Recurrent genomic structure and context-dependent biological divergence across hydrologically fragmented landscapes in African annual killifishes

Geographic isolation and environmental heterogeneity jointly structure evolutionary divergence, but how similar landscape features generate recurrent patterns of genomic differentiation across independently evolving lineages, and the extent to which such patterns extend to functional, phenotypic, and host-associated biological variation, remains less clear. We integrated whole-genome sequencing, phylogenomic inference, multivariate morphology, and gut microbiome composition across two annual killifish species occupying hydrologically fragmented landscapes in Malawi. Both species exhibited pronounced geographic organization of genome-wide variation associated with drainage structure, revealing recurrent spatial structuring of genomic differentiation despite their independent evolutionary histories. However, the functional genomic signatures of divergence differed between lineages and evolutionary scales. Within Nothobranchius kirki, exon-level differentiation was associated primarily with translation-related functions, whereas divergence within N. wattersi involved ATP biosynthesis and physiological homeostasis. Interspecific divergence was enriched for transcription factor activity and transcription factor binding, implicating regulatory differentiation at the deeper evolutionary scale. Morphological variation was also strongly structured among populations and broadly associated with drainage systems and geographic regions, but morphological distance was unrelated to genome-wide genetic differentiation. Gut microbiome composition retained significant species- and population-associated structure but exhibited substantially greater overlap among populations than either genomic or morphological variation. Together, these results show that recurrent spatial organization of genomic differentiation across fragmented landscapes does not necessarily imply recurrent functional or organismal outcomes. Instead, similar constraints imposed by hydrological connectivity and isolation can be associated with recurrent population genomic structure while functional genomic, phenotypic, and host-associated variation remain strongly contingent on lineage and biological context.