Microbial diversity across spatial and temporal scales in high mountain watersheds of the Teton Range, U.S.A.

Abstract

Mountain ranges cover approximately 30% of Earth’s terrestrial surface and supply freshwater to human populations, globally (Milner et al. 2017; Moser et al. 2019). Beyond supporting humans, mountain watersheds (i.e. streams, rivers, lakes and upstream meltwater sources) are hotspots of biodiversity. In particular, mountain streams and lakes host diverse microbial communities in biofilms (Battin et al. 2016). These biofilms are primary sites of biogeochemistry and contribute to energy flow in montane food webs (Battin et al. 2016; Hotaling et al. 2017; Jorgenson et al. 2024; Michoud et al. 2025). Mountain stream biofilm communities are also increasingly threatened by climate change. Specifically, changes in meltwater supplies, temperature, and nutrient (e.g., nitrogen and phosphorus) concentrations threaten to reduce the presence of taxa adapted to historically cold and low nutrient conditions (Huss et al. 2017; Ren et al. 2019; Bourquin et al. 2025). Such changes may exert long-term declines in biofilm diversity and impact mountain ranges, globally (Ren et al. 2019; Oleksy et al. 2021; Brahney et al. 2022; Bourquin et al. 2025). A loss or shift in microbial diversity in mountain lakes and streams has far-reaching implications including: collapses in food web diversity, changes in water quality, loss of unique mountain specific adaptations, and altered biogeochemical cycles (Battin et al 2016; Hotaling et al. 2019; Busi et al. 2022; Kohler et al. 2022; Jansen et al. 2024; Ezzat et al. 2025). Yet, predicting how biofilm diversity will respond to climate change remains difficult because mountain lakes and streams are heterogenous ecosystems with strong spatial and temporal variation in environmental conditions that impact community structure and function (Wilhelm et al. 2013; Fell et al. 2021). For instance, differences in meltwater contributions (Hotaling et al. 2019; Brighenti et al. 2021), underlying bedrock porosity (Miller et a. 2021), and seasonality (Pernthaler et al. 1998) contribute to localized conditions that likely impact how microbial communities respond to change. Our ability to understand these ecosystems is further complicated by the fact accessing mountain lakes and streams is logistically challenging limiting the frequency and spatial resolution of sampling campaigns. Fortunately, due to accumulated datasets and collaborative efforts to conserve these regions (e.g. Hotaling et al. 2019; Jorgenson et al. 2024; Ezzat et al. 2025; Bourquin et al. 2025), we are now able to address and explore temporal and spatial processes influencing biofilm diversity. My dissertation contributes to these ongoing efforts by investigating biofilm diversity across space and time in alpine streams and mountain lakes of the Teton Range, Wyoming, USA. For the first chapter of my dissertation, I investigated interannual biofilm diversity and composition in the Teton Range. From 2019 to 2022, I collected biofilms, in August, across alpine streams that may receive differing meltwater inputs. These meltwater inputs are a combination of ice, snow, rain, and groundwater that were categorized into three major stream types in the Teton Range by Hotaling et al. (2019). These stream types included glacier-fed, snow-fed, and icy seeps (i.e. subterranean ice melt). To assess interannual diversity, I used targeted amplicon sequencing for both bacteria and eukaryotes. The results from this chapter indicated both bacterial and eukaryotic diversity are correlated with environmental conditions in stream sites similar to other studies (Wilhelm et al. 2013; Hotaling et al. 2019). However, bacterial and eukaryotic composition did not change by site or with environmental conditions. I further analyzed the core microbiome to understand stable interannual taxa within each site. I found that Cyanobacteria and Ochrophyta were stable and abundant community members potentially contributing to the year-to-year similarities and environmental correlations. Collectively, the results from Chapter 1 suggest that microbial biofilm diversity may be structured by site specific environmental conditions interannually. I encourage futures studies to continuously monitor diversity through time as alpine streams continue to be impacted by climate change and to more robustly explore the interconnections between streams, hydrology, geochemistry, and microbial ecology. In my second chapter, I characterized the evolutionary selection pressures and putative adaptations of nitrogenase in cyanobacteria. To do this, I collected biofilms from mountain lakes in the Teton Range and used amplicon based approaches to analyze a fragment of the gene encoding the Fe-protein, nifH, of nitrogenase. Afterwards, I filtered my data to only cyanobacteria and calculated evolutionary selection pressures. I further describe putative biochemical changes in NifH that could signal minor adaptations to the Teton Range. I did this by comparing Teton Range amplicon sequence variants (ASVs) to non-Teton Range sequences from NCBI. The results of this chapter indicate nifH is strongly conserved in cyanobacteria and that there may be biochemical differences in the Fe-protein in the Teton Range for heterocystous and non-heterocystous families of cyanobacteria. Collectively, the results of this chapter reveal the nifH gene is strongly conserved yet there may be minor NifH protein adaptations promoting flexibility across cyanobacterial families. In this chapter, I further suggest consideration of broader sequencing efforts to encompass the full nitrogenase gene complex and to assess non-cyanobacterial diazotroph adaptations. In the third chapter of my dissertation, I conducted nutrient enrichment experiments in mountain lakes of the Teton Range to investigate how increasing nutrients impact biofilm biomass and community compositions. To do this, I used nutrient diffusing substrates to increase nitrogen (N) and phosphorus (P) concentrations. After the experiment, I quantified photosynthetic and fungal biomass and characterized community composition of cyanobacteria, algae, and fungi with amplicon sequencing. The results of this chapter did not support my expectation of colimitation of N and P in either biomass or community composition. Rather, I observed significant correlations that differed for biomass and the community composition of cyanobacterial, algal, and fungal communities. The results of this chapter highlight that mountain lake photosynthetic and fungal biofilm communities may be limited by other factors such as temperature or micronutrients rather than colimitation. I encourage future studies to assess the interactions between temperature and nutrients on nutrient enrichment biofilm responses. Together, each chapter of my dissertation helps shed light into the temporal and spatial patterns of diversity, adaptations, and sensitivity of microbial communities to nutrients across alpine streams and mountain lakes of the Teton Range. These results underscore the need to continuously assess microbial diversity across space and time in these habitats