The afterlife of a horizontally transferred gene: Expansion and functional diversification of a C1A peptidase in wild Hordeum species

Horizontal gene transfer (HGT) can accelerate plant adaptation, but the evolutionary fate of newly acquired nuclear genes is often unclear. Here, we analyzed a papain-like cysteine peptidase horizontally transferred from Panicoideae to wild Hordeum (Poaceae). Using chromosome-level assemblies of 21 diploid Hordeum species, we assessed presence/absence, copy-number variation, coding diversity, selection, predicted protein functionality, and expression. To infer donor relationships, we screened 77 Panicoideae accessions (48 species, 20 genera) by polymerase chain reaction and sequenced positive amplicons with Nanopore. The transferred locus, likely acquired as part of a larger panicoid DNA segment, is retained across Hordeum sect. Stenostachys but shows strong post-transfer diversification, with 1-8 copies per species, extensive transposable-element insertions, and numerous coding-sequence variants. Close homologs in Panicoideae were rare and restricted to Panicum and Paspalum; gene trees identified Panicum bergii as the closest sampled relative. Selection analyses indicated heterogeneous constraints and episodic positive selection in some Hordeum lineages. Only a minority of copies retained an intact catalytic triad, whereas most were predicted to be pseudopeptidases. Transcripts were detected across species and tissues. These results indicate that an HGT-derived DNA block can persist, amplify, and diversify in recipient genomes, generating raw material for later functional evolution.