Evolutionary Diversification and Functions of the Candidate Male Killing Gene wmk

Symbiont-mediated male killing (MK) is a mechanism that selectively eliminates male offspring, often by disrupting sex-specific developmental processes. In Drosophila melanogaster, the WO-mediated killing gene wmk from Wolbachia prophage WO transgenically reproduces the MK phenotype, yet how the gene evolves and functions across diverse Wolbachia has not been systematically investigated. We analyzed 32 Wolbachia genomes available in the NCBI database to study wmk homologs across different arthropod hosts, reproductive parasitism functions, and Wolbachia supergroups. First, we report at least five distinct wmk phylogenetic clusters (Types I to V), often organized in multigenic dyads or triads. Second, among MK Wolbachia, there is a significantly higher number of wmk genes and diversity in Lepidoptera strains than in Drosophila strains, which exclusively harbor wmk Types I and III. Third, there are three patterns of wmk sequence and genomic organizational changes in Drosophila MK strains that associate with different evolutionary trajectories underpinning the MK phenotype. Fourth, single and combinatory transgenic expression of Types I and III in D. melanogaster uncovers male-biased lethality associated with Type I; however, dual expression of the Types together elicits a major reduction in offspring number. Fifth, wmk genes have low expression level across D. melanogaster developmental stages relative to the cifA and cifB genes, which could explain why cytoplasmic incompatibility is expressed in this system. These findings establish a complex and phylogenetically informed genetic basis of wmk-induced lethality, highlighting the role of gene copy number and expression, wmk Types, and host background in shaping the phenotype.