A targeted next-generation sequencing toolkit for exon-based cichlid phylogenomics

Abstract

Cichlid fishes (family Cichlidae) are models for evolutionary and ecological research. Massively parallel sequencing approaches have been successfully applied to study relatively recent diversification in groups of African and Neotropical cichlids, but such technologies have yet to be used for addressing larger-scale phylogenetic questions of cichlid evolution. Here, we describe a process for identifying putative single-copy exons from five African cichlid genomes and sequence the targeted exons for a range of divergent (>tens of millions of years) taxa with probes designed from a single reference species (Oreochromis niloticus, Nile tilapia). Targeted sequencing of 923 exons across 10 cichlid species that represent the family's major lineages and geographic distribution resulted in a complete taxon matrix of 564 exons (649,549 bp), representing 559 genes. Maximum likelihood and Bayesian analyses in both species tree and concatenation frameworks yielded the same fully resolved and highly supported topology, which matched the expected backbone phylogeny of the major cichlid lineages. This work adds to the body of evidence that it is possible to use a relatively divergent reference genome for exon target design and successful capture across a broad phylogenetic range of species. Furthermore, our results show that the use of a third-party laboratory coupled with accessible bioinformatics tools makes such phylogenomics projects feasible for research groups that lack direct access to genomic facilities. We expect that these resources will be used in further cichlid evolution studies and hope the protocols and identified targets will also be useful for phylogenetic studies of a wider range of organisms.

Keywords: Cichlidae; Cichlinae; Neotropical cichlids; exons; phylogenomics; targeted next-generation sequencing.