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.

     

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.     

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.     

Elevated mtDNA diversity in introduced populations of Cynotilapia afra (Günther 1894) in Lake Malawi National Park is evidence for multiple source populations and hybridization

Genetic variation in many invasive species shows little or no signs of a founder event, suggesting that high genetic diversity may facilitate establishment success. The rocky‐shore, plankton‐feeding cichlid fish Cynotilapia afra is endemic to Lake Malawi, but naturally absent from many suitable sites. In the 1960s, this species was introduced to the southern areas of the lake, presumably as a result of the aquarium fish trade. It has now become established on a number of rocky areas within the Lake Malawi National Park. Here, we analysed DNA sequence variation in the mitochondrial control region of six native and four introduced populations of C. afra, and three populations of the closely‐related and hybridizing Pseudotropheus zebra. In contrast to previous studies of Lake Malawi rock dwelling cichlids, network analyses suggested that native populations of C. afra showed high levels of lineage sorting in mtDNA. Introduced populations showed higher sequence and haplotype diversity than their native counterparts. Our analyses suggested that the elevated gene diversity was largely attributed to the fact that the introduced C. afra populations were derived from several genetically distinct and geographically separate populations, and to a lesser extent because of introgressive hybridization with native P. zebra. The establishment and spread of C. afra may be partly because of its ability to occupy a vacant ecological niche, but it may also have been facilitated by its enhanced genetic diversity.     

Microsatellites reveal high levels of population substructuring in the species-poor Eretmodine cichlid lineage from Lake Tanganyika

This study investigated fine-scale population substructuring in an apparently monogamous, biparental mouth-brooding cichlid. Microsatellite allele frequencies were determined at four polymorphic loci for nine populations of Eretmodus cyanostictus. We provide empirical support for the hypothesis that a species employing this breeding strategy should exhibit high levels of population substructuring. Stretches of sand represent considerable barriers to dispersal and, in contrast to the rock-dwelling cichlids of Lake Malawi, distance alone, along a continuous rocky shoreline, is sufficient to reduce gene flow significantly There was a significant pattern of isolation by distance both along the whole study area and over the stretch of continuous shoreline, suggesting that this species has poor dispersal capabilities and that juveniles establish territories close to their natal site. Despite limited dispersal, E. cyanostictus populations are not significantly more inbred than a more-widely dispersing rock-dwelling cichlid from Lake Malawi. This finding may cast doubt on the hypothesis that polyandry has evolved as a mechanism for maintaining genetic diversity in Lake Malawi cichlids. High levels of substructuring may not always promote high levels of speciation, and other factors, such as the intensity of sexual selection, may be more important in determining the speciation potential of a lineage.     

Body shape differences in a pair of closely related Malawi cichlids and their hybrids: Effects of genetic variation,phenotypic plasticity,and transgressive segregation

Phenotypic differences may have genetic and plastic components. Here, we investigated the contributions of both for differences in body shape in two species of Lake Malawi cichlids using wild‐caught specimens and a common garden experiment. We further hybridized the two species to investigate the mode of gene action influencing body shape differences and to examine the potential for transgressive segregation. We found that body shape differences between the two species observed in the field are maintained after more than 10 generations in a standardized environment. Nonetheless, both species experienced similar changes in the laboratory environment. Our hybrid cross experiment confirmed that substantial variation in body shape appears to be genetically determined. The data further suggest that the underlying mode of gene action is complex and cannot be explained by simple additive or additive‐dominance models. Transgressive phenotypes were found in the hybrid generations, as hybrids occupied significantly more morphospace than both parentals combined. Further, the body shapes of transgressive individuals resemble the body shapes observed in other Lake Malawi rock‐dwelling genera. Our findings indicate that body shape can respond to selection immediately, through plasticity, and over longer timescales through adaptation. In addition, our results suggest that hybridization may have played an important role in the diversification of Lake Malawi cichlids through creating new phenotypic variation.     

Recent speciation between sympatric Tanganyikan cichlid colour morphs

Lake Tanganyika, Africa's oldest lake, harbours an impressive diversity of cichlid fishes. Although diversification in its radiating groups is thought to have been initially rapid, cichlids from Lake Tanganyika show little evidence for ongoing speciation. In contrast, examples of recent divergence among sympatric colour morphs are well known in haplochromine cichlids from Lakes Malawi and Victoria. Here, we report genetic evidence for recent divergence between two sympatric Tanganyikan cichlid colour morphs. These Petrochromis morphs share mitochondrial haplotypes, yet microsatellite loci reveal that their sympatric populations form distinct genetic groups. Nuclear divergence between the two morphs is equivalent to that which arises geographically within one of the morphs over short distances and is substantially smaller than that among other sympatric species in this genus. These patterns suggest that these morphs diverged only recently, yet that barriers to gene flow exist which prevent extensive admixture despite their sympatric distribution. The morphs studied here provide an unusual example of active diversification in Lake Tanganyika's generally ancient cichlid fauna and enable comparisons of speciation processes between Lake Tanganyika and other African lakes.     

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.     

Geographical ancestry of Lake Malawi's cichlid fish diversity

The Lake Malawi haplochromine cichlid flock is one of the largest vertebrate adaptive radiations. The geographical source of the radiation has been assumed to be rivers to the south and east of Lake Malawi, where extant representatives of the flock are now present. Here, we provide mitochondrial DNA evidence suggesting the sister taxon to the Lake Malawi radiation is within the Great Ruaha river in Tanzania, north of Lake Malawi. Estimates of the time of divergence between the Lake Malawi flock and this riverine sister taxon range from 2.13 to 6.76 Ma, prior to origins of the current radiation 1.20–4.06 Ma. These results are congruent with evaluations of 2–3.75 Ma fossil material that suggest past faunal connections between Lake Malawi and the Ruaha. We propose that ancestors of the Malawi radiation became isolated within the catchment during Pliocene rifting that formed both Lake Malawi and the Kipengere/Livingstone mountain range, before colonizing rivers to the south and east of the lake region and radiating within the lake basin. Identification of this sister taxon allows tests of whether standing genetic diversity has predisposed Lake Malawi cichlids to rapid speciation and adaptive radiation.     

Ancient hybridization and phenotypic novelty within Lake Malawi's cichlid fish radiation

Does hybridization play a broad innovative role in evolution? Many studies have shown hybrid origins of individual species, particularly in major adaptive radiations, but this may be a consequence, rather than a cause, of the existence of many closely related species. Cases of hybridization in the early stages of major adaptive radiations are comparatively rare. Here, we report phylogenetic evidence for ancient introgression between distinct lineages of the species-rich Lake Malawi haplochromine cichlid fishes. Mitochondrial DNA (mtDNA) sequences indicated surprisingly close relationships between the shallow-water rocky habitat "Mbuna" species and a group of dark-adapted "Deep-Benthic" species specialized for feeding in low-light conditions (dawn/dusk, under overhangs, and deep water). By contrast, analyses of nuclear amplified fragment length polymorphism data demonstrated that these Deep-Benthic cichlids were more closely related to shallow water "Shallow-Benthic" soft-sediment feeders, a group that shares similar head and body morphology. A coalescent-based computer simulation indicated that the mtDNA similarity of rocky habitat Mbuna species and dark-adapted Deep-Benthic species was due to hybridization rather than incomplete lineage sorting. Comparisons of morphology indicated that some Deep-Benthic species possessed novel morphology not present in other Lake Malawi species groups. Thus, these analyses support the hypothesis that ancient hybridization occurred within the Lake Malawi cichlid radiation, that the event occurred before the radiation of a species group adapted to low-light benthic habitats, and that this group went on to dominate the deep-water regions of Lake Malawi. The results of this study contribute to a growing literature consistent with a creative role of hybridization in the evolution of species diversity and adaptive radiations.     

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.     

Axes of differentiation in the bower-building cichlids of Lake Malawi

The 500-1000 cichlid species endemic to Lake Malawi constitute one of the most rapid and extensive radiations of vertebrates known. There is a growing debate over the role natural and sexual selection have played in creating this remarkable assemblage of species. Phylogenetic analysis of the Lake Malawi species flock has been confounded by the lack of appropriate morphological characters and an exceptional rate of speciation, which has allowed ancestral molecular polymorphisms to persist within species. To overcome this problem we used amplified fragment length polymorphism (AFLP) to reconstruct the evolution of species within three genera of Lake Malawi sand-dwelling cichlids that construct elaborate male display platforms, or bowers. Sister taxa with distinct bower morphologies, and that exist in discrete leks separated by only 1-2 m of depth, are divergent in both sexually selected and ecological traits. Our phylogeny suggests that the forces of sexual and ecological selection are intertwined during the speciation of this group and that specific bower characteristics and trophic morphologies have evolved repeatedly. These results suggest that trophic morphology and bower form may be inappropriate characters for delineating taxonomic lineages. Specifically the morphological characters used to describe the genera Lethrinops and Tramitichromis do not define monophyletic clades. Using a combination of behavioural and genetic characters, we were able to identify several cryptic cichlid species on a single beach, which suggests that sand dweller species richness has been severely underestimated.     

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.     

Species-Specific Population Structure in Rock-Specialized Sympatric Cichlid Species in Lake Tanganyika,East Africa

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. [Reviewing Editor: Dr. Rafael Zardoya]     

Hybridization Promotes Evolvability in African Cichlids: Connections Between Transgressive Segregation and Phenotypic Integration

Hybridization is a potential source of novel variation through (1) transgressive segregation, and (2) changes in the patterns and strength of phenotypic integration. We investigated the capacity of hybridization to generate novel phenotypic variation in African cichlids by examining a large F2 population generated by hybridizing two Lake Malawi cichlid species with differently shaped heads. Our morphometric analysis focused on the lateral and ventral views of the head. While the lateral view exhibited marked transgressive segregation, the ventral view showed a limited ability for transgression, indicating a difference in the genetic architecture and selective history between alternate views of the head. Moreover, hybrids showed a marked reduction in integration, with a lower degree of integration observed in transgressive individuals. In all, these data offer novel insights into how hybridization can promote evolvability, and provide a possible explanation for how broad phenotypic diversity may be achieved in rapidly evolving groups.     

PhyloFinder: An intelligent search engine for phylogenetic tree databases

Background

Evolutionary biologists want to explain the origin of novel features and functions. Two recent but separate lines of research address this question. The first describes one possible outcome of hybridization, called transgressive segregation, where hybrid offspring exhibit trait distributions outside of the parental range. The second considers the explicit mapping of form to function and illustrates manifold paths to similar function (called many to one mapping, MTOM) when the relationship between the two is complex. Under this scenario, functional novelty may be a product of the number of ways to elicit a functional outcome (i.e., the degree of MTOM). We fuse these research themes by considering the influence of MTOM on the production of transgressive jaw biomechanics in simulated hybrids between Lake Malawi cichlid species.

Results

We characterized the component links and functional output (kinematic transmission, KT) of the 4-bar mechanism in the oral jaws of Lake Malawi cichlids. We demonstrated that the input and output links, the length of the lower jaw and the length of the maxilla respectively, have consistent but opposing relationships with KT. Based on these data, we predicted scenarios in which species with different morphologies but similar KT (MTOM species) would produce transgressive function in hybrids. We used a simple but realistic genetic model to show that transgressive function is a likely outcome of hybridization among Malawi species exhibiting MTOM. Notably, F₂ hybrids are transgressive for function (KT), but not the component links that contribute to function. In our model, transgression is a consequence of recombination and assortment among alleles specifying the lengths of the lower jaw and maxilla.

Conclusion

We have described a general and likely pervasive mechanism that generates functional novelty. Simulations of hybrid offspring among Lake Malawi cichlids exhibiting MTOM produce transgressive function in the majority of cases, and at appreciable frequency. Functional transgression (i) is a product of recombination and assortment between alleles controlling the lengths of the lower jaw and the maxilla, (ii) occurs in the absence of transgressive morphology, and (iii) can be predicted from the morphology of parents. Our genetic model can be tested by breeding Malawi cichlid hybrids in the laboratory and examining the resulting range of forms and functions.     

Genital morphology and allometry differ by species and sex in Malawi cichlid fishes

The African cichlid fishes show great diversity in mating displays and reproductive strategies, yet species differences in genital morphology have been little studied. Observational notes have described broad sex differences in external genital shape between males and females, but these differences have not been quantified. We examined three aspects of genital morphology (relative anogenital distance, relative vent length, and relative external genital area) in two riverine and eleven Lake Malawi African cichlid species from eight genera. We find the most sexually distinct morphology in the Lake Malawi rock cichlids and the least sexual dimorphism in the riverine outgroup; additionally, diversity in metrics within genus indicates that these traits are recently evolving. Sexual dimorphism in morphology is present in most species, and, in the Lake Malawi species, multivariate discriminant analysis allows for accurate assignment of gonadal sex based on genital morphology and body size. This will serve as a useful method for sexing fish in a nonlethal fashion and provides a starting point for further examination of the evolution of genital morphology in this diverse group of fishes.     

Establishment and expansion of Lake Malawi rock fish populations after a dramatic Late Pleistocene lake level rise

Major environmental events that fragment populations among multiple island habitats have potential to drive large-scale episodes of speciation and adaptive radiation. A recent palaeolimnological study of sediment cores indicated that Lake Malawi underwent major climate-driven desiccation events 75 000–135 000 years ago that lowered the water level to at least 580 m below the present state and severely reduced surface area. After this period, lake levels rose and stabilized, creating multiple discontinuous littoral rocky habitats. Here, we present evidence supporting the hypothesis that establishment and expansion of isolated philopatric rock cichlid populations occurred after this rise and stabilization of lake level. We studied the Pseudotropheus ( Maylandia ) species complex, a group with both allopatric and sympatric populations that differ in male nuptial colour traits and tend to mate assortatively. Using coalescent analyses based on mitochondrial DNA, we found evidence that populations throughout the lake started to expand and accumulate genetic diversity after the lake level rise. Moreover, most haplotypes were geographically restricted, and the greatest genetic similarities were typically among sympatric or neighbouring populations. This is indicative of limited dispersal and establishment of assortative mating among populations following the lake level rise. Together, this evidence is compatible with a single large-scale environmental event being central to evolution of spatial patterns of genetic and species diversity in P. ( Maylandia ) and perhaps other Lake Malawi rock cichlids. Equivalent climate-driven pulses of habitat formation and fragmentation may similarly have contributed to observed rapid and punctuated cladogenesis in other adaptive radiations.