New genes play a prominent role in evolution of new sperm classes of Drosophila

The widespread and regulated production of infertile parasperm assisting fertilization of eusperm provides a compelling model for elucidating the mechanisms of cellular innovation and altruism. Here we identify two specific parasperm lineages in Drosophila pseudoobscura that are segregated in development from the eusperm lineage by their single-cell transcriptomes. Most X-linked genes, except for species-specific new genes, show testis-specific repressive epigenetic modifications and become downregulated upon meiotic entry, supporting meiotic X chromosome inactivation. Between sperm classes, nearly half of the known meiotic arrest genes and their downstream targets are differentially transcribed, with over 200 genes displaying a gradient of transcription levels correlated with that of tail lengths. New genes are overrepresented throughout the hierarchical regulatory network of sperm divergence and over 60% of them exhibit a new cell-level expression compared to their parental copies. These results together indicate that new genes play a prominent role in the emergence of low-cost parasperm that shield the eusperm until fertilization.