Bacterial community adaptation after freshwater and seawater coalescence

Abstract Microbial community coalescence, the merging of entire microbial communities, is common across ecosystems, particularly in estuaries where freshwater and seawater mix. The complexity of these habitats makes the in situ study of community dynamics after coalescence challenging, highlighting the need for controlled experiments to unravel the factors influencing the coalescence in the estuary. To study these processes, we combined natural freshwater and seawater bacterial communities at five different mixing ratios and incubated them in parallel microcosms containing freshwater or seawater incubation media. Forty mixed communities were tracked over six passages using Nanopore full-length 16S rRNA gene sequencing. In the original field samples, freshwater hosted more diverse communities than seawater. While the communities were structurally distinct, shared bacterial families accounted for approximately 95% of total reads. Many low-abundance taxa were lost upon laboratory incubation, while potentially faster-growing ones were enriched. We found that the coalescence outcome was strongly shaped by the incubation media, whereas the mixing ratio had a minor influence. Mixed communities converged toward the source community native to the incubation media, with increasing similarity at a higher source community proportion. In freshwater, a 25% inoculum of the freshwater community was sufficient to re-establish a near-native freshwater community, whereas in seawater, similarity to the native seawater community depended on the inoculation ratio. Network analysis showed a tightly connected module of seawater families, reflecting their shared habitat preference or cooperation, whereas freshwater families were more loosely connected. We also observed that most families were unaffected by mixing ratios or temporal dynamics, with only a few showing mixing ratio dependence. For instance, the freshwater family Comamonadaceae and seawater families Marinomonadaceae and Pseudoalteromonadaceae were dominant in their respective native environments, and increased in mixed communities in proportion to their initial source proportion. Overall, environmental filtering had a stronger impact than the mixing ratio of source communities on coalesced communities, with habitat-specific taxa further modulating the outcome. These findings advanced our understanding of microbial responses to coalescence and provided insights into microbial community assembly in dynamic estuarine systems. Highlights Environmental filtering outweighs the microbial source community ratio in shaping coalescence outcomes. Asymmetric resilience: freshwater communities require a lower inoculum to re-establish than seawater communities. Modularity of the seawater source community was observed during coalescence. Full-length 16S rRNA gene profiling with a custom dual-barcoding Nanopore protocol enables cost-effective bacterial community tracking. Controlled coalescence experiments offer mechanistic insights into estuarine microbial community transitions.