A deleterious effect associated with UNH159 is attenuated in twin embryos of an inbred line of blue tilapia Oreochromis aureus.

Offspring of a highly inbred gynogenetic line of Oreochromis aureus displayed 12-fold increase in twinning rate compared to the outbred population. Asymmetric conjoined twins, which consist of a normal embryo attached to a malformed-atrophic twin, were frequently encountered in both gynogenetic (90·7%) and outbred (38·2%) embryos. The monozygotic origin of these twins was determined using five microsatellite markers. Progeny of heterozygous parents for the microsatellite UNH159 were separated into sub-sets of twins and normal full-sibs. Consistent with previous reports, the normal embryo sub-set exhibited elimination of both types of homozygotes for the UNH159 genetic marker at 2-8 days after fertilization. Unexpectedly, this elimination was less frequent in twins. The UNH159 marker as well as RNA-binding motif protein, X-linked (rbmx), SRY-box containing gene 3 (sox3) and alpha-thalassemia/mental retardation syndrome X-linked (atrx) genes were mapped to linkage group 2. These gene orthologues are all located on the mammalian X chromosome and atrx is necessary for the X-chromosome inactivation.

Chromosome evolution in African cichlid fish: contributions from the physical mapping of repeated DNAs.

Cichlid fishes have been the subject of increasing scientific interest because of their rapid adaptive radiation that has led to extensive ecological diversity and because of their enormous importance to tropical and subtropical aquaculture. To further understanding of chromosome evolution among cichlid species, we have comparatively mapped the SATA satellite DNA, the transposable element ROn-1, and repeated sequences in the bacterial artificial chromosome clone BAC-C4E09 on the chromosomes of a range of African species of Cichlidae, using fluorescence in situ hybridization. The SATA satellite DNA was mapped in almost all the centromeres of all tilapiine and haplochromine species studied. The maintenance and centromeric distribution of the SATA satellite DNA in African cichlids suggest that this sequence plays an important role in the organization and function of the centromere in these species. Furthermore, analysis of SATA element distribution clarifies that chromosome fusions occurred independently in Oreochromis and Tilapia genera, and led to the reduced chromosome number detected in O. karongae and T. mariae. The comparative chromosome mapping of the ROn-1 SINE-like element and BAC-C4E09 shows that the repeated sequences have been maintained among tilapiine, haplochromine and hemichromine fishes and has demonstrated the homology of the largest chromosomes among these groups. Furthermore, the mapping of ROn-1 suggested that different chromosomal rearrangements could have occurred in the origin of the largest chromosome pairs of tilapiines and non-tilapiines.

Copy number variation of lipocalin family genes for male-specific proteins in tilapia and its association with gender.

Lipocalins are involved in the binding of small molecules like sex steroids. We show here that the previously reported tilapia male-specific protein (MSP) is a lipocalin encoded by a variety of paralogous and homologous genes in different tilapia species. Exon-intron boundaries of MSP genes were typical of the six-exon genomic structure of lipocalins, and the transcripts were capable of encoding 200 amino-acid polypeptides that consisted of a putative signal peptide and a lipocalin domain. Cysteine residues are conserved in positions analogous to those forming the three disulfide bonds characteristic of the ligand pocket. The calculated molecular mass of the secreted MSP (20.4 kDa) was less than half of that observed, suggesting that it is highly glycosylated like its homologue tributyltin-binding protein. Analysis of sequence variations revealed three types of paralogs MSPA, MSPB and MSPC. Expression of both MSPA and MSPB was detected in testis. In haploid Oreochromis niloticus embryos, each of these types consisted of two closely related paralogs, and asymmetry between MSP copy numbers on the maternal (six copies) and the paternal (three copies) chromosomes was observed. Using this polymorphism we mapped MSPA and MSPC to linkage group 12 of an F(2) mapping family derived from a cross between O. niloticus and Oreochromis aureus. Females with high MSP copy number were more frequent by more than twofold than males. Gender-MSPC combinations showed significant deviation from expected Mendelian segregation (P=0.009) suggesting elimination of males with MSPC copies. We discuss different hypotheses to explain this elimination, including possibility for allelic conflict resulted by the hybridization.

Genetics of sex determination in tilapiine species.

We identified DNA markers linked to sex determining genes in six closely related species of tilapiine fishes. The mode of sex determination differed among species. In Oreochromis karongae and Tilapia mariae the sex-determining locus is on linkage group (LG) 3 and the female is heterogametic (WZ-ZZ system). In O. niloticus and T. zillii the sex-determining locus is on LG1 and the male is heterogametic (XX-XY system). A more complex pattern was observed in O. aureus and O. mossambicus, in which markers on both LG1 and LG3 were associated with sex. We found evidence for sex-linked lethal effects on LG1, as well as interactions between loci in the two linkage groups. Comparison of genetic and physical maps demonstrated a broad region of recombination suppression harboring the sex-determining locus on LG3. Sex-specific recombination suppression was found in the female heterogametic sex. Sequence analysis showed the accumulation of repetitive elements in this region. Phylogenetic analysis suggests that at least two transitions in the mode of sex determination have occurred in this clade. This variation in sex determination mechanisms among closely related species makes tilapias an excellent model system for studying the evolution of sex chromosomes in vertebrates.

Evolutionary biology: evidence for sympatric speciation?

Sympatric speciation is difficult to demonstrate in nature and remains a hotly debated issue. Barluenga et al. present a case of putative sympatric speciation for two cichlid species in the Nicaraguan crater lake Apoyo, but they overlook or reinterpret some key published information on the system. Although sympatric speciation is possible in theory, we show here that, when this information is taken into account, the results of Barluenga et al. do not provide conclusive evidence for sympatric speciation: this is because the null hypothesis of multiple invasion with introgression cannot be rejected.

Genetic and developmental basis of cichlid trophic diversity.

Cichlids have undergone extensive evolutionary modifications of their feeding apparatus, making them an ideal model to study the factors that underlie craniofacial diversity. Recent studies have provided critical insights into the molecular mechanisms that have contributed to the origin and maintenance of cichlid trophic diversity. We review this body of work, which shows that the cichlid jaw is regulated by a few genes of major additive effect, and is composed of modules that have evolved under strong divergent selection. Adaptive variation in cichlid jaw shape is evident early in development and is associated with allelic variation in and expression of bmp4. Modulating this growth factor in the experimentally tractable zebrafish model reproduces natural variation in cichlid jaw shape, supporting a role for bmp4 in craniofacial evolution. These data demonstrate the utility of the cichlid jaw as a model for studying the genetic and developmental basis of evolutionary changes in craniofacial morphology.

Hybridization and contemporary evolution in an introduced cichlid fish from Lake Malawi National Park.

Rapidly evolving systems offer the chance to observe genetic and phenotypic change in real time. We exploit a well-characterized introduction of cichlid fish into Lake Malawi National Park to document a short history of habitat colonization and the evolution of genes and colour pattern. In the early 1960s, a fish exporter introduced individuals of Cynotilapia afra to a single site (Mitande Point) of Thumbi West Island and, as late as 1983, the species was confined to this location. In 2001, C. afra had colonized the entire perimeter of Thumbi West. In July of that year, we sampled C. afra individuals from six sites around the island and scored variation in dorsal fin colour as well as allelic diversity at six microsatellite loci. We found that, in two decades, C. afra had diverged into genetically distinct, phenotypically different northern and southern populations. We observed a high proportion of hybrids between the introduced C. afra and the native Metriaclima zebra on the southern coast of Thumbi West, and speculate that hybridization is facilitated by low water clarity at these windward sites. The short history of C. afra at Thumbi West is a microcosm of contemporary evolutionary divergence and may provide the opportunity to study the process from start to finish in genetic detail.

Two unlinked loci controlling the sex of blue tilapia (Oreochromis aureus).

Sex determination in the blue tilapia (Oreochromis aureus) is thought to be a WZ-ZZ (female heterogametic) system controlled by a major gene. We searched for DNA markers linked to this major gene using the technique of bulked segregant analysis. We identified 11 microsatellite markers on linkage group 3 which were linked to phenotypic sex. The putative W chromosome haplotype correctly predicts the sex of 97% of male and 85% of female individuals. Our results suggest the W locus lies within a few centimorgans of markers GM354, UNH168, GM271 and UNH131. Markers on LG1 also showed a strong association with sex, and indicate the segregation of a male-determining allele in this region. Analysis of epistatic interactions among the loci suggests the action of a dominant male repressor (the W haplotype on LG 3) and a dominant male determiner (the Y haplotype on LG1). These markers have immediate utility for studying the strength of different sex chromosome alleles, and for identifying broodstock carrying copies of the W haplotype.

Identification of a sex-determining region in Nile tilapia (Oreochromis niloticus) using bulked segregant analysis.

Sex determination in the Nile tilapia (Oreochromis niloticus) is thought to be an XX-XY (male heterogametic) system controlled by a major gene. We searched for DNA markers linked to this major locus using bulked segregant analysis. Ten microsatellite markers belonging to linkage group 8 were found to be linked to phenotypic sex. The putative Y-chromosome alleles correctly predict the sex of 95% of male and female individuals in two families. Our results suggest a major sex-determining locus within a few centimorgans of markers UNH995 and UNH104. A third family from the same population showed no evidence for linkage of this region with phenotypic sex, indicating that additional genetic and/or environmental factors regulate sex determination in some families. These markers have immediate utility for studying the strength of different Y chromosome alleles, and for identifying broodstock carrying one or more copies of the Y haplotype.

Genetic basis of adaptive shape differences in the cichlid head.

East African cichlids exhibit an extraordinary level of morphological diversity. Key to their success has been a dramatic radiation in trophic biology, which has occurred rapidly and repeatedly in different lakes. In this report we take the first step in understanding the genetic basis of differences in cichlid oral jaw design. We estimate the effective number of genetic factors that control differences in the cichlid head through a comprehensive morphological assessment of two Lake Malawi cichlid species and their F(1) and F(2) hybrid progeny. We estimate that between one and 11 factors underlie shape difference of individual bony elements. We show that many of the skeletal differences in the head and oral jaw apparatus are inherited together, suggesting a degree of pleiotropy in the genetic architecture of this character complex. Moreover, we find that cosegregation of shape differences in different elements corresponds to developmental, rather than functional, units.

The cusp of evolution and development: a model of cichlid tooth shape diversity.

Tooth shape is a hallmark of repeated evolutionary radiations among cichlid fishes from East Africa. Cusp shape and number vary both within populations and among closely related species with different feeding behaviors and ecologies. Here, we use histology and scanning electron microscopy to chart the developmental trajectory of tooth shape differences in fishes from Lake Malawi. We demonstrate that species with bi- or tricuspid adult (replacement) teeth initially possess a first-generation unicuspid dentition. Notably, the timing of turnover from first-generation to replacement teeth differs among species and is correlated with feeding ecology. Next, we use field data for cichlid species with adult unicuspid, bicuspid, and tricuspid teeth to demonstrate a strong and positive relationship between the number of teeth in a row and tooth shape. We discuss cichlid tooth ontogeny in the context of morphogenetic models designed to explain the developmental basis of tooth shape variation in mammals. We suggest that the dramatic differences in cichlid dentitions can be explained by variation in the expression of common activators and inhibitors acting at multiple stages of odontogenesis.

Rapid isolation of CA microsatellites from the tilapia genome.

We have developed (CA)n microsatellite markers for the cichlid fish, Oreochromis niloticus using a variation of the hybrid capture method. The resulting genomic library was highly enriched in repetitive DNA with 96% of clones containing CA repeats. The number of repeats ranged from four to 45 with an average of 19. Two-thirds of the sequenced clones had 12 or more repeats and sufficient flanking sequence to design primers. The resulting markers were tested in an F2 cross of O. niloticus x O. aureus. Nearly 90% of the markers amplified in this cross and 74% of these were informative. This work demonstrates the importance of minimizing the number of polymerase chain reaction (PCR) amplification cycles before and after the enrichment steps to reduce PCR recombination and the generation of chimaeric clones.

Microsatellite variation associated with prolactin expression and growth of salt-challenged tilapia.

Biologists have long argued that runs of alternating purines and pyrimidines could form alternative DNA structures, which might regulate transcription. Here, we report that simple sequence repeat polymorphisms in the tilapia prolactin 1 (prl 1) promoter are associated with differences in prl 1 gene expression and the growth response of salt-challenged fishes. Individuals homozygous for long microsatellite alleles express less prl 1 in fresh water but more prl 1 in half-seawater than fishes with other genotypes. Our work provides the first in vivo evidence that differences in microsatellite length among individuals may indeed affect gene expression and that variance in expression has concomitant physiological consequences. These results suggest that dinucleotide microsatellites represent an under-appreciated source of genetic variation for regulatory evolution.

Cone opsin genes of african cichlid fishes: tuning spectral sensitivity by differential gene expression.

Spectral tuning of visual pigments is typically accomplished through changes in opsin amino acid sequence. Within a given opsin class, changes at a few key sites control wavelength specificity. To investigate known differences in the visual pigment spectral sensitivity of the Lake Malawi cichlids, Metriaclima zebra (368, 488, and 533 nm) and Dimidiochromis compressiceps (447, 536, and 569 nm), we sequenced cone opsin genes from these species as well as Labeotropheus fuelleborni and Oreochromis niloticus. These cichlids have five distinct classes of cone opsin genes, including two unique SWS-2 genes. Comparisons of the inferred amino acid sequences from the five cone opsin genes of M. zebra, D. compressiceps, and L. fuelleborni show the sequences to be nearly identical. Therefore, evolution of key opsin sites cannot explain the differences in visual pigment sensitivities. Real-time PCR demonstrates that different cichlid species express different subsets of the available cone opsin genes. Metriaclima zebra and L. fuelleborni express a complement of genes which give them UV-shifted visual pigments, while D. compressiceps expresses a different set to produce a red-shifted visual system. Thus, variations in cichlid spectral sensitivity have arisen through evolution of gene regulation, rather than through changes in opsin amino acid sequence.

Assessing morphological differences in an adaptive trait: a landmark-based morphometric approach.

East African cichlid fishes have evolved a stunning array of oral jaw morphologies. To better understand the adaptive evolution of this trait, we performed a morphological analysis of the jaws of two closely related species from Lake Malawi that have very different modes of feeding. Labeotropheus fuelleborni forages along the substrate with a "biting" mode of feeding, while Metriaclima zebra feeds in the water column with a "sucking" mode. We analyzed each of the four skeletal elements that make up the oral jaws: the dentary, articular, premaxilla, and maxilla. In addition, we performed the same analysis on the neurocranium, an element closely associated with the oral jaws. We used the thin-plate spline method to quantify morphological differences, which allowed us to relate our results to the functional biology of the species. We find many aspects of shape change that relate directly to the functional design of the cichlid head. The same series of measurements was made on hybrids between Labeotropheus and Metriaclima. For every character, hybrid progeny are statistically different from both parental species. These results suggest an additive mode of action of the alleles responsible for these phenotypes.

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.

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.