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BackgroundCarotenoids contribute significantly to animal body coloration, including the spectacular color pattern diversity among fishes. Fish, as other animals, derive carotenoids from their diet. Following uptake, transport and metabolic conversion, carotenoids allocated to body coloration are deposited in the chromatophore cells of the integument. The genes involved in these processes are largely unknown. Using RNA-Sequencing, we tested for differential gene expression between carotenoid-colored and white skin regions of a cichlid fish,Tropheus duboisi“Maswa”, to identify genes associated with carotenoid-based integumentary coloration. To control for positional gene expression differences that were independent of the presence/absence of carotenoid coloration, we conducted the same analyses in a closely related population, in which both body regions are white.ResultsA larger number of genes (n = 50) showed higher expression in the yellow compared to the white skin tissue than vice versa (n = 9). Of particular interest was the elevated expression level ofbco2ain the white skin samples, as the enzyme encoded by this gene catalyzes the cleavage of carotenoids into colorless derivatives. The set of genes with higher expression levels in the yellow region included genes involved in xanthophore formation (e.g.,pax7andsox10), intracellular pigment mobilization (e.g.,tubb,vim,kif5b), as well as uptake (e.g.,scarb1) and storage (e.g.,plin6) of carotenoids, and metabolic conversion of lipids and retinoids (e.g.,dgat2, pnpla2, akr1b1,dhrs). Triglyceride concentrations were similar in the yellow and white skin regions. Extracts of integumentary carotenoids contained zeaxanthin, lutein and beta-cryptoxanthin as well as unidentified carotenoid structures.ConclusionOur results suggest a role of carotenoid cleavage by Bco2 in fish integumentary coloration, analogous to previous findings in birds. The elevated expression of genes in carotenoid-rich skin regions with functions in retinol and lipid metabolism supports hypotheses concerning analogies and shared mechanisms between these metabolic pathways. Overlaps in the sets of differentially expressed genes (includingdgat2,bscl2,faxdc2andretsatl) between the present study and previous, comparable studies in other fish species provide useful hints to potential carotenoid color candidate genes.

African cichlids are well known for their adaptive radiations, but it is now apparent that they also harbor an extraordinary diversity of sex chromosome systems. In this study, we sequenced pools of males and females from species in 3 different genera of cichlids from Lake Tanganyika. We then searched for regions that were differentiated following the patterns expected for sex chromosomes. We report 2 novel sex chromosomes systems, an XY system on LG19 in Tropheus sp. "black" and a ZW system on LG7 in Hemibates stenosoma. We also identify a ZW system on LG5 in Cyprichromis leptosoma that may be convergent with a system previously described in Lake Malawi cichlids. Our data also identify candidate single nucleotide polymorphisms for the blue/yellow tail color polymorphism observed among male C. leptosoma.

Carotenoid pigments play a major role in animal body colouration, generating strong interest in the genes involved in the metabolic processes that lead from their dietary uptake to their storage in the integument. Here, we used RNA sequencing (RNA-Seq) to test for differentially expressed genes in a taxonomically replicated design using three pairs of related cichlid fish taxa from the genera Tropheus and Aulonocara . Within each pair, taxa differed in terms of red and yellow body colouration, and high‐performance liquid chromatography (HPLC) analyses of skin extracts revealed different carotenoid profiles and concentrations across the studied taxa. Five genes were differentially expressed in all three yellow–red skin contrasts ( dhrsx , nlrc3 , tcaf2 , urah and ttc39b ), but only the tetratricopeptide repeat protein-coding gene ttc39b , whose gene product is linked to mammalian lipid metabolism, was consistently expressed more highly in the red skin samples. The RNA-Seq results were confirmed by quantitative PCR. We propose ttc39b as a compelling candidate gene for variation in animal carotenoid colouration. Since differential expression of ttc39b was correlated with the presence/absence of yellow carotenoids in a previous study, we suggest that ttc39b is more likely associated with the concentration of total carotenoids than with the metabolic formation of red carotenoids.

Selection arising from social competition over non-mating resources, i.e. resources that do not directly and immediately affect mating success, offers a powerful alternative to sexual selection to explain the evolution of conspicuous ornaments, particularly in females. Here, we address the hypothesis that competition associated with the territoriality exhibited by both males and females in the cichlid fish Tropheus selects for the display of a conspicuous colour pattern in both sexes. The investigated pattern consists of a vertical carotenoid-coloured bar on a black body. Bar width affected the probability of winning in size-matched female–female, but not male–male, contests for territory possession. Our results support the idea that the emergence of female territoriality contributed to the evolution of sexual monomorphism from a dimorphic ancestor, in that females acquired the same conspicuous coloration as males to communicate in contest competition.

Food quality determines the growth rate of primary consumers and ecosystem trophic efficiencies, but it is not clear whether variation in primary consumer densities control, or is controlled by, variation in food quality. We quantified variation in the density and condition of an abundant algae-eating cichlid, Tropheus brichardi, with respect to the quality and productivity of algal biofilms within and across rocky coastal sites in Lake Tanganyika, East Africa. Adjacent land use and sediment deposition in the littoral zone varied widely among sites. Tropheus brichardi maximized both caloric and phosphorus intake at the local scale by aggregating in shallow habitats: algivore density decreased with depth, tracking attached algae productivity (rETRMAX) remarkably well (r2 = 0.84, P = 0.00033). In contrast, algivore density was unrelated to among-site variation in algal productivity. Rather, there was significant increase in algal quality (r2 = 0.44, P = 0.011) and decrease in algal biomass (r2 = 0.53, P = 0.0068) with T. brichardi density across sites, consistent with strong top-down control of primary producers. The amount of inorganic sediment on rock surfaces was the strongest predictor of among-site variation in algivore density (r2 = 0.69, P = 0.00096), and algivore gut length increased with sedimentation (r2 = 0.36, P = 0.034). These patterns indicate extrinsic and top-down forcing of algal food quality and quantity across coastal landscapes, combined with adaptive habitat selection by fish at the local scale. Factors that degrade food quality by decreasing algal nutrient content or diluting the resource with indigestible material are likely to depress grazer densities, potentially dampening top-down control in high-light, low-nutrient aquatic ecosystems.

The establishment of hybrid taxa relies on reproductive isolation from the parental forms, typically achieved by ecological differentiation. Here, we present an alternative mechanism, in which shifts in the strength and location of dispersal barriers facilitate diversification by hybridisation. Our case study concerns the highly diverse, stenotopic rock-dwelling cichlids of the African Great Lakes, many of which display geographic colour pattern variation. The littoral habitat of these fish has repeatedly been restructured in the course of ancient lake level fluctuations. Genetic data and an experimental cross support the hybrid origin of a distinct yellow-coloured variant of Tropheus moorii from ancient admixture between two allopatric, red and bluish variants. Deficient assortative mating preferences imply that reproductive isolation continues to be contingent on geographic separation. Linking paleolimnological data with the establishment of the hybrid variant, we sketch a selectively neutral diversification process governed solely by rearrangements of dispersal barriers.

Whether premating isolation is achieved by male-specific, female-specific or sex-independent assortative preferences often depends on the underlying evolutionary processes. Here we test mate preferences of males presented with females of different allopatric colour variants of the cichlid fish Tropheus sp., a Lake Tanganyika endemic with rich geographical colour pattern variation, in which the strength of sexual isolation varies between populations. We conducted two-way mate choice experiments to compare behaviour of males of a red-bodied morph (population Moliro) towards females from their own population with behaviour towards females from four allopatric populations at different stages of phylogenetic and phenotypic divergence. Males courted same-population females significantly more intensely than females of other populations, and reduced their heteromorphic courtship efforts both with increasing genetic and increasing phenotypic distinctness of the females. In particular, females of a closely related red-bodied population received significantly more courtship than either genetically distinct, similarly coloured females ('Kirschfleck' morph) or genetically related, differently coloured females ('yellow-blotch' morph), both of which were courted similarly. Genetically and phenotypically distinct females (Tropheus polli) were not courted at all. Consistent with previous female-choice experiments, female courtship activity also decreased with increasing genetic distance from the males' population. Given successful experimental and natural introgression between colour morphs and the pervasive allopatry of related variants, we consider it unlikely that assortative preferences of both sexes were driven by direct selection during periods of secondary contact or, in turn, drove colour pattern differentiation in allopatry. Rather, we suggest that sexual isolation evolved as by-product of allopatric divergence.

Species richness and geographical phenotypic variation in East African lacustrine cichlids are often correlated with ecological specializations and limited dispersal. This study compares mitochondrial and microsatellite genetic diversity and structure among three sympatric rock-dwelling cichlids of Lake Tanganyika, Eretmodus cyanostictus, Tropheus moorii, and Ophthalmotilapia ventralis. The species represent three endemic, phylogenetically distinct tribes (Eretmodini, Tropheini, and Ectodini), and display divergent ecomorphological and behavioral specialization. Sample locations span both continuous, rocky shoreline and a potential dispersal barrier in the form of a muddy bay. High genetic diversity and population differentiation were detected in T. moorii and E. cyanostictus, whereas much lower variation and structure were found in O. ventralis. In particular, while a 7-km-wide muddy bay curtails dispersal in all three species to a similar extent, gene flow along mostly continuous habitat appeared to be controlled by distance in E. cyanostictus, further restricted by site philopatry and/or minor habitat discontinuities in T. moorii, and unrestrained in O. ventralis. In contrast to the general pattern of high gene flow along continuous shorelines in rock-dwelling cichlids of Lake Malawi, our study identifies differences in population structure among stenotopic Lake Tanganyika species. The amount of genetic differentiation among populations was not related to the degree of geographical variation of body color, especially since more phenotypic variation is observed in O. ventralis than in the genetically highly structured E. cyanostictus.

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.

The species flocks of cichlid fishes in the East African Lakes Tanganyika, Malawi and Victoria are prime examples of adaptive radiation and explosive speciation. Several hundreds of endemic species have evolved in each of the lakes over the past several thousands to a few millions years. Sexual selection via colour-assortative mating has often been proposed as a probable causal factor for initiating and maintaining reproductive isolation. Here, we report the consequences of human-mediated admixis among differentially coloured populations of the endemic cichlid fishTropheus mooriifrom several localities that have accidentally been put in sympatry in a small harbour bay in the very south of Lake Tanganyika. We analysed the phenotypes (coloration) and genotypes (mitochondrial control region and five microsatellite loci) of almost 500 individuals, sampled over 3 consecutive years. Maximum-likelihood-based parenthood analyses and Bayesian inference of population structure revealed that significantly more juveniles are the product of within-colour-morph matings than could be expected under the assumption of random mating. Our results clearly indicate a marked degree of assortative mating with respect to the different colour morphs. Therefore, we postulate that sexual selection based on social interactions and female mate choice has played an important role in the formation and maintenance of the different colour morphs inTropheus, and is probably common in other maternally mouthbrooding cichlids as well.

The spectacularly diverse cichlid fish species flocks of the East African Rift Lakes have elicited much debate on the potential evolutionary mechanisms responsible for the origin of these adaptive radiations. An historical perspective on population structure may offer insights into the processes driving population differentiation and possibly speciation. Here, we examine mitochondrial DNA (mtDNA) sequence variation in two endemic species of rock-dwelling cichlids, Simochromis babaulti and S. diagramma, from Lake Tanganyika. Phylogeographic analyses were used to infer what factors might have been important in the genetic structuring of Simochromis populations. Patterns of mtDNA differentiation in Simochromis were compared to those of other rock-dwelling cichlids to distinguish between competing hypotheses concerning the processes underlying their evolution. In striking contrast to previous findings, populations of Simochromis, even those separated by up to 300 km, were found to share mitochondrial DNA haplotypes. There is no correspondence between mtDNA genealogies and the geographical distribution of populations. Only S. babaulti, but not S. diagramma was found to have a significant association between genetic and geographical distance. These phylogeographic patterns suggest that the evolutionary effects of abiotic and biotic factors shaping population genetic structure may differ substantially even among closely related species of rock-dwelling cichlids. Physical events and barriers to gene flow that are believed to have had a major impact on the geographical distribution and intralacustrine speciation of Tropheus do not seem to have equally strongly affected its close relative Simochromis. These findings emphasize that no single mechanism can be responsible for the formation of population structure, speciation, and the adaptive radiation of all cichlid fishes.

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.

Since their discovery at the turn of the century, the species assemblages of cichlid fishes in the East African Lakes Victoria, Malawi and Tanganyika have fascinated evolutionary biologists. Many models have attempted to account for the 'explosive' evolution of several hundred species within these lakes. Here we report a case of surprisingly large genetic divergence among populations of the endemic Tropheus lineage of Lake Tanganyika. This lineage of six species contains twice as much genetic variation as the entire morphologically highly diverse cichlid assemblage of Lake Malawi and six times more variation than the Lake Victoria species flock. Although it is highly variable in coloration, this group of species and its closest relatives have not undergone appreciable morphological change. The observed geographic pattern of genetic variation suggests that major lake level fluctuations affected the distribution and speciation of this lineage of cichlid fishes.

Mating behaviour affects reproductive isolation and phenotypic differentiation. In Lake Tanganyika, the cichlid fish Tropheus moorii diversified into numerous, currently allopatric colour variants. Allopatric isolation is periodically interrupted by dispersal and secondary contact during lake level fluctuations, making long-term differentiation partly dependent on assortative mating. Laboratory experiments with two moderately distinct morphs revealed assortative female preferences in one (Nakaku), but random mate choice in the other morph (Mbita). No discrimination was apparent between two subtly differentiated morphs (Chimba and Moliro). Tested against each other in a previous study, the highly distinct Moliro and Nakaku exhibited strong assortative preferences. The correlation between colour pattern similarity and mate discrimination suggests that allopatry and philopatric behaviour are less crucial for the maintenance of differentiation between highly distinct morphs than for more similar morphs. Interestingly, the asymmetric isolation in one pair of morphs is congruent with a pattern of unidirectional mitochondrial introgression between populations.

Lake Victoria, together with its satellite lakes, harbours roughly 200 endemic forms of cichlid fishes that are classified as 'haplo-chromines' and yet the lake system is less than a million years old. This 'flock' has attracted attention because of the possibility that it evolved within the lake from one ancestral species and that biologists are thus presented with a case of explosive evolution. Within the past decade, however, morphology has increasingly emphasized the view that the flock may be polyphyletic. We sequenced up to 803 base pairs of mitochondrial DNA from 14 representative Victorian species and 23 additional African species. The flock seems to be monophyletic, and is more akin to that from Lake Malawi than to species from Lake Tanganyika; in addition, it contains less genetic variation than does the human species, and there is virtually no sharing of mitochondrial DNA types among species. These results confirm that the founding event was recent.

Water level fluctuations are important modulators of speciation processes in tropical lakes, in that they temporarily form or break down barriers to gene flow among adjacent populations and/or incipient species. Time estimates of the most recent major lowstands of the three African Great Lakes are thus crucial to infer the relative timescales of explosive speciation events in cichlid species flocks. Our approach combines geological evidence with genetic divergence data of cichlid fishes from the three Great East African Lakes derived from the fastest-evolving mtDNA segment. Thereby, we show for each of the three lakes that individuals sampled from several populations which are currently isolated by long geographic distances and/or deep water form clusters of equally closely related haplotypes. The distribution of identical or equally closely related haplotypes in a lake basin allows delineation of the extent of lake level fluctuations. Our data suggest that the same climatic phenomenon synchronized the onset of genetic divergence of lineages in all three species flocks, such that their most recent evolutionary history seems to be linked to the same external modulators of adaptive radiation. A calibration of the molecular clock of the control region was elaborated by gauging the age of the Lake Malawi species flock through the divergence among the utaka-cichlid and the mbuna-cichlid lineages to minimally 570,000 years and maximally 1 Myr. This suggests that the low-lake-level period which established the observed patterns of genetic relatedness dates back less than 57,000 years, probably even to 17,000-12,400 years ago, when Lake Victoria dried up and Lakes Malawi and Tanganyika were also low. A rapid rise of all three lakes about 11,000 years ago established the large-scale population subdivisions observed today. Over that period of time, a multitude of species originated in Lakes Malawi and Victoria with an impressive degree of morphological and ecological differentiation, whereas the Tanganyikan taxa that were exposed to the same habitat changes hardly diverged ecologically and morphologically. Our findings also show that patterns of genetic divergences of stenotopic organisms provide valuable feedback on geological and sedimentological time estimates for lake level changes.

Of the three cichlid species flocks in eastern Africa, Lake Tanganyika harbors the oldest species assemblage, which is also the most diverse morphologically and behaviorally. For 12 species (20 individuals) of 12 genera of the tribe Ectodini, 852 bp from two segments (cytochrome b and control region) of the mitochondrial genome were sequenced. In addition, orthologous sequences were obtained from eight species ( 11 individuals) representing other mouthbrooding lineages from Lake Tanganyika. Comparisons of sequence divergences revealed that the single Tanganyikan tribe Ectodini appears to be approximately five times older than the whole Lake Malawi cichlid species flock, suggesting that the radiation of the Tanganyikan mouthbrooding lineages took place long before the species flocks of Lakes Malawi and Victoria evolved. Seven of nine surveyed tribes of Tanganyikan cichlids appear to be approximately equally divergent, and this seems to corroborate the hypothesis of a rapid simultaneous formation of lineages at an early stage in the history of the Lake Tanganyika species flock. The close genetic relationship between the endemic Trupheus lineage and a nonendemic Haplochromine, Astatotilapia burtoni, indicates that members of the tribe Tropheini may be the sister group of the cichlid flocks of Lakes Malawi and Victoria. The phylogenetic analyses demonstrate the monophyly of the Ectodini and identify the Cyprichromini as their sister group among the Tanganyikan cichlids. Within the tribe Ectodini the molecular data suggest both a branching pattern different than that previously proposed and a subdivision of the Ectodini into four clades, instead of the two originally described. The previously postulated model of morphological transformations believed to be responsible for the drastically different types of ecological specialization found among the Ectodini might therefore be in need of reinterpretation. Characters immediately related to foraging and nutrition seem to be particularly prone to homoplasy, even among members of a single lineage of cichlid fishes.

Necrotic and granulomatous gastritis is described in Lake Tanganyika cichlids. Clostridium hastiforme and flagellated protozoa were both associated with the reaction but the significance of either is unknown. Nevertheless, treatment of surviving fish with ampicillin was carried out and mortalities ceased. The possible involvement of an unsuitable diet as a predisposing factor is discussed.

I. Sound production is studied in Halochromis murtoni, Simochromis diagramma, S. babatiltz, Tropheus moorii, T. brichardi and T. duboisi (Pisces, Cichlidae). The recorded sounds are analysed and visualized as sonograms. 2. As far as communication is concerned, Tropheus is specialized in sound production while Simochromis is specialized in colour patterns. 3. These ways of evolution from an Haplochromis-like ancestor could be correlated with the fact that Tropheus became night-active, and Simochromis day-active.

A phylogeny of the principal lineages of cichlid fishes and two other fish families of the suborder Labroidei was based on phylogenetic information from DNA sequences of the flanking region of a (CA)$_{\text{n}}$ microsatellite locus. Microsatellite (CA)$_{\text{n}}$ containing clones from a genomic library of an African cichlid fish from Lake Tanganyika, Tropheus moorii, were sequenced and primers for the polymerase chain reaction designed. All primers amplified the homologous microsatellite loci in many more than the source species and one microsatellite flanking locus (TmoM27) was particularly conserved and amplified in several lineages of perciform fishes that diverged more than 80-100 million years ago. Despite the extensive level of evolutionary conservation of this microsatellite flanking region (MFR), this nuclear region contained reliable phylogenetic information in the form of both point and length mutations. A phylogeny of cichlids based on this MFR agrees with other phylogenetic hypotheses based on morphological, mitochondrial, and anonymous nuclear DNA. Madagascan and Indian cichlids are found to be paraphyletic and the most basal group in the family Cichlidae. African and Neotropical cichlids are both monophyletic and sistergroups. Within African lineages, the East African cichlids are most likely to be monophyletic and the West African cichlids are probably paraphyletic and basal to all African species. The focal microsatellite locus contained much variation in (CA)$_{\text{n}}$ repeats in African cichlids and in surfperches (up to 64 repeats), but was short (with only 2-4 repeats) and almost invariant in Neotropical cichlids. The design of phylogenetically highly versatile MFR-primers will be of use not only for phylogeny reconstruction among families of perciform fishes, but also for population-level work in the thousands of species belonging to this highly species-rich suborder of fishes.

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.