Large-scale investigation of orphan genes in the human gut microbiome elucidates their evolutionary origins

Abstract Orphan genes, i.e., genes that lack homologs outside a given species, are ubiquitous in all domains of life. In prokaryotes, orphans can originate (i) in the native genome via de novo evolution from non-genic regions or alternative frames of existing genes, or by rapid divergence and remodeling, or (ii) in a foreign genome, including viruses, followed by horizontal transfer. However, quantitative evidence supporting either scenario is lacking. Here we performed a systematic, large-scale analysis of orphan genes from human gut prokaryotes. After exhaustive filtering, we identified more than 3 million orphans in 4,644 species which lack similarity to other prokaryotes. We find that a given species pangenome contains on average 2.6% orphan genes, which are mostly rare within a species. Overall, orphan genes use optimal codons less frequently, and their proteins are more disordered than those of conserved (i.e., non-orphan) genes. Importantly, the GC content of orphan genes in a given genome closely matches that of conserved ones. In contrast, the 5% of orphans that share similarity to known viral sequences have distinct characteristics that set them apart from the rest of the orphans, including lower GC content. By identifying the genomic region from which they evolved in closely related species, we provide evidence for native origination for a small subset of orphan genes and find that these orphans also differ in their properties from the remaining orphans. Finally, predicting orphan function by examining functional annotations in operon-like arrangements suggests that some orphan genes are membrane-related and involved in spore germination. Our results support that orphans emerge due to multiple routes, challenging the notion that external elements such as phages and plasmids are the primary source of prokaryotic genetic novelty. Importantly, origination in the native genome might provide a constant inflow of mostly transient genes into the cloud fraction of prokaryotic pangenomes, where some orphans may prove adaptive, facilitating evolutionary innovation.