Construction of a BAC library for Haplochromis chilotes,a cichlid fish from Lake Victoria

Cichlid fishes in Lake Victoria are model organisms for studying rapid radiation and speciation. On the way to examine the molecular basis of how these cichlid fishes achieved such a remarkable morphological diversification, we constructed a bacterial artificial chromosome (BAC) library derived from a cichlid species, Haplochromis chilotes, from Lake Victoria. The library includes 157,056 clones with the average insert size of 128 kb, corresponding to a 10-fold coverage of the H. chilotes genome. Given that the cichlid fishes endemic to Lake Victoria are closely related to one another phylogenetically and their genomes are nearly identical, this BAC library can be utilized to isolate genes from the more than 200 Haplochromine cichlid species in Lake Victoria.     

Shaping of the lower jaw bone during growth of Nile tilapia Oreochromis niloticus and a Lake Victoria cichlid Haplochromis chilotes: a geometric morphometric approach

East African cichlids have evolved feeding apparatus morphologies to adapt to diverse feeding environments. However, little is known about how the morphologies are formed during development. Here, we assessed the shape changes of the lower jaw bone during growth of the Nile tilapia Oreochromis niloticus and a Lake Victoria cichlid Haplochromis chilotes using geometric morphometric methods. 'Early Juvenile to Late Juvenile' and 'Late Juvenile to Adult' transitions of the shape change during growth of both O. niloticus and H. chilotes were detected. The 'Early Juvenile to Late Juvenile' transition of the shape change in H. chilotes occurred slightly earlier than in O. niloticus. We also compared the shape changes during growth of the two cichlids. Principal component analysis showed both commonalities and differences in the morphological changes between the cichlids. Our data suggest that most shape change may have a similar pattern during the growth of O. niloticus and H. chilotes, and that the differences in adult shapes may be due to differences arising early in development, not to the difference of shape change during growth. These data provide a basis for understanding the developmental mechanisms underlying this adaptive trait of East African cichlids.     

PERMANENT GENETIC RESOURCES: Isolation and characterization of microsatellite loci in the cichlid fish in Lake Victoria, Haplochromis chilotes

Twelve short tandem repeat markers were successfully isolated from a cichlid, Haplochromis chilotes, in Lake Victoria, and characterized in Haplochromis pyrrhocephalus. The microsatellite regions of these markers were found to have between two and 48 alleles with heterozygosity ranging from 0.07 to 0.97. No loci showed significant departures from the Hardy–Weinberg or linkage equilibrium after the Bonferroni correction (P > 0.05). Cross‐species amplification in other cichlids of Lake Victoria, Haplochromis laparogramma, Lithochromis rubripinnis, L. rufus and Haplochromis sp. ‘rockkribensis’, was successful.     

Gene flow between species of Lake Victoria haplochromine fishes

The haplochromine cichlid fishes of Lake Victoria (LV), East Africa, are a textbook example of adaptive radiation-a rapid divergence of multiple morphologically distinguishable forms from a few founding lineages. The forms are generally believed to constitute a "flock" of several hundred reproductively isolated species in a dozen or so genera. This belief has, until now, not been subjected to a test, however. Here, we compare genetic variation at 11 loci in 10 haplochromine populations of 6 different species. Although the genetic diversity in the populations is quite high, using a variety of statistical tests, we find no evidence of genetic differentiation among the populations of LV haplochromines. On genetic distance trees, populations of the same species intermingle with those of different species. At the molecular level, the species are indistinguishable from one another. Genetic comparisons with closely related species in 2 crater lakes indicate that the species within LV continue exchanging genes. These observations have important implications for phylogenetic reconstruction. The approach used in this study is applicable to other instances of adaptive radiation.     

Genomic Signatures for Species-Specific Adaptation in Lake Victoria Cichlids Derived from Large-Scale Standing Genetic Variation

The cichlids of Lake Victoria are a textbook example of adaptive radiation, as >500 endemic species arose in just 14,600 years. The degree of genetic differentiation among species is very low due to the short period of time after the radiation, which allows us to ascertain highly differentiated genes that are strong candidates for driving speciation and adaptation. Previous studies have revealed the critical contribution of vision to speciation by showing the existence of highly differentiated alleles in the visual opsin gene among species with different habitat depths. In contrast, the processes of species-specific adaptation to different ecological backgrounds remain to be investigated. Here, we used genome-wide comparative analyses of three species of Lake Victoria cichlids that inhabit different environments—Haplochromis chilotes, H. sauvagei, and Lithochromis rufus—to elucidate the processes of adaptation by estimating population history and by searching for candidate genes that contribute to adaptation. The patterns of changes in population size were quite distinct among the species according to their habitats. We identified many novel adaptive candidate genes, some of which had surprisingly long divergent haplotypes between species, thus showing the footprint of selective sweep events. Molecular phylogenetic analyses revealed that a large fraction of the allelic diversity among Lake Victoria cichlids was derived from standing genetic variation that originated before the adaptive radiation. Our analyses uncovered the processes of species-specific adaptation of Lake Victoria cichlids and the complexity of the genomic substrate that facilitated this adaptation.     

The synonymy of Haplochromis pharyngalis and Haplochromis petronius (Cichlidae)

Haplochromis pharyngalis and Haplochromis petronius, two endemic cichlids from the Lake Edward system (Uganda, Democratic Republic of the Congo), are very similar in general morphology but have been reported to differ in pharyngeal jaw morphology and distribution. This study analysed 51 morphometrics and various qualitative characteristics of 48 specimens from different localities. The morphological traits of both species strongly overlap, and differences in the pharyngeal jaw morphology correspond to a geographic morphocline. We conclude that all specimens belong to one valid species, H. pharyngalis, and consider H. petronius to be a synonym.     

B chromosomes have a functional effect on female sex determination in Lake Victoria cichlid fishes

The endemic cichlid fishes in Lake Victoria are a model system for speciation through adaptive radiation. Although the evolution of the sex-determination system may also play a role in speciation, little is known about the sex-determination system of Lake Victoria cichlids. To understand the evolution of the sex-determination system in these fish, we performed cytogenetic analysis in 11 cichlid species from Lake Victoria. B chromosomes, which are present in addition to standard chromosomes, were found at a high prevalence rate (85%) in these cichlids. In one species, B chromosomes were female-specific. Cross-breeding using females with and without the B chromosomes demonstrated that the presence of the B chromosomes leads to a female-biased sex ratio in this species. Although B chromosomes were believed to be selfish genetic elements with little effect on phenotype and to lack protein-coding genes, the present study provides evidence that B chromosomes have a functional effect on female sex determination. FISH analysis using a BAC clone containing B chromosome DNA suggested that the B chromosomes are derived from sex chromosomes. Determination of the nucleotide sequences of this clone (104.5 kb) revealed the presence of several protein-coding genes in the B chromosome, suggesting that B chromosomes have the potential to contain functional genes. Because some sex chromosomes in amphibians and arthropods are thought to be derived from B chromosomes, the B chromosomes in Lake Victoria cichlids may represent an evolutionary transition toward the generation of sex chromosomes.     

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.     

Two sequences encoding chalcone synthase in yellow lupin (Lupinus luteus l.) may have evolved by gene duplication

Two full copy cDNA sequences encoding chalcone synthase (CHS) were selected from a yellow lupin (Lupinus luteus L.) root and nodule cDNA library, and sequenced. Analysis of their open reading frames gave evidence that both encode the functional enzyme. Sequence alignment and phylogenetic studies on the DNA and protein level of these clones compared to the sequences of chalcone synthases from 54 other plant species reveal the possibility that lupin chalcone synthase is encoded by a multigene family consisting of at least two distinct genes that probably diverged by gene duplication. The duplication event is estimated to have taken place about 16 million years ago.     

High prevalence of non-synonymous substitutions in mtDNA of cichlid fishes from Lake Victoria

When a population size is reduced, genetic drift may fix slightly deleterious mutations, and an increase in nonsynonymous substitution is expected. It has been suggested that past aridity has seriously affected and decreased the populations of cichlid fishes in Lake Victoria, while geographical studies have shown that the water levels in Lake Tanganyika and Lake Malawi have remained fairly constant. The comparably stable environments in the latter two lakes might have kept the populations of cichlid fishes large enough to remove slightly deleterious mutations. The difference in the stability of cichlid fish population sizes between Lake Victoria and the Lakes Tanganyika and Malawi is expected to have caused differences in the nonsynonymous/synonymous ratio, ω (= d_N/d_S), of the evolutionary rate. Here, we estimated ω and compared it between the cichlids of the three lakes for 13 mitochondrial protein-coding genes using maximum likelihood methods. We found that the lineages of the cichlids in Lake Victoria had a significantly higher ω for several mitochondrial loci. Moreover, positive selection was indicated for several codons in the mtDNA of the Lake Victoria cichlid lineage. Our results indicate that both adaptive and slightly deleterious molecular evolution has taken place in the Lake Victoria cichlids' mtDNA genes, whose nonsynonymous sites are generally conserved.     

Evolution of the ribosomal RNA internal transcribed spacer one (ITS-1) in cichlid fishes of the Lake Victoria region

The nucleotide sequences of the first internal transcribed spacer (ITS-1) of the ribosomal RNA gene cluster have been determined for 11 species of closely related endemic cichlid fishes of the Lake Victoria region (LVR) and 6 related East African cichlids. The ITS-1 sequences confirmed independently derived basal phylogenies, but provide limited insight within this species flock. The line leading to Pseudocrenilabrus multicolor arose early, close to the divergence event that separated the tilapiine and haplochromine tribes of the "African Group" of the family Cichlidae. In this phylogeny, Astatoreochromis alluaudi and the riverine Astatotilapia burtoni are sister taxa, which together are a sister group to a monophyletic assemblage including both Lake Victoria and Lake Edward taxa. The ITS-1 data support the monophyly of haplochromine genera across lakes. Since Lake Victoria is believed to have been dry between 14, 500 and 12,400 BPE, the modern assemblage must have been derived from reinvasion by the products of earlier cladogenesis events. Thus, although the regional superflock is monophyletic, the haplochromines of Lake Victoria itself did not evolve in situ from a single ancestor.     

Morphometric differentiation among haplochromine cichlid fish species of a satellite lake of Lake Victoria

Lake Victoria holds a young but species‐rich assemblage of cichlid fishes, which form a monophyletic assemblage with additional species from surrounding water bodies, termed the Lake Victoria superflock. Lake Victoria is surrounded by smaller lakes that are somewhat disconnected from the main lake. Lake Kanyaboli is such a small lake, having markedly reduced species diversity, in part comprised of Lake Victoria species and endemics. Here, we studied the modern haplochromine component of the cichlid fauna, represented by Lipochromis maxillaris, Astatotilapia nubila, Xystichromis phytophagus and Astatotilapia sp. ‘Bigeye’, as well as a number of unidentified modern haplochromine specimens. We used landmark‐based geometric morphometrics to study the degree of morphological divergence among those young entities. Twenty landmarks and 14 interlandmark distances were used for shape analysis. Multivariate analysis revealed significant differences between all four species, but principal component analysis and canonical variate analysis did not clearly discriminate between A. nubila and X. phytophagus, demonstrating great overall morphological similarity despite clear dietary differences. Besides coloration there was sexual dimorphism in body proportions, so that only male individuals were analysed further. In all four species, the observed similarities and differences in body shape conform to the type of ecological specialization of the fish. Most unidentified specimens overlapped the range of A. nubila and X. phytophagus, while the assignment test based on the canonical variate analysis suggested 70% of the three overlapping entities as separate units. To test their reproductive distinctness and to demonstrate potential hybridization, nuclear genetic data are needed.     

Genetic distinction of four haplochromine cichlid fish species in a satellite lake of Lake Victoria,East Africa

Lake Victoria is famous for its in evolutionary terms young but species‐rich assemblage of cichlid fishes. This ‘superflock’ also includes additional species from adjacent water systems. Lake Victoria is surrounded by several smaller lakes that are connected to the main water body of Lake Victoria only through swampy areas. Lake Kanyaboli is one such lake, harbouring a much poorer species diversity, mostly comprised of Lake Victoria endemics, some of which are now considered extirpated from the main lake. The focus of this study was on the modern haplochromine component of the cichlid fauna, represented by Lipochromis maxillaris, Astatotilapia nubila, Xystichromis phytophagus and Astatotilapia sp. ‘Bigeye’, as well as a number of morphologically distinct haplochromine specimens that could not be assigned to any of the recognized species. We used five microsatellite markers to distinguish these five taxa. Genetically, L. maxillaris was clearly differentiated from all other taxa, and A. sp. ‘Bigeye’ was moderately differentiated from the remaining three. Astatotilapia nubila, X. phytophagus and the unidentified specimens constituted a partially overlapping cluster. As each of the clusters had several (5–14) private alleles, extremely recent divergence is suggested. As all taxa except for A. sp. ‘Bigeye’ and the unidentified specimens also occur or at least occurred in Lake Victoria, it is likely that they evolved as part of the Lake Victoria superflock, while A. sp. ‘Bigeye’ and the unidentified specimens may have currently evolved in situ. The observation of slightly distinct albeit overlapping body shapes and the extremely close genetic relationship between three of the five taxa are fully compatible and in support of the hybrid swarm theory of adaptive radiation.     

Photopic adaptations to a changing environment in two Lake Victoria cichlids

During the past 30 years, Lake Victoria cichlid fishes have encountered severe environmental and ecological changes including an introduced predator and new prey types. Furthermore, increased eutrophication has led to reduced water transparency and shifted the spectral composition of underwater light to longer wavelengths. In the present study, collections of two cichlid species, Haplochromis pyrrhocephalus and Haplochromis tanaos, from before and after the environmental changes, were compared with respect to their photopic resolution and sensitivity. Eyes of both species were dissected and retinal features were measured from tangential sections. In both species, the eyes became smaller, independently of body size. This decrease possibly occurred to make space for other structures that increased in size. In H. pyrrhocephalus, a significantly lower resolution was found. However, the size, and thus photon‐catching ability of the double cones, remained unchanged, despite the smaller eyes. In the modern populations of H. tanaos, the double cone size increased in relation to eye size, so that the photon‐catching ability of the smaller fish remained the same. However, no significant decrease in resolution was found. Shortwave sensitivity was found to be lower in both modern populations because of a reduction or the complete absence of single cones. The results obtained in the present study imply that these resurgent zooplanktivores are capable of adapting their eye morphology to the changed environmental conditions without losing crucial aspects used for survival and reproduction. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106 , 328–341.     

The food of Bagrus docmac (Forsk.) (Pisces: Siluriformes) and its relationship with Haplochromis Hilgendorf (Pisces: Cichlidae), in Lake Victoria, East Africa

The diet of the siluriform fish Bagrus docmac (Forsk.) is described in relation to various localities in Lake Victoria, East Africa. Invertebrates which are the principal food of young fish, but generally of little significance in adults, are particularly poorly represented in fishes from Tanzanian waters and most important in those from the Kavirondo Gulf where the ontogenetic changes in diet differ from those found elsewhere in the Lake. B. docmac starts feeding piscivorously at about 10 cm standard length with the transition from a principally invertebrate to a primarily piscivorous diet occurring at 15 cm S.L. In adult B. docmac fish are found in over 95 % of individuals with food. Haplochromis is the all important food type, and Tilapia , the most valuable commercial fish in the lake, is not preyed upon at all. Bathymetric changes with respect to the invertebrate food are noted, and it is shown that piscivorous feeding occurs mainly by day and invertebrate feeding by night. Quantitative estimates of consumption of Haplochromis by B, docmac are computed from the results of a series of 24 h fishing surveys and found to exceed published data from elsewhere. The formula for the computation of a daily ration is given as Log R =2.973 Log L —3.36089 and it is calculated that the mean annual consumption of Haplochromis by B. docmac is approximately 75% of the mean standing stock of the prey fish, as compared with a commercial catch of about 5% of the mean standing stock.     

H3 and H4 histone cDNA sequences from Xenopus: a sequence comparison of H4 genes

Ovarian poly (A) + RNA from Xenopus laevis and Xenopus borealis was used to construct two cDNA libraries which were screened for histone sequences. cDNA clones to H4 mRNA were obtained from both species and an H3 cDNA clone from Xenopus laevis. The complete DNA sequences of these clones have been determined and are presented. These new sequences are compared with other H3 and H4 DNA sequences both in the coding and 3' noncoding regions. We find that there is considerable non-random codon usage in ten H4 genes. In addition there are some sequence similarities in the 3' noncoding regions of H3 and H4 genes.     

Testing clove oil as an anaesthetic for long-distance transport of live fish: the case of the Lake Victoria cichlid Haplochromis obliquidens

Clove oil can be used as an anaesthetic in the handling of marine and freshwater fish. Few studies report on its use for periods up to 48 h, for example, under long‐distance transport conditions. This study tested the effect of different clove oil concentrations for 1–48 h on recovery and survival of the cichlid Haplochromis obliquidens, an ornamental fish species endemic to Lake Victoria. Haplochromis obliquidens were anaesthetized for 1 h using 5–25 μl L^?1 clove oil. There was no correlation between clove oil concentration and post‐anaesthesia recovery time (P = 0.15). On average, fish recovered within 9.5 ± 2 min, and no fish died within 24 h after recovery. Results from exposure of fish to 18–20 μl L^?1 clove oil for up to 48 h suggested a narrow margin of safety as this concentration range induced mortality. At 18 μl L^?1 recovery times ranged from 3 to 43 min between 24 and 36 h exposure, while fish exposed longer than 36 h recovered within 1–10 min, or within 1–2 min after 44–48 h. At the end of a 48‐h transport experiment total ammonia levels were higher in transport water containing anaesthetized fish than for non‐anaesthetized fish (1.65 ± 0.19 and 0.54 ± 0.08 mg L^?1 NH + NH₃, respectively). The combined use of clove oil and the selective ammonium ion exchanger zeolite was considered feasible as ammonia levels could be reduced by up to 82% compared to control bags without zeolite.