Fungi dominate eukaryotic microbial assemblages in hot spring biofilms of East and Southeast Asia.

Terrestrial hot springs are globally distributed extreme environments, and these systems have long served as natural laboratories for studying microbial life under thermal stress. While much of the research to date has focused on thermophilic bacteria and archaea, there is a growing appreciation for the diversity and ecological significance of eukaryotic microorganisms in these habitats. In this study, we used metagenomic sequencing to assess inter-domain microbial diversity in biofilms from 47 circumneutral hot springs across East and Southeast Asia, with a specific focus on resolving eukaryotic taxa and their ecology. Whilst all biofilm communities were dominated by bacteria, the microbial eukaryotes represented approximately 10% of the taxonomic diversity and accounted for 1.3% of overall taxa abundance, indicating a small but significant presence. We provide the first comprehensive inter-domain checklist of over 14,500 microbial taxa in hot springs. Patterns in diversity were significantly correlated with temperature, hydrogen sulfide, and pH in hot springs. Fungi emerged as the most abundant and prevalent eukaryotic group, indicating an important role as eukaryotic saprotrophs, with Ascomycota yeasts comprising the most individually abundant taxa. Among other microbial eukaryotic phyla, the photosynthetic Chlorophyta and Bacillariophyta were most abundant. Predatory/grazing microbial eukaryotes were relatively less diverse and abundant. Network co-occurrence analysis was used to indicate extensive and specific biotic interactions between eukaryotes and bacteria in biofilms. We further employed metatranscriptomics to identify putatively active taxa, revealing that most detected eukaryotes were transcriptionally active. While fungi accounted for most transcripts, the highest RNA:DNA ratios were observed among predatory and photosynthetic taxa, suggesting elevated activity in these functional groups. Overall, our findings highlight the diversity, interactions, and activity of eukaryotes in Southeast Asian hot spring biofilms, underscoring their potential importance in shaping microbial community structure and function in extreme environments.