Saccharomyces yeasts are emerging as model organisms for ecology and evolution, and researchers need environmental Saccharomyces isolates to test ecological and evolutionary hypotheses. However, methods for isolating Saccharomyces from nature have not been standardized, and isolation methods may influence the genotypes and phenotypes of studied strains. We compared the effectiveness and potential biases of an established enrichment culturing method against a newly developed direct plating method for isolating forest floor Saccharomyces spp. In a European forest, enrichment culturing was both less successful at isolating Saccharomyces paradoxus per sample collected and less labour intensive per isolated S. paradoxus colony than direct isolation. The two methods sampled similar S. paradoxus diversity: The number of unique genotypes sampled (i.e., genotypic diversity) per S. paradoxus isolate and average growth rates of S. paradoxus isolates did not differ between the two methods, and growth rate variances (i.e., phenotypic diversity) only differed in one of three tested environments. However, enrichment culturing did detect rare Saccharomyces cerevisiae in the forest habitat and also found two S. paradoxus isolates with outlier phenotypes. Our results validate the historically common method of using enrichment culturing to isolate representative collections of environmental Saccharomyces. We recommend that researchers choose a Saccharomyces sampling method based on resources available for sampling and isolate screening. Researchers interested in discovering new Saccharomyces phenotypes or rare Saccharomyces species from natural environments may also have more success using enrichment culturing. We include step‐by‐step sampling protocols in the supplemental materials.

17Saccharomyces yeasts are emerging as model organisms for ecology and 18 evolution, and researchers need environmental Saccharomyces isolates to test 19 ecological and evolutionary hypotheses. However, methods for isolating 20Saccharomyces from nature have not been standardized and isolation methods can 21 influence the genotypes and phenotypes of studied strains. We developed a direct 22 isolation method for forest floor Saccharomyces and compared its success and 23 phenotypic biases to a previously published enrichment-based isolation method. In a 24European forest, direct isolation was more successful at isolating S. paradoxus, but 25 also more labor intensive, than enrichment culturing. Average growth rates of S. 26 paradoxus isolates collected using the two methods did not differ at the enrichment 27 isolation temperature, but variances in growth rates did: direct isolation produced a 28 collection of S. paradoxus isolates with less variation in growth rates than enrichment 29 culturing. In other words, enrichment culturing sampled more phenotypic diversity 30 than direct isolation. Enrichment culturing also sampled more Saccharomyces species 31 diversity than direct isolation, including our only isolations of rare S. cerevisiae. 32

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Microorganisms are famous for adapting quickly to new environments. However, most evidence for rapid microbial adaptation comes from laboratory experiments or domesticated environments, and it is unclear how rates of adaptation scale from human-influenced environments to the great diversity of wild microorganisms. We examined potential monthly-scale selective pressures in the model forest yeast Saccharomyces paradoxus. Contrary to expectations of seasonal adaptation, the S. paradoxus population was stable over four seasons in the face of abiotic and biotic environmental changes. While the S. paradoxus population was diverse, including 41 unique genotypes among 192 sampled isolates, there was no correlation between S. paradoxus genotypes and seasonal environments. Consistent with observations from other S. paradoxus populations, the forest population was highly clonal and inbred. This lack of recombination, paired with population stability, implies that S. paradoxus evolved the phenotypic plasticity needed to resist seasonal environmental fluctuations long ago, and that individual S. paradoxus are generalists with regard to seasonal environments. Similarly, while the forest population included diversity among phenotypes related to intraspecific interference competition, there was no evidence for active coevolution among these phenotypes. At least ten percent of the forest S. paradoxus individuals produced “killer toxins”, which kill sensitive Saccharomyces cells, but the presence of a toxin-producing isolate did not predict resistance to the toxin among nearby isolates. How forest yeasts acclimate to changing environments remains an open question, and future studies should investigate the physiological responses that allow microbial cells to cope with environmental fluctuations in their native habitats.