List of plasmids used in this study.

The identification of traits that affect adaptation of microbial species to external abiotic factors, such as temperature, is key for our understanding of how biodiversity originates and can be maintained in a constantly changing environment. The Saccharomyces genus, which includes eight species with different thermotolerant profiles, represent an ideal experimental platform to study the impact of adaptive alleles in different genetic backgrounds. Previous studies identified a group of adaptive genes for maintenance of growth at lower temperatures. Here, we carried out a genus-wide assessment of the role of genes partially responsible for cold-adaptation in all eight Saccharomyces species for six candidate genes. We showed that the cold tolerance trait of S. kudriavzevii and S. eubayanus is likely to have evolved from different routes, involving genes important for the conservation of redox-balance, and for the long-chain fatty acid metabolism, respectively. For several loci, temperature- and species-dependent epistasis was detected, underscoring the plasticity and complexity of the genetic interactions. The natural isolates of S. kudriavzevii, S. jurei and S. mikatae had a significantly higher expression of the genes involved in the redox balance compared to S. cerevisiae, suggesting a role at transcriptional level. To distinguish the effects of gene expression from allelic variation, we independently replaced either the promoters or the coding sequences (CDS) of two genes in four yeast species with those derived from S. kudriavzevii. Our data consistently showed a significant fitness improvement at cold temperatures in the strains carrying the S. kudriavzevii promoter, while growth was lower upon CDS swapping. These results suggest that transcriptional strength plays a bigger role in growth maintenance at cold temperatures over the CDS and supports a model of adaptation centred on stochastic tuning of the expression network.

解析影响微生物物种适应外部非生物因子（如温度）的性状，是理解生物多样性如何在持续变化的环境中起源并得以维持的关键所在。酵母属（Saccharomyces）包含8种具有不同耐热特性谱的物种，是研究适应性等位基因在不同遗传背景下功能的理想实验平台。既往研究已鉴定出一组参与低温下维持生长的适应性基因。本研究针对6个候选基因，在全部8种酵母属物种中开展全属范围的功能评估，解析其在低温适应中的部分作用机制。研究表明，库德里阿兹威酵母（S. kudriavzevii）与巴亚努斯酵母（S. eubayanus）的耐寒性状可能通过不同路径演化：前者依赖维持氧化还原平衡的关键基因，后者则与长链脂肪酸代谢通路相关。在多个基因座中检测到温度与物种依赖性的上位性互作，凸显了遗传相互作用的可塑性与复杂性。与酿酒酵母（S. cerevisiae）相比，库德里阿兹威酵母、尤雷酵母（S. jurei）及米卡塔酵母（S. mikatae）的天然分离株中，参与氧化还原平衡的基因表达量显著更高，提示其耐寒性存在转录层面的调控作用。为区分基因表达与等位基因变异的效应，本研究将4种酵母的2个基因的启动子或编码序列（CDS）独立替换为库德里阿兹威酵母的对应序列。实验数据一致显示，携带库德里阿兹威酵母启动子的菌株在低温环境下的适应度显著提升，而经编码序列替换的菌株生长能力则有所下降。上述结果表明，相较于编码序列，转录强度在低温维持生长过程中发挥了更为关键的作用，同时支持了以表达网络随机调控为核心的适应模型。