Mitochondrial genome primers for Lake Malawi cichlids

Resolving the evolutionary history of rapidly diversifying lineages like the Lake Malawi Cichlid Flock demands powerful phylogenetic tools. Although this clade of over 500 species of fish likely diversified in less than two million years, the availability of extensive sequence data sets, such as complete mitochondrial genomes, could help resolve evolutionary patterns in this group. Using a large number of newly developed primers, we generated whole mitochondrial genome sequences for 14 Lake Malawi cichlids. We compared sequence divergence across protein‐coding regions of the mitochondrial genome and also compared divergence in the mitochondrial loci to divergence at two nuclear protein‐coding loci, Mitfb and Dlx2. Despite the widespread sharing of haplotypes of identical sequences at individual loci, the combined use of all protein‐coding mitochondrial loci provided a bifurcating phylogenetic hypothesis for the exemplars of major lineages within the Lake Malawi cichlid radiation. The primers presented here could have substantial utility for evolutionary analyses of mitochondrial evolution and hybridization within this diverse clade.     

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.     

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.     

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.     

Intraspecific cone opsin expression variation in the cichlids of Lake Malawi

The expression of cone opsin genes is a primary determinant of the characteristics of colour vision. Interspecific variation in opsin expression is common in African cichlids. It is correlated with foraging among cichlids from Lake Malawi, and with ambient light environment among cichlids from Lake Victoria. In this study, we tested whether gene expression varied within species such that it might be important in contributing to divergence. We hypothesized that light attenuation with depth would be correlated with predictable changes in gene expression in Lake Malawi, and that this variation would tune visual sensitivities to match the ambient light environment. We observed significant differences in cone opsin expression in three different comparisons among populations of the same species. Higher LWS expression was found in shallow versus deep Copadichromis eucinostomus. In Metriaclima zebra, individuals from Zimbawe Rock expressed significantly more SWS2B than those from Thumbi West Island, although these locales have similar ambient light environments. Finally, Tropheops gracilior from deeper water had significantly more variation in expression than their shallow counterparts. These results support that gene expression varies significantly between populations of the same species. Surprisingly, these results could not be explained by predicted visual performance as models predicted that differential expression patterns did not confer sensitivity advantages at different depths. This suggested that expression variation did not confer a local sensitivity advantage. Therefore, our findings were contrary to a primary requirement of the sensory bias hypothesis. As such, other explanations for intraspecific gene expression variation need to be tested.     

Divergence with gene flow in the rock-dwelling cichlids of Lake Malawi

Within the past two million years, more than 450 species of haplochromine cichlids have diverged from a single common ancestor in Lake Malawi. Several factors have been implicated in the diversification of this monophyletic clade, including changes in lake level and low levels of gene flow across limited geographic scales. The objectives of this study were to determine the effect of recent lake-level fluctuations on patterns of allelic diversity in the genus Metriaclima, to describe the patterns of population structure within this genus, and to identify barriers to migration. This was accomplished through an analysis of allele frequencies at four microsatellite loci. Twelve populations spanning four species within Metriaclima were surveyed. The effect of lake-level fluctuations can be seen in the reduced genetic diversity of the most recently colonized sites; however, genetic diversity is not depressed at the species level. Low levels of population structure exist among populations, yet some gene flow persists across long stretches of inhospitable habitat. No general barrier to migration was identified. The results of this study are interpreted with respect to several speciation models. Divergence via population bottlenecks is unlikely due to the large allelic diversity observed within each species. Genetic drift and microallopatric divergence are also rejected because some gene flow does occur between adjacent populations. However, the reduced levels of gene flow between populations does suggest that minor changes in the selective environment could cause the divergence of populations.     

Distribution and breeding biology of offshore cichlids in Lake Malawi/ Niassa

Synopsis Lake Malawi/Niassa is the second largest rift valley lake in Africa, with an area of 28 800 km², and an average and maximum depth of 292 m and>700 m, respectively. The lake is well known for the great diversity of fish occurring in the inshore zone. However, the offshore fish community is poorly documented. To rectify this, regular sampling was undertaken over two years, using trawl and gillnets at six offshore locations. This paper reports on the species composition, spatial distribution and breeding biology of the dominant cichlids species from the offshore pelagic zone. Cichlids formed approximately 88% of the offshore fish biomass. Most abundant were two species of zooplanktivores in the genus Diplotaxodon that made up 71% of the offshore fish biomass. An undescribed species, given the cheironym D. bigeye, was mainly found at a depth of 220 m during the day, but moved into near surface waters at night when the moon was full. This species was absent from the shallow regions of the lake. The most abundant offshore species was D. limnothrissa, which was distributed evenly throughout the lake to depths of 220 m. A less common offshore zooplanktivore was Copadichromis quadrimaculatus that formed 5% of the biomass and was confined to the upper 100 m of the water column. The main piscivores were in the genus Rhamphochromis and formed approximately 10% of the offshore fish biomass. The two dominant taxa were R. longiceps and the large Rhamphochromis group, and both were more common in the southern half of the lake. The former occurred mainly in the upper 100 m of the water column and the latter mainly at depths of 100–150 m. The length at maturity and fecundity for the dominant offshore species were estimated and seasonal breeding cycles determined from gonad activity and gonado-somatic indices.     

Sensory basis for detection of benthic prey in two Lake Malawi cichlids

The adaptive radiations of African cichlids resulted in a diversity of feeding morphologies and strategies, but the role of sensory biology in prey detection and feeding ecology remains largely unexplored. Two endemic Lake Malawi cichlid genera, Tramitichromis and Aulonocara, feed on benthic invertebrates, but differ in lateral line morphology (narrow and widened lateral line canals, respectively) and foraging strategy. The hypothesis that they use their lateral line systems differently was tested by looking at the relative contribution of the lateral line system and vision in prey detection by Tramitichromis sp. and comparing results to those from a complementary study using Aulonocara stuartgranti (Schwalbe et al., 2012). First, behavioral trials were used to assess the ability of Tramitichromis sp. to detect live (mobile) and dead (immobile) benthic prey under light and dark conditions. Second, trials were run before, immediately after, and several weeks after chemical ablation of the lateral line system to determine its role in feeding behavior. Results show that Tramitichromis sp. is a visual predator that neither locates prey in the dark nor depends on lateral line input for prey detection and is thus distinct from A. stuartgranti, which uses its lateral line or a combination of vision and lateral line to detect prey depending on light condition. Investigating how functionally distinctive differences in sensory morphology are correlated with feeding behavior in the laboratory and determining the role of sensory systems in feeding ecology will provide insights into how sensory capabilities may contribute to trophic niche segregation.     

Comparative analysis reveals signatures of differentiation amid genomic polymorphism in Lake Malawi cichlids

Background  

Cichlid fish from East Africa are remarkable for phenotypic and behavioral diversity on a backdrop of genomic similarity. In 2006, the Joint Genome Institute completed low coverage survey sequencing of the genomes of five phenotypically and ecologically diverse Lake Malawi species. We report a computational and comparative analysis of these data that provides insight into the mechanisms that make closely related species different from one another.     

Genetic population structure indicates sympatric speciation of Lake Malawi pelagic cichlids.

Allopatric processes of speciation have routinely been presented to explain the extraordinary radiation of the East African Great Lakes cichlid fish species flocks. The 21 or more species of pelagic cichlids within the Lake Malawi flock appear to have lake-wide distributions that challenge such a concept. Data from six microsatellite DNA loci indicate single, panmictic populations across the lake of three Diplotaxodon species. Levels of variability at these loci suggest that populations have been large and stable. Mitochondrial DNA sequence data (872 bp of control region + 981 bp of the NADH-2) from 90 species, representing all major clades within the Lake Malawi flock, indicate reciprocal monophyly of the pelagic clade. We suggest that these data support a hypothesis that speciation in sympatry is more plausible (and widespread) within the cichlid species flocks than previously thought.     

Secondary contact during adaptive radiation: a community matrix for Lake Malawi cichlids

We used a community of Lake Malawi rock‐dwelling cichlids to study secondary contact during adaptive radiation. Using abundance data from survey plots we constructed a matrix of pair‐wise interaction coefficients for males of 21 native and eight transplanted species. After controlling for the effects of habitat variation, correlations among residual male abundances suggest that coevolved species compete less than those brought into artificial secondary contact 30 years ago and that species with the same body colour compete more than those with different body colours. The latter result provides evidence that a trait related to reproductive isolation affects competitive interactions and the distribution of individuals throughout an entire community. Our results further suggest lake level fluctuations that divide and reconnect communities act to increase local (alpha), as well as total (gamma) diversity, in this adaptive radiation. The communities are not, however, unsaturated in the simplest sense; new species can enter a community, but they disproportionately reduce the abundance of original community members.     

Homing and home-site fidelity in rock-dwelling cichlids (Pisces: Teleostei) of Lake Malawi,Africa

Synopsis Existence of home site fidelity and homing ability was established for Pseudotropheus aurora. Territorial males stayed up to 1 1/2 years within the same territories. Females showed long-term fidelity to certain places. Transplantation experiments revealed that P. aurora males home over distances of up to at least 2500 m but would not cross deep water. The territorial males of five other sympartic species returned from 1000 m to their territories. The mechanisms and functions of homing in cichlids are discussed.     

Sexually dimorphic levels of color trait integration and the resolution of sexual conflict in Lake Malawi cichlids

East African cichlids are renowned for their propensity to radiate, and variation in color patterns accounts for much of endemic cichlid diversity. Sexual dimorphism in color among cichlid species likely represents the outcome of different selective regimes acting on each sex, and is a classic example of sexual conflict. It is generally assumed that this conflict has been mitigated through the evolution of sex-linked color polymorphisms. Here, we propose that the evolution of sex-specific differences in levels of color trait integration may represent an additional mechanism through which sexual conflict has been resolved in this group. Specifically, we predict: (1) that general patterns of integration are influenced by early developmental events and thus conserved across sexes and (2) that male color is less integrated than females, and thus more evolvable in terms of producing an elaborate palette (i.e., in response to sexual selection), whereas female color is more integrated, facilitating wholesale shifts in color for background matching (i.e., in response to natural selection for crypsis). We tested these hypotheses using an F(2) design to compare the segregation of male and female color patterns. Both exploratory methods and hypothesis-driven analyses of integration demonstrate that the covariance structure of color traits in males and females is distinct, and that males are significantly less integrated than females. We suggest that the ability of species to promote different levels, and to a lesser extent patterns, of phenotypic integration between males and females may have contributed to the evolutionary success of this group.     

Repeated trans-watershed hybridization among haplochromine cichlids (Cichlidae) was triggered by Neogene landscape evolution

The megadiverse haplochromine cichlid radiations of the East African lakes, famous examples of explosive speciation and adaptive radiation, are according to recent studies, introgressed by different riverine lineages. This study is based on the first comprehensive mitochondrial and nuclear DNA dataset from extensive sampling of riverine haplochromine cichlids. It includes species from the lower River Congo and Angolan (River Kwanza) drainages. Reconstruction of phylogenetic hypotheses revealed the paradox of clearly discordant phylogenetic signals. Closely related mtDNA haplotypes are distributed thousands of kilometres apart and across major African watersheds, whereas some neighbouring species carry drastically divergent mtDNA haplotypes. At shallow and deep phylogenetic layers, strong signals of hybridization are attributed to the complex Late Miocene/Early Pliocene palaeohistory of African rivers. Hybridization of multiple lineages across changing watersheds shaped each of the major haplochromine radiations in lakes Tanganyika, Victoria, Malawi and the Kalahari Palaeolakes, as well as a miniature species flock in the Congo basin (River Fwa). On the basis of our results, introgression occurred not only on a spatially restricted scale, but massively over almost the whole range of the haplochromine distribution. This provides an alternative view on the origin and exceptional high diversity of this enigmatic vertebrate group.     

Phylogeography of Lake Malawi cichlids of the genus Pseudotropheus: significance of allopatric colour variation

One of the most compelling features of the cichlid fishes of the African Great Lakes is the seemingly endless diversity of male coloration. Colour diversification has been implicated as an important factor driving cichlid speciation. Colour has also been central to cichlid taxonomy and, thus, to our concept of species diversity. We undertook a phylogeographical examination of several allopatric populations of the Lake Malawi cichlid Pseudotropheus zebra in order to reconstruct the evolutionary history of the populations, which exhibit one of two dorsal fin colours. We present evidence that populations with red dorsal fins (RT) are not monophyletic. The RT population defining the northern limit of the distribution has evidently originated independently of the southern RT populations, which share a common ancestry. This evidence of species-level colour convergence is an important discovery in our understanding of cichlid evolution. It implies that divergence in coloration may accompany speciation, and that allopatric populations with similar coloration cannot be assumed to be conspecific. In addition to this finding, we have observed evidence for introgression, contributing to current evidence that this phenomenon may be extremely widespread. Thus, in species-level phylogenetic reconstructions, including our own, consideration must be given to the potential effects of introgression.     

Microsatellite paternity analysis on captive Lake Malawi cichlids supports reproductive isolation by direct mate choice

Three sympatrically occurring members of a cichlid fish species complex from Lake Malawi have previously been reported to show assortative mating in the field. Offspring from three experimental tanks each housing both sexes of all three members of this complex were screened at one to four microsatellite loci to determine paternity. Reproductive isolation was found to persist without exception under laboratory conditions, providing strong evidence for behavioural barriers to mating. Direct mate choice is suggested as the process preventing interbreeding. The results are interpreted with reference to the rapid and explosive evolution of the cichlid group.     

Mix and match color vision: tuning spectral sensitivity by differential opsin gene expression in Lake Malawi cichlids

Cichlid fish of the East African Rift Lakes are renowned for their diversity and offer a unique opportunity to study adaptive changes in the visual system in rapidly evolving species flocks. Since color plays a significant role in mate choice, differences in visual sensitivities could greatly influence and even drive speciation of cichlids. Lake Malawi cichlids inhabiting rock and sand habitats have significantly different cone spectral sensitivities. By combining microspectrophotometry (MSP) of isolated cones, sequencing of opsin genes, and spectral analysis of recombinant pigments, we have established the cone complements of four species of Malawi cichlids. MSP demonstrated that each of these species predominately expresses three cone pigments, although these differ between species to give three spectrally different cone complements. In addition, rare populations of spectrally distinct cones were found. In total, seven spectral classes were identified. This was confirmed by opsin gene sequencing, expression, and in vitro reconstitution. The genes represent the four major classes of cone opsin genes that diverged early in vertebrate evolution. All four species possess a long-wave-sensitive (LWS), three spectrally distinct green-sensitive (RH2), a blue-sensitive (SWS2A), a violet-sensitive (SWS2B), and an ultraviolet-sensitive (SWS1) opsin. However, African cichlids determine their spectral sensitivity by differential expression of primarily only three of the seven available cone opsin genes. Phylogenetic analysis suggests that all percomorph fish have similar potential.     

Extensive homoplasy, nonstepwise mutations, and shared ancestral polymorphism at a complex microsatellite locus in Lake Malawi cichlids

Recent studies have suggested that size homoplasy is a prevalent feature of microsatellites and is expected to increase with time of divergence among populations and taxa. In this study, we performed sequence analysis of alleles from a complex microsatellite locus (Pzeb4, initially isolated from Pseudotropheus (Maylandia) zebra) from 1 midwater-feeding and 10 rock-dwelling cichlid fish species from Lake Malawi, East Africa, to investigate how widespread size homoplasy is among closely related taxa at this locus. All cichlid fishes endemic to this lake are believed to have originated within the last 700,000 years, and some species may be less than 200 years old. The number of eletromorphs found per species varied from 3 to 13. Sequence analysis of 95 cloned Pzeb4 PCR products (representing 18 electromorphs) revealed 13 new alleles. Ten of the 13 electromorphs (77%) were found to show size homoplasy due to either single nucleotide substitutions/indels or large indels. To investigate how well this locus fits the single-step mutation model (SMM), the minimum number of mutations required to explain the length differences between pairs of alleles was plotted against their size differences. Of the 300 comparisons, 166 (55.3%) corresponded to SMM expectations and 86 (28.7%) required a smaller number of mutations, and for 48 (16.0%) pairwise comparisons, a larger number of mutations were required to explain the length differences as compared with SMM expectations. Finally, a large deletion in the microsatellite sequence observed in the three rock-dwelling species Pseudotropheus lucerna, Pseudotropheus (Tropheops) 'band,' and Pseudotropheus (Tropheops) 'rust' and the midwater-feeding species Copadichromis sp. is believed to represent a shared ancestral polymorphism.     

A genetic demographic analysis of Lake Malawi rock‐dwelling cichlids using spatio‐temporal sampling

We estimated the effective population sizes (N_e) and tested for short‐term temporal demographic stability of populations of two Lake Malawi cichlids: Maylandia benetos, a micro‐endemic, and Maylandia zebra, a widespread species found across the lake. We sampled a total of 351 individuals, genotyped them at 13 microsatellite loci and sequenced their mitochondrial D‐loop to estimate genetic diversity, population structure, demographic history and effective population sizes. At the microsatellite loci, genetic diversity was high in all populations. Yet, genetic diversity was relatively low for the sequence data. Microsatellites yielded mean N_e estimates of 481 individuals (±99 SD) for M. benetos and between 597 (±106.3 SD) and 1524 (±483.9 SD) individuals for local populations of M. zebra. The microsatellite data indicated no deviations from mutation–drift equilibrium. Maylandia zebra was further found to be in migration–drift equilibrium. Temporal fluctuations in allele frequencies were limited across the sampling period for both species. Bayesian Skyline analyses suggested a recent expansion of M. zebra populations in line with lake‐level fluctuations, whereas the demographic history of M. benetos could only be estimated for the very recent past. Divergence time estimates placed the origin of M. benetos within the last 100 ka after the refilling of the lake and suggested that it split off the sympatric M. zebra population. Overall, our data indicate that micro‐endemics and populations in less favourable habitats have smaller N_e, indicating that drift may play an important role driving their divergence. Yet, despite small population sizes, high genetic variation can be maintained.