Naturally and sexually selected traits in haplochromine cichlid fishes

Humankind seeks for explanations to describe the evolution of the astonishing biodiversity surrounding us. To understand organismal diversity, we first need to understand the evolutionary processes underlying it. However, we are still struggling with Darwin’s ‘mystery of mysteries’ (Darwin 1859), that is to understand how and why new species form (Coyne & Orr 2004). The establishment of reproductive isolation between divergent populations can evolve through barriers in post- (Snook et al. 2009) and pre-copulatory sexual selection (Darwin 1871). The two fundamental modes of Darwinian sexual selection are contests for mates (intrasexual selection) and mate choice by the opposite sex (intersexual selection) (Darwin 1871). Even though reproductive isolation could arise through sexual selection alone, it was hypothesized that it functions most effectively in conjunction with selection for species recognition or ecological selection (Ritchie 2007). Speciation through ecological selection drives adaptive diversification into a variety of ecological niches, which is described as ‘adaptive radiation’ in evolutionary groups that have exhibited exceptional extent of diversification (Schluter 2000). A textbook example of adaptive radiations and, therefore, an ideal system to study diversification are the perciform fishes of the family Cichlidae (e.g. Maan et al. 2006; Seehausen et al. 2008; Salzburger 2009). Their rapid speciation resulted in an estimated number of around 3’000 species (Snoeks 1994; Turner et al. 2001), turning cichlids into the most species-rich family of vertebrates (Salzburger & Meyer 2004; Salzburger 2009). Cichlids are distributed across South and Central America, Africa and parts of India. This distribution suggests a Gondwanian origin of the group (Salzburger 2009). Their centre of diversity, however, lies in the East African Great Lakes, which harbour extremely diverse and species-rich flocks of cichlid fishes and are therefore a prime model system in evolutionary biology (Meyer 1993; Turner et al. 2001; Seehausen 2006). In addition to the extrinsic environmental factors such as geologic and climatic events creating novel ecological niches (Fryer & Iles 1972; Sturmbauer 1998; Sturmbauer et al. 2001), several evolutionary key innovations have been hypothesized to have played a role in their rapid speciation and adaptations to a variety of ecological niches. Of particular importance are the special pharyngeal jaw apparatus (Fryer & Iles 1972; Liem 1973), the highly complex reproductive behaviour (Fryer & Iles 1972; Goodwin et al. 1998; Kornfield & Smith 2000) and the wealth of colour morphs. It was shown that colour and pigmentation patterns seem to play a central role in the explosively radiating cichlid fish lineages in the East African Great Lakes in general, and in haplochromine cichlids in particular (Seehausen et al. 1999; Kocher 2004; Turner 2007; Salzburger 2009). Haplochromines comprise about 80% of East African cichlid species including the entire species flocks of lakes Victoria and Malawi, the tribe Tropheini from Lake Tanganyika and many riverine species (e.g. Turner et al. 2001; Salzburger et al. 2005). Interestingly, all haplochromines are maternal mouthbrooders, with females incubating their offspring – until fully developed – in their buccal cavities (e.g. Fryer & Iles 1972; Salzburger et al. 2005). This special breeding behaviour evolved several times during cichlid evolution (Goodwin et al. 1998), but only the ‘modern haplochromines’ show a derived polygynous or polygynandrous maternal mouthbrooding system with males displaying the so-called egg-spots on their anal fins (Fryer & Iles 1972; Salzburger et al. 2005, 2007). These ovoid markings consist of a transparent outer ring encircling a brightly coloured yellow, orange or reddish centre (Wickler 1962; Fryer & Iles 1972). The conspicuous central area is formed by two chromatophore cell types, xanthophores and iridophores (Salzburger et al. 2007; Santos et al. 2014). Even though this trait is proposed to be a putative key innovation mediating the evolutionary success of haplochromines (Salzburger et al. 2005; Salzburger 2009), their function is not fully understood. Several hypotheses exist that seek to explain the function of egg-spots: Wickler (1962) associated the function of egg-spots with the special mouthbrooding behaviour, and suggested that egg-spots mimic real eggs and function as an attracting signal during courtship and as releasers for egg-uptake and, hence, to maximize fertilization. Support for Wickler’s hypothesis was only found with respect to the function in courtship since females of the species Astatotilapia elegans and Pseudotropheus (Maylandia) aurora preferred to lay batches with males with many egg-spots (Hert 1989, 1991), whereas females of Pseudotropheus (Maylandia) lombardoi preferably chose males with an artificially enlarged egg-spot over males with one natural or many egg-spots (Couldridge 2002). However, there was no influence of egg-spots on fertilization rate (Hert 1989). Further doubts about the egg mimicry hypothesis arose because egg-spots often do not resemble size, shape and colour of a species’ actual eggs (Jackson & van Lier Ribbink 1975; Goldschmidt 1991). This mismatch between real eggs and egg-spots may be due to a trade-off between attractiveness towards females and conspicuousness for predators (Goldschmidt 1991). An alternative explanation could be that egg-spots serve as species recognition signal (Axelrod & Burgess 1973). So far, the results from studies that aimed to evaluate the function and selection pressures on egg-spots are scarce, rather inconsistent and raise the necessity for new experimental work on their mode of action and their evolutionary origin. Part 1 of my thesis is therefore dedicated to the evolution and function of egg-spots. The first manuscript focuses on the evolutionary origin of anal fin egg-spots, more specifically, we tested the hypothesis whether a sensory bias has triggered the evolution of egg-spots in cichlid fishes (1.1). Mate choice trials were conducted to see if females of the basal haplochromine Pseudocrenilabrus multicolor (naturally showing no true egg-spot on its anal fin) prefer computer-animated photographs of males with an artificially added egg-spot. Additionally, colour preferences (outside a mating context) were tested in a phylogenetically representative set of East African cichlids. The next two chapters focus on the putative function of egg-spots in sexual selection in the two haplochromine species Astatotilapia burtoni (1.2 The function of anal fin egg-spots in the cichlid fish Astatotilapia burtoni) and Astatotilapia calliptera (1.3 Egg-spot pattern and body size asymmetries influence male aggression in haplochromine cichlid fishes), which both exhibit several egg-spots on their anal fin. In both species, mate choice trials were conducted to test if females prefer to lay eggs with males with many egg-spots over males with fewer or no egg-spots. Since carotenoid based colouration can be indicative for the health and strength of its bearer (e.g. Endler 1978, 1980; Hill 1992), egg-spots are also a prime example to examine if there is a function in intrasexual selection. Therefore, male aggression experiments were conducted in both species to test if egg-spots could play a role in the assessment of an opponent’s strength. Visual signals will most probably not only diverge due to sexual selection, but might be influenced by their environment and are therefore expected to evolve to a point where viability costs balance mating advantage (Darwin 1871; Zahavi 1975; Endler 1978; Andersson 1994). To examine how the egg-spot phenotype can be influenced by sexual and ecological selection, the next manuscript examines the variation of anal fin egg-spots along an environmental gradient in a haplochromine cichlid fish (2.1). This project constitutes the first of two studies of Part 2 describing adaptive divergence in lake-stream systems in A. burtoni. This species represents an ideal model organism to address questions about adaptive divergence in lake-stream systems in cichlids, since A. burtoni is one of only few cichlid species, which inhabits shallow zones of one of the East African Great Lakes as well as rivers and streams surrounding it (Fernald & Hirata 1977; Kullander & Roberts 2011). Populations of lacustrine and riverine habitats of four lake-stream systems were examined with regards to sex- and habitat-specific differences in egg-spot characteristics such as number, size and colouration. Finally, we tested for an association between the conspicuousness of male egg-spots and underwater light environment as well as the status of the immune system. However, not only visual signals - like egg-spots - can adapt to the respective environmental conditions, but lake-stream systems are also a unique system to study how populations experiencing different environmental conditions may diverge in general. So far, adaptive divergence in cichlids has mainly been investigated within lakes, e.g. along depth or habitat gradients (see e.g. Barluenga et al. 2006; Seehausen et al. 2008). The A. burtoni setting should therefore be established as the first lake-stream system in cichlids, which is described in the second study of Part 2 (2.2 Adaptive divergence between lake and stream populations of an East African cichlid fish). Here, we first established phylogeographic relationships and assessed the population structure as well as body shape differences in over 20 A. burtoni populations from the southern part of Lake Tanganyika. In a second step, we focused on four lake-stream systems in detail (the same systems as in chapter 2.1) and, in addition to the body shape and population-genetic surveys, we quantified other ecologically relevant traits (gill raker and lower pharyngeal jaw) as well as stomach contents. To test whether the shifts in the examined traits reflect ecologically based adaptive divergence (Berner et al. 2009; Harrod et al. 2010), we tested for an association between morphological variation and environmental factors, such as resource use and water velocity. Finally, a mating experiment was conducted to test for reproductive isolation among lake and stream populations. Adults and offspring from this common garden setting were further used to evaluate levels of phenotypic plasticity in the traits body shape and gill raker morphology. During the sampling trips for the study mentioned above, we observed a clear-cut barrier for the occurrence of A. burtoni in the streams surrounding Lake Tanganyika. At a certain elevation A. burtoni was absent and seemed to be replaced by another riverine cichlid, namely a species of the Pseudocrenilabrus philander complex. Interestingly, they both were found to co-occur in Lake Chila, a small lake 20 km south of Lake Tanganyika. The first side project of Part 3 concentrates on this P. philander complex with the manuscript about the phylogeographic and phenotypic assessment of a basal haplochromine cichlid fish from Lake Chila, Zambia (3.1). Here we report the discovery of a population of the normally riverine P. cf. philander in Lake Chila. We examined this lake population for increased morphological variation compared to riverine populations of P. cf. philander. With this dataset we wanted to test whether ecological opportunity in the form of a greater number and more diverse ecological niches promotes diversification in lakes compared to rivers (as seen in e.g. Stelkens & Seehausen 2009). The phenotypic variability of this Lake Chila population was evaluated in relation to other lacustrine and riverine populations by quantifying colouration and body shape. Additionally, phylogeographic history was investigated with attention to a case of hybridization of two distinct lineages. The second side project focuses on a special case of morphological variation, namely mouth asymmetry, by performing a field based assessment of attack strategy and feeding success in the scale- eating cichlid fish Perissodus microlepis (3.2 A fitness benefit for mouth dimorphism in a scale-eating cichlid fish). Perissodus microlepis is the most common and perhaps the most specialized lepidophagous cichlid in Lake Tanganyika (Takahashi et al. 2007) and exhibits a pronounced asymmetry with individuals that feature a mouth slightly bent to the right or to the left side in order to optimize feeding successes (Hori 1993). In this study the lateralisation dynamics in P. microlepis were reassessed in a semi-natural environment in order to confirm laboratory based findings about asymmetrical attack strategies and to test if dimorphic experimental populations of P. microlepis ultimately are more successful and show a higher feeding success than monomorphic experimental populations. All together, we aimed to disentangle causalities in the evolution of this system and to demonstrate the selective advantage of dimorphic mouth opening and attack strategy in scale-eaters. This is necessary to explain how such asymmetries have evolved and can be maintained in natural populations. In summary, my thesis consists of two main parts and a third part comprising two side projects. Part 1 investigates the trait egg-spots, which were mentioned to be a key innovation of haplochromines, the most species-rich tribe of cichlids. Three manuscripts deal with their mode of action as well as their evolutionary origin. Part 2 examines the divergence among lake and stream populations with respect to egg-spots and in a second project with respect to body shape and other ecologically relevant traits. Additionally, the phylogeographic relationships of A. burtoni populations from the southern part of Lake Tanganyika were established

Egg-spot pattern and body size asymmetries influence male aggression in haplochromine cichlid fishes

In male-male encounters, visual ornaments are used to assess the strength of an opponent and to determine a subsequent attack strategy. Our study suggests that both the ornament egg-spot and body size influence the strength assessment in a cichlid fish. Furthermore, we show that egg-spot differences become more important when body size differences between contestants are small. This provides evidence that egg-spots serve as an honest visual signal reflecting a male's qualit

Inhibition of Aromatase Induces Partial Sex Change in a Cichlid Fish: Distinct Functions for Sex Steroids in Brains and Gonads

Sex steroids are major drivers of sexual development and also responsible for the maintenance of the established gender. Especially fishes exhibit great plasticity and less conservation in sex determination and sexual development compared to other vertebrate groups. In addition, fishes have a constant sex steroid production throughout their entire lifespan, which makes them particularly susceptible to interferences with the endogenous sex steroid system. This susceptibility has recently been used to show that inhibition of the key enzyme of estrogen synthesis, aromatase Cyp19a1, can induce functional sex reversal even in adult fish. Here, we investigated the impact of the aromatase inhibitor (AI) fadrozole in adult females of the East African cichlid fish Astatotilapia burtoni. Using gene expression, phenotypic measurements, behavioral experiments, and hormone measurements, we assessed if females treated with fadrozole develop a male-like phenotype. We found that AI treatment has a different effect on gene expression in the gonad compared to the brain, the 2 tissues mostly implicated in sexual development. In contrast to observations in other gonochoristic species, A. burtoni ovaries cannot be transformed into functional testis by AI. However, rapid changes towards a male-like phenotype can be induced with AI in coloration, hormone levels, and behavior

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 towards conspecifics versus inconspicuousness towards 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 (i) males possess larger and more conspicuous egg-spots than females, which is likely explained by their function in sexual selection; (ii) riverine fish generally feature fewer but larger and/or more intensively colored egg-spots, which is probably to maintain signal efficiency in intraspecific interactions in long-wavelength shifted riverine light conditions; and (iii) 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 syste

A Sensory Bias Has Triggered the Evolution of Egg-Spots in Cichlid Fishes

Although, generally, the origin of sex-limited traits remains elusive, the sensory exploitation hypothesis provides an explanation for the evolution of male sexual signals. Anal fin egg-spots are such a male sexual signal and a key characteristic of the most species-rich group of cichlid fishes, the haplochromines. Males of about 1500 mouth-brooding species utilize these conspicuous egg-dummies during courtship – apparently to attract females and to maximize fertilization success. Here we test the hypothesis that the evolution of haplochromine egg-spots was triggered by a pre-existing bias for eggs or egg-like coloration. To this end, we performed mate-choice experiments in the basal haplochromine Pseudocrenilabrus multicolor, which manifests the plesiomorphic character-state of an egg-spot-less anal fin. Experiments using computer-animated photographs of males indeed revealed that females prefer images of males with virtual (‘in-silico’) egg-spots over images showing unaltered males. In addition, we tested for color preferences (outside a mating context) in a phylogenetically representative set of East African cichlids. We uncovered a strong preference for yellow, orange or reddish spots in all haplochromines tested and, importantly, also in most other species representing more basal lines. This pre-existing female sensory bias points towards high-quality (carotenoids-enriched) food suggesting that it is adaptive

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

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

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

Random asymmetry, that is the co-existence 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 (Boulenger, 1898), 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

Indermaur_et_al_preyfish

Scale counts on the prey fis

Egg-spot pattern and body size asymmetries influence male aggression in haplochromine cichlid fishes

Assessing an opponent’s strength is an important component of attack strategies in territorial combats between males. Body size is often considered to directly influence an individual’s strength, but other honest visual signals may also affect the assessment of opponents. Among such visual signals are the so-called egg-spots, a conspicuous ovoid marking on the anal fin of male haplochromine cichlid fishes, made up of carotenoid-containing and other pigment cells. It has long been assumed that egg-spots are mainly relevant in courtship and spawning behavior, and previous work has focused primarily on their function in intersexual selection. Recently, however, both body size and egg-spots have been suggested to play a role in male–male interactions. To test whether egg-spots function in female choice or whether egg-spots and/or body size function as a predictor of strength and the subsequent attack strategy in male–male interactions, we performed a series of behavioral experiments in the haplochromine cichlid Astatotilapia calliptera. The trials revealed a limited involvement of egg-spots in female choice, yet a much stronger influence in male interactions. Territorial males combined information from the strength assessment based on body size and egg-spots to adopt their attack strategies. They launched more attacks against the larger intruder with many egg-spots compared with the smaller intruder without or with fewer egg-spots. Our study provides evidence that egg-spots serve as honest visual signal and that the level of asymmetries in egg-spot pattern and body size determines the relative impact of each trait in strength assessment