Hikoyu Suzuki

Lineage-Specific Expansion of Vomeronasal Type 2 Receptor-Like (OlfC) Genes in Cichlids May Contribute to Diversification of Amino Acid Detection Systems

Fish use olfaction to sense a variety of nonvolatile chemical signals in water. However, the evolutionary importance of olfaction in species-rich cichlids is controversial. Here, we determined an almost complete sequence of the vomeronasal type 2 receptor-like (OlfC: putative amino acids receptor in teleosts) gene cluster using the bacterial artificial chromosome library of the Lake Victoria cichlid, Haplochromis chilotes. In the cluster region, we found 61 intact OlfC genes, which is the largest number of OlfC genes identified among the seven teleost fish investigated to date. Data mining of the Oreochromis niloticus (Nile tilapia) draft genome sequence, and genomic Southern hybridization analysis revealed that the ancestor of all modern cichlids had already developed almost the same OlfC gene repertoire, which was accomplished by lineage-specific gene expansions. Furthermore, comparison of receptor sequences showed that recently duplicated paralogs are more variable than orthologs of different species at particular sites that were predicted to be involved in amino acid selectivity. Thus, the increase of paralogs through gene expansion may lead to functional diversification in detection of amino acids. This study implies that cichlids have developed a potent capacity to detect a variety of amino acids (and their derivatives) through OlfCs, which may have contributed to the extraordinary diversity of their feeding habitats.

Characterization of V1R receptor (ora) genes in Lake Victoria cichlids.

Although olfaction could play a crucial role in underwater habitats by allowing fish to sense a variety of nonvolatile chemical signals, the importance of olfaction in species-rich cichlids is still controversial. In particular, examining whether cichlids rely on olfaction for reproduction is of primary interest to understand the mechanisms of speciation. In the present study, we explored the V1R (also known as ora) genes, which are believed to encode reproductive pheromone receptors in fish, in the genomes of Lake Victoria cichlids. By screening a bacterial artificial chromosome library, we identified all six intact V1R genes (V1R1 to V1R6) that have been reported in other teleost fish. Furthermore, RT-PCR and in situ hybridization analyses showed that all of the V1R genes were expressed in the olfactory epithelium, indicating that these receptors are functional in cichlids. These observations indicate that cichlids use V1R-mediated olfaction in some ways for their social behaviors.

Multiple Episodic Evolution Events in V1R Receptor Genes of East-African Cichlids

Fish use olfaction to detect a variety of nonvolatile chemical signals, and thus, this sense is key to survival and communication. However, the contribution of the olfactory sense to social—especially reproductive—interactions in cichlids is still controversial. To obtain insights into this issue, we investigated the genes encoding V1Rs—possible candidates for reproductive pheromone receptors—among East-African cichlids. Interestingly, we found an excess of nonsynonymous over synonymous substitutions in four of six V1R genes in multiple cichlid lineages. First, we found that highly dimorphic V1R2 allele groups were shared among the cichlids inhabiting all East-African Great Lakes emerged through the episodic accumulation of the nonsynonymous substitutions prior to the radiation of the Lake Tanganyika species flock. We further detected such episodic events in V1R1 of the tribe Tropheini, and in V1R3 and V1R6 of the tribe Trematocarini. The excess of nonsynonymous substitutions in these examples were indicated as dN/dS > 1, which were all statistically significant by Fisher’s exact test. Furthermore, we speculate that the amino acid changes in these episodic events are likely functional switch because they occurred in the putative ligand-binding pocket. Our finding of the occurrence of multiple episodic events and the unexpected gene diversity in one unique gene family is suggestive of the contribution of the V1R to the species diversification and the social interaction in cichlids.

A microsatellite-based genetic linkage map and putative sex-determining genomic regions in Lake Victoria cichlids.

Cichlid fishes in East Africa have undergone extensive adaptive radiation, which has led to spectacular diversity in their morphology and ecology. To date, genetic linkage maps have been constructed for several tilapias (riverine), Astatotilapia burtoni (Lake Tanganyika), and hybrid lines of Lake Malawi cichlids to facilitate genome-wide comparative analyses. In the present study, we constructed a genetic linkage map of the hybrid line of Lake Victoria cichlids, so that maps of cichlids from all the major areas of East Africa will be available. The genetic linkage map shown here is derived from the F2 progeny of an interspecific cross between Haplochromis chilotes and Haplochromis sauvagei and is based on 184 microsatellite and two single-nucleotide polymorphism (SNP) markers. Most of the microsatellite markers used in the present study were originally designed for other genetic linkage maps, allowing us to directly compare each linkage group (LG) among different cichlid groups. We found 25 LGs, the total length of which was 1133.2cM with an average marker spacing of about 6.09cM. Our subsequent linkage mapping analysis identified two putative sex-determining loci in cichlids. Interestingly, one of these two loci is located on cichlid LG5, on which the female heterogametic ZW locus and several quantitative trait loci (QTLs) related to adaptive evolution have been reported in Lake Malawi cichlids. We also found that V1R1 and V1R2, candidate genes for the fish pheromone receptor, are located very close to the recently detected sex-determining locus on cichlid LG5. The genetic linkage map study presented here may provide a valuable foundation for studying the chromosomal evolution of East African cichlids and the possible role of sex chromosomes in generating their genomic diversity.

Distinct functions of two olfactory marker protein genes derived from teleost-specific whole genome duplication

BackgroundWhole genome duplications (WGDs) have been proposed to have made a significant impact on vertebrate evolution. Two rounds of WGD (1R and 2R) occurred in the common ancestor of Gnathostomata and Cyclostomata, followed by the third-round WGD (3R) in a common ancestor of all modern teleosts. The 3R-derived paralogs are good models for understanding the evolution of genes after WGD, which have the potential to facilitate phenotypic diversification. However, the recent studies of 3R-derived paralogs tend to be based on in silico analyses. Here we analyzed the paralogs encoding teleost olfactory marker protein (OMP), which was shown to be specifically expressed in mature olfactory sensory neurons and is expected to be involved in olfactory transduction.ResultsOur genome database search identified two OMPs (OMP1 and OMP2) in teleosts, whereas only one was present in other vertebrates. Phylogenetic and synteny analyses suggested that OMP1 and 2 were derived from 3R. Both OMPs showed distinct expression patterns in zebrafish; OMP1 was expressed in the deep layer of the olfactory epithelium (OE), which is consistent with previous studies of mice and zebrafish, whereas OMP2 was sporadically expressed in the superficial layer. Interestingly, OMP2 was expressed in a very restricted region of the retina as well as in the OE. In addition, the analysis of transcriptome data of spotted gar, a non-teleost fish, revealed that single OMP gene was expressed in the eyes.ConclusionWe found distinct expression patterns of zebrafish OMP1 and 2 at the tissue and cellular level. These differences in expression patterns may be explained by subfunctionalization as the model of molecular evolution. Namely, single OMP gene was speculated to be originally expressed in the OE and the eyes in the common ancestor of all Osteichthyes (bony fish including tetrapods). Then, two OMP gene paralogs derived from 3R-WGD reduced and specialized the expression patterns. This study provides a good example for analyzing a functional subdivision of the teleost OE and eyes as revealed by 3R-derived paralogs of OMPs.

Evolution of vomeronasal receptor 1 ( V1R ) genes in the common marmoset ( Callithrix jacchus )

Pheromones are crucial for eliciting innate responses and inducing social and sexual behaviors in mammals. The vomeronasal receptor 1 genes, V1Rs, encode members of a pheromone receptor family that are mainly expressed in the vomeronasal organ (VNO). The V1R family shows extraordinary variety in gene number among vertebrates owing to successive gene gains and losses during evolution. Such diversity is believed to reflect a degree of dependence on the VNO. We investigated V1R evolution in primate lineages closely related to humans because these VNOs show a trend toward degeneration. We performed extensive phylogenetic analyses for V1Rs from a broad range of primate species. Although the decline of intact genes was evident in anthropoids (hominoids, Old World monkeys and New World monkeys), we found that a certain number of intact genes persist in New World monkeys. In one New World monkey species, the common marmoset (Callithrix jacchus), we examined seven putatively functional V1Rs using in situ hybridization and reverse transcription-PCR. Based on their mRNA expression patterns in the VNO and other organs, two types of V1Rs emerged: the canonical class with VNO-specific expression, and a second group having more ubiquitous expression in various organs as well as VNO. Furthermore, phylogenetic analysis revealed that the class with the more widespread expression had been retained longer in evolution than the VNO-specific type. We propose that the acquisition of a novel non-VNO-related function(s) may have led to the survival of a small but persistent number of V1Rs in anthropoid primates.

A long-range cis -regulatory element for class I odorant receptor genes

Individual olfactory sensory neurons express a single odorant receptor gene from either class I genes residing in a single cluster on a single chromosome or class II genes spread over multiple clusters on multiple chromosomes. Here, we identify an enhancer element for mouse class I genes, the J element, that is conserved through mammalian species from the platypus to humans. The J element regulates most class I genes expression by exerting an effect over ~ 3 megabases within the whole cluster. Deletion of the trans J element increases the expression frequencies of class I genes from the intact J allele, indicating that the allelic exclusion of class I genes depends on the activity of the J element. Our data reveal a long-range cis-regulatory element that governs the singular class I gene expression and has been phylogenetically preserved to retain a single cluster organization of class I genes in mammals.“Each olfactory sensory neuron expresses a single odorant receptor gene from either class I or class II genes. Here, the authors identify an enhancer for mouse class I genes, that is highly conserved, and regulates most class I genes expression by acting over ~ 3 megabases within the whole cluster.”