Skin microbiome mirrors habitat divergence in amphibious combtooth blenny fish (Teleostei, Blenniidae)

Host-associated microbiomes play vital roles in organismal health, ecological interactions, and evolution, yet little is known about how microbial communities shift during the transition from aquatic to increasingly terrestrial habitats. Here, we characterize the skin microbiomes of three combtooth blenny species (Blenniella paula, Praealticus labrovittatus, and Alticus arnoldorum) that occupy distinct positions along the intertidal gradient in Guam--from fully subtidal (and exclusively aquatic) to intertidal (amphibious) and supratidal environments (exclusively terrestrial). Using 16S rRNA amplicon sequencing, we compared skin-associated bacterial communities with those in surrounding seawater and substrate biofilms to assess habitat influences on microbiome structure. Skin microbiomes were distinct from environmental microbial communities, indicating strong ecological filtering by the host. The divergence between skin and substrate microbiomes in the three species parallels their distribution along progressively higher zones of the intertidal gradient. The most divergent skin microbiome was that of the supratidal fish A. arnoldorum, characterized by higher Gammaproteobacteria abundance and enrichment of epiphytic and mucus-associated taxa. Across all species, we identified 32 microbial orders significantly enriched on the skin relative to environmental samples, including taxa commonly associated with fish mucosa (e.g., Vibrio, Alteromonas, Cetobacterium) and others rarely reported in aquatic marine fish (e.g., Rubritalea, Granulosicoccus). Several rare taxa with potential pathogenicity were also detected at low abundances. Together, these findings suggest that habitat-specific selective pressures strongly shape fish skin microbiomes along subtidal (aquatic) to supratidal (terrestrial) habitats and suggest that microbial symbionts may contribute to the ecological and physiological adaptations enabling amphibious lifestyles. This study provides the first comparative assessment of skin microbiome divergence across amphibious fish species along an intertidal gradient and offers a framework for predicting microbiome responses to environmental change.