Abstract
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 can influence the genotypes and phenotypes of studied strains. We developed a direct isolation method for forest floor Saccharomyces and compared its success and phenotypic biases to a previously published enrichment-based isolation method. In a European forest, direct isolation was more successful at isolating S. paradoxus, but also more labor intensive, than enrichment culturing. Average growth rates of S. paradoxus isolates collected using the two methods did not differ at the enrichment isolation temperature, but variances in growth rates did: direct isolation produced a collection of S. paradoxus isolates with less variation in growth rates than enrichment culturing. In other words, enrichment culturing sampled more phenotypic diversity than direct isolation. Enrichment culturing also sampled more Saccharomyces species diversity than direct isolation, including our only isolations of rare S. cerevisiae. Enrichment culturing may sample higher Saccharomyces phenotypic and species diversity because of variations in interactions between yeasts and the other microbes that were present in the soil and leaf litter samples. We recommend direct culturing for researchers interested in randomly sampling their study habitats and enrichment culturing for researchers interested in discovering new Saccharomyces phenotypes or rare Saccharomyces species from natural environments. We include step-by-step sampling protocols in the supplemental materials.
Introduction
Naturally-occurring Saccharomyces populations are models for ecology and evolution (Boynton & Greig, 2014). Use of these models has led to exciting discoveries about the ecology and evolution of microbial phenotypes; for example, adaptation to climate can lead to speciation (Leducq et al., 2014), domesticated S. cerevisiae is more phenotypically diverse than wild S. paradoxus (Warringer et al., 2011), and interspecific hybrids can have high fitnesses in stressful environments (Bernardes, Stelkens, & Greig, 2017; Stelkens, Brockhurst, Hurst, Miller, & Greig, 2014). These studies made inferences based on the phenotypes and genotypes of isolates collected from wild and domesticated substrates. And Saccharomyces substrates are diverse: wild substrates include tree bark, insect guts, leaf litter, soil, fruits, and parasitic Cyttaria galls, (Kowallik & Greig, 2016; Libkind et al., 2011; Mortimer & Polsinelli, 1999; Sampaio & Goncalves, 2008; Stefanini et al., 2012), and domesticated substrates include wine, beer, bread, kimchi, kombucha, palm wine, and pulque, among many other substrates (Boynton & Greig, 2016; Carbonetto, Ramsayer, Nidelet, Legrand, & Sicard, 2018; Estrada-Godina et al., 2001; Ezeronye & Okerentugba, 2001; Gallone et al., 2016; Greenwalt, Steinkraus, & Ledford, 2000; Jeong, Jung, Lee, Jin, & Jeon, 2013). One challenge of environmental yeast sampling is to minimize sampling biases so researchers can assure that differences among source environments, not sampling techniques, are responsible for observed phenotypic patterns.
Enrichment culturing is a reliable and frequently-used method for isolating difficult-to-culture microbes, including Saccharomyces, from natural environments (Schlegel & Jannasch, 1967; Sniegowski, Dombrowski, & Fingerman, 2002) (Figure 1A). Microbiologists have been relying on enrichment cultures for over a century (Beijernick, 1961), and have used enrichment culturing to isolate many of the model Saccharomyces strains commonly used in laboratory studies (Johnson et al., 2004; Liti et al., 2009; Sniegowski et al., 2002). To isolate a microbe using enrichment culturing, a researcher adds a small amount of natural material to a growth medium designed to be hospitable to the target microbe and inhospitable to other microbes (Liti, Warringer, & Blomberg, 2017; Schlegel & Jannasch, 1967). If the enrichment medium is well-designed, the target microbe is expected to grow in abundance, and after some incubation time, this enrichment culture can be streaked to solid media and colonies of the target microbe can be easily isolated. An alternative to enrichment culturing is to spread a microbial substrate directly onto a selective solid medium, with or without dilution, and to pick colonies which morphologically resemble the target microbe (Stefanini et al., 2012) (Figure 1B).
Because it can be difficult to isolate Saccharomyces from natural substrates, many investigations of wild Saccharomyces rely on enrichment culturing, usually in high-sugar, acidic media (Charron, Leducq, Bertin, Dube, & Landry, 2014; Robinson, Pinharanda, & Bensasson, 2016; Sniegowski et al., 2002; Sweeney, Kuehne, & Sniegowski, 2004). Comparative studies of Saccharomyces genomes have been carried out using Saccharomyces strains isolated using disparate strategies, including both enrichment and direct culturing (Liti et al., 2009; Peter et al., 2018). However, isolation strategy can influence the genotypes and phenotypes of isolated microbes (Stefani et al., 2015), and we were concerned about the biases that might be introduced during enrichment culturing of Saccharomyces yeasts. Specifically, we were concerned that isolation method might bias results in studies of Saccharomyces phenotypes. For example, enrichment culturing might select for individuals with high relative fitness in the enrichment medium. Such potential biases in sampled yeast phenotypes are likely to lead to biases in sampled genotypes because genetic information is responsible for expressed phenotypes. Isolation biases have also been suggested as potential explanations for differences between results of culture-dependent and culture-independent studies of environmental Saccharomyces (Alsammar et al., 2018).
This study’s goals were to compare isolation success between enrichment culturing and a direct culturing strategy, and to quantify biases in Saccharomyces phenotypes (and therefore genotypes) that might be introduced when sampling a forest environment. We tested the assumption that it is easier to sample Saccharomyces from forest substrates using enrichment cultures than direct plating. We also compared growth rates between S. paradoxus isolated using enrichment and direct strategies. Enrichment culturing might decrease or increase sampled S. paradoxus phenotypic diversity compared to direct plating, thereby decreasing or increasing variance among S. paradoxus growth rates. For example, variance among growth rates would be low (and average growth rates high) among S. paradoxus isolated using enrichment cultures if the enrichment temperature and media select for the fastest growing S. paradoxus present in every sample. Conversely, variance among growth rates would be high for S. paradoxus isolated using enrichment cultures if diversity in the non-Saccharomyces microbial communities present on sampled substrates select for diverse S. pardoxus among samples.
To test these predictions, we compared Saccharomyces sampling success and isolates’ growth rates among soil and leaf litter samples from a well-studied northern German forest (Kowallik & Greig, 2016; Kowallik, Miller, & Greig, 2015). A previous study showed that S. paradoxus, the wild sister species of the lab model S. cerevisiae, is readily isolated using enrichment cultures from oak leaf litter in this forest (Kowallik & Greig, 2016). We were also previously able to isolate S. paradoxus directly from these forest substrates without enrichment (Kowallik, 2015).
Methods
Field sampling and yeast isolation
All isolates were sampled from a mixed hardwood and conifer forest in Nehmten, Schleswig-Holstein, northern Germany (Nehmtener Forst). We sampled leaf litter and soil material from close to the bases of ten oak trees at four sampling dates (Table 1), although not all trees were sampled at every date. Trees were between 12 and 744 m from one another. At each date, samples were collected from leaf litter and the top organic layer of soil within one meter of the base of each tree. Paired leaf litter and soil samples were collected on the north, south, east, and west side of each tree at all collection days except 7 April, when samples were collected at an arbitrary two of the four cardinal directions.
Material was collected simultaneously for the direct plating and enrichment collections at each sampling point (Figure 1). First, leaf litter was collected by aseptically transferring litter into sterile collection tubes: approximately 5 ml of compressed leaf litter was collected for the direct plating method and approximately 2 ml for the enrichment method. Then, the remaining leaf litter was removed from the soil surface and the top approximate 2 cm of soil (mostly composed of soil organic layer) were aseptically transferred into sterile collection tubes. As for leaf litter, approximately 5 ml of compressed soil was collected for the direct plating method and approximately 2 ml for the enrichment method. Instruments were sterilized between samples using 70% ethanol. Samples were transported between the field and lab at ambient temperature and processed within four hours of collection.
For direct plating (Figure 1A), material was mixed with 20 ml sterile water in a sterile 50 ml tube, the mixture was vigorously mixed for at least 10 seconds with a vortex mixer on its highest setting, and 0.2 ml of the resulting dirty liquid was pipetted on each of two plates containing solid modified selective media PIM1 (3 g yeast extract, 5 g peptone, 10 g sucrose, 3 g malt extract, 1 mg chloramphenicol, 80 ml ethanol, 5.2 ml 1 M HCl, and 20 g agar per liter) (Kowallik & Greig, 2016; Sniegowski et al., 2002). Liquid was spread on plates using sterile glass beads, and plates were left open in a laminar flow hood until dry. Plates were incubated for three days at 30 °C before colonies were picked.
For enrichments (Figure 1B), material was mixed with 10 ml liquid selective media PIM1 (composition as for solid PIM1 but without agar) in a 15-ml sterile tube, mixtures were inverted, and tubes were incubated, slightly open and without shaking, at 30 °C. After 10 days, a sterile wooden stick was inserted into each enrichment tube and a small amount of liquid was streaked onto a single plate with solid selective media PIM2 (20 g Methyl-(alpha)-D-glucopyranoside, 1 ml 5% Antifoam Y-30 emulsion, 6.7 g Yeast Nitrogen Base without amino acids, 4 ml 1M HCl, and 20 g agar per liter) (Kowallik & Greig, 2016; Sniegowski et al., 2002), and plates were incubated 4 days at 30°C before colonies were picked.
We include these procedures as step-by-step protocols for the convenience of future researchers in the supplementary materials (Supplemental File 1).
Yeast identification
After incubation, we streaked colonies with yeast-like morphology to fresh YPD media (10 g yeast extract, 20 g peptone, 20 g dextrose, and 25 g agar per liter). For each method, up to 6 (March and April sampling days) or 12 (June and July sampling days) colonies per sample were selected. After one day of growth on YPD at 30 °C, cultures were frozen at −80 °C in 20% glycerol and a small amount of each culture was transferred to sporulation media (20 g potassium acetate, 2.2g yeast extract, 0.5 g dextrose, 870 mg complete amino acid mixture, and 25 g agar per liter). Any cultures with bacteria-like morphology on YPD media (slimy culture and/or cells smaller than 1 micron across) were not frozen and were discarded. Sporulation cultures were incubated for at least three days at room temperature before being screened under a compound microscope for Saccharomyces-like asci (tetrads).
All cultures producing tetrads were identified using sequencing of the internal transcribed sequence (ITS), a region neighboring rRNA-coding DNA (Schoch et al., 2012). We sequenced every strain using the ITS1/ITS4 primer pair (White, Bruns, Lee, & Taylor, 1990). PCR mixes were 7-15 µl in volume and contained one yeast colony, 0.5 µM each primer, and either 50% Phusion® High-Fidelity PCR master mix with HF buffer or 1x HF-buffer, 100 µM dNTP mix, 3% DMSO, and 1 U/50 µl Phusion DNA polymerase. PCR reactions were cycled at 98 °C for 30 s, then 35 cycles of 98 °C for 5 s, 62 °C for 20 s, and 72 °C for 30 s, plus a 10 min terminal extension at 72 °C. PCR products were cleaned using illustra™ ExoProStar™ according to the manufacturer’s instructions, and sequenced on an ABI 3130xl sequencer.
ITS sequences were compared to sequences from the type or neotype strains of S. paradoxus, S. cerevisiae, S. kudriavzevii, and S. mikatae (Genbank accession numbers NR_138272.1, NR_111007.1, KY105195.1, and KY105198.1). If a sequence did not align with Saccharomyces sequences, we compared the sequence with all sequences in the NCBI database from type strains using BLAST (Zhang, Schwartz, Wagner, & Miller, 2000). If the sequence aligned with Saccharomyces sequences but had more than one base pair different from its closest match, we supplemented ITS sequences with sequences from the gene for translation elongation factor 1 using primers EF1-983F and EF1-2212R (Rehner & Buckley, 2005) using the protocols above, but with a PCR annealing temperature of 57 °C. In some cases, cultures originating from apparent single colonies were in fact mixtures of two yeast species. We counted these colonies as Saccharomyces if sequences from one of the species was Saccharomyces.
Growth rates
We compared the distributions of maximum growth rates between two groups of S. paradoxus strains: strains collected using enrichment culturing and strains collected using direct plating. To avoid confounding effects of environmental source (i.e., combination of substrate, date collected, and tree), we compared growth rates for pairs of S. paradoxus strains originating from the same environmental source. In other words, we collected a dataset of S. paradoxus growth rates from two groups of strains with equal representations of combinations of substrate, date collected, and tree, and differing only in the method used to isolate the strains. To ensure that all isolates were pure S. paradoxus cultures, we streaked all isolates used for growth rate measurements to single-colony cultures a second time and reidentified these cultures by mating them with a S. paradoxus tester strain (NCYC 3708, α, ura3::KANMX, ho::HYGMX). In total, 110 isolates (55 from each sampling method) were measured.
Growth rates were measured using an Epoch 2 microplate reader (Biotek Instrument, Inc., Winooski, VT, USA) and calculated using the included Gen5 software version 3.03.14 (Biotek Instrument, Inc., Winooski, VT, USA). We first inoculated strains in 0.2 ml liquid YPD media (composition as for solid YPD, but without agar) in a 96-well microplate and incubated cultures without shaking or measurement in the microplate reader at 30 °C for 24 hours to condition strains to microplate reader conditions. We then transferred 2 µl from each culture to 198 µl fresh liquid YEPD in a new microplate and incubated the new microplate under the same conditions for 20-24 hours. OD660 was measured during the second incubation every ten minutes, and maximum growth rate (mOD660/min) was calculated from the maximum slope of each growth curve over four points (30 min total) using Gen5 software. Reported growth rates for each isolate are means of three replicates.
Statistical analyses
We compared sampling success across substrates (leaf litter or soil) and methods (direct plating or enrichment) using a generalized linear mixed-effects model with probability of isolating Saccharomyces (including S. paradoxus and S. cerevisiae) as the response variable, substrate and method as fixed effects, and tree and date as random effects. We selected the best model using a top-down strategy, comparing Akaike’s Information Criteria (AIC) after removing predictors from a full model one by one.
We compared growth rate distributions by first comparing variances using Levene’s test (Levene, 1960) for homogeneity of variance, and then comparing medians using a paired Wilcoxon signed rank test. Statistics were computed using R version 3.3.1 (R Development Core Team, 2016) and the car and lme4 packages (Bates, Machler, Bolker, & Walker, 2015; R Development Core Team, 2016). Graphics were produced using the ggplot2 package (Wickham, 2016).
Results
Influence of sampling method on success isolating Saccharomyces
Direct plating was more successful than enrichment culturing for isolating Saccharomyces from natural substrates (z = 6.1, p < .001) (Tables 2, 3, Figure 2). We found Saccharomyces isolates in 45% of direct plating cultures and 19% of enrichment cultures. However, enrichment culturing produced the only S. cerevisiae found in this study: we found six S. cerevisiae isolates from a single enrichment culture from tree 3 in March of 2017. All other Saccharomyces isolates found in this study were S. paradoxus. Other detected yeast species included Saccharomycodes ludwigii, Torulaspora delbrueckii, Pichia membranifaciens, and Hanseniaspora osmophila, all of which have previously been found alongside Saccharomyces yeasts in beverage fermentations (Domizio et al., 2011; Gschaedler, 2017).
While the direct plating method was more successful than the enrichment method, it was also more labor-intensive (Table 4). We screened 3.4 times as many colonies for tetrads when using the direct plating method than we did using the enrichment method. Only 32% of the processed direct plating colonies were S. paradoxus, compared to 74% of enrichment colonies.
Both methods isolated Saccharomyces colonies from both substrates, most trees, and all timepoints (Figure 2). We had significantly more sampling success on soil than leaf litter substrates (z = 5.7, p < .001, Table 3), but other relationships among sampling success, sampling method, and sampling environments were idiosyncratic. For example, direct plating did not produce any Saccharomyces isolates from tree 6, while three enrichment samples from this tree isolated S. paradoxus, and enrichments produced more Saccharomyces isolates in March than direct plating did (Figure 2). Because our sampling effort was not the same for all trees at all months, we did not model tree habitat or sampling month as fixed effects; instead, we modeled these parameters as random effects, and found that models including tree and month fit the data better than models without tree and month (Table 2). A list of sequenced yeasts from each sample is included in the supplemental materials (Supplemental File 2).
Phenotypes of sampled S. paradoxus
Variances in growth rate, but not median growth rates, were different between strains isolated using the two methods (Figure 3). Growth rates of S. paradoxus isolated using enrichment culturing had a larger variance than growth rates of S. paradoxus isolated using direct plating (Levine’s test F1,108 = 5.42, p = .02). Median growth rates for the two groups of S. paradoxus strains did not differ (Wilcoxon signed rank test V = 6320, p = .39). Measured growth rates of each strain are included in the supplemental materials (Supplemental File 2).
Discussion
Direct plating detects S. paradoxus more frequently than enrichment culturing
Enrichment culturing did not increase Saccharomyces sampling success from forest leaf litter and soil over direct plating, in spite of researchers’ long history of using enrichment culturing to isolate Saccharomyces from forest environments (Kowallik & Greig, 2016; Naumov, Naumova, & Sniegowski, 1998; Sniegowski et al., 2002). We expect reliable Saccharomyces isolation from this forest using direct plating to be a result of high S. paradoxus abundance on forest floor substrates. Indeed, it is common to find hundreds to tens of thousands of S. paradoxus cells per gram of leaf litter near the bases of oak trees in this forest (Kowallik & Greig, 2016). We expect direct plating to be less successful in environments in which Saccharomyces are rarer, and note that enrichment culturing is frequently used to isolate Saccharomyces from tree bark, which may be a habitat with lower Saccharomyces density than the forest floor habitats we sampled (Kowallik et al., 2015; Sniegowski et al., 2002).
Isolation using enrichment culturing samples more phenotypic diversity than direct plating
Conditions in enrichment cultures resulted in isolating a different (albeit higher) phenotypic diversity than random colony selection on plate cultures did (Figure 3). There are several potential methodological and ecological explanations for the high phenotypic diversity in enrichment isolates. But we expect that interactions with microbes during the enrichment culturing are most likely to be responsible for the increased phenotypic diversity among enrichment-sourced S. paradoxus isolates.
Microbes that potentially interact with S. paradoxus were doubtless present in our enrichment cultures, and it is realistic to expect these microbes to influence the phenotypes of the S. paradoxus that were ultimately recovered. Our enrichment cultures contained all of the microbes that were present in the soil and leaf litter samples, and soil and leaf litter include a wide diversity of bacterial and fungal taxa that can interact with fungi (Curd, Martiny, Li, & Smith, 2018; Glassman et al., 2018; Santonja et al., 2018). For example, some agricultural soils contain Bacillus and Pseudomonas species that secrete compounds toxic to phytopathogenic fungi (Islam, Jeong, Lee, & Song, 2012; Petatán-Sagahón et al., 2011). Conversely, some soil bacteria promote mycorrhizal fungal growth and host plant colonization (Xie et al., 2018).
We expect a variety of similar inhibition and facilitation interactions to determine the identities of the Saccharomyces strains that ultimately reached high frequencies in enrichment cultures. Bacteria and fungi co-occurring with S. paradoxus on oak bark can both inhibit and facilitate S. paradoxus growth (Kowallik et al., 2015). These effects are dependent on temperature and interacting microbe identities, and likely also depend on other environmental conditions and S. paradoxus genotype. The Saccharomyces strains recovered at the end of our enrichment cultures were probably the fittest Saccharomyces strains present in the cultures, but this high relative fitness was as likely to be a result of interactions with co-occurring microbes that occur by chance in the same enrichment cultures as it was to be the result of intrinsic growth rate. Microbial diversity during enrichment may similarly explain our idiosyncratic sampling success across months and trees (Figure 2). For example, it is possible that a bacterium that facilitates rare S. paradoxus growth in the enrichment medium was more common in spring than summer months, resulting in higher enrichment sampling success in spring than summer. Unfortunately, we did not measure the microbial diversity of the enrichment cultures and do not know which microbes may have interacted with Saccharomyces.
It is also possible, although in our opinion less likely, that methodological biases resulted in relatively low phenotypic diversity among directly plated S. paradoxus compared to enrichment S. paradoxus. While we aimed to randomly select colonies with Saccharomyces-like morphologies from direct culture plates, biases in colony picking could have selected for low variance in S. paradoxus phenotypes. For example, if a S. paradoxus colony had an unusual morphology, it might have been mistaken for a bacterial colony and not isolated. However, direct culturing isolated more non-Saccharomyces yeast isolates than enrichment culturing did, and we expect interspecific morphological variation to be higher than intraspecific morphological variation. We therefore consider it unlikely that biases in colony picking decreased phenotypic variance among S. paradoxus isolates.
We continue to expect plate culturing to be a more random sampling of Saccharomyces diversity than enrichment culturing. Colonies are physically isolated on a plate and unlikely to influence each other’s growth, and plated colonies come from a well-mixed mixture of environmental substrate and sterile water. We therefore expect Saccharomyces colonies on plates to accurately reflect the diversity of Saccharomyces strains present in nature, as long as all strains are able to grow on the selective media chosen. Biases in sample success can still be introduced to direct plating samples by the presence of morphologically similar yeast species. On petri dishes, a high density of non-Saccharomyces yeasts with morphologies similar to Saccharomyces could have prevented us from detecting Saccharomyces. This issue can be mitigated by designing a more selective isolation medium, picking more colonies per plate (we picked up to 6 or 12 colonies across two plates), or replica plating selective plates to media containing a color indicator for the target yeast (e.g., Wallerstein media) (Hall, 1971).
Recommendations for future yeast sampling
Researchers should consider both resources available for sampling and study goals when choosing a Saccharomyces field sampling strategy. Our results identified a tradeoff between resources spent on sampling and resources spent on sequencing: enrichment culturing was less successful than direct plating at finding Saccharomyces per sample collected, but more successful per ITS region sequenced (Figure 2, Table 4). Researchers with a few precious samples are therefore better off isolating Saccharomyces using direct plating than enrichments, especially if Saccharomyces is common on their substrates. Conversely, if samples are easy to get but funds available for sequencing are limited, researchers may prefer to use enrichment culturing. Researchers with limited time or freezer space who would like assurances that most picked colonies are Saccharomyces may also prefer enrichment culturing.
However, the scientific question to be answered by environmental Saccharomyces samples may be more important than sampling and sequencing resource limitations, especially as sequencing becomes cheaper. Direct plating samples a more random collection of Saccharomyces cells from environmental samples than enrichment culturing, and we recommend that researchers who need random environmental samples to answer ecological and evolutionary questions rely on direct plating. Researchers targeting phenotypic diversity, especially for applied yeast biology (e.g., food microbiology, drug discovery) may uncover more diversity by isolating environmental Saccharomyces using enrichment cultures. Researchers interested in detecting rare Saccharomyces species in an environment (e.g., S. cerevisiae from our study forest, S. mikatae and S. eubayanus from European forests) (Alsammar et al., 2018) may also have more success using enrichment culturing or a combination of enrichment and direct plating strategies.
Conclusions
Isolation protocols do indeed influence characteristics of isolated microbes. As researchers continue to develop Saccharomyces yeasts as model organisms for ecology and evolution, they must also consider how isolation history of environmental strains can influence the ecological stories the strains can tell. The results of this study highlight the need for consistent sampling within studies, and are a warning to researchers comparing phenotypes (and perhaps also genotypes) among Saccharomyces strains from different sources. Much of what we know about Saccharomyces evolution has come from culture collections (Strope et al., 2015; Warringer et al., 2011)—these collections are invaluable, as are the data and conclusions they enable, but sampling information about deposited strains is not always available. As technology improves and both genome sequencing and phenotype assays become cheaper and more accessible (Porter & Hajibabaei, 2018; Stewart et al., 2018; van Dijk, Auger, Jaszczyszyn, & Thermes, 2014; Zackrisson et al., 2016), environmental sampling may emerge as a limiting step to studying Saccharomyces ecology and evolution. Researchers should carefully consider the consequences of their chosen sampling strategies as they conduct this exciting microbiological research.
Data Accessibility
All data for this project are included in Supplementary File 2.
Acknowledgements
We would like to thank Danielle Stevens and Tjorben Nawroth for help in the field, Jenna Gallie and the Gallie lab for help with growth curves, and Amine Hassani for helpful conversations on microbial diversity in enrichment cultures. Thank you to Christoph Freiherr von Fürstenberg-Plessen for permission to work in the Nehmten forest. This work was supported through a Max Planck Fellowship to Eva H. Stukenbrock.