Exon-based Phylogenomics and the Relationships of African Cichlid Fishes: Tackling the Challenges of Reconstructing Phylogenies with Repeated Rapid Radiations

Abstract

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 the 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 the 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. [African cichlids; Blenniiformes; Gene tree heterogeneity; Hybrid assembly; Phylogenetic network; Pseudocrenilabrinae; Species tree.].

Figure 1.

(Color online) Cichlidae and Pseudocrenilabrinae relationships prior to this work (a) and distribution of Pseudocrenilabrinae tribes (b–e). a) Pseudocrenilabrinae relationships are based on combined evidence from existing molecular phylogenies. Bifurcating branches indicate robust relationships. Polytomies show conflicting or poorly supported nodes. Dashed (red) branches are tribes that were not sampled in this study. Square brackets to the right of the phylogeny denote commonly recovered tribal assemblages or groupings discussed throughout the text: WCA, West/Central African Lineages; EAR, East African Radiations Clade; MVhL, Malawi, Victoria, H-Lineage and Lamprologini Clade; H-Lineage, Haplochromini and remaining Tanganyikan tribes; C-Lineage, H-Lineage minus Eretmodini. Tribes with Tanganyikan endemics. The Malawi and Victoria system radiations are nested within Haplochromini. *Tribes with disputed taxonomic status. Rectangles next to tribe names indicate tribes with riverine and/or lacustrine lineages. Numbering of tribes matches numbers in the distribution maps (b–e). b) EAR tribes 15–28. c) Former-tilapiine tribes 10–14. d) Etiini and former-tilapiine tribes 7–9. e) WCA tribes 1–5. See Figure S1 in Appendix S1 of the Supplementary material available on Dryad for individual tribal distribution maps.

Figure 2.

(Color online) Proportion of parsimony informative sites (a), gene tree resolution (b,c), and phylogenies (d,e) for Pseudocrenilabrinae and close relatives. b) Bootstrap support at various gene tree depths. The first four (blue) boxes correspond to increasing distances from the root, while the last (yellow) box corresponds to the shallowest 20 of tree depth. c) Bootstrap support and 95 confidence interval (shaded area) for gene trees inferred from loci of various lengths. d) Concatenated RAxML tree showing branch lengths; see Figure S6 of the Supplementary material available on Dryad for full annotations. e) ASTRAL-II species tree. Numbers above branches are bootstrap and local posterior probability support. Pie charts show gene tree support for the main (dark gray), and first (light gray) and second (white) alternative quartet topologies (i.e., ASTRAL’s q1–q3 support values). Pies encircled with a thick (red) line indicate nodes that also had low gene concordance, below 50. Figure S8 of the Supplementary material available on Dryad contains full concordance factor annotations. Branches that differ between the species and concatenated phylogenies are lighter (red). Shaded (colored online) boxes with round edges and black names denote tribes, while nested boxes with square edges and white names indicate subclades discussed in the text. Square brackets to the right show tribal assemblages and noncichlid groupings. Gray and white vertical bars furthest to the right denote cichlid subfamilies and noncichlid Blenniiformes. Superscript numbers next to some species names indicate illustrated species to the right of the phylogeny. Illustrations by VAC.

Figure 3.

(Color online) Best phylogenetic networks estimated with SNaQ for eight taxonomic subsets. a) Sub1: Pseudocrenilabrinae tribes; b) Sub2: WCA lineages; c) Sub3: Chromidotilapiini; d) Sub4: former-tilapiine tribes; e) Sub5: Barombi Mbo, Bermin and Natron/Magadi flocks, and riverine relatives; f) Sub6: EAR, former-tilapiine and other Tanganyikan lineages; g) Sub7: EAR clade; h) Sub8: Lamprologini and close relatives. Tribes are differentiated by numbers in square brackets adjacent to species names and also by color in the online version of this figure. Vertical lines to the right of phylogenies denote multitribal groupings (a–d and f–h) or species flocks (e). Gray numbers around nodes show bootstrap support for species relationships. Diagonal black lines connecting lineages show hybrid networks. Black and italicized numbers indicate bootstrap support and inheritance probabilities (i.e., proportion of genes transferred during hybridization) of estimated networks.

Figure 4.

(Color online) Tree spaces comparing Pseudocrenilabrinae hypotheses across concatenated, species, hybrid network, and gene trees based on the tip–tip path difference metric. a) Comparison of full phylogeny across concatenated, species, and gene trees. b–i) Comparison of concatenated, species, hybrid network, and gene trees, restricted to taxa in hybrid network subsets. Tree spaces inferred with the Kendal–Colijn metric showed comparable similarity patterns across concatenated, species, and hybrid network trees and are thus included in Fig. S7 of Appendix S2 of the Supplementary material available on Dryad.