Persistence of Resident and Transplanted Genotypes of the Undomesticated Yeast, Saccharomyces Paradoxus in Forest Soil

bioRxiv preprint doi: https://doi.org/10.1101/305060; this version posted April 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Persistence of Resident and Transplanted Genotypes of the

2 Undomesticated Yeast, Saccharomyces paradoxus in Forest Soil

4 James B. Anderson, Dahlia Kasimer, Clara Xia, Nicolas C. H. Shröeder, Patrick

5 Cichowicz, Silvio Lioniello, Rudrackshi Chakrabarti, Eashwar Mohan, and Linda M.

6 Kohn

9 Department of Biology, 3359 Mississauga Road, University of Toronto, Mississauga

10 Ontario L5L 1C6 Canada

12 Running title: Persistence of Saccharomyces paradoxus in forest soils 13

15 Key words: fungus, population, dispersal, genetic drift, 5FOA resistance

17 Corresponding Author: James B. Anderson [email protected]

18 bioRxiv preprint doi: https://doi.org/10.1101/305060; this version posted April 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Anderson et al., 2

1 ABSTRACT

2 One might expect yeasts in soil to be highly dispersed via water or insects, forming

3 ephemeral, genetically heterogeneous populations subject to competition and

4 environmental stochasticity. Here, we report years-long persistence of genotypes of the

5 yeast Sacchaormyces paradoxus in space and time. Within 1 km2 in a mixed hardwood

6 forest on scales from centimeters to tens of meters, we detect persistence over 3 years

7 of native genotypes, identified by SNPs genome-wide, of the wild yeast,

8 Saccharomyces paradoxus around Quercus rubra and Q. alba. Yeasts were recovered

9 by enrichment in ethanol-containing medium, which measures only presence or

10 absence, not abundance. Additional transplantation experiments employed strains

11 marked with spontaneous defects in the URA3 gene, which also confer resistance to 5-

12 Fluoroorotic acid (5FOA). Plating soil suspensions from transplant sites on 5FOA

13 medium permitted one-step quantification of yeast colony-forming units, with no

14 interference from other unmarked yeasts or microorganisms. After an initial steep

15 decrease in abundance, the yeast densities fluctuated over time, increasing in

16 association with rainfall and decreasing in association with drought. After 18 months,

17 the transplanted yeasts remain in place on the nine sites. In vitro transplantation

18 experiments into non-sterile soil in petri dishes showed similar patterns of persistence

19 and response to moisture and drought. To determine whether S. cerevisiae, not

20 previously recovered from soils regionally, can persist in our cold-climate sites, we

21 transplanted marked S. cerevisiae alone and in mixture with S. paradoxus in fall, 2017.

22 Five months on, S. cerevisiae persist to the same extent as S. paradoxus. bioRxiv preprint doi: https://doi.org/10.1101/305060; this version posted April 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Anderson et al., 3

2 IMPORTANCE

3 Saccharomyces yeasts are intensively studied in biological research and in their

4 domesticated roles of brewing and baking. And yet, remarkably little is known about

5 their mode of life in forest soils. We report here that resident genotypes of the yeast S.

6 paradoxus are persistent of a time scale of years in their micro-habitats in forest soils.

7 We also show that resident genotypes can be replaced by transplanted yeast

8 genotypes. The high inoculum levels in experimental transplantations rapidly

9 decreased over time, but the transplanted genotypes persisted at low abundance. We

10 conclude that, in forest soils, Saccharomyces yeasts exist at very low abundance and

11 that dispersal events are rare.

12 bioRxiv preprint doi: https://doi.org/10.1101/305060; this version posted April 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Anderson et al., 4

1 In their review of the ecology and evolution of non-domesticated Saccharomyces

2 species, Boynton and Greig (1) encouraged further investigation in nature to place them

3 in ecological context with reference to the scientific model, S. cerevisiae, long-

4 domesticated for food and beverage fermentation. Our focus here was on S.

5 paradoxus, the most tractable comparator of wild to domesticated Saccharomyces. S.

6 paradoxus is associated with, but not limited to, oak leaf litter in forest soils (2, 3). In

7 this yeast, progress has been made on understanding the relative importance of

8 dispersal, genetic drift, and local adaptation in populations (4), and the effects of

9 substrate utilization on metabolism and fitness (5,6). Saccharomyces yeasts are

10 present in soil and plant material, especially bark of deciduous trees where they utilize

11 exudates such as sap. Filteau et al. (6) have elucidated the genetic basis of a fitness

12 determinant in S. paradoxus: degradation of allantoate, the major nitrogen source in

13 maple sap. However, there has been little information on absolute abundance of these

14 yeasts in their natural habitat. Most information on the occurrence of wild yeasts in soil

15 has come through enrichment culture in ethanol-containing medium, which cannot be

16 used to estimate abundance. A major goal of this study was to measure the

17 abundance over time of yeasts transplanted to their natural substrates at high initial

18 densities.

19 Dispersal of Saccharomyces species between substrates is poorly understood

20 (1), but the recent work of Boynton et al. (4) suggests that lack of dispersal and

21 prominence of genetic drift are more important in shaping local populations of S.

22 paradoxus than fitness differences among genotypes. S. paradoxus may be dispersed bioRxiv preprint doi: https://doi.org/10.1101/305060; this version posted April 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Anderson et al., 5

1 by rainwater and by insects such as Drosophila (7, 8), from which it has been isolated.

2 Despite these potential dispersal mechanisms, there is genetic differentiation among S.

3 paradoxus populations that is roughly proportional to distance on a scale from

4 centimeters to thousands of kilometers (9). Despite progress in describing population-

5 genetic structure and in documenting potential dispersal mechanisms, the extent to

6 which wild yeasts actually grow and persist in stasis in their habitats in soil is unknown.

7 In this study, we first followed naturally occurring genotypes of S. paradoxus over

8 a three-year period on a fine geographical scale of marked sites in a natural woodland.

9 This extended the time frame covered by our earlier study (10) by two years. This part

10 of the study included only enrichment culturing and therefore registered only presence

11 or absence, not abundance of yeasts. We then initiated transplantation experiments

12 with yeast strains marked with spontaneous mutations. The transplanted yeasts in our

13 study allowed quantification of colony-forming units per unit of soil and of change over

14 time from an initially high level of inoculum. We found that abundance fluctuated, with

15 an overall downward trend over time and that rainfall events were associated with a

16 temporary increase in abundance. Even when abundance counts approached zero in

17 some transplant sites, enrichment culture invariably recovered the transplanted

18 genotype. Although our experiments focused on S. paradoxus, we show that marked

19 strains of S. cerevisiae, although not among the residents found in this locality, persist

20 in proximity to transplanted S. paradoxus.

22 bioRxiv preprint doi: https://doi.org/10.1101/305060; this version posted April 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Anderson et al., 6

2 RESULTS & DISCUSSION

4 In 2014 and 2015, yeasts were collected by enrichment culture in ethanol medium from

5 72 sites around the bases of three oak trees and genotyped for SNP sites across their

6 genomes. A remarkable pattern of persistence of genotypes in their sites of origin was

7 observed over this one-year time span. In the present study, we sampled these 72

8 sites again in 2016 and a limited selection of eight sites again in 2017 (Table 1),

9 extending the sampling times of 2014 and 2015 reported by Xia et al. (10) by two years.

10 At Oak 1, all sites were occupied in all years by a single S. paradoxus genotype,

11 clade a. Here, the rate of recovery of S. paradoxus was remarkable in its completeness

12 across all sites. Oak 2 showed an entirely different pattern of occupancy from that of

13 Oak 1. Overall, less than half the sites at Oak 2 returned yeasts, but those that did

14 included a broad diversity of genotypes, including: S. paradoxus clade d, a hybrid

15 lineage between two divergent lineages; an isolate of clade b, also a hybrid, but

16 between more recently diverged lineages (clades a and c); clade e, a genotype of

17 European origin, and three other yeast species, Lachancea thermotolerans,

18 Torulaspora delbrueckii, and Pichia mandshurica. Oak 2 is also notable for the

19 complete absence of clades a and c among the recovered yeast samples.

20 Oak 3 and Oak 4 presented partial occupancy of sites, a predominance of clade

21 c among the positive sites, and minority of clades a, b. There were three additional

22 yeast species: Pichia kudriavezeyii, P. mandshurica, and Candida californica, a species bioRxiv preprint doi: https://doi.org/10.1101/305060; this version posted April 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Anderson et al., 7

1 reported to be consistently vectored by Drosophila melanogaster (11). The final year of

2 limited sampling yielded one isolate of clade c. Overall, the pattern of persistence is

3 extended at least through 2016, and is consistent with persistence in the limited sample

4 into 2017.

5 The previous re-sampling experiments examined yeasts in natural sites around

6 the bases of oak trees by enrichment in ethanol containing medium; this culture method

7 is not quantitative and only registers presence or absence, with some unknown

8 threshold for detecting presence. In studying natural populations of S. paradoxus,

9 there has been a clear need for a method to quantify transplanted yeasts in situ. A

10 recently reported method for quantifying yeasts in their natural habitats (4) used digital

11 droplet PCR, which sensitively measures the ratios of the abundance of transplanted

12 genotypes, but not their absolute abundance. In this study, we used strains marked

13 with spontaneous mutations in the URA3 gene, which conferred a Ura-, 5FOA-resistant

14 phenotype. With this method, absolute abundance, colony forming units per g of soil,

15 could be measured in one step by dilution plating on agar medium containing 5FOA,

16 with no interference by growth of other soil microorganisms.

17 We transplanted yeast populations of high density into nine sites around the

18 bases of three additional oak trees (Oak 6, Oak 7, and Oak 8) in order to measure their

19 abundance over time in the natural habitat. Figure 1 shows the change in the

20 composite abundance of all transplant populations over time. After a steep decrease in

21 abundance over the first two weeks after transplantation, the levels fluctuated over time

22 with seven of nine sites still registering counts one full year after transplantation and two bioRxiv preprint doi: https://doi.org/10.1101/305060; this version posted April 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Anderson et al., 8

1 of nine sites returned no CFUs. However, enrichment cultures at the end of the

2 experiment, including those two sites registering no CFUs in the final plating on 5FOA

3 medium, were all positive for the transplanted strains. The original resident yeasts on

4 these sites sampled before transplantation (Table S1) would not be expected to appear

5 on these 5FOA plates; whether or not the original resident strains remain on the

6 transplant sites at low frequency is not known.

7 In addition to abundance, Figure 1 also indicates rainfall over time. For the first

8 and larger of the two conspicuous rainfall events, between weeks four and six, all (nine

9 of nine) populations increased in abundance (under the null hypothesis that increase in

10 CFUs, or not, is a random behavior, the probability of the same directionality of

11 response in all nine sites: P ≤ 1/ 29, or 0.002). In the second event, between weeks 8

12 and 10, eight of the nine populations increased in abundance. At this stage, we

13 interpret the apparent increase in population abundance with rainfall as a possible

14 association, which is addressed below in a laboratory experiment. This transplantation

15 experiment was not expected to distinguish whether the increase was due to

16 reproduction of cells or to separation of aggregated cells because of moisture (driving

17 up CFUs on the detection plates), or to some other mechanism.

18 The outdoor transplants introduced yeast populations to a spot about 5 cm in

19 diameter. At week 39, we recovered samples along six transects extending outward

20 from three transplant sites (Figure 2). Here the abundance values clearly show that the

21 plume of transplanted yeast cells had spread laterally. How this happened cannot be

22 distinguished in this experiment. The yeast cells may have been washed outward from bioRxiv preprint doi: https://doi.org/10.1101/305060; this version posted April 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Anderson et al., 9

1 their central location with rainfall. This lateral spread may in part explain rapid initial

2 decrease in abundance in the central location after transplantation. Actual reduction in

3 viable cells may also contribute to the reduction in observed CFUs.

4 The next experiment was devised to reproduce the outdoor transplant

5 experiment in vitro, with petri dishes containing a limited volume of non-sterile soil. The

6 pattern of abundance over time in the indoor experiment paralleled the outdoor

7 experiment. Yeast abundance initially decreased sharply and then fluctuated over time,

8 gradually approaching low levels by week 41 (Figure 3). A final enrichment culturing at

9 week 48, followed by testing on 5FOA medium, revealed that all nine plates harbored

10 viable representatives of the originally transplanted strains. Under these conditions, the

11 decline in CFUs could not have been due to spreading of the yeast populations as

12 occurred in the outdoor transplantation sites. The decline of CFUs must have been due

13 to a decrease in the proportion of viable cells or in their propensity to form aggregates

14 (in which multiple viable cells that were aggregated would plate as one CFU).

15 After the indoor transplantation experiment was completed, the soil plates were

16 re-purposed for another experiment to test for the effect of moisture after extended

17 drought. The plates were allowed to dry out at room temperature for three months. At

18 this point, soil samples were taken for dilution plating on 5FOA medium and for

19 enrichment culture. No CFUs were registered for this initial sampling; the transplanted

20 yeasts had become rare. Nonetheless, the enrichment cultures were all positive for the

21 transplanted yeasts (which had the 5FOA phenotype). Water was then added to the

22 plates so that the soil became saturated, but without free water. Water was added to bioRxiv preprint doi: https://doi.org/10.1101/305060; this version posted April 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Anderson et al., 10

1 the plates twice per week to keep the soil saturated. Samples were taken at week 1

2 and week 2 two after the initial sampling. All plates registered CFUs and, for all

3 replicates, the CFUs per g soil increased from initial sampling (0.0 ± 0.0 SE) to week 1

4 (1367 ± 519 SE), and to week 2 (2156 ± 764 SE). The effect in this in-vitro experiment

5 was remarkably consistent with response to rainfall after a period of dryness in the

6 outdoor transplantation experiment (null hypothesis, increase in CFUs, or not, is

7 random; probability of the same directionality of response in all nine plates: P ≤1/ 29, or

8 0.002).

9 In a final experiment, we addressed the question of whether S. cerevisiae would

10 persist in outdoor transplant sites, as did S. paradoxus. The rationale for this

11 experiment is that S. cerevisiae is not found in our study site or in northern latitudes in

12 woodlands (12,13). Furthermore, genotypes of S. cerevisiae do not appear to spread

13 from vineyards to forests (14). Here, we transplanted a marked genotype of S.

14 cerevisiae in proximity to S. paradoxus. Both yeast species and the mix declined in

15 abundance over the first six weeks to very low CFU counts at the end of the winter

16 (week 18, Figure 4). The ratio of the two yeast species fluctuated around 1:1. At the

17 end of the experiment CFUs were low (Table S2); in the mixed population, in total, there

18 were six CFUs of S. cerevisiae and six of S. paradoxus. In enrichment culture at week

19 19, all nine sites recovered the original genotype transplanted. At this stage, S.

20 cerevisiae appears to persist about as well as S. paradoxus (5) in the study locality,

21 where they have not been detected previously. bioRxiv preprint doi: https://doi.org/10.1101/305060; this version posted April 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Anderson et al., 11

1 There are two main contributions from this study. The first contribution is

2 technical. Although our focus here was on persistence of yeast strains in their habitats,

3 our transplantation experiments demonstrate the feasibility of long-term measurements

4 of fitness effects in mixtures of strains in the field and in the laboratory. An advantage

5 of transplantation to indoor microcosms with non-sterile soil is that the markers used to

6 distinguish strains need not be limited to spontaneous mutations as was the case here

7 for outdoor transplantation experiments. Targeted comparisons might involve specific

8 gene deletions or modifications that would not be appropriate for outdoor

9 transplantation.

10 The second contribution is biological. Our observations that resident yeast

11 populations persist over time with infrequent dispersal is entirely consistent with the

12 scenario proposed by Boynton et al. (4). Our transplantation experiments are also

13 consistent with a low level of dispersal, contributing a new dimension to our

14 understanding of the carrying capacity of soil habitats for yeast populations. The initial

15 high levels of inoculum in the transplantations were not sustainable. The CFUs per g of

16 soil decreased precipitously in the first two weeks after transplantation and then more

17 slowly, finally registering few or no CFUs on the 5FOA selective medium. And yet,

18 enrichment culturing indicated that the original, transplanted genotypes were still on

19 their sites in a viable state. Although the initial high levels of inoculum were not

20 maintained, the transplanted genotypes persist and the transplants replace the naturally

21 occurring yeast residents on those sites. Only by sampling the transplant sites in

22 subsequent years can the limits of persistence be measured. Overall, our results are bioRxiv preprint doi: https://doi.org/10.1101/305060; this version posted April 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Anderson et al., 12

1 consistent with the idea that Saccharomyces yeasts exist at extremely low levels in the

2 soil, with the possibility for limited population growth when temperature and moisture

3 conditions in the soil permit. Against this prevalent pattern of stasis, the sudden

4 availability of higher nutrient conditions, for example from sap flows, could lead to

5 substantial growth in yeast populations with subsequent decrease in population density

6 over time; our transplantation experiments were designed to simulate these high-growth

7 events. We speculate that such chance amplifications in yeast populations would have

8 more effect on long term presence than fitness differences among genotypes, especially

9 where those fitness differences only relate to slight differences in growth rate measured

10 under rich nutrient conditions in laboratory culture.

12 MATERIALS AND METHODS

14 Enrichment recovery of naturally occurring yeasts. Our initial goal was to identify

15 naturally occurring genotypes of S. paradoxus and to track their distributions over time.

16 The sampling area included the same sites studied via enrichment culture by Xia et al.

17 (10) in 2014 and 2015 and this study extended the earlier sampling into 2016 and 2017

18 (Table 1). The sample encompassed four oak trees, with three sites around the base of

19 each tree separated by buttress roots, with six microsites in each site (Fig. 1, See Xia et

20 al., 10). Collection of soil samples, enrichment culture with 8% ethanol, DNA isolation,

21 Illumina sequencing, read alignment against a standard genome, and variant discovery bioRxiv preprint doi: https://doi.org/10.1101/305060; this version posted April 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Anderson et al., 13

1 were exactly as described by Xia et al. (10). Each newly isolated strain was identified to

2 clades a – e, which were described earlier by Xia et al. (10)

4 Marked strains for transplantation experiments. Transplantation experiments were

5 done with spontaneously arising ura3 mutants. We found these mutants in S. paradoxus

6 by allowing a diploid culture to sporulate and then plating large numbers of spores in

7 tetrads on 5FOA medium. The sporulation step was done to facilitate homozygosity for

8 any newly arising mutants. The resistant mutants appearing on 5FOA medium had a

9 Ura- phenotype (15). Three ura3 mutants of S. paradoxus were used: one frameshift

10 (Sce5003, c.260_261insC), one nonsense (Sce5006 c.553C>T, GLU to stop), and one

11 missense mutation (Sce5010, c. 801G>A, GLY to ASP).

12 For a marked version of S. cerevisiae, we crossed a wild type strain (Sce13) with

13 another strain carrying multiple auxotrophies (Sce695). From the offspring of this cross,

14 we identified haploid strains of complementary mating type that were 5FOA resistant

15 (and Ura-) and His-. These two haploids were then mated to form a diploid that was

16 homozygous for ura3 and his3. The his3 mutation was used to distinguish S.

17 cerevisiae from S. paradoxus (His+) in mixed transplant sites. None of the transplanted

18 strains of either S. paradoxus or S. cerevisiae contained any genetically engineered, or

19 recombinant DNA, constructs.

21 Outdoor transplantation of S. paradoxus. Oak 6, Oak 7, and Oak 8, were selected

22 for transplantation. At each tree, there were three transplant sites separated by buttress bioRxiv preprint doi: https://doi.org/10.1101/305060; this version posted April 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Anderson et al., 14

1 roots (total of nine sites). The three sites at each tree were inoculated with a different

2 one of the three ura3- marked strains of S. paradoxus (23 Sept., 2016). Immediately

3 before transplantation, any yeast residents were identified by enrichment culture as

4 described (Table S1).

5 At each site, we applied 100 ml of water with a suspension of 1010 cells through

6 the perforated cap of a large saltshaker. A spot on the soil surface of about 5 cm in

7 diameter was sprinkled with the cell suspension, allowing sufficient time for the fluid to

8 soak in without running across the surface. At the time of sampling, ca. 0.5 g of soil

9 was scraped into a 15 ml plastic tube, weighed, and suspended in either 10 ml of water

10 or, at the end of the experiment, in 10 ml of enrichment medium (6). After vigorous

11 agitation, 100 µl of the suspension, or a dilution thereof, was spread onto 9 cm petri

12 dishes with 5FOA medium (15). After three days of incubation at 30 C, colonies were

13 counted and the number of CFUs per g of soil calculated. Raw CFU data are given in

14 Table S2. At the end of the experiment, and after enrichment culturing, colonies were

15 tested for 5FOA resistance and the URA- phenotype. Sites without transplantation

16 invariably registered no 5FOA resistant cultures.

18 Indoor transplantation of S. paradoxus. These experiments were constructed to

19 mimic the outdoor experiments, except that the available volume was limited and the

20 inoculum was initially mixed into the non-sterile soil, which had been collected near the

21 bases of several oak trees, pooled, and sieved to a fine particle size. Deep petri dishes

22 (2.5 X 9 cm) containing ca. 50 g soil mix were inoculated with 10 ml of water containing bioRxiv preprint doi: https://doi.org/10.1101/305060; this version posted April 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Anderson et al., 15

1 a suspension of 109 cells (12 Jan., 2017). Each soil plate was weighed initially and then

2 weekly thereafter; the reduction in weight over the preceding week was compensated

3 by the addition of sterile water. After this experiment was completed, the soil plates

4 were re-purposed to test the effect of moisture addition after a period of extended

5 dryness as described below. The plates were allowed to dry out at RT for three months

6 and CFUs were measured before and after the addition water to the plates.

8 Outdoor transplantation of S. cerevisiae. We selected three new trees (Oak 9, Oak

9 10, and Oak 11) with three sites around the base of each tree separated from one

10 another by buttress roots. At each tree, S. cerevisiae was transplanted to one site, S.

11 paradoxus to another site, and an equal mix of the two species to a third site.

12 Transplantation yeast cells was done exactly as in the outdoor transplantation

13 experiment. Approximately 1010 cells suspended in water were applied to each site (26

14 October, 2017).

16 Data availability. A comprehensive variant (.vcf) file for all S. paradoxus strains in

17 Table 1 is available in the Dryad Digital Repository (accession number pending

18 acceptance). Alignments of Illumina reads of the 2014 and 2015 collections with a

19 reference S. paradoxus genome (.bam files) are accessible through NCBI’s SRA

20 (http://www.ncbi.nlm.nih.gov/sra) as accession PRJNA324830 (see ref. no. 10).

1 SUPPLEMENTAL MATERIAL

2 Table S1. Summary of resident yeast species on the sites for outdoor transplantation.

3 Table S2. Raw data (CFUs / g soil) for Figures 1-4.

5 ACKNOWLEDGEMENTS

6 This study was supported by Individual Discovery Grants from the Natural Sciences and

7 Engineering Research Council of Canada (NSERC) to JBA and LMK.

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14 11. Stamps JA, Yang LH, Marales VM, Boundyt-Mills KL. 2012. Drosophila regulate 15 yeast density and increase yeast community similarity in a natural substrate. PLoS One 16 https://doi.org/10.1371/journal.pone.0042238

17 12. Charron G, Leducq JB, Bertin C, Dube AK, Landry, CR. 2014. Exploring the 18 northern limit of the distribution of Saccharomyces cerevisiae and Saccharomyces 19 paradoxus in North America. FEMS Yeast Research 14:281–288. 20 21 22 13. Robinson HA, Pinharanda A, and Bensasson D. 2016. Summer temperature can 23 predict the distribution of wild yeast populations. Ecology and Evolution 6:1236-1250. 24 doi: 10.1002/ece3.1919

25 14. Hyma KE and Fay JC. 2013. Mixing of vineyard and oak-tree ecotypes of 26 Saccharomyces cerevisiae in North American vineyards. Molecular Ecology 22: 2917- 27 2930. doi 10.111/mec.12155 28 29 15. Boeke JD, LaCroute F, Fink GR. 1984. A positive selection for mutants lacking 30 orotidine-5’-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. 31 Molecular and General Genetics 197:345-346. 32 33 bioRxiv preprint doi: https://doi.org/10.1101/305060; this version posted April 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Anderson et al., 18

1 Table 1. Identity of yeast strains recovered by enrichment culture from sites near the

2 bases of four oak trees.

3 4 Tree-Site 2014 2015 2016 2017 5 -microsite 6 7 1-1-1 WX1/a R1/a DK1/a 8 1-1-2 WX2/a R2/a DK2/a JBA1/a 9 1-1-3 WX3/a R3/a DK3/a 10 1-1-4 WX4/a R4/a DK4/a 11 1-1-5 WX5/a R5/a DK5/a 12 1-1-6 WX6/a R6/a DK6/a 13 1-2-1 WX7/a R7/a DK7/a 14 1-2-2 WX8/a R8/a DK8/a 15 1-2-3 WX9/a R9/a DK9/a 16 1-2-4 WX10/a R10/a DK10/a JBA2/a 17 1-2-5 WX11/a R11/a DK11/a 18 1-2-6 WX12/a R12/a DK12/a 19 1-3-1 WX13/a R13/a DK13/a 20 1-3-2 WX14/a R14/a DK14/a 21 1-3-3 WX15/a R15/a DK15/a 22 1-3-4 WX16/a R16/a DK16/a 23 1-3-5 WX17/a R17/a DK17/a 24 1-3-6 WX18/a R18/a DK18/a JBA3/a 25 26 2-1-1 - - - 27 2-1-2 - R19/d - 28 2-1-3 - - - 29 2-1-4 WX19/d&WX20/d - 30 2-1-5 - R20/d - 31 2-1-6 - R21/d - 32 2-2-1 - - - 33 2-2-2 - R22/d - 34 2-2-3 WX21/d R23/d DK19/Pm 35 2-2-4 - R24/d - 36 2-2-5 - - - 37 2-2-6 WX22/b - - 38 2-3-1 - - DK20/e 39 2-3-2 - Lt DK21/e 40 2-3-3 - - DK22/d 41 2-3-4 - Td DK23/d 42 2-3-5 Lt - - 43 2-3-6 - Td - 44 45 3-1-1 - R28/a DK24/Pk - 46 3-1-2 - R29/c DK25/c bioRxiv preprint doi: https://doi.org/10.1101/305060; this version posted April 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Anderson et al., 19

1 3-1-3 - - DK26/c 2 3-1-4 WX25/a Td DK27/c 3 3-1-5 - - DK28/c 4 3-1-6 - R31/c DK29/c 5 3-2-1 - - - 6 3-2-2 - - - 7 3-2-3 - - DK30/c 8 3-2-4 WX26/b - - 9 3-2-5 WX27/c - - 10 3-2-6 - - - 11 3-3-1 - - - 12 3-3-2 - R32/c DK31/c - 13 3-3-3 - - DK32/c 14 3-3-4 WX28/c R33/c DK33/c 15 3-3-5 - - DK34/c 16 3-3-6 WX29/c R34/c - 17 18 4-1-1 WX30/c R35/a - 19 4-1-2 WX31/c R36/c - 20 4-1-3 WX32/c R37/c DK36/a - 21 4-1-4 WX33/c R38/c DK37/c 22 4-1-5 - - DK38/c 23 4-1-6 WX34/c R39/c DK39/c 24 4-2-1 WX35/c R40/c DK40/c 25 4-2-2 WX36/c R41/c DK41/c JBA4/c 26 4-2-3 WX37/c R42/c DK42/c 27 4-2-4 WX38/c R43/c DK43/c 28 4-2-5 WX39/c R44/c DK44/Pm 29 4-2-6 WX40/c R45/c DK45/c 30 4-3-1 - - DK46/c 31 4-3-2 WX41/c - DK47/c 32 4-3-3 WX42/a - DK48/c 33 4-3-4 WX43/c R46/c DK49/c - 34 4-3-5 - R47/c DK50/c 35 4-3-6 WX44/c Cc DK51/c 36 37 38 Note: Sites are annotated by Tree-Site-Microsite. Strains are represented as strain

39 name/clade membership (in bold). Minus (-), sample taken, but no yeast recovered. In

40 2014, 2015, and 2016 all 72 oak microsites were sampled; in 2017, eight samples were

41 taken. The data for 2014 and 2015 are from the report of Xia et al. (10) and the data

42 from 2016 and 2017 are from this study. Abbreviations for non-Saccharomyces species bioRxiv preprint doi: https://doi.org/10.1101/305060; this version posted April 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Anderson et al., 20

1 names: Cc, Candida californica; Lt, Lachancea thermotolerans; Pk, Pichia kudriavezeyii;

2 Pm, P. mandshurica; and Td, Torulaspora delbrueckii.

3 bioRxiv preprint doi: https://doi.org/10.1101/305060; this version posted April 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Anderson et al., 21

2.0 50 wk 1 - 705 ± 431 wk 2 - 14 ± 11 40 1.5

30 1.0 20

Normalizedabundance 0.5 10 Rainfallin week (mm) before sample

0.0 0 3 4 5 6 7 8 9 10 30 57 75 Time in weeks after transplant 2

3 Figure 1. Outdoor transplantation of yeast strains and abundance over time. Three

4 strains were each transplanted to three sites around the bases of three oak trees (three

5 strains per tree). The abundance values for each site were normalized against the

6 average counts of colony forming units (CFU) per gram of soil between weeks 3 and 8

7 plus or minus standard error. The average value for normalization (over the nine sites)

8 was 373,440 CFU / g soil ± 76,254 (SE). In the initial sampling, the colonies were too

9 numerous to count on the dilution plates. The average relative abundance values for all

10 nine sites, plus or minus standard error for weeks 1 and 2, appear in the upper left;

11 these values are off the scale of this graph. The bars represent rainfall amount in the

12 week prior to sampling.

13 bioRxiv preprint doi: https://doi.org/10.1101/305060; this version posted April 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Anderson et al., 22

1 2 3 4 5 6 7 4.0

3.0

2.0

1.0 Normalizedabundance

0.0 2.5 5.0 7.5 12.5 17.5 22.5 27.5 Distance in cm from transplant center 8 9 10 11 12 Figure 2. Outdoor transplantation and relative abundance with distance in cm from the

13 transplant site. On three of the nine sites for outdoor transplantation (each at a

14 different tree and each a different strain), six diameters were measured at distance

15 intervals (two intervals per site) at week 39 after transplantation. The values for each

16 site were normalized against CFU / g soil for each of the six diameters plus or minus

17 standard error. The average value for normalization (over the six diameters) was

18 58,449 CFU / g soil ± 14,644 (SE).

19 bioRxiv preprint doi: https://doi.org/10.1101/305060; this version posted April 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Anderson et al., 23

1 2 2.5 wk 0 - 69 ± 10 wk 1 - 7.1 ± 2.5 2.0

1.5

1.0

Normailizedabundance 0.5

0.0 2 3 4 5 6 7 9 11 13 15 17 19 21 25 29 33 41 Time in weeks 3 4 5 6 7 Figure 3. Indoor transplantation of yeast strains and abundance over time. Three

8 strains were transplanted to each of three petri dishes containing 50 g of forest soil

9 (nine plates total). The values for each site were normalized against CFU / g soil

10 between weeks 2 and 41 plus or minus standard error. The average value for

11 normalization was 612,118 CFU / g soil ± 74,280 (SE). The values for the initial

12 sampling and week 1 after transplantation appear in the upper right. bioRxiv preprint doi: https://doi.org/10.1101/305060; this version posted April 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Anderson et al., 24

a S. cerevisiae b S. paradoxus 4.0 4.0 wk 0 - 6.0 ± 1.9 wk 0 - 71.5 ± 45.8 3.0 wk 1 - 2.1 ± 0.8 3.0 wk 1 - 29.5 ± 23.4

2.0 2.0

1.0 1.0 Normalizedabundance 0.0 Normailizedabundance 0.0 2 3 4 5 6 18 2 3 4 5 6 18 Time in weeks after transplant Time in weeks after transplant

c Mix of S. cerevisiae and S. paradoxus d Ratio of S. cerevisiae to S. paradoxus 0.8

4.0 S. wk 0 - 6.3 ± 2.4 0.7 3.0 wk 1 - 6.5 ± 3.4 0.6 0.5 2.0 S. cerivisaeS. /

paradoxus 0.4 1.0 0.3 Ratio 0.0 0.2 Normalizedabundance 2 3 4 5 6 18 0 1 2 3 4 5 6 18 Time in weeks after transplant Time in weeks after transplant

1 2 3 4 5 Figure 4. Outdoor transplantation of S. cerevisiae and S. paradoxus and relative

6 abundance over time. Inocula containing (a) S. cerevisiae only, (b) S. paradoxus only,

7 and (c) a mix of S. cerevisiae and S. paradoxus, were transplanted to three sites at the

8 bases of three oak trees; each tree had one site of each type of inoculum. (d) the ratio

9 between S. cerevisiae and S. paradoxus for the mixed population; note that counts on

10 5FOA test plates were low for the 18-week time point, total of 12 CFUs on nine plates.

11 The values for each site were normalized against CFU / g soil between weeks 3 and 10

12 plus or minus standard error. The average values for normalization were: S. paradoxus,

13 738,722 ± 632,755 (SE); S. cerevisiae 922,523 ± 592,478, and the mix of the two

14 yeasts 1,287,051 ± 315,193 (SE). The average relative abundance values (all nine

15 sites) for weeks 1 and 2, which were off scale on the graph, appear in the upper right.