A collection of barcoded natural isolates of Saccharomyces paradoxus to study microbial evolutionary ecology

Abstract

While the use of barcoded collections of laboratory microorganisms and the development of barcode-based cell tracking are rapidly developing in genetics and genomics research, tools to track natural populations are still lacking. The yeast Saccharomyces paradoxus is an emergent microbial model in ecology and evolution. More than five allopatric and sympatric lineages have been identified and hundreds of strains have been isolated for this species, allowing to assess the impact of natural diversity on complex traits. We constructed a collection of 550 barcoded and traceable strains of S. paradoxus, including all three North American lineages SpB, SpC, and SpC*. These strains are diploid, many have their genome fully sequenced and are barcoded with a unique 20 bp sequence that allows their identification and quantification. This yeast collection is functional for competitive experiments in pools as the barcodes allow to measure each lineage's and individual strains' fitness in common conditions. We used this tool to demonstrate that in the tested conditions, there are extensive genotype-by-environment interactions for fitness among S. paradoxus strains, which reveals complex evolutionary potential in variable environments. This barcoded collection provides a valuable resource for ecological genomics studies that will allow gaining a better understanding of S. paradoxus evolution and fitness-related traits.

Keywords: Saccharomyces paradoxus; barcoded yeast collection; competition assay; wild yeast.

Figure 1

The S. paradoxus population structure and geographical distribution in North America. (a) Representation of the evolutionary history of the S. paradoxus North American lineages (Leducq et al., 2016). The European SpA and American lineages diverged about 200,000 years ago. It is hypothesized that SpB and SpC were in allopatry during the last glaciation from 110,000 to 12,000 before present (BP). A secondary contact between SpB and SpC would have occurred after the glacial retreats, leading to the formation of SpC* by hybridization. The SpD clade was identified recently, and its origin is not yet elucidated (Xia et al., 2017). (b) Geographical distribution of the S. paradoxus strains used in this study. Circle size is proportional to the number of strains at the location

Figure 2

Integration protocol to barcode natural S. paradoxus strains. Two different barcodes (Tag1 and Tag2) were assigned to each individual strain according to its associated antibiotic resistance cassette, hygromycin B (HPH), or nourseothricin (NAT). The integration method involves three steps: (I) Barcode amplification from the S. cerevisiae deletion collection and antibiotic resistance cassette amplification from plasmids pFA6a‐hph‐NT1 for hygromycin B (HPH) and from pFA6a‐nat‐NT2 for nourseothricin (NAT); (II) Barcode fusion by PCR with the antibiotic resistance cassettes; (III) Barcode insertion in S. paradoxus by transformation and homologous recombination. Each strain was barcoded in two copies, one with the Tag1‐HPH module and the other with the Tag2‐NAT module, each time with a unique barcode (Tag)

Figure 3

Competition assay using the barcoded strains. The initial pool contains all barcoded strains with either the Tag1‐HPH (red border) or the Tag2‐NAT (green border) module from the SpB (red), SpC (blue), SpC* (purple), and SpD (beige) lineages. Wells of a deep‐well plate were inoculated with the pool in three conditions: YPD at 25°C, YPD at 35°C, and proline medium at 25°C. Until the cultures reach 18 generations, each well was diluted to keep cells in exponential growth. At the end of the experiment, wells were pooled by group of four to extract DNA. Samples were collected at t ₁ and points t ₅ for the YPD medium and t ₇ for the proline medium

Figure 4

Barcoded strains show growth similar to that of parental strains on solid medium. Growth is inferred from colony size measurements after a 19‐hr incubation period on solid YPD medium at 25°C and 35°C. Eight replicates were performed for each strain. No significant differences were observed between the hygromycin B (HPH) barcode, the nourseothricin (NAT) barcode, and the WT strains at both temperatures for all lineages (Kruskal–Wallis tests, p‐values > 0.2)

Figure 5

Relative fitness of strains and lineages of S. paradoxus assessed by barcode sequencing. (a) Correlation of relative fitness values between replicates and conditions. (b) The fitness estimates across experiments strongly correlate between the two modules Tag1‐HPH and Tag2‐NAT (Spearman's ρ: 0.9024, p‐value < 2.2e⁻¹⁶). (c) Average fitness of lineages in each condition. A Kruskal–Wallis test followed by a Dunn posthoc test was performed to compare the fitness of the SpB, SpC, and SpC* lineages. Numbers represent the fitness classes within each experimental condition among the lineages (see Supporting Information Table S7 for details). (d) Individual fitness values in each condition for the different lineages. Although absolute values cannot be compared between conditions, the relative values can be compared within conditions. The lines connect the same strains in two conditions. The average value of the estimates for the two tag modules is shown

Figure 6

Genotype‐by‐environment interaction for fitness. The interaction between genotype and environment was investigated by analyzing the correlation between the strain fitness within (a, b, and c) and between (d, e, and f) conditions, considering the two tag modules as biological replicates. Fitness correlation is systematically high between tag modules within condition comparisons. This shows the extent of noise caused by strain transformations and/or biases or noise in barcode quantification (a, b, and c) and the maximum correlation possible between conditions. Correlations between conditions are systematically lower, showing a major effect of growth conditions in relative fitness among strains