Abstract
The larval stages of the central European sibling caddisfly species Sericostoma personatum (Spence in Kirby and Spence, 1826) and S. flavicorne Schneider, 1845 are morphologically similar and can only be distinguished by differences in coloration in late larval instars. Identification using the mitochondrial barcoding gene, i.e., the Cytochrome c Oxidase 1, is impossible, as both species share the same highly differentiated haplotypes due to introgression. Nuclear gene markers obtained through double digest restriction site associate sequencing (ddRAD seq), however, can reliably distinguish both species, yet the method is expensive as well as time-consuming and therefore not practicable for species determination. To facilitate accurate species identification without sequencing genome-wide markers, we developed nine diagnostic nuclear RFLP markers based on ddRAD seq data. The markers were successfully tested on geographically distinct populations of the two Sericostoma species in western Germany, on known hybrids, and on another sericostomatid caddisfly species, Oecismus monedula (Hagen, 1859) that sometimes shares the habitat and can be morphologically confounded with Sericostoma. We describe a simple and fast protocol for reliable species identification of S. personatum and S. flavicorne independent of the life cycle stage of the specimens.
Highlights
Macroinvertebrate species are important indicators for the ecological status and water quality of freshwater ecosystems
We developed and tested nine restriction fragment length polymorphism (RFLP) markers to distinguish the two sibling caddisfly species S. flavicorne and S. personatum in Germany, which cannot be identified using COI barcoding
An advantage of the RFLP approach is that no sequencing is required and species assignment is possible directly after polymerase chain reaction (PCR) and a short restriction incubation, followed by simple agarose gel visualization
Summary
Macroinvertebrate species are important indicators for the ecological status and water quality of freshwater ecosystems. Reliable morphological species identification can be difficult or impossible, especially for closely-related species or early larval stages, because diagnostic characters are lacking or not yet visible. To deal with this problem, DNA-based methods have been developed for species identification, i.e., DNA barcoding (Hebert et al 2003). Species identification is based on comparing sequences of standardized marker genes, in the case of macroinvertebrates typically the mitochondrial Cytochrome c Oxidase 1 gene (CO1), with a reference database. Weigand et al 2017), or selection on maternally inherited traits (Toews et al 2014; Morales et al 2015) has led to mito-nuclear discordance patterns Using only mitochondrial genes can lead to wrong species delimitation when speciation has occurred relatively recently and rapidly (e.g. Schlick-Steiner et al 2010; Weiss et al 2018) or when incomplete lineage sorting (Toews and Brelsford 2012), hybridization and introgression (e.g. Weigand et al 2017), or selection on maternally inherited traits (Toews et al 2014; Morales et al 2015) has led to mito-nuclear discordance patterns
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