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Small streams and their headwaters are key sources of microbial diversity in fluvial systems and serve as an entry point for bacteria from surrounding environments. Community assembly processes occurring in these streams shape downstream population structure and nutrient cycles. To elucidate the development and stability of microbial communities along the length of a first-through third-order stream, fine-scale temporal and spatial sampling regimes were employed along Mc-Nutt Creek in Athens, GA, USA. 16S rRNA amplicon libraries were constructed from samples collected on a single day from 19 sites spanning the first 16.76 km of the stream. To provide context for this spatial study and evaluate temporal variability, selected sites at the stream's upper, mid, and lower reaches were sampled daily for 5 days preceding and following the spatial study. In a second study, three sites at and near the creek's headwaters were sampled daily for 11 days to understand initial bacterioplankton community assembly. Both studies revealed decreasing alpha and beta diversity with increasing downstream distance. These trends were accompanied by the enrichment of a small fraction of taxa found at low abundance in headwaterproximal sites. Similar sets of taxa consistently increased in relative abundance in downstream samples over time scales ranging from 1 day to 1 year, many of which belong to clades known to be abundant in freshwater environments. These results underpin the importance of headwaters as the site of rapid in-stream selection that results in the reproducible establishment of a highly stable community of freshwater riverine bacteria. IMPORTANCE Headwater streams are critical introduction points of microbial diversity for larger connecting rivers and play key roles in the establishment of taxa that partake in in-stream nutrient cycling. We examined the microbial community composition of a first-through third-order stream using fine-scale temporal and spatial regimes. Our results show that the bacterioplankton community develops rapidly and predictably from the headwater population with increasing total stream length. Along the length of the stream, the microbial community exhibits substantial diversity loss and enriches repeatedly for select taxa across days and years, although the relative abundances of individual taxa vary over time and space. This repeated enrichment of a stable stream community likely contributes to the stability and flexibility of downstream communities.

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Summary Stemflow is a spatially concentrated input of rainwater at the base of trees, resulting from precipitation draining down tree branches to the stem. Depending on tree shape, stemflow can represent a significant fraction of the total rainfall that contacts the tree's canopy area, and can become chemically enriched along its drainage path. As a result, stemflow has been hypothesized to influence microbial communities in the receiving soil proximal to the stem. However, previous studies have (i) yielded conflicting results on the significance of stemflow as a driver in bacterial community composition, and (ii) not directly compared communities in soils with and without stemflow receipt. In this study, a stemflow diversion system was employed on Quercus virginiana trees in Skidaway Island (Georgia, USA) to directly compare soil bacterial communities receiving no stemflow to those beneath trees with no diversion system in place. In both treatments, sample distance from the stem significantly influenced bacterial community structure. However, the absence of stemflow resulted in increased bacterial community diversity across all samples. Stemflow diversion also significantly altered longitudinal patterns in the abundance of multiple taxonomic groups. These results support the hypothesis that Q. virginiana stemflow has a significant impact on bacterial soil inhabitants and is a key factor in taxon selection in stem‐proximal communities.

microbial community composition of a first-through third-order stream using fine-47 scale temporal and spatial regimes. Our results show that the bacterioplankton community 48 develops rapidly and predictably from the headwater population with increasing total stream 49 length. Along the length of the stream, the microbial community exhibits substantial diversity 50 loss and enriches repeatedly for select taxa across days and years, although the relative 51 abundances of individual taxa vary over time and space. This repeated enrichment of a stable 52 stream community likely contributes to the stability and flexibility of downstream communities. 53