Determining phylogenetic relationships among recently diverged species has long been a challenge in evolutionary biology. Cytoplasmic markers, which have been widely used notably in the context of molecular barcoding, have not always proved successful in resolving such phylogenies, but phylogenies for closely related species have been resolved at a much higher detail in the last couple of years with the advent of next-generation-sequencing technologies and associated techniques of reduced genome representation. Here we examine the potential and limitations of one of such techniques – Restriction-site Associated DNA (RAD) sequencing, a method that produces thousands of (mostly) anonymous nuclear markers, in disentangling the phylogeny of the fly genus Chiastocheta (Diptera: Anthomyiidae). This genus encompasses seven described species of seed predators, which have been widely studied in the context of their ecological and evolutionary interactions with the plant Trollius europaeus (Ranunculaceae). So far, phylogenetic analyses using mitochondrial markers failed to resolve monophyly of most of the species from this recently diversified genus, suggesting that their taxonomy may need to be revised. However, relying on a single, non-recombining molecule and ignoring potential incongruences between mitochondrial and nuclear loci may provide incomplete account of a lineage history. In this study, we apply both classical Sanger sequencing of three mtDNA regions and RAD-sequencing, for reconstructing the phylogeny of the genus. Contrasting with results based on mitochondrial markers, RAD-sequencing analyses retrieved the monophyly of all seven species, in agreement with the morphological species assignment. We found robust nuclear-based species assignment of individual samples, and low levels of estimated contemporary gene flow among them. However, despite recovering species’ monophyly, interspecific relationships varied depending on the set of RAD loci considered, producing contradictory topologies. Moreover, coalescence-based phylogenetic analyses revealed low supports for most of the interspecific relationships. Our results indicate that despite the higher performance of RAD-sequencing in terms of species trees resolution compared to cytoplasmic markers, reconstructing inter-specific relationships may lie beyond the possibilities offered by large sets of RAD-sequencing markers in cases of strong gene tree incongruence.

Copyright © 2017. Published by Elsevier Inc.