African cichlids (subfamily: Pseudocrenilabrinae) are among the most diverse vertebrates, and their propensity for repeated rapid radiation has made them a celebrated model system in evolutionary research. Nonetheless, despite numerous studies, phylogenetic uncertainty persists, and riverine lineages remain comparatively underrepresented in higher-level phylogenetic studies. Heterogeneous gene histories resulting from incomplete lineage sorting (ILS) and hybridization are likely sources of uncertainty, especially during episodes of rapid speciation. We investigate relationships of Pseudocrenilabrinae and its close relatives while accounting for multiple sources of genetic discordance using species tree and hybrid network analyses with hundreds of single-copy exons. We improve sequence recovery for distant relatives, thereby extending the taxonomic reach of our probes, with a hybrid reference guided/de novo assembly approach. Our analyses provide robust hypotheses for most higher-level relationships and reveal widespread gene heterogeneity, including in riverine taxa. ILS and past hybridization are identified as sources of genetic discordance in different lineages. Sampling of various Blenniiformes (formerly Ovalentaria) adds strong phylogenomic support for convict blennies (Pholidichthyidae) as sister to Cichlidae, and points to other potentially useful protein-coding markers across the order. A reliable phylogeny with representatives from diverse environments will support ongoing taxonomic and comparative evolutionary research in the cichlid model system.

The dataset associated with this article consists of alignments for 588 single-copy exons for 178 taxa, most of which are African cichlids from the subfamily Pseudocrenilabrinae: 165 cichlids (Pseudocrenilabrinae: 150, Cichlinae: 3, Etroplinae: 5, Ptychochrominae: 7); 13 non-cichlid Blenniiformes). Raw reads were obtained through targeted exon capture with probes designed based on an Oreochromis niloticus genome. Target sequences were assembled using a combination of Bowtie2 guided, hybrid guided/de novo and reduced-sensitivity approaches. Target sequences were aligned in MUSCLE.