Gene expression remodelling and immune response during adaptive divergence in an African cichlid fish.

Variation in gene expression contributes to ecological speciation by facilitating population persistence in novel environments. Likewise, immune responses can be of relevance in speciation driven by adaptation to different environments. Previous studies examining gene expression differences between recently diverged ecotypes have often relied on only one pair of populations, targeted the expression of only a subset of genes or used wild-caught individuals. Here, we investigated the contribution of habitat-specific parasites and symbionts and the underlying immunological abilities of ecotype hosts to adaptive divergence in lake-river population pairs of the cichlid fish Astatotilapia burtoni. To shed light on the role of phenotypic plasticity in adaptive divergence, we compared parasite and microbiota communities, immune response, and gene expression patterns of fish from natural habitats and a lake-like pond set-up. In all investigated population pairs, lake fish were more heavily parasitized than river fish, in terms of both parasite taxon composition and infection abundance. The innate immune response in the wild was higher in lake than in river populations and was elevated in a river population exposed to lake parasites in the pond set-up. Environmental differences between lake and river habitat and their distinct parasite communities have shaped differential gene expression, involving genes functioning in osmoregulation and immune response. Most changes in gene expression between lake and river samples in the wild and in the pond set-up were based on a plastic response. Finally, gene expression and bacterial communities of wild-caught individuals and individuals acclimatized to lake-like pond conditions showed shifts underlying adaptive phenotypic plasticity.

Demography and genome divergence of lake and stream populations of an East African cichlid fish.

Disentangling the processes and mechanisms underlying adaptive diversification is facilitated by the comparative study of replicate population pairs that have diverged along a similar environmental gradient. Such a setting is realized in a cichlid fish from southern Lake Tanganyika, Astatotilapia burtoni, which occurs within the lake proper as well as in various affluent rivers. Previously, we demonstrated that independent lake and stream populations show similar adaptations to the two habitat regimes. However, little is known about the evolutionary and demographic history of the A. burtoni populations in question and the patterns of genome divergence among them. Here, we apply restriction site-associated DNA sequencing (RADseq) to examine the evolutionary history, the population structure and genomic differentiation of lake and stream populations in A. burtoni. A phylogenetic reconstruction based on genome-wide molecular data largely resolved the evolutionary relationships among populations, allowing us to re-evaluate the independence of replicate lake-stream population clusters. Further, we detected a strong pattern of isolation by distance, with baseline genomic divergence increasing with geographic distance and decreasing with the level of gene flow between lake and stream populations. Genome divergence patterns were heterogeneous and inconsistent among lake-stream population clusters, which is explained by differences in divergence times, levels of gene flow and local selection regimes. In line with the latter, we only detected consistent outlier loci when the most divergent lake-stream population pair was excluded. Several of the thus identified candidate genes have inferred functions in immune and neuronal systems and show differences in gene expression between lake and stream populations.

A complex mode of aggressive mimicry in a scale-eating cichlid fish.

Aggressive mimicry is an adaptive tactic of parasitic or predatory species that closely resemble inoffensive models in order to increase fitness via predatory gains. Although similarity of distantly related species is often intuitively implicated with mimicry, the exact mechanisms and evolutionary causes remain elusive in many cases. Here, we report a complex aggressive mimicry strategy in Plecodus straeleni, a scale-eating cichlid fish from Lake Tanganyika, which imitates two other cichlid species. Employing targeted sequencing on ingested scales, we show that P. straeleni does not preferentially parasitize its models but­contrary to prevailing assumptions­targets a variety of co-occurring dissimilar looking fish species. Combined with tests for visual resemblance and visual modelling from a prey perspective, our results suggest that complex interactions among different cichlid species are involved in this mimicry system.

Adaptive divergence between lake and stream populations of an East African cichlid fish.

Divergent natural selection acting in different habitats may build up barriers to gene flow and initiate speciation. This speciation continuum can range from weak or no divergence to strong genetic differentiation between populations. Here, we focus on the early phases of adaptive divergence in the East African cichlid fish Astatotilapia burtoni, which occurs in both Lake Tanganyika (LT) and inflowing rivers. We first assessed the population structure and morphological differences in A. burtoni from southern LT. We then focused on four lake-stream systems and quantified body shape, ecologically relevant traits (gill raker and lower pharyngeal jaw) as well as stomach contents. Our study revealed the presence of several divergent lake-stream populations that rest at different stages of the speciation continuum, but show the same morphological and ecological trajectories along the lake-stream gradient. Lake fish have higher bodies, a more superior mouth position, longer gill rakers and more slender pharyngeal jaws, and they show a plant/algae and zooplankton-biased diet, whereas stream fish feed more on snails, insects and plant seeds. A test for reproductive isolation between closely related lake and stream populations did not detect population-assortative mating. Analyses of F1 offspring reared under common garden conditions indicate that the detected differences in body shape and gill raker length do not constitute pure plastic responses to different environmental conditions, but also have a genetic basis. Taken together, the A. burtoni lake-stream system constitutes a new model to study the factors that enhance and constrain progress towards speciation in cichlid fishes.

Introgressive hybridization between color morphs in a population of cichlid fishes twelve years after human-induced secondary admixis.

In the extremely species-rich haplochromine cichlid fishes of the East African Great Lakes, prezygotic isolation between closely related species is often maintained by color-assortative mating. In 1998, local fisherman working for the ornamental fish trade released different color morphs of the cichlid genus Tropheus into a small harbor basin in the southern part of Lake Tanganyika. This artificial amalgamation of color morphs provides a unique possibility to study mating patterns in cichlids in a natural environment over time. In a precursor study, we analyzed genotypes and phenotypes of almost 500 individuals sampled between 1999 and 2001 and uncovered a marked degree of color-assortative mating, which depended on the level of color pattern dissimilarity between morphs. Twelve years after introduction of nonindigenous morphs, we again sampled Tropheus individuals from the harbor basin and an adjacent, originally pure population and analyzed phenotypes (coloration) and genotypes (mitochondrial control region and 9 microsatellite loci) to assess the current status of the admixed population. Principal component analyses of color score data and population assignment tests demonstrate an increasing level of introgressive hybridization between morphs but also some ongoing color-assortative mating within morphs. The observed mating pattern might have been influenced by fluctuating environmental conditions such as periodic algal blooms or increased sedimentation causing turbid conditions in an otherwise clear lake.

Speciation dynamics and extent of parallel evolution along a lake-stream environmental contrast in African cichlid fishes.

Understanding the dynamics of speciation is a central topic in evolutionary biology. Here, we investigated how morphological and genomic differentiation accumulated along the speciation continuum in the African cichlid fish Astatotilapia burtoni. While morphological differentiation was continuously distributed across different lake-stream population pairs, we found that there were two categories with respect to genomic differentiation, suggesting a "gray zone" of speciation at ~0.1% net nucleotide divergence. Genomic differentiation was increased in the presence of divergent selection and drift compared to drift alone. The quantification of phenotypic and genetic parallelism in four cichlid species occurring along a lake-stream environmental contrast revealed parallel and antiparallel components in rapid adaptive divergence, and morphological convergence in species replicates inhabiting the same environments. Furthermore, we show that the extent of parallelism was higher when ancestral populations were more similar. Our study highlights the complementary roles of divergent selection and drift on speciation and parallel evolution.

Rapid radiation, ancient incomplete lineage sorting and ancient hybridization in the endemic Lake Tanganyika cichlid tribe Tropheini.

The evolutionary history of the endemic Lake Tanganyika cichlid tribe Tropheini, the sister group of the species flocks of Lake Malawi and the Lake Victoria region, was reconstructed from 2009 bp DNA sequence of two mitochondrial genes (ND2 and control region) and from 1293 AFLP markers. A period of rapid cladogenesis at the onset of the diversification of the Tropheini produced a multitude of specialized, predominantly rock-dwelling aufwuchs-feeders that now dominate in Lake Tanganyika's shallow habitat. Nested within the stenotopic rock-dwellers is a monophyletic group of species, which also utilize more sediment-rich habitat. Most of the extant species date back to at least 0.7 million years ago. Several instances of disagreement between AFLP and mtDNA tree topology are attributed to ancient incomplete lineage sorting, introgression and hybridization. A large degree of correspondence between AFLP clustering and trophic types indicated fewer cases of parallel evolution of trophic ecomorphology than previously inferred from mitochondrial data.

Where Am I? Niche constraints due to morphological specialization in two Tanganyikan cichlid fish species.

Food resource specialization within novel environments is considered a common axis of diversification in adaptive radiations. Feeding specializations are often coupled with striking morphological adaptations and exemplify the relation between morphology and diet (phenotype-environment correlations), as seen in, for example, Darwin finches, Hawaiian spiders, and the cichlid fish radiations in East African lakes. The cichlids' potential to rapidly exploit and occupy a variety of different habitats has previously been attributed to the variability and adaptability of their trophic structures including the pharyngeal jaw apparatus. Here we report a reciprocal transplant experiment designed to explore the adaptability of the trophic structures in highly specialized cichlid fish species. More specifically, we forced two common but ecologically distinct cichlid species from Lake Tanganyika, Tropheus moorii (rock-dweller), and Xenotilapia boulengeri (sand-dweller), to live on their preferred as well as on an unpreferred habitat (sand and rock, respectively). We measured their overall performance on the different habitat types and explored whether adaptive phenotypic plasticity is involved in adaptation. We found that, while habitat had no effect on the performance of X. boulengeri, T. moorii performed significantly better in its preferred habitat. Despite an experimental duration of several months, we did not find a shift in the morphology of the lower pharyngeal jaw bone that would be indicative of adaptive phenotypic plasticity in this trait.

Stable species boundaries despite ten million years of hybridization in tropical eels.

Genomic evidence is increasingly underpinning that hybridization between taxa is commonplace, challenging our views on the mechanisms that maintain their boundaries. Here, we focus on seven catadromous eel species (genus Anguilla) and use genome-wide sequence data from more than 450 individuals sampled across the tropical Indo-Pacific, morphological information, and three newly assembled draft genomes to compare contemporary patterns of hybridization with signatures of past introgression across a time-calibrated phylogeny. We show that the seven species have remained distinct for up to 10 million years and find that the current frequencies of hybridization across species pairs contrast with genomic signatures of past introgression. Based on near-complete asymmetry in the directionality of hybridization and decreasing frequencies of later-generation hybrids, we suggest cytonuclear incompatibilities, hybrid breakdown, and purifying selection as mechanisms that can support species cohesion even when hybridization has been pervasive throughout the evolutionary history of clades.

Repeated Evolution Versus Common Ancestry: Sex Chromosome Evolution in the Haplochromine Cichlid Pseudocrenilabrus philander.

Why sex chromosomes turn over and remain undifferentiated in some taxa, whereas they degenerate in others, is still an area of ongoing research. The recurrent occurrence of homologous and homomorphic sex chromosomes in distantly related taxa suggests their independent evolution or continued recombination since their first emergence. Fishes display a great diversity of sex-determining systems. Here, we focus on sex chromosome evolution in haplochromines, the most species-rich lineage of cichlid fishes. We investigate sex-specific signatures in the Pseudocrenilabrus philander species complex, which belongs to a haplochromine genus found in many river systems and ichthyogeographic regions in northern, eastern, central, and southern Africa. Using whole-genome sequencing and population genetic, phylogenetic, and read-coverage analyses, we show that one population of P. philander has an XX-XY sex-determining system on LG7 with a large region of suppressed recombination. However, in a second bottlenecked population, we did not find any sign of a sex chromosome. Interestingly, LG7 also carries an XX-XY system in the phylogenetically more derived Lake Malawi haplochromine cichlids. Although the genomic regions determining sex are the same in Lake Malawi cichlids and P. philander, we did not find evidence for shared ancestry, suggesting that LG7 evolved as sex chromosome at least twice in haplochromine cichlids. Hence, our work provides further evidence for the labile nature of sex determination in fishes and supports the hypothesis that the same genomic regions can repeatedly and rapidly be recruited as sex chromosomes in more distantly related lineages.

The puzzling phylogeography of the haplochromine cichlid fish Astatotilapia burtoni.

Astatotilapia burtoni is a member of the "modern haplochromines," the most species-rich lineage within the family of cichlid fishes. Although the species has been in use as research model in various fields of research since almost seven decades, including developmental biology, neurobiology, genetics and genomics, and behavioral biology, little is known about its spatial distribution and phylogeography. Here, we examine the population structure and phylogeographic history of A. burtoni throughout its entire distribution range in the Lake Tanganyika basin. In addition, we include several A. burtoni laboratory strains to trace back their origin from wild populations. To this end, we reconstruct phylogenetic relationships based on sequences of the mitochondrial DNA (mtDNA) control region (d-loop) as well as thousands of genomewide single nucleotide polymorphisms (SNPs) derived from restriction-associated DNA sequencing. Our analyses reveal high population structure and deep divergence among several lineages, however, with discordant nuclear and mtDNA phylogenetic inferences. Whereas the SNP-based phylogenetic hypothesis uncovers an unexpectedly deep split in A. burtoni, separating the populations in the southern part of the Lake Tanganyika basin from those in the northern part, analyses of the mtDNA control region suggest deep divergence between populations from the southwestern shoreline and populations from the northern and southeastern shorelines of Lake Tanganyika. This phylogeographic pattern and mitochondrial haplotype sharing between populations from the very North and the very South of Lake Tanganyika can only partly be explained by introgression linked to lake-level fluctuations leading to past contact zones between otherwise isolated populations and large-scale migration events.

Mouth dimorphism in scale-eating cichlid fish from Lake Tanganyika advances individual fitness.

Random asymmetry, that is the coexistence of left- and right-sided (or -handed) individuals within a population, is a particular case of natural variation; what triggers and maintains such dimorphisms remains unknown in most cases. Here, we report a field-based cage experiment in the scale-eating Tanganyikan cichlid Perissodus microlepis, which occurs in two morphs in nature: left-skewed and right-skewed individuals with respect to mouth orientation. Using underwater cages stocked with scale-eaters and natural prey fish, we first confirm that, under semi-natural conditions, left-skewed scale-eaters preferentially attack the right flank of their prey, whereas right-skewed individuals feed predominantly from the left side. We then demonstrate that scale-eaters have a higher probability for successful attacks when kept in dimorphic experimental populations (left- and right-skewed morphs together) as compared to monomorphic populations (left- or right-skewed morphs), most likely because prey fishes fail to accustom to strikes from both sides. The significantly increased probability for attacks appears to be the selective agent responsible for the evolution and maintenance of mouth dimorphism in P. microlepis, lending further support to the hypothesis that negative frequency-dependent selection is the stabilizing force balancing the mouth dimorphism at quasi-equal ratios in scale-eating cichlids.

Adaptive phenotypic plasticity contributes to divergence between lake and river populations of an East African cichlid fish.

Adaptive phenotypic plasticity and fixed genotypic differences have long been considered opposing strategies in adaptation. More recently, these mechanisms have been proposed to act complementarily and under certain conditions jointly facilitate evolution, speciation, and even adaptive radiations. Here, we investigate the relative contributions of adaptive phenotypic plasticity vs. local adaptation to fitness, using an emerging model system to study early phases of adaptive divergence, the generalist cichlid fish species Astatotilapia burtoni. We tested direct fitness consequences of morphological divergence between lake and river populations in nature by performing two transplant experiments in Lake Tanganyika. In the first experiment, we used wild-caught juvenile lake and river individuals, while in the second experiment, we used F1 crosses between lake and river fish bred in a common garden setup. By tracking the survival and growth of translocated individuals in enclosures in the lake over several weeks, we revealed local adaptation evidenced by faster growth of the wild-caught resident population in the first experiment. On the other hand, we did not find difference in growth between different types of F1 crosses in the second experiment, suggesting a substantial contribution of adaptive phenotypic plasticity to increased immigrant fitness. Our findings highlight the value of formally comparing fitness of wild-caught and common garden-reared individuals and emphasize the necessity of considering adaptive phenotypic plasticity in the study of adaptive divergence.

Immigrant and extrinsic hybrid inviability contribute to reproductive isolation between lake and river cichlid ecotypes.

Understanding how reproductive barriers evolve and which barriers contribute to speciation requires the examination of organismal lineages that are still in the process of diversification and the study of the full range of reproductive barriers acting at different life stages. Lake and river ecotypes of the East African cichlid fish Astatotilapia burtoni show habitat-specific adaptations, despite different levels of genetic differentiation, and thus represent an ideal model to study the evolution of reproductive barriers. To evaluate the degree of reproductive isolation between genetically divergent lake and river populations, we performed a mesocosm mating experiment in a semi-natural setting at Lake Tanganyika. We assessed reproductive isolation in the presence of male-male competition by analyzing survival and growth rates of introduced adults and their reproductive success from genetic parentage of surviving offspring. The genetically divergent river population showed reduced fitness in terms of survival, growth rate, and mating success in a lake-like environment. Hybrid offspring between different populations showed intermediate survival consistent with extrinsic postzygotic reproductive barriers. Our results suggest that both prezygotic (immigrant inviability) and postzygotic reproductive barriers contribute to divergence, and highlight the value of assessing multiple reproductive barriers acting at different stages and in natural contexts to understand speciation mechanisms.

Nuclear and mitochondrial data reveal different evolutionary processes in the Lake Tanganyika cichlid genus Tropheus.

BACKGROUND: Cichlid fishes are notorious for their wealth of intra- and interspecific colour pattern diversity. In Lake Tanganyika, the endemic genus Tropheus represents the most impressive example for geographic variation in the pattern and hue of integument colouration, but the taxonomy of the over 100 mostly allopatric colour morphs remains to a large degree unresolved. Previous studies of mitochondrial DNA sequence data revealed polyphyly of the six nominally described species and complex phylogeographic patterns influenced by lake level fluctuations and population admixture, and suggested the parallel evolution of similar colour patterns in divergent evolutionary lineages. A gene tree of a rapidly radiating group may be subject to incomplete and stochastic lineage sorting, and to overcome this problem we used multi-locus, nuclear AFLP data in comparison with mtDNA sequences to study diversification, migration and introgression in Tropheus colour morphs in Lake Tanganyika. RESULTS: Significant incongruence between phylogenetic reconstructions from mitochondrial and AFLP data suggested incomplete sorting of mitochondrial haplotypes as well as frequent introgression between differentiated lineages. In contrast to the mitochondrial phylogeny, the AFLP phenogram was largely congruent with species classifications, colour pattern similarities, and in many cases also with the current geographic distribution of populations, and did not produce evidence of convergent colour pattern evolution. Homoplasy in the AFLP data was used to identify populations that were strongly affected by introgression. CONCLUSION: Different evolutionary processes were distinguished by the combination of mitochondrial and AFLP data. Mitochondrial phylogeographic patterns retained signals of large-scale migration events triggered by historical, major lake level fluctuations, whereas AFLP data indicated genetic cohesion among local groups of populations resulting from secondary contact of adjacent populations in the course of the more frequently occurring, minor lake level fluctuations. There was no support for the parallel evolution of similar colour patterns in the AFLP data. Genetic signatures of introgression and hybridisation detected in several populations suggest that lake level fluctuations drove the stunning diversification of Tropheus morphs not only through population fragmentation, but also by promoting hybridisation between differentiated morphs in secondary contact.

Reticulate phylogeny of gastropod-shell-breeding cichlids from Lake Tanganyika--the result of repeated introgressive hybridization.

BACKGROUND: The tribe Lamprologini is the major substrate breeding lineage of Lake Tanganyika's cichlid species flock. Among several different life history strategies found in lamprologines, the adaptation to live and breed in empty gastropod shells is probably the most peculiar. Although shell-breeding arose several times in the evolutionary history of the lamprologines, all obligatory and most facultative shell-breeders belong to the so called "ossified group", a monophyletic lineage within the lamprologine cichlids. Since their distinctive life style enables these species to live and breed in closest vicinity, we hypothesized that these cichlids might be particularly prone to accidental hybridization, and that introgression might have affected the evolutionary history of this cichlid lineage. RESULTS: Our analyses revealed discrepancies between phylogenetic hypotheses based on mitochondrial and nuclear (AFLP) data. While the nuclear phylogeny was congruent with morphological, behavioral and ecological characteristics, several species--usually highly specialized shell-breeders--were placed at contradicting positions in the mitochondrial phylogeny. The discordant phylogenies strongly suggest repeated incidents of introgressive hybridization between several distantly related shell-breeding species, which reticulated the phylogeny of this group of cichlids. Long interior branches and high bootstrap support for many interior nodes in the mitochondrial phylogeny argue against a major effect of ancient incomplete lineage sorting on the phylogenetic reconstruction. Moreover, we provide morphological and genetic (mtDNA and microsatellites) evidence for ongoing hybridization among distantly related shell-breeders. In these cases, the territorial males of the inferred paternal species are too large to enter the shells of their mate, such that they have to release their sperm over the entrance of the shell to fertilize the eggs. With sperm dispersal by water currents and wave action, trans-specific fertilization of clutches in neighboring shells seem inevitable, when post-zygotic isolation is incomplete. CONCLUSION: From the direct observation of hybrids we conclude that hybridization between distantly related gastropod-shell-breeding cichlids of Lake Tanganyika follows inevitably from their ecological specialization. Moreover, the observed incongruence between mtDNA and nuclear multilocus phylogeny suggests that repeated hybridization events among quite distantly related taxa affected the diversification of this group, and introduced reticulation into their phylogeny.

Variation of anal fin egg-spots along an environmental gradient in a haplochromine cichlid fish.

Male secondary sexual traits are targets of inter- and/or intrasexual selection, but can vary due to a correlation with life-history traits or as by-product of adaptation to distinct environments. Trade-offs contributing to this variation may comprise conspicuousness toward conspecifics versus inconspicuousness toward predators, or between allocating resources into coloration versus the immune system. Here, we examine variation in expression of a carotenoid-based visual signal, anal-fin egg-spots, along a replicate environmental gradient in the haplochromine cichlid fish Astatotilapia burtoni. We quantified egg-spot number, area, and coloration; applied visual models to estimate the trait's conspicuousness when perceived against the surrounding tissue under natural conditions; and used the lymphocyte ratio as a measure for immune activity. We find that (1) males possess larger and more conspicuous egg-spots than females, which is likely explained by their function in sexual selection; (2) riverine fish generally feature fewer but larger and/or more intensely colored egg-spots, which is probably to maintain signal efficiency in intraspecific interactions in long wavelength shifted riverine light conditions; and (3) egg-spot number and relative area correlate with immune defense, suggesting a trade-off in the allocation of carotenoids. Taken together, haplochromine egg-spots feature the potential to adapt to the respective underwater light environment, and are traded off with investment into the immune system.

Monogamy in the maternally mouthbrooding Lake Tanganyika cichlid fish Tropheus moorii.

Supported by evidence for assortative mating and polygynandry, sexual selection through mate choice was suggested as the main force driving the evolution of colour diversity of haplochromine cichlids in Lakes Malawi and Victoria. The phylogenetically closely related tribe Tropheini of Lake Tanganyika includes the genus Tropheus, which comprises over 100 colour variants currently classified into six morphologically similar, polyphyletic species. To assess the potential for sexual selection in this sexually monochromatic maternal mouthbrooder, we used microsatellite-based paternity inference to investigate the mating system of Tropheus moorii. In contrast to haplochromines in Lake Malawi, multiple paternity is rare or even absent in broods of T. moorii. Eighteen of the 19 analysed families were consistent with genetic monogamy, while either a mutation or more than one sire explained the genotype of one offspring in another brood. We discuss the differences in breeding behaviour between T. moorii and the Lake Malawi haplochromines, and evaluate additional factors or alternatives to sexual selection as promoters of colour diversification. A preliminary survey of other Tropheini species suggested that multiple paternity is infrequent in the entire tribe.

Back to Tanganyika: a case of recent trans-species-flock dispersal in East African haplochromine cichlid fishes.

The species flocks of cichlid fishes in the East African Great Lakes are the largest vertebrate adaptive radiations in the world and illustrious textbook examples of convergent evolution between independent species assemblages. Although recent studies suggest some degrees of genetic exchange between riverine taxa and the lake faunas, not a single cichlid species is known from Lakes Tanganyika, Malawi and Victoria that is derived from the radiation associated with another of these lakes. Here, we report the discovery of a haplochromine cichlid species in Lake Tanganyika, which belongs genetically to the species flock of haplochromines of the Lake Victoria region. The new species colonized Lake Tanganyika only recently, suggesting that faunal exchange across watersheds and, hence, between isolated ichthyofaunas, is more common than previously thought.

The function of anal fin egg-spots in the cichlid fish Astatotilapia burtoni.

Color and pigmentation patterns of animals are often targets of sexual selection because of their role in communication. Although conspicuous male traits are typically implicated with intersexual selection, there are examples where sex-specific displays play a role in an intrasexual context, e.g. when they serve as signals for aggression level and/or status. Here, we focus on the function of a conspicuous male ornament in the most species-rich tribe of cichlid fishes, the haplochromines. A characteristic feature of these ca. 1500 species are so-called egg-spots in form of ovoid markings on the anal fins of males, which are made up of carotenoid based pigment cells. It has long been assumed that these yellow, orange or reddish egg-spots play an important role in the courtship and spawning behavior of these maternal mouth-brooding fishes by mimicking the eggs of a conspecific female. The exact function of egg-spots remains unknown, however, and there are several hypotheses about their mode of action. To uncover the function of this cichlid-specific male ornament, we used female mate choice experiments and a male aggression test in the haplochromine species Astatotilapia burtoni. We manipulated the number and arrangement of egg-spots on the anal fins of males, or removed them entirely, and tested (1) female preference with visual contact only using egg-traps, (2) female preference with free contact using paternity testing with microsatellites and (3) male aggression. We found that females did not prefer males with many egg-spots over males with fewer egg-spots and that females tended to prefer males without egg-spots over males with egg-spots. Importantly, males without egg-spots sired clutches with the same fertilization rate as males with egg-spots. In male aggression trials, however, males with fewer egg-spots received significantly more attacks, suggesting that egg-spots are an important signal in intrasexual communication.