Peripheral Isolate Speciation of a Lake Malawi Cichlid Fish from Shallow Muddy Habitats

Much of the exceptional diversity of cichlid fishes in the African Great Lakes can be explained by geographic variation among isolated populations of species specialised to live on patchily distributed rocky habitat. However, there are also many endemic species that are not specialised for rocky shores. These appear to experience weaker geographic isolation. Major decreases in lake volume may have segregated such populations in isolated refugia in the distant past, but subsequent range changes have likely eliminated most of the phylogeographic signal of these events. Divergence in currently isolated peripheral water bodies may be more amenable to the study of recent processes of allopatric divergence. We investigate a haplochromine cichlid fish, here referred to as Lethrinops sp. ‘chilingali’, isolated in a small satellite lake near Lake Malawi, and the candidate sister taxon, Lethrinops lethrinus, which inhabits shallow muddy habitats in the main lake and associated water bodies. The satellite lake form from Lake Chilingali showed significant morphological differentiation, with a less ventrally-placed mouth and shorter snout, associated with a shift in diet from a diverse range of benthic invertebrates towards specialisation on mid-water chaoborid larvae and pupae. The Lake Chilingali population showed substantially reduced mitochondrial DNA diversity and no haplotype sharing was observed with populations from the main lake system. In laboratory experiments, putative species showed a high degree of assortative mating and territorial males were significantly more aggressive towards intruders of their own population. This study adds to the evidence that rapid evolution of novel phenotypes in peripheral habitats can add to the diversity of lacustrine cichlids through the evolution of at least partial reproductive isolation in allopatry.     

Quantitative Genetic Analyses of Male Color Pattern and Female Mate Choice in a Pair of Cichlid Fishes of Lake Malawi,East Africa

The traits involved in sexual selection, such as male secondary sexual characteristics and female mate choice, often co-evolve which can promote population differentiation. However, the genetic architecture of these phenotypes can influence their evolvability and thereby affect the divergence of species. The extraordinary diversity of East African cichlid fishes is often attributed to strong sexual selection and thus this system provides an excellent model to test predictions regarding the genetic architecture of sexually selected traits that contribute to reproductive isolation. In particular, theory predicts that rapid speciation is facilitated when male sexual traits and female mating preferences are controlled by a limited number of linked genes. However, few studies have examined the genetic basis of male secondary sexual traits and female mating preferences in cichlids and none have investigated the genetic architecture of both jointly. In this study, we artificially hybridized a pair of behaviorally isolated cichlid fishes from Lake Malawi and quantified both melanistic color pattern and female mate choice. We investigated the genetic architecture of both phenotypes using quantitative genetic analyses. Our results suggest that 1) many non-additively acting genetic factors influence melanistic color patterns, 2) female mate choice may be controlled by a minimum of 1–2 non-additive genetic factors, and 3) F₂ female mate choice is not influenced by male courting effort. Furthermore, a joint analysis of color pattern and female mate choice indicates that the genes underlying these two traits are unlikely to be physically linked. These results suggest that reproductive isolation may evolve rapidly owing to the few genetic factors underlying female mate choice. Hence, female mate choice likely played an important role in the unparalleled speciation of East African cichlid fish.     

Comparative Morphometrics and Associated Growth Trends of Two Benthophagous Cichlid Species from Lake Malawi (Pisces, Perciformes)

Body shape differences between two benthophagous, sympatric cichlid species, Ctenopharynx pictus and Otopharynx sp. “heterodon nankhumba” were examined for 14 morphometric characters using measurements obtained from 11 morphological landmarks and additional 6 characters from the head region using principal component and regression analyses. The two species, though not closely related, superficially resemble each other and, being benthic feeders, directly compete for food resources unless morphological differences lead to trophic specialization. Hence, the present study explores the presence of such morphological variations. Principal component analysis (PCA), revealed that only four characters loaded highly on PC II and, therefore, account for most of the variations in the external morphology between the two species. C. pictus has longer lower jaws reflecting longer gape, and a shorter abdominal cavity than O. sp. “heterodon nakhumba”. The regression analyses showed significant differences in the allometric coefficients of the lower jaw and abdominal cavity, which might have contributed to differences in overall shape detected between the two species. The difference in gape size between the two species may be related to different feeding habits employed by each species, and such specializations are suggested to reduce interspecific competition for food resources. We suggest that the ensuing ecological separation promotes sympatry of these two species.     

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     

Molecular Characterization of Two Endothelin Pathways in East African Cichlid Fishes

The adaptive radiations of cichlid fishes in East Africa have been associated with the acquisition of evolutionary novelties as well as the ecological opportunities existing in the East African Great lakes. Two remarkable evolutionary innovations are the pharyngeal jaw apparatus, found in all cichlid species, and the anal fin egg-spots of mouthbrooding cichlids. Based on their conserved functions during the development of both the jaw apparatus and pigmentation, the endothelin ligands and receptors form a putative link between these naturally and sexually selected traits. Here we study the evolutionary history of four members of two endothelin pathways (Edn1/EdnrAa and Edn3b/EdnrB1a) to elucidate their possible roles during the evolution and development of key innovations in East African cichlids species. The analyses performed on partial sequences (ca. 6,000 bp per taxon) show that all four endothelin family members evolved under purifying selection, although both ligands are characterized by an accelerated rate of protein evolution in comparison to the receptors. In accordance with earlier findings, we show that the mature protein sequence of Edn1 and Edn3 are highly conserved, also in cichlids, whereas the preproendothelin parts are variable indicating relaxed selective constraints. In the receptors, nonsynonymous substitutions were mainly found in the ligand-binding domains suggesting functional divergence. Gene expression assays with Real-Time PCR indeed reveal that the two studied endothelin pathways are expressed in the cichlid pharyngeal jaw and in the haplochromine egg-spot (among other pigment-cell containing tissues), suggesting their involvement during morphogenesis of naturally and sexually selected traits in cichlids.     

Allelic Variation in Malawi Cichlid Opsins: A Tale of Two Genera

The role of sequence variation in the spectral tuning of color vision is well established in many systems. This includes the cichlids of Lake Victoria where sequence variation has been linked to environmental light gradients and speciation. The cichlids of Lake Malawi are a similar model for visual evolution, but the role of gene sequence variation in visual tuning between closely related species is unknown. This work describes such variation in multiple species of two rock-dwelling genera: Metriaclima and Labidochromis. Genomic DNA for seven cone opsin genes was sequenced and the structure of the opsin proteins was inferred. Retinal binding pocket polymorphisms were identified and compared to available data regarding spectral absorbance shifts. Sequence variation with known or potential effects on absorbance spectra were found in four genes: SWS1 (UV sensitive), SWS2B (violet sensitive), RH2Aβ (green sensitive), and LWS (red sensitive). Functional variation was distributed such that each genus had both a variable short-wavelength and long-wavelength sensitive opsin. This suggests spectral tuning is important at the margins of the cichlid visual spectrum. Further, there are two SWS1 opsin alleles that differ in sensitivity by 10 nm and are >2 MY divergent. One of these occurs in a haplotype block >1 kb. Potential haplotype blocks were found around the RH2 opsin loci. These data suggest that molecular diversification has resulted in functionally unique alleles and changes to the visual system. These data also suggest that opsin sequence variation tunes spectral sensitivities between closely related species and that the specific regions of spectral tuning are genus-specific.     

Complete Mitochondrial DNA Sequences of the Threadfin Cichlid (Petrochromis trewavasae) and the Blunthead Cichlid (Tropheus moorii) and Patterns of Mitochondrial Genome Evolution in Cichlid Fishes

The cichlid fishes of the East African Great Lakes represent a model especially suited to study adaptive radiation and speciation. With several African cichlid genome projects being in progress, a promising set of closely related genomes is emerging, which is expected to serve as a valuable data base to solve questions on genotype-phenotype relations. The mitochondrial (mt) genomes presented here are the first results of the assembly and annotation process for two closely related but eco-morphologically highly distinct Lake Tanganyika cichlids, Petrochromis trewavasae and Tropheus moorii. The genomic sequences comprise 16,588 bp (P. trewavasae) and 16,590 bp (T. moorii), and exhibit the typical mitochondrial structure, with 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and a non-coding control region. Analyses confirmed that the two species are very closely related with an overall sequence similarity of 96%. We analyzed the newly generated sequences in the phylogenetic context of 21 published labroid fish mitochondrial genomes. Consistent with other vertebrates, the D-loop region was found to evolve faster than protein-coding genes, which in turn are followed by the rRNAs; the tRNAs vary greatly in the rate of sequence evolution, but on average evolve the slowest. Within the group of coding genes, ND6 evolves most rapidly. Codon usage is similar among examined cichlid tribes and labroid families; although a slight shift in usage patterns down the gene tree could be observed. Despite having a clearly different nucleotide composition, ND6 showed a similar codon usage. C-terminal ends of Cox1 exhibit variations, where the varying number of amino acids is related to the structure of the obtained phylogenetic tree. This variation may be of functional relevance for Cox1 synthesis.     

Lipid Vesicle Shape Analysis from Populations Using Light Video Microscopy and Computer Vision

We present a method for giant lipid vesicle shape analysis that combines manually guided large-scale video microscopy and computer vision algorithms to enable analyzing vesicle populations. The method retains the benefits of light microscopy and enables non-destructive analysis of vesicles from suspensions containing up to several thousands of lipid vesicles (1–50 µm in diameter). For each sample, image analysis was employed to extract data on vesicle quantity and size distributions of their projected diameters and isoperimetric quotients (measure of contour roundness). This process enables a comparison of samples from the same population over time, or the comparison of a treated population to a control. Although vesicles in suspensions are heterogeneous in sizes and shapes and have distinctively non-homogeneous distribution throughout the suspension, this method allows for the capture and analysis of repeatable vesicle samples that are representative of the population inspected.     

Different Sources of Allelic Variation Drove Repeated Color Pattern Divergence in Cichlid Fishes

The adaptive radiations of East African cichlid fish in the Great Lakes Victoria, Malawi, and Tanganyika are well known for their diversity and repeatedly evolved phenotypes. Convergent evolution of melanic horizontal stripes has been linked to a single locus harboring the gene agouti-related peptide 2 (agrp2). However, where and when the causal variants underlying this trait evolved and how they drove phenotypic divergence remained unknown. To test the alternative hypotheses of standing genetic variation versus de novo mutations (independently originating in each radiation), we searched for shared signals of genomic divergence at the agrp2 locus. Although we discovered similar signatures of differentiation at the locus level, the haplotypes associated with stripe patterns are surprisingly different. In Lake Malawi, the highest associated alleles are located within and close to the 5′ untranslated region of agrp2 and likely evolved through recent de novo mutations. In the younger Lake Victoria radiation, stripes are associated with two intronic regions overlapping with a previously reported cis-regulatory interval. The origin of these segregating haplotypes predates the Lake Victoria radiation because they are also found in more basal riverine and Lake Kivu species. This suggests that both segregating haplotypes were present as standing genetic variation at the onset of the Lake Victoria adaptive radiation with its more than 500 species and drove phenotypic divergence within the species flock. Therefore, both new (Lake Malawi) and ancient (Lake Victoria) allelic variation at the same locus fueled rapid and convergent phenotypic evolution.     

Adaptive Significance of Intra- and Interspecific Differences in the Feeding Repertoires of Cichlid Fishes

Intraspecific modulatory multiplicity in the feeding mechanismof cichlids as elucidated by electromyography has profound implicationson small-scale trophic events the sum of which is the very coreof such large-scale population and evolutionary phenomena asefficiency of trophic exploitation niche width and overlap competitionand adaptation. The greatest paradox emerging from the studyon intra and interspecific differences in feeding repertoiresof cichlid fishes is that the most specialized taxa are notonly remarkable specialists in a narrow sense but also jacks-of-all-trades.If specialists are simultaneously jacks of-all-trades how couldthey have evolved according to the widely accepted hypothesisthat broadening the range of usable resources prevents speciesfrom specializing on individual types? The organism can be considered to be composed of structuralelements and functional components that exert mutual influences.As a result, a network of interacting constraints is set up.The nature of the network determines the direction and rangeof evolutionary changes the possibilities of optimizing adaptationsand built-in variability of the trait. A change in the networkcan put a static trait into a different context to become dynamic.These changes in variability due to changes in the structureof the network cannot be explained in terms of simple adaptationto the external environment. Fitness is considered to dependon the nature of a network of interacting constraints. The possibilitiesof optimization the sensitivity of the phenotypic traits toenvironmental differences and the correlated evolutionary responsesof different traits are all related to this network of interactingconstraints between the elements and components of the organism.Thus the experimental and comparative studies of these networksof interacting constraints should become an increasingly moreimportant focus for morphologists as they attempt to refinetheir understanding of adaptation.     

Plasticity of opsin gene expression in cichlids from Lake Malawi

Sensory systems play crucial roles in survival and reproduction. Therefore, sensory plasticity has important evolutionary implications. In this study, we examined retinal plasticity in five species of cichlid fish from Lake Malawi. We compared the cone opsin expression profiles of wild‐caught fish to lab‐reared F₁ that had been raised in a UV minus, reduced intensity light environment. All of the opsin genes that were expressed in wild‐caught fish were also expressed in lab‐reared individuals. However, we found statistically significant differences in relative opsin expression among all five species. The most consistent difference was in the SWS2B (violet) opsin, which was always expressed at higher levels in lab‐reared individuals. Estimates of visual pigment quantum catch suggest that this change in expression would increase retinal sensitivity in the light environment of the lab. We also found that the magnitude of plasticity varied across species. These findings have important implications for understanding the genetic regulation of opsin expression and raise many interesting questions about how the cichlid visual system develops. They also suggest that sensory plasticity may have facilitated the ecological diversification of cichlids in Lake Malawi.     

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.     

Geography disentangles introgression from ancestral polymorphism in Lake Malawi cichlids

Phenotypically diverse Lake Malawi cichlids exhibit similar genomes. The extensive sharing of genetic polymorphism among forms has both intrigued and frustrated biologists trying to understand the nature of diversity in this and other rapidly evolving systems. Shared polymorphism might result from hybridization and/or the retention of ancestrally polymorphic alleles. To examine these alternatives, we used new genomic tools to characterize genetic differentiation in widespread, geographically structured populations of Labeotropheus fuelleborni and Metriaclima zebra. These phenotypically distinct species share mitochondrial DNA (mtDNA) haplotypes and show greater mtDNA differentiation among localities than between species. However, Bayesian analysis of nuclear single nucleotide polymorphism (SNP) data revealed two distinct genetic clusters corresponding perfectly to morphologically diagnosed L. fuelleborni and M. zebra. This result is a function of the resolving power of the multi‐locus dataset, not a conflict between nuclear and mitochondrial partitions. Locus‐by‐locus analysis showed that mtDNA differentiation between species (F_CT) was nearly identical to the median single‐locus SNP F_CT. Finally, we asked whether there is evidence for gene flow at sites of co‐occurrence. We used simulations to generate a null distribution for the level of differentiation between co‐occurring populations of L. fuelleborni and M. zebra expected if there was no hybridization. The null hypothesis was rejected for the SNP data; populations that co‐occur at rock reef sites were slightly more similar than expected by chance, suggesting recent gene flow. The coupling of numerous independent markers with extensive geographic sampling and simulations utilized here provides a framework for assessing the prevalence of gene flow in recently diverged species.     

Polygynandry inPseudotropheus zebra, a cichlid fish from Lake Malawi

Synopsis Parental care in the Malawian cichlid fishPseudotropheus zebra BB is extensive and exclusively maternal; males contribute only genetic material. The costs of searching for multiple mates (in this case risk of predation on orally incubated eggs) suggested that females should be monandrous; microsatellite genetypes of seven brooding females and their young, however, reveal extensive multiple paternity in this species, with a mean of 3.8 paternal individuals per brood. Polygynandry inP. zebra is probably not maintained by selection for genetically diverse offspring; potential explanations include avoidance of inbreeding, and bet-hedging on other male characteristics that females are unable to evaluate when selecting a mate. The observed degree of multiple paternity strongly suggests that females are free to choose mates as they will, a prerequisite of many theories positing sexual selection as a key element in Malawi chichlid evolution. It should also result in elevation of effective population sizes, and thus be antagonistic to runaway evolution of male secondary sexual characteristics, but not necessarily to other modes of sexual selection.     

Depth equilibration by two predatory cichlid fish from Lake Malawi

The cichlid fish, Haplochromis polystigma and Haplochromis livingstonii are piscivorous ambush predators endemic to Lake Malawi, Africa. Experimentally it was found that large adult H. livingstonii could equilibrate to 38 m, but that juveniles were restricted to shallower depths. Mean depth attained by 8 H. polystigma was 45 m but two individuals exceeded the maximum simulated depth (59 m). H. livingstonii had a mean equilibration rate of 4–7 m day⁻¹ for the first 10 m declining to 2·4 m day⁻¹ thereafter. The mean rate of descent for H. polystigma was 3·8 m day⁻¹ for the first 10 m and 2·8 m day⁻¹ thereafter. The fish were decompressed at a rate of a 20% decrease every 12 h. Although cichlid fishes occur down to 200 m depth in Lake Malawi, experimental and field data indicate that the depth distribution of individual species is restricted and that the restriction is largely related to swimbladder physiology. All cichlid species so far tested equilibrate slowly to depth (2 to 5 m a day) and are not capable of rapid decompression.     

Speciation in rapidly diverging systems: lessons from Lake Malawi

Rapid evolutionary radiations provide insight into the fundamental processes involved in species formation. Here we examine the diversification of one such group, the cichlid fishes of Lake Malawi, which have radiated from a single ancestor into more than 400 species over the past 700 000 years. The phylogenetic history of this group suggests: (i) that their divergence has proceeded in three major bursts of cladogenesis; and (ii) that different selective forces have dominated each cladogenic event. The first episode resulted in the divergence of two major lineages, the sand- and rock-dwellers, each adapted to a major benthic macrohabitat. Among the rock-dwellers, competition for trophic resources then drove a second burst of cladogenesis, which resulted in the differentiation of trophic morphology. The third episode of cladogenesis is associated with differentiation of male nuptial colouration, most likely in response to divergent sexual selection. We discuss models of speciation in relation to this observed pattern. We advocate a model, divergence with gene flow, which reconciles the disparate selective forces responsible for the diversification of this group and suggest that the nonadaptive nature of the tertiary episode has significantly contributed to the extraordinary species richness of this group.     

Identification of the Lake Malawi Oreochromis (Nyasalapia) spp. using multivariate morphometric techniques

With the aim of reliably distinguishing these commercially important species on the basis of external characteristics alone, morphometric techniques were employed on a sample of the four species of Oreochromis ( Nyasalapia ) described from Lake Malawi: O. karongae, O. lidole, O. saka and O. squamipinnis . Univariate analysis of variance on the ratios of 23 variables to standard length indicated many differences among all species, but there was considerable individual variation, and consequent overlap. Residuals from a regression of each variable on length were employed for multivariate analysis. Cluster analysis on the means of the residuals was used to construct a phenogram which formed the basis for denning a series of dichotomous discriminant analyses. In each discriminant analysis, variables were successively eliminated in the reverse order of the magnitude of their correlation with the discriminant function. The combination of variables producing 95% accuracy of classification was selected, and the discriminant function equations for each step calculated. Some further variables were eliminated by checking for redundancy through analysis of correlations. The resulting equations enable O. karongae to be separated using eight measurements, O. lidole using 10, and O. saka and O. squamipinnis to be distinguished by a combination of 13 measurements.     

Some effects of demersal trawling in Lake Malawi (Lake Nyasa) from 1968 to 1974

Exploratory trawl data were used to define demersal fish stocks of Lake Malawi in terms of abundance by depth and area. The mean weight of the catch generally declined with increasing depth. A comparison of initial standing stock estimates and the yield from a commercial fishery over a 7 year period suggests a potential yield of less than half of the initial stock. Over 160 species of fish have been identified in the trawl catch of which over 80% by weight were cichlids. Some of the commonest species of cichlids are listed. The composition of the catch in the heavily exploited southern end of the lake has changed from one dominated by large species of Haplochromis sp. and Lethrinops sp. to one dominated by smaller species.     

Evolutionary relationships of the Lake Malawi Oreochromis species: evidence from allozymes

Three tilapiine species belonging to the endemic Lake Malawi species flock known as' chambo' Oreochromis ( Nyasalapia ) karongae , O. (N.) lidole and O. (N.) squamipinnis and the species Oreochromis (Oreochromis) shiranus were collected from the wild. These four species were analysed at 43 enzyme loci using starch gel electrophoresis. No significant deviations from Hardy-Weinberg equilibrium were observed after correcting for multiple simultaneous testing. The expected heterozygosity ( H _e) was lowest in O. (O.) shiranus ( H _e=0.082). The three chambo species had higher levels: H _e=0.103 to H _e=0.116. The three chambo species as a whole were polymorphic (99%) at 16 different loci sharing 10 of these in common. No fixed differences between the three chambo species were observed but highly significant allele frequency differences existed between all the chambo species. The F _ST calculated for the three chambo species was 0051, closer to intra-specific than to inter-specific levels. O. (O.) shiranus could be clearly separated from all chambo species at five fixed loci. Comparison of the allozyme data from these species with five species from the same sub genera support the hypothesis that the chambo form a monophyletic group and that O. (N.) macrochir or a related species represents the sister taxon.