Molecular Characterization of Two Endothelin Pathways in East African Cichlid Fishes

Abstract

The adaptive radiations of cichlid fishes in East Africa have been associated with the acquisition of evolutionary novelties as well as the ecological opportunities existing in the East African Great lakes. Two remarkable evolutionary innovations are the pharyngeal jaw apparatus, found in all cichlid species, and the anal fin egg-spots of mouthbrooding cichlids. Based on their conserved functions during the development of both the jaw apparatus and pigmentation, the endothelin ligands and receptors form a putative link between these naturally and sexually selected traits. Here we study the evolutionary history of four members of two endothelin pathways (Edn1/EdnrAa and Edn3b/EdnrB1a) to elucidate their possible roles during the evolution and development of key innovations in East African cichlids species. The analyses performed on partial sequences (ca. 6,000 bp per taxon) show that all four endothelin family members evolved under purifying selection, although both ligands are characterized by an accelerated rate of protein evolution in comparison to the receptors. In accordance with earlier findings, we show that the mature protein sequence of Edn1 and Edn3 are highly conserved, also in cichlids, whereas the preproendothelin parts are variable indicating relaxed selective constraints. In the receptors, nonsynonymous substitutions were mainly found in the ligand-binding domains suggesting functional divergence. Gene expression assays with Real-Time PCR indeed reveal that the two studied endothelin pathways are expressed in the cichlid pharyngeal jaw and in the haplochromine egg-spot (among other pigment-cell containing tissues), suggesting their involvement during morphogenesis of naturally and sexually selected traits in cichlids.

Sequence analyses of the distal-less homeobox gene family in East African cichlid fishes reveal signatures of positive selection

“…In the developing pharyngeal teeth and the anal fin dlx3b, dlx4a (not in anal fin) and dlx5a , the exact loci for which we found strong patterns of purifying selection, are expressed at high levels. Although this observation seems to contradict other cases in which candidate genes showed accelerated rates of protein evolution (see [31,53,84]), they do not stand alone (see e.g., [36]). It has been shown that minor changes in the complex genetic pathways underlying the development of morphological structures can lead to the evolution of novelties (see e.g., [85]).…”

“…This was done for eight dlx loci: dlx1a , dlx2a , dlx3a , dlx3b , dlx4a , dlx4b , dlx5a and dlx6a (see Additional file 3 for primer sequences). Standard PCR reactions, purification steps and sequencing reactions were set up and performed as described elsewhere [36]. PCR products of the partially sequenced loci were visualized with GelRed (Biotium) on a 1.5% agarose gel and sequenced on a 3130 xl capillary sequencer (Applied Biosystems).…”

“…To test whether the dlx genes evolved under non-neutral evolution in specific lineages a LRT between the null model (ω s  = 1) and the alternative model (ω s  ≥ 1) was performed in the branch-site analyses. Branches of interest, or so-called foreground branches, were chosen based on the results of the phylogenetic analyses and branch tests performed in Hyphy ([69], following [36]).…”

“…Furthermore, species-specific jaw shapes of different cichlid species correlate with differences in early bmp4 expression patterns, a gene which has also the potential to change the mandibular morphology in zebrafish [40]. Many of these candidate genes belong to larger gene families such as the endothelin family of ligands and receptors that are putatively involved in the morphogenesis of the pharyngeal jaw apparatus and pigmentation [36], and the above mentioned Hox gene clusters [41]. …”

“…Teleost and cichlid-specific phylogenies were examined to compare the rate of evolution of both between and within dlx gene trees. Several studies have shown that loci putatively involved in evolutionary innovations are characterized by adaptive protein evolution in cichlids [31,36,53]. Therefore, all loci were screened for elevated rates of protein evolution by means of d N / d S analyses.…”