Comparative genomics reveals a genomic and functional continuum among secondary replicons in Halobacteriota

A significant fraction of archaeal genomes have their genomic content distributed across two or more DNA molecules, yet the characteristics and evolution of these different replicons remain poorly understood. Here, we investigated the genomic and functional organization of secondary replicons in Halobacteriota using 211 complete genomes, comprising 766 replicons, including 211 primary chromosomes (chr1), and 555 secondary replicons. Comparative analyses of genomic features, including replicon size, GC content, and codon usage, revealed a continuous distribution of secondary replicons from plasmid-like to chromosome-like characteristics. Larger secondary replicons showed progressively more chromosome-like genomic and functional properties, including greater protein-family overlap with chr1 and a higher proportion of conserved gene families. At the phylum level, functional profiles substantially overlapped among replicon types, providing further support for a continuum rather than discrete functional classes. Larger replicons also showed increased protein-family duplication, while pangenome analysis revealed a greater proportion of conserved gene families, with accessory families most abundant at intermediate sizes and rare and singleton families declining with increasing replicon size. Together, these results support a model in which secondary replicons in Halobacteriota span a genomic and functional continuum from plasmid-like to chromosome-like states , with increasing size associated with greater conservation, gene duplication and progressively more chromosome-like gene content and functional profile.