The origin of novel phenotypic characters is a key component in organismal diversification; yet, the mechanisms underlying the emergence of such evolutionary novelties are largely unknown. Here we examine the origin of egg-spots, an evolutionary innovation of the most species-rich group of cichlids, the haplochromines, where these conspicuous male fin colour markings are involved in mating. Applying a combination of RNAseq, comparative genomics and functional experiments, we identify two novel pigmentation genes, fhl2a and fhl2b, and show that especially the more rapidly evolving b-paralog is associated with egg-spot formation. We further find that egg-spot bearing haplochromines, but not other cichlids, feature a transposable element in the cis-regulatory region of fhl2b. Using transgenic zebrafish, we finally demonstrate that this region shows specific enhancer activities in iridophores, a type of pigment cells found in egg-spots, suggesting that a cis-regulatory change is causally linked to the gain of expression in egg-spot bearing haplochromines.

Summary1. The Tasty Chick Hypothesis (TCH) proposes that hatching asynchrony evolved as an antiparasite strategy. Hosts would benefit if ectoparasites aggregate more on the offspring that are of lowest reproductive value within a brood, i.e. on the last-hatched chicks, because offspring reproductive value generally decreases with hatching rank. The poor body condition of the later-hatched chicks would impair parasite resistance and render them especially attractive to ectoparasites. Thus, the TCH predicts a decline in chick parasite load with hatching order in avian broods. 2. We investigated the main assumption of the TCH that junior chicks are less immunocompetent than their senior siblings. We also examine the prediction of the TCH that junior chicks bear more ectoparasites than their senior siblings. 3. Conform to the assumption of the TCH for hosts, junior chicks in broods of the barn owl ( Tyto alba L.) showed a lower humoral immune response than their senior siblings. In contrast, the cell-mediated immune response of senior chicks in broods of the great tit ( Parus major L.) was not significantly greater than that of their junior siblings. 4. In line with the prediction of the TCH for the distribution of parasites among hosts, the fly Carnus haemapterus Nitzsch infested junior chicks in larger numbers than senior chicks in both barn owl and kestrel ( Falco tinnunculus L.) broods. 5. In conflict with the TCH, ticks ( Ixodes ricinus L.) were distributed randomly with respect to hatching rank in broods of the barn owl and the great tit. Moreover, louseflies Crataerina melbae Rondani infested mainly senior chicks instead of junior chicks in the Alpine swift ( Apus melba L.). 6. Summarizing, the present descriptive study indicates that the distribution of ectoparasites within-broods is not generally governed by rank-related variation in host defence of chicks as initially suggested by the TCH. We argue that specific aspects of the morphology, life history and ecological requirements of various ectoparasite species need more consideration as to explain the dynamics and evolution of host-parasite interactions.

The evolution of convergent phenotypes is one of the most interesting outcomes of replicate adaptive radiations. Remarkable cases of convergence involve the thick-lipped phenotype found across cichlid species flocks in the East African Great Lakes. Unlike most other convergent forms in cichlids, which are restricted to East Africa, the thick-lipped phenotype also occurs elsewhere, for example in the Central American Midas Cichlid assemblage. Here, we use an ecological genomic approach to study the function, the evolution and the genetic basis of this phenotype in two independent cichlid adaptive radiations on two continents. We applied phylogenetic, demographic, geometric morphometric and stomach content analyses to an African (Lobochilotes labiatus) and a Central American (Amphilophus labiatus) thick-lipped species. We found that similar morphological adaptations occur in both thick-lipped species and that the 'fleshy' lips are associated with hard-shelled prey in the form of molluscs and invertebrates. We then used comparative Illumina RNA sequencing of thick vs. normal lip tissue in East African cichlids and identified a set of 141 candidate genes that appear to be involved in the morphogenesis of this trait. A more detailed analysis of six of these genes led to three strong candidates: Actb, Cldn7 and Copb. The function of these genes can be linked to the loose connective tissue constituting the fleshy lips. Similar trends in gene expression between African and Central American thick-lipped species appear to indicate that an overlapping set of genes was independently recruited to build this particular phenotype in both lineages.

Sex determination mechanisms are highly variable across teleost fishes and sexual development is often plastic. Nevertheless, downstream factors establishing the two sexes are presumably conserved. Here, we study sequence evolution and gene expression of core genes of sexual development in a prime model system in evolutionary biology, the East African cichlid fishes. Using the available five cichlid genomes, we test for signs of positive selection in 28 genes including duplicates from the teleost whole-genome duplication, and examine the expression of these candidate genes in three cichlid species. We then focus on a particularly striking case, the A- and B-copies of the aromatase cyp19a1, and detect different evolutionary trajectories: cyp19a1A evolved under strong positive selection, whereas cyp19a1B remained conserved at the protein level, yet is subject to regulatory changes at its transcription start sites. Importantly, we find shifts in gene expression in both copies. Cyp19a1 is considered the most conserved ovary-factor in vertebrates, and in all teleosts investigated so far, cyp19a1A and cyp19a1B are expressed in ovaries and the brain, respectively. This is not the case in cichlids, where we find new expression patterns in two derived lineages: the A-copy gained a novel testis-function in the Ectodine lineage, whereas the B-copy is overexpressed in the testis of the speciest-richest cichlid group, the Haplochromini. This suggests that even key factors of sexual development, including the sex steroid pathway, are not conserved in fish, supporting the idea that flexibility in sexual determination and differentiation may be a driving force of speciation.

Changes in gene expression play a fundamental role in phenotypic evolution. Transcriptome evolutionary dynamics have so far mainly been compared among distantly related species and remain largely unexplored during rapid organismal diversification, in which gene regulatory changes have been suggested as particularly effective drivers of phenotypic divergence. Here, we studied gene expression evolution in a model system of adaptive radiation, the cichlid fishes of African Lake Tanganyika. By comparing gene expression profiles of six different organs in 74 cichlid species representing all subclades of this radiation, we demonstrate that the rate of gene expression evolution varies among organs, transcriptome parts, and the subclades of the radiation, indicating different strengths of selection. We found that the non-coding part of the transcriptome evolved more rapidly than the coding part, and that the gonadal transcriptomes evolved more rapidly than the somatic ones, with the exception of liver. We further show that the rate of gene-expression change was not constant over the course of the radiation but accelerated at its later phase. Finally, we show that – at the per-gene level – the evolution of expression patterns is dominated by stabilizing selection.