITS reads per Gb of data (56 000 reads out of 1.8 Gb and 165 000 out of 2.7 Gb for each of the two nanopore runs). The adult dataset contained about 17 000 ITS reads out of 0.8 Gb, which would be about 21 000 reads per Gb. If only specimens with at least 100 plant ITS reads are considered, 120 specimens out of 192 were successfully sequenced in the two runs comprising mainly larvae. Three cases in the larval dataset were clear contaminations (e.g., a mixture of Urtica L. and Cucumis L. for an Euura vittata larva) or a likely contamination ( Salix?phylicifolia L. for an adult Euura hartigi , although this host is possible). In the mostly adult dataset, 32 specimens could be considered successfully sequenced (at least 100 plant ITS reads), but most of these (30) can be considered contaminations (mostly Salix and Betula , but only one third of these matched the expected larval host genus of the species). Two larvae included in the mostly adult dataset yielded sequences of the expected host ( Betula for Euura cadderensis and Scolioneura betuleti larvae).

Although contaminations in the lab or field (e.g., pollen) are clearly likely, we consider the first host plant evidence for Euura leptostigma and E. sordidiapex based on these results to be sufficient. For three E. leptostigma larvae identical Salix consensus sequences were recovered with high coverage (about 120, 4500 and 8000×) and without indication of other variants. For two E. sordidiapex larvae, three different Betula haplotypes were recovered with high coverage (900 and 1700×) and without indication of other plant species. One of the sequenced E. leptostigma larvae was collected from a grey Salix ( S. glauca L. or S. lapponum L.), although in the rearing lab other species of Salix may have been offered. The other two specimens of E. leptostigma were collected from a mixture of Salix . Additionally, three adults of E. leptostigma have been reared from Salix phylicifolia and unidentified Salix . The two sequenced E. sordidiapex larvae were collected from Betula nana L. and B. pubescens Ehrh.

Figures

Fig. 1. Maximum likelihood tree of Tenthredinidae Latreille, 1803 based on combined COI and 9 nuclear genes. Number of base pairs available for each specimen in the alignment is shown at the end of each label.

Fig. 2. Maximum likelihood tree of Nematinae Thomson, 1871 based on combined COI and 9 nuclear genes. Number of base pairs available for each specimen in the alignment is shown at the end of each label.

Fig. 3A. Part of maximum likelihood tree of Euura Newman, 1837 based on 9 nuclear genes (Euura punicea group s. lat.). Number of base pairs available for each specimen in the alignment is shown at the end of each label.

Fig. 3B. Part of maximum likelihood tree of Euura Newman, 1837 based on 9 nuclear genes. Number of base pairs available for each specimen in the alignment is shown at the end of each label.

Fig. 3C. Part of maximum likelihood tree of Euura Newman, 1837 based on 9 nuclear genes. Number of base pairs available for each specimen in the alignment is shown at the end of each label.

Fig. 3D. Part of maximum likelihood tree of Euura Newman, 1837 based on 9 nuclear genes. Number of base pairs available for each specimen in the alignment is shown at the end of each label.