Detection of cross-contamination and strong mitonuclear discordance in two species groups of sawfly genus Empria (Hymenoptera, Tenthredinidae)

Abstract

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 suggesting different relationships. As previous studies were based on only few nuclear genes, the causes and the degree of mitonuclear discordance remain ambiguous. Here, we obtain genomic-scale ddRAD data together with Sanger sequencing of mitochondrial COI and two to three nuclear protein coding genes to investigate species limits and mitonuclear discordance in two closely related species groups within 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 previous and 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 E. immersa group are not recovered as monophyletic also based on nuclear data, but this could partly be because of unresolved taxonomy. Preliminary analyses of ddRAD data did not recover monophyly of E. japonica within 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 due to specimen identification tag jumps during sequencing library preparation of pooled specimens 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 cross-contamination rate is not unusually high for our ddRAD dataset as a whole (based on counting identical sequences between immersa and longicornis groups that 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 small number of recovered loci (~1000) compared to most other specimens (>10 000 in some cases) because of poor sequencing results. We caution 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 unique combination of limited number of tags (less than the number of specimens).

First microsatellite markers for the pine catkin sawflyXyela concava(Hymenoptera, Xyelidae) and their application in phylogeography and population genetics

“…One explanation could be the different effective population sizes ( N e ) of the mitochondrial and nuclear genome, which will cause differences in the diversity of the genetic structure of populations over time (incomplete lineage sorting; Harrison, 1989; Funk & Omland, 2003). However, this non-congruent pattern also might have been caused by biased mitochondrial introgression as often found in “Symphyta” as recently discussed by Prous, Lee & Mutanen (2019, preprint). The authors assume that mitochondrial introgression in sawflies might be promoted by a combination of the haplodiploid reproduction system of Hymenoptera (Aron et al., 2005) and the low mitochondrial mutation rates in sawflies (Tang et al., 2019).…”

On Bulgarian sawflies, including a new species of Empria (Hymenoptera, Symphyta)

“…The newly obtained DNA sequences were sequenced as described previously (Prous 2012;Prous et al 2017Prous et al , 2019. Additional primers used for sequencing that are not mentioned in Prous (2012) and Prous et al (2017Prous et al ( , 2019 were as follows:…”

“…Penis valves are not distinguishable from other E. tridens and genetic data (based on one male DEI-GISHym88776, Fig. 16) does not indicate the existence of an additional species either (based on three genes, closest specimens are always other specimens of E. tridens that were studied by Prous et al 2019). The females of Endelomyia filipendulae are morphologically not easily separable from those of E. aethiops (Gmelin, 1790) using the ovipositor characters illustrated by Lacourt (1998).…”

Taxonomic review of the genus Empria Lepeletier & Serville (Hymenoptera, Tenthredinidae) in South Korea: morphological and molecular identification of two new species

“…We complemented this default option with an SH-like approximate likelihood ratio (SH-aLRT) test (Guindon et al 2010) and ultrafast bootstrap (Hoang et al 2018) with 1000 replicates to estimate robustness of reconstructed splits. Analyzed sequences are from Prous and Heidemaa (2012), Liston et al (2019), Prous et al (2019), and this study.…”