Quantifying stream periphyton assemblage responses to nutrient amendments with a molecular approach

Nutrient (nitrogen [N] and phosphorus [P]) pollution is a pervasive water quality issue in the USA for small streams and rivers. The effect of nutrients on the biotic condition of streams is often evaluated with biological indicators such as macroinvertebrate assemblages or periphyton assemblages, particularly diatoms. Molecular approaches facilitate the use of periphyton assemblages as bioindicators because periphyton is diverse and its composition as a whole, rather than just diatoms, soft-bodied algae, or any single group, may convey additional information about responses to nutrients. To further develop the concept that a taxonomically-broad evaluation of periphyton assemblages could be useful for developing stream bioindicators, we examined microbial assemblage composition with both 16S and 18S rRNA genes, enabling us to evaluate composition in 3 domains. We measured otherwise unknown nutrient responses of different periphyton groups in situ with experiments that used glass fiber filters to allow diffusion of amended nutrients into a stream. We deployed these experimental setups in 2 streams that differ in the extent of agricultural land-use in their catchments in the southeastern USA. Experiments consisted of controls, N amendments, P amendments, and both N and P amendments. Periphyton assemblages that grew on the filters differed significantly by stream, date or season, and nutrient treatment. Assemblage differences across treatments were more consistent among Bacteria and Archaea than among eukaryotes. Effects of nutrient amendments were more pronounced in the stream with less agricultural land use and, therefore, lower nutrient loading than in the stream with more agricultural land use and higher nutrient loading. Combined N and P amendments decreased species richness and evenness for Bacteria and Archaea by ∼36 and ∼9%, respectively, compared with controls. Indicator species analysis revealed that specific clades varied in their response to treatments. Indicators based on the responses of these indicator clades were related to nutrient treatments across sites and seasons. Analyses that included all the taxa in a domain did not resolve differences in responses to N vs P. Instead, better resolution was achieved with an analysis focused on diatoms, which responded more strongly to P than N. Overall, our results showed that in situ nutrient-diffusing substrate experiments are a useful approach for describing assemblage responses to nutrients in streams. This type of molecular approach may be useful to environmental agencies and stakeholders responsible for assessing and managing stream water quality and biotic condition.