A population genetic assessment of taxonomic species: The case of Lake Malawi cichlid fishes

Organisms sampled for population‐level research are typically assigned to species by morphological criteria. However, if those criteria are limited to one sex or life stage, or the organisms come from a complex of closely related forms, the species assignments may misdirect analyses. The impact of such sampling can be assessed from the correspondence of genetic clusters, identified only from patterns of genetic variation, to the species identified using only phenotypic criteria. We undertook this protocol with the rock‐dwelling mbuna cichlids of Lake Malawi, for which species within genera are usually identified using adult male coloration patterns. Given high local endemism of male colour patterns, and considerable allele sharing among species, there persists considerable taxonomic uncertainty in these fishes. Over 700 individuals from a single transect were photographed, genotyped and separately assigned: (a) to morphospecies using photographs; and (b) to genetic clusters using five widely used methods. Overall, the correspondence between clustering methods was strong for larger clusters, but methods varied widely in estimated number of clusters. The correspondence between morphospecies and genetic clusters was also strong for larger clusters, as well as some smaller clusters for some methods. These analyses generally affirm (a) adult male‐limited sampling and (b) the taxonomic status of Lake Malawi mbuna, as the species in our study largely appear to be well‐demarcated genetic entities. More generally, our analyses highlight the challenges for clustering methods when the number of populations is unknown, especially in cases of highly uneven sample sizes.     

Variation in habitat preference and population structure among three species of the Lake Malawi cichlid genus Protomelas

Several studies have demonstrated strong population structuring over small distances in the rocky-shore mbuna cichlid fishes from Lake Malawi, suggesting the potential for allopatric speciation. However, many endemic Lake Malawi cichlids are neither mbuna, nor confined to rocky shores. Using microsatellites, we investigated the population structure in three species of the non-mbuna genus Protomelas. The rocky-shore P. taeniolatus showed high levels of population structure even over distances of less than 1 km, while the sandy-shore species P. similis showed no significant structure over distances up to 21 km. Protomelas fenestratus, which is generally found at the interface between rocks and sand, also showed low levels of population structure. Our results suggest that the model of allopatric speciation based on habitat fragmentation within the current lake basin may be equally applicable to rocky-shore non-mbuna as to mbuna, but that an alternative model is required to explain speciation among sandy-shore species as well as the deep-water and pelagic species.     

Aggression in closely related Malawi cichlids varies inversely with habitat complexity

Within the past 2 million years, the cichlids of Lake Malawi have diversified into well over 500 species resulting in one of the worlds largest lacustrine fish radiations. As a result, many of the habitats within the lake support a high diversity of species. In these highly species rich communities, male cichlids must acquire and defend a territory to successfully reproduce. Within the rock-dwelling cichlids of Lake Malawi (mbuna), this has resulted in the formation of polyspecific leks on the heterogeneous rocky benthos. Aggression is fairly common in these leks and has been tied not only to individual reproduction but to the larger phenomenon of community assembly and the maintenance of biological diversity. In this study, I examined the patterns of aggressive acts of four species within the mbuna genus Maylandia at two locations in the southern Lake Malawi. The number of aggressive acts of two sympatric species was examined at each location. At each site, one species defends territories over bedrock and the other over cobble. The number of aggressive acts across the four species was compared. The influence of habitat type on male aggression was examined and the targets of male aggression were identified to evaluate several hypotheses concerning the evolution of male aggression. The results show that aggression quantitatively varied among species, was largely directed towards heterospecifics, and was strongly influenced by habitat type. The aggressive behavior of one sympatric species pair, Maylandia benetos and Maylandia zebra, was observed under controlled laboratory conditions. Laboratory results support field observations: the bedrock associated species performed more aggressive acts and aggressive acts were directed equally at con- and heterospecifics. The results of this study suggest that habitat complexity plays a larger role in shaping aggressive behavior than other suggested factors such as competition for resources.     

Nuclear gene variation and molecular dating of the cichlid species flock of Lake Malawi

The cichlid fishes of Lake Malawi are famously diverse. However, evolutionary studies have been difficult because of their recent and uncertain phylogenetic history. Portions of 12 nuclear loci were sequenced in nine rock-dwelling species (mbuna) and three representatives of pelagic nonmbuna species. In contrast to the pattern of variation at mitochondrial genes, which do provide phylogenetic resolution at the level of mbuna versus nonmbuna, and among some genera, the nuclear loci were virtually devoid of phylogenetic signal. Only a small minority of variable positions were phylogenetically informative, and no phylogenetic branches are supported by more than one site. From the nuclear gene perspective the Malawian radiation appears to be a star phylogeny, as if the founding of the lake was accompanied by a partial bottleneck. The pattern is different from that found in Lake Victoria, in which nuclear loci share large amounts of ancestral variation. In the case of nuclear genes of Lake Malawi, the absence of phylogenetically informative variation suggests a relative absence of ancestral variation. Nuclear genes also differed from the mitochondria in having nearly twice the amount of divergence from Oreochromis (tilapia). An approximate splitting time between mbuna and nonmbuna lineages was estimated as 0.7 Myr. Oreochromis is estimated to have diverged from the cichlids in Lake Malawi and Lake Tanganyika about 18 MYA.     

The distribution and dispersal of introduced fish at Thumbi West Island in Lake Malawi, Africa

The rock-dwelling cichlids, or mbuna, of Lake Malawi are exceptionally diverse. Explanations of this diversity have used limited dispersal by the mbuna as the basis for allopatric models of speciation. However, there have been no studies that have attempted to define the extent and rates of dispersal of mbuna populations in the field. The present work took advantage of some translocations in which several species of mbuna were introduced to the Cape Maclear region of Lake Malawi from elsewhere in the lake. A detailed series of transects involving observations at four depths was undertaken at Thumbi West Island to define the distribution of the introduced species. There are large differences between species in the extent to which they have spread. Some species are now present all round the island while others have dispersed only a limited distance from the site of the initial introductions. This survey provides the first comprehensive data set on the distribution of the introduced species and it raises a number of important questions regarding their dispersal ability and their likely impact on the endemic community.     

Sexual selection,reproductive behavior,and speciation in the mbuna species flock of Lake Malawi (Pisces: Cichlidae)

Synopsis The cichlid species flocks of the African Great Lakes represent the most extreme case of adaptive radiation among vertebrates. Recently, attention has focused on the potential for sexual selection to drive or accelerate speciation in these fishes. Cichlids as a whole are social in nature and display complex behavior, particularly during courtship and spawning; however, the extent to which changes in species recognition cues may account for species diversity among haplochromine lineages has remained speculative. Our investigations have indicated that oral incubating haplochromines show a reduction in diversity and extent of courtship relative to substrate brooding cichlids, and apparently retain aspects of a primitive specific mate recognition system. Laboratory observations of courtship in the Malawian endemicPseudotropheus zebra suggest that organization of the spawning bout is loose, and lacking in any well defined stimulus-response chain. Interspecific comparisons of in situ courtship behavior among male mbuna, lithophilous haplochromines of Lake Malawi, revealed only one potential example of species-specific behavior, and indicated that mate choice occurred prior to the onset of intense courtship. Courtship display by male mbuna does not appear critical to species recognition and may represent an evolutionary relict. Alternatively, male courtship display may have an indirect effect on successful reproduction or may be maintained through pleiotropic effects. The mbuna display no evidence of behavioral innovation and show limited interspecific differentiation in behavioral expression. More generally, there is no evidence at present to suggest that epigamic sexual selection, acting on courtship behavior, has been a major mechanism in the diversification of the haplochromine species flocks.     

Extreme microallopatric divergence in a cichlid species from Lake Malawi

We demonstrate significant population structuring on an extremely small spatial scale between adjacent demes of a Lake Malawi haplochromine cichlid species of the mbuna group, Pseudotropheus callainos, separated by only 35 m of habitat discontinuity. This substantiates further the notion that intralacustrine allopatric divergence may help to explain the high level of species richness of the mbuna in comparison to other Malawian cichlids, as well as of the Malawian haplochromines as a whole.     

Hybrid origin of a cichlid population in Lake Malawi: implications for genetic variation and species diversity

The importance of species recognition to taxonomic diversity among Lake Malawi cichlids has been frequently discussed. Hybridization - the apparent breakdown of species recognition - has been observed sporadically among cichlids and has been viewed as both a constructive and a destructive force with respect to species diversity. Here we provide genetic evidence of a natural hybrid cichlid population with a unique colour phenotype and elevated levels of genetic variation. We discuss the potential evolutionary consequences of interspecific hybridization in Lake Malawi cichlids and propose that the role of hybridization in generating both genetic variability and species diversity of Lake Malawi cichlids warrants further consideration.     

Aggressive biases towards similarly coloured males in Lake Malawi cichlid fishes

In haplochromine cichlids, female mate choice based on male nuptial coloration has played an important role in speciation. Recent studies suggest that male coloration strongly influences the distribution of these fishes based on male-male aggression; males direct more aggression towards similarly coloured opponents while tolerating differently coloured individuals. We explored the role of male nuptial colour in aggression among the mbuna of Lake Malawi, examining aggression by male Metriaclima mbenjii, the red top cobalt zebra, towards conspecific opponents, similarly coloured heterospecific opponents and differently coloured heterospecifics. In trials in which focal males were offered a single opponent, while the total number of aggressive behaviours did not vary among opponent species, the types of behaviours did; focal males directed more lateral displays towards conspecifics than towards the other opponent species. When focal males were offered two opponents simultaneously, M. mbenjii directed more aggressive behaviours and more lateral displays towards similarly coloured opponents, regardless of species. Furthermore, when offered a conspecific and a similarly coloured opponent simultaneously, there were no differences in behaviour towards either opponent. Thus, nuptial coloration is used by males to identify competitors, and it suggests that male-male aggression may have also been an important diversifying force in speciation in rock-dwelling Lake Malawi cichlids.     

Lake Malawi cichlid evolution along a benthic/limnetic axis

Divergence along a benthic to limnetic habitat axis is ubiquitous in aquatic systems. However, this type of habitat divergence has largely been examined in low diversity, high latitude lake systems. In this study, we examined the importance of benthic and limnetic divergence within the incredibly species‐rich radiation of Lake Malawi cichlid fishes. Using novel phylogenetic reconstructions, we provided a series of hypotheses regarding the evolutionary relationships among 24 benthic and limnetic species that suggests divergence along this axis has occurred multiple times within Lake Malawi cichlids. Because pectoral fin morphology is often associated with divergence along this habitat axis in other fish groups, we investigated divergence in pectoral fin muscles in these benthic and limnetic cichlid species. We showed that the eight pectoral fin muscles and fin area generally tended to evolve in a tightly correlated manner in the Lake Malawi cichlids. Additionally, we found that larger pectoral fin muscles are strongly associated with the independent evolution of the benthic feeding habit across this group of fish. Evolutionary specialization along a benthic/limnetic axis has occurred multiple times within this tropical lake radiation and has produced repeated convergent matching between exploitation of water column habitats and locomotory morphology.     

Identification of Cichlid Fishes from Lake Malawi Using Computer Vision

Background

The explosively radiating evolution of cichlid fishes of Lake Malawi has yielded an amazing number of haplochromine species estimated as many as 500 to 800 with a surprising degree of diversity not only in color and stripe pattern but also in the shape of jaw and body among them. As these morphological diversities have been a central subject of adaptive speciation and taxonomic classification, such high diversity could serve as a foundation for automation of species identification of cichlids.

Methodology/Principal Finding

Here we demonstrate a method for automatic classification of the Lake Malawi cichlids based on computer vision and geometric morphometrics. For this end we developed a pipeline that integrates multiple image processing tools to automatically extract informative features of color and stripe patterns from a large set of photographic images of wild cichlids. The extracted information was evaluated by statistical classifiers Support Vector Machine and Random Forests. Both classifiers performed better when body shape information was added to the feature of color and stripe. Besides the coloration and stripe pattern, body shape variables boosted the accuracy of classification by about 10%. The programs were able to classify 594 live cichlid individuals belonging to 12 different classes (species and sexes) with an average accuracy of 78%, contrasting to a mere 42% success rate by human eyes. The variables that contributed most to the accuracy were body height and the hue of the most frequent color.

Conclusions

Computer vision showed a notable performance in extracting information from the color and stripe patterns of Lake Malawi cichlids although the information was not enough for errorless species identification. Our results indicate that there appears an unavoidable difficulty in automatic species identification of cichlid fishes, which may arise from short divergence times and gene flow between closely related species.     

No evidence for parallel sympatric speciation in cichlid species of the genus Pseudotropheus from north-western Lake Malawi

To test the hypothesis of parallel speciation by sexual selection, we examined length variation at six microsatellite loci of samples from four sites of four to six putative species belonging to two subgenera of rocky shore mbuna cichlids from Lake Malawi. Almost all fixation indices were significantly different from zero, suggesting that there is presently little or no gene flow among allopatric populations or sympatric species. Analysis of variance indicated that genetic distances among allopatric populations of putative conspecifics were significantly lower than among sympatric populations of heterospecifics. The topology of trees based on distance matrices was also largely consistent with the hypothesis that the putative species are monophyletic and have thus not evolved in parallel in their present locations. If parallel speciation does occur in Malawi cichlids, it may be on a larger spatial scale than investigated in our study.     

Signal and preference divergence among populations of the non‐endemic basal Lake Malawi cichlid fish Astatotilapia calliptera (Perciformes: Cichlidae)

In Lake Malawi and Lake Victoria, cichlid fishes have diversified into hundreds of species, many reproductively isolated by mate choice. Territorial males tend to be more aggressive to similar‐coloured males, facilitating coexistence of divergent colour morphs or species. Behavioural mate choice and aggression biases of species and allopatric populations of specialized rocky shore cichlids are influenced by divergent signals such as male colour. Believed to be basal to the Lake Malawi haplochromine radiation, and inhabiting shallow weedy areas of the lake and neighbouring water bodies, Astatotilapia calliptera also shows allopatric variation in colour. Here, it is demonstrated that such signal divergence is associated with tendencies of females to mate with males of their own population and also for males to preferentially attack males of their own population, indicating that preference divergence related to signal divergence in allopatry may have operated throughout the adaptive radiation of the Malawian cichlids. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 180–188.     

Evidence for male-biased dispersal in lake malawi cichlids from microsatellites

This study addressed within-population dispersal patterns among the species-rich Lake Malawi cichlids, specifically among the rock-dwelling mbuna group. Relatedness values were calculated for 160 individuals belonging to two species from known locations in the field by screening six highly polymorphic microsatellite loci. The results from both species indicate that spatially adjacent females have higher average relatedness values than those separated by larger distances, but that this pattern is reversed in males. This therefore provides firm evidence for male-biased dispersal within the Malawian cichlid flock.     

Adult plasticity in African cichlids: Rapid changes in opsin expression in response to environmental light differences

Phenotypic plasticity allows organisms to adapt quickly to local environmental conditions and could facilitate adaptive radiations. Cichlids have recently undergone an adaptive radiation in Lake Malawi where they inhabit diverse light environments and tune their visual sensitivity through differences in cone opsin expression. While cichlid opsin expression is known to be plastic over development, whether adults remain plastic is unknown. Adult plasticity in visual tuning could play a role in cichlid radiations by enabling survival in changing environments and facilitating invasion into novel environments. Here we examine the existence of and temporal changes in adult visual plasticity of two closely related species. In complementary experiments, wild adult Metriaclima mbenji from Lake Malawi were moved to the lab under UV‐deficient fluorescent lighting; while lab raised M. benetos were placed under UV‐rich lighting designed to mimic light conditions in the wild. Surprisingly, adult cichlids in both experiments showed significant changes in the expression of the UV‐sensitive single cone opsin, SWS1, in only 3 days. Modeling quantum catches in the light environments revealed a possible link between the light available to the SWS1 visual pigment and SWS1 expression. We conclude that adult cichlids can undergo rapid and significant changes in opsin expression in response to environmental light shifts that are relevant to their habitat and evolutionary history in Lake Malawi. This could have contributed to the rapid divergence characteristic of these fantastic fishes.     

Retroposition of the AFC Family of SINEs (Short Interspersed Repetitive Elements) Before and During the Adaptive Radiation of Cichlid Fishes in Lake Malawi and Related Inferences About Phylogeny

Lake Malawi is home to more than 450 species of endemic cichlids, which provide a spectacular example of adaptive radiation. To clarify the phylogenetic relationships among these fish, we examined the presence and absence of SINEs (short interspersed repetitive elements) at orthologous loci. We identified six loci at which a SINE sequence had apparently been specifically inserted by retroposition in the common ancestor of all the investigated species of endemic cichlids in Lake Malawi. At another locus, unique sharing of a SINE sequence was evident among all the investigated species of endemic non-Mbuna cichlids with the exception of Rhamphochromis sp. The relationships were in good agreement with those deduced in previous studies with various different markers, demonstrating that the SINE method is useful for the elucidation of phylogenetic relationships among cichlids in Lake Malawi. We also characterized a locus that exhibited transspecies polymorphism with respect to the presence or absence of the SINE sequence among non-Mbuna species. This result suggests that incomplete lineage sorting and/or interspecific hybridization might have occurred or be occurring among the species in this group, which might potentially cause misinterpretation of phylogenetic data, in particular when a single-locus marker, such as a sequence in the mitochondrial DNA, is used for analysis. Received: 15 December 2000 / Accepted: 30 January 2001     

Acoustic diversity in Lake Malawi’s rock-dwelling cichlids

The cichlids of Lake Malawi are one of the world’s most species rich and phenotypically diverse groups of extant vertebrates. The extraordinary variability of this group’s color patterns, reproductive behaviors, and trophic morphologies are well documented. More recently, an additional axis of phenotypic diversity has been identified. Lake Malawi cichlids have been shown to use species-specific acoustic communication in both aggressive and reproductive encounters. However, documentation of acoustic signals used by this group is limited to a small number of taxa observed within the confines of the laboratory. This study examines the acoustic signals produced by six species spanning four genera of rock-dwelling cichlids recorded in their natural habitat, the shallow waters surrounding Thumbi West Island, Lake Malawi. Four acoustic parameters were quantified and compared between species: trill duration, number of pulses per trill, pulse duration, and pulse period. Using these characteristics, sympatric species within the genus Maylandia were easily distinguished. Furthermore, a comparison of this data to previously published acoustic data reveals possible geographic dialects within species.     

Identification of a novel sex determining chromosome in cichlid fishes that acts as XY or ZW in different lineages

Sex determination systems are highly conserved among most vertebrates with genetic sex determination, but can be variable and evolve rapidly in some. Here, we study sex determination in a clade with exceptionally high sex chromosome turnover rates. We identify the sex determining chromosomes in three interspecific crosses of haplochromine cichlid fishes from Lakes Victoria and Malawi. We find evidence for different sex determiners in each cross. In the Malawi cross and one Victoria cross the same chromosome is sex-linked but while females are the heterogametic sex in the Malawi species, males are the heterogametic sex in the Victoria species. This chromosome has not previously been reported to be sex determining in cichlids, increasing the number of different chromosomes shown to be sex determining in cichlids to 12. All Lake Victoria species of our crosses are less than 15,000 years divergent, and we identified different sex determiners among them. Our study provides further evidence for the diversity and evolutionary flexibility of sex determination in cichlids, factors which might contribute to their rapid adaptive radiations.

     

Assortative aggression among males in a sympatric pair of Labeotropheus from Lake Malaŵi,Africa

Female mate choice has strong experimental support as a diversifying force in the speciation of the haplochromine cichlid fishes of Lake Malaŵi, Africa. Somewhat less understood is the role that male–male aggression might have played in the evolution of new species of these fishes. In the rock-dwelling haplochromines of Lake Malaŵi, primarily territory-holding males successfully court females; by determining which males gain territories, male–male aggression could support speciation by excluding less-fit males from the breeding population. To test the hypothesis that males should direct more aggression towards conspecific rivals, the aggression directed towards conspecific and heterospecific opponents was compared in a sympatric pair of cichlids of the genus Labeotropheus Ahl 1927 (Labeotropheus fuelleborni Ahl 1927 and Labeotropheus trewavasae Fryer 1956). It was found that when presented with a pair of rivals, males of both species did direct more aggression towards the conspecific opponent, and the amount of aggression increased when the conspecific opponent was larger than the heterospecific opponent. In addition, this study found a difference in the behavioural repertoire of the species: L. fuelleborni tends to rely on displays to intimidate opponents, whereas L. trewavasae employs more physical attacks to drive away opponents. Males of both species can thus recognize conspecifics and assess an opponent's relative threat to their ability to successfully reproduce, and use species-specific strategies to intimidate opponents.