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

Abstract

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.

Figure 1. Sampling localities in Lake Victoria.

Scale bar, 20 km.

Figure 2. B chromosomes in the pedigree of L. rubripinnis from the Matumbi Island population.

(A–D) The karyotypes of individuals in one clutch of the pedigree. The larger and smaller B chromosomes are indicated as B₁ and B₂, respectively. STT, subtelocentric and telocentric chromosomes; MSM, metacentric and submetacentric chromosomes. (E, F) FISH analysis using B₁ (E) and B₂ (F) probes on metaphase spreads from individuals of this pedigree. Arrowheads indicate B₁ and B₂ chromosomes. The FISH images were obtained by merging the DAPI-stained patterns (blue) and the signals from the FISH probes (green). Scale bar, 5 µm.

Figure 3. Demonstration of variation in the number of B chromosomes in Lake Victoria cichlids using painting FISH.

(A–D) Painting FISH was carried out using the B₁ probe on karyotypes of wild-caught L. rubripinnis individuals that possess 0 (A), 1 (B), 2 (C), and 3 (D) B chromosomes. The images were obtained by merging the DAPI-stained patterns (blue) and FISH signals from the B₁ probe (green). Scale bar, 5 µm.

Figure 4. A female-biased sex ratio resulting from the presence of female-specific B chromosomes in L. rubripinnis.

(A) Correlation between sex and karyotype in the wild-caught individuals and F1 and F2 individuals of L. rubripinnis and in offspring from cross-breeding experiments. The number below each histogram indicates the number of individuals. Asterisks indicate the significant association between B chromosomes and females of this species (Fisher's exact test: **P<0.01, *P<0.05). (B) The sex-ratio distortion induced by the presence of the female-specific B chromosome in cross-breeding families. The number of individuals that died as fry is indicated in parentheses. Statistical significance of deviation of sex ratio from 1∶1 was tested by binominal test. P-values are shown in the table below a bar graph.

Figure 5. The presence of several protein-coding genes derived from chromosome 1 in B chromosomes.

(A) A pie graph showing the composition of the 104.5 kb B-BAC sequence. The percentages of genes and total repetitive sequences are indicated. (B) The copy number of the ihhb region as determined by real-time PCR in F2 individuals of L. rubripinnis from Matumbi Island population that have different numbers of B chromosomes. The mean value is shown; error bars indicate the standard deviation. (C) FISH analysis using B-BAC as a probe. The images were obtained by merging DAPI-stained patterns (blue) and the signals from the FISH probe (green). Arrowheads indicate B chromosomes. Arrows indicate chromosome 1. Scale bar, 5 µm. Since the B-BAC probe showed stronger signals than the probe using microdissected B chromosome DNA, we detected the homologous autosome region only when we used B-BAC probe.