Abstract
The genus Blepharisma (Alveolata, Ciliophora) is a unicellular organism distributed worldwide, even in extreme environments, and comprises numerous species. While usually proliferating through cell division, Blepharisma undergoes sexual reproduction (conjugation) when cells are moderately starved. Conjugation is initiated by mating pheromones (gamone 1 and gamone 2) secreted by complementary mating-type cells. Gamone 1, a glycoprotein, functions in a species-specific manner, while gamone 2, an amino acid derivative, is a common molecule across species. The specific function of gamone 1 suggests the possibility that mutations in gamone 1 might have led to reproductive isolation during the evolutionary process, triggering species diversification. In this study, by comparing the amino acid sequences of gamone 1 homologs from 15 strains (representing five species), we found that mutations resulting in distinct amino acid properties occur across species boundaries and are mainly concentrated at two specific regions within gamone 1. These mutations potentially alter the binding affinity of gamone 1 to its receptors, suggesting their effect in causing reproductive isolation. The interspecies artificial conjugation conducted previously and the molecular phylogenetic tree constructed using the gamone 1 homolog sequences in this study provide insights into the speciation process within the genus Blepharisma.