Document Type

Thesis

Date of Degree Completion

Winter 2026

Degree Name

Master of Science (MS)

Department

Biology

Committee Chair

James Johnson

Second Committee Member

Wayne Quirk

Third Committee Member

Geoffrey Sasaki

Abstract

Yeasts are ubiquitous unicellular fungi that play critical roles in decomposition, nutrient cycling, and fermentation processes across diverse ecosystems. Despite their ecological and biotechnological importance, the diversity and distribution of wild yeast populations in many temperate regions remain poorly characterized. This study evaluated how classifier thresholds influence species assignment and functional inference from internal transcribed spacer 2 (ITS2) Sanger sequences of cultured yeasts collected from diverse habitats in central Washington. The project aimed to assess the stability of taxonomic calls across classifier settings and to illustrate how methodological decisions shape downstream ecological conclusions. Samples from each habitat were enriched under selective conditions (pH 3.5, 5% ethanol, reduced oxygen) and incubated at 8 °C and 20 °C to capture yeasts across thermal niches. Seventy-one morphologically distinct isolates were purified and sequenced at the ITS2 locus. Taxonomic classification was performed using the UNITE database at 99%, 97%, and dynamic thresholds in QIIME 2, and ecological guilds were inferred using FunGuildR. Community composition and categorical variables were evaluated by PERMANOVA to test for effects of incubation temperature and habitat. iii Thirteen genera were recovered, spanning Ascomycota and Basidiomycota, with Ascomycota predominating overall. Incubation temperature did not significantly affect total isolate richness, although Basidiomycota were proportionally more common at 8 °C. Across classifier thresholds, 16.9% of isolates changed species-level or genus-level assignment, most frequently within Basidiomycota, where sequence similarity to reference data was lower. Although 76% of isolates exhibited ≥99% identity, and 86% of the isolates exhibited ≥97% identity to a UNITE reference, the modification of threshold choice altered both species names and inferred ecological guilds. The majority of isolates were classified as saprotrophic. These findings demonstrate that small adjustments in classifier thresholds can dramatically change how culture-based ITS2 datasets may interpret and assign taxonomic compositions. Because species-level determinations and guild assignments proved sensitive to classifier settings, future surveys should explicitly report database versions and threshold parameters. Further recommendations include validation of ambiguous identifications with secondary loci such as LSU or TEF1, and, where possible, employing both culture-based sequencing and high-throughput approaches to stabilize ecological inference.

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