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

Species-specific genes, also known as orphans, are ubiquitous across life’s domains. In prokaryotes, species-specific orphan genes (SSOGs) are mostly thought to originate in external elements such as viruses, followed by horizontal gene transfer, while the scenario of native origination, through rapid divergence or de novo, is mostly dismissed. However, quantitative evidence supporting either scenario is lacking. Here, we systematically analyzed genomes from 4,644 human gut microbiome species and identified over 600,000 unique SSOGs, representing an average of 2.6% of a given species’ pangenome. These sequences are mostly rare within each species yet show signs of purifying selection. Overall, SSOGs use optimal codons less frequently, and their proteins are more disordered than those of conserved genes (i.e., non-SSOGs). Importantly, across species, the GC content of SSOGs closely matches that of conserved ones. In contrast, the ∼5% of SSOGs that share similarity to known viral sequences have distinct characteristics, including lower GC content. Thus, SSOGs with similarity to viruses differ from the remaining SSOGs, contrasting an external origination scenario for most SSOGs. By examining the orthologous genomic region in closely related species, we show that a small subset of SSOGs likely evolved natively de novo and find that these genes also differ in their properties from the remaining SSOGs. Our results challenge the notion that external elements are the dominant source of prokaryotic genetic novelty and will enable future studies into the biological role and relevance of species-specific genes in the human gut. ### Competing Interest Statement The authors have declared no competing interest.