Quantitative stable isotope probing to measure in situ growth of protist populations

Soil protists shape microbial food webs and nutrient cycling, yet methods for measuring their population growth in soil have lagged behind the taxonomic resolution available from 18S rRNA gene sequencing. Microscopy-based approaches can estimate abundance and growth, but with limited taxonomic resolution. Here, we tested whether quantitative stable isotope probing (qSIP) can provide reproducible, sequencing-resolved growth measurements for soil protists. We incubated soil with natural-abundance or {superscript 1} O-labeled water and measured taxon-specific 18O enrichment in DNA using three common 18S rRNA gene primer sets. 18O enrichment values were positively correlated across datasets, with relationships closest to 1:1 after poorly resolved taxonomic assignments were excluded, indicating that qSIP provides reproducible population-level growth signals across marker choices. We then compared 18S amplicon profiles from unfractionated DNA with qSIP-derived growth measurements across a soil moisture gradient. Amplicon profiles showed small shifts in relative abundances of major protist groups, whereas qSIP revealed a large moisture response in the growing community: 7 ASVs were growing at 20% field capacity compared with 143 at 80% field capacity, representing 1.6% and 63.3% of total protist relative abundance, respectively. Average growth was <1% day{square}1 in the two driest treatments, increasing to 2.1% day{square}1 at 60% and 5.6% day{square}1 at 80% field capacity; among growing taxa, rates averaged 8.7% and 8.3% day{square}1 in the two wetter treatments. By pairing taxonomic resolution with isotope-based growth estimates, qSIP with 18O-H2O moves soil protist ecology toward quantitative population dynamics: identifying which taxa grow, how fast, and how growth responds to the environment.