Hybrid enrichment of poorly preserved museum specimens refines homology hypotheses in a group of minute litter bugs (Hemiptera: Dipsocoromorpha: Schizopteridae)

Abstract

Although they are a valuable source of specimens, insect natural history collections continue to be under‐utilized in molecular systematics, mostly due to difficulties in obtaining DNA sequences. Old specimens or specimens stored under suboptimal conditions are intractable for traditional Sanger sequencing. In this study we use an inexpensive hybrid capture with in‐house generated baits to retrieve commonly utilized ribosomal and mitochondrial loci from old museum specimens and combine them with a Sanger‐generated dataset comprising recently collected material. We focus on the Corixidea genus group (Schizopteridae), which comprises rarely collected, small (1–2 mm) and primarily tropical insects of which only c. 10–20% of the species have been described. A molecular phylogeny is needed to resolve relationships and revise the genus‐level classification to correctly place the c. 150 yet to be described species. Applying this approach, we constructed a dataset, containing 101 taxa, 11 of which were preserved in low‐percentage ethanol, 48 are dry and point‐mounted, and 40 are > 20 years old at DNA extraction. The obtained data proved sufficient for reconstructing a well‐supported phylogeny with c. 50% of the predicted diversity, and for the oldest successfully sequenced specimen (95 years) to be unambiguously placed in that phylogeny. We confirmed monophyly of the Corixidea genus group, showed paraphyly of the genus Corixidea, and recovered nine well‐supported clades within the group. Ancestral character states of selected morphological features were inferred and used to re‐examine primary homology hypotheses and inform an upcoming taxonomic revision.

A phylogenomic tree inferred with an inexpensive PCR‐generated probe kit resolves higher‐level relationships among Neptis butterflies (Nymphalidae: Limenitidinae)

“…One of these reduced representation methods for isolating homologous loci is sequence capture, also known as targeted capture or hybrid enrichment. This method can isolate many orthologous loci, even from low quality or degraded DNA samples (Hawkins et al, 2016;Knyshov et al, 2019), and has been increasingly used in phylogenetic research of many different groups (Haddad et al, 2018;Courcelle et al, 2019;Liu et al, 2019;Van Dam et al, 2019;Zhang et al, 2019). This technique is particularly useful when working with rare or geographically isolated species represented only by dried specimens in museum collections that would be difficult to collect as fresh specimens.…”

Phylogenetic relationships and revised classification of the true bug infraorder Dipsocoromorpha (Insecta: Hemiptera: Heteroptera)

“…Although our recent studies using hybrid capture methods successfully incorporated both ribosomal and mitochondrial sequence data (Knyshov et al, 2019a(Knyshov et al, , 2019b, initial analyses showed that the mitochondrial sequence generated for this dataset contained substantial amounts of missing data and their inclusion in analyses did not positively impact support values. Thus, the final analyses and results presented here are based on ribosomal (Sanger + Illumina datasets) and morphological data.…”

“…We used the protocol of Knyshov et al (2019aKnyshov et al ( , 2019b) to obtain substantially larger molecular datasets for a subset of taxa. Using the primers listed in Knyshov et al (2019aKnyshov et al ( , 2019b, we selected eight specimens with good DNA quality (one dipsocorid, one ceratocombid, two "Ogeriinae" and two Schizopterinae) and used long-range PCR to amplify the nuclear ribosomal operon and the mitochondrion. PCR products were turned into Illumina libraries and sequenced on 5% of a MiSeq V3 lane at IIGB UCR.…”

“…PCR products were turned into Illumina libraries and sequenced on 5% of a MiSeq V3 lane at IIGB UCR. For hybrid capture following the pooled protocol of Knyshov et al (2019aKnyshov et al ( , 2019b, the amplicons also were turned into biotinylated bait to enrich DNA of 18 crucial taxa for which only extracts of suboptimal quality were available. Two pools with nine libraries each were enriched for the fragments of interest and subsequently sequenced on 13% of a MiSeq V3 lane at IIGB UCR.…”

“…We suspect these structures to produce glandular secretions of unknown function. These MSOs that in some species recognizably distort the habitus of a specimen are unknown in other Heteroptera but occur in a substantial number of schizopterid species (Hill, 1990(Hill, , 2004(Hill, , 2013Weirauch, 2012;Knyshov et al, 2016Knyshov et al, , 2019bFrankenberg et al, 2018). Finally, male genitalic features of Dipsocoromorpha demonstrate enormous structural diversity and variation in the degree and direction of asymmetry (Knyshov et al, 2018).…”