Synonymous mutations in protein-coding genes do not alter protein sequences and are thus generally presumed to be neutral or nearly neutral^1,2,3,4,5. Here, to experimentally verify this presumption, we constructed 8,341 yeast mutants each carrying a synonymous, nonsynonymous or nonsense mutation in one of 21 endogenous genes with diverse functions and expression levels and measured their fitness relative to the wild type in a rich medium. Three-quarters of synonymous mutations resulted in a significant reduction in fitness, and the distribution of fitness effects was overall similar—albeit nonidentical—between synonymous and nonsynonymous mutations. Both synonymous and nonsynonymous mutations frequently disturbed the level of mRNA expression of the mutated gene, and the extent of the disturbance partially predicted the fitness effect. Investigations in additional environments revealed greater across-environment fitness variations for nonsynonymous mutants than for synonymous mutants despite their similar fitness distributions in each environment, suggesting that a smaller proportion of nonsynonymous mutants than synonymous mutants are always non-deleterious in a changing environment to permit fixation, potentially explaining the common observation of substantially lower nonsynonymous than synonymous substitution rates. The strong non-neutrality of most synonymous mutations, if it holds true for other genes and in other organisms, would require re-examination of numerous biological conclusions about mutation, selection, effective population size, divergence time and disease mechanisms that rely on the assumption that synoymous mutations are neutral.

蛋白质编码基因的同义突变不会改变蛋白质序列，因此通常被认为是中性或接近中性的^1,2,3,4,5. 在这里，为了通过实验验证这一假设，我们构建了 8,341 个酵母突变体，每个突变体在具有不同功能和表达水平的 21 个内源基因之一中携带同义、非同义或无义突变，并在丰富的培养基中测量它们相对于野生型的适应性。四分之三的同义突变导致适应度显着降低，并且适应度效应的分布在同义突变和非同义突变之间总体上相似（尽管不相同）。同义突变和非同义突变都经常干扰突变基因的 mRNA 表达水平，干扰的程度部分预测了适应度效应。在其他环境中的调查显示，尽管非同义突变体在每个环境中的适应度分布相似，但非同义突变体的跨环境适应度变化比同义突变体更大，这表明在不断变化的环境中，与同义突变体相比较小比例的非同义突变体总是无害的，以允许固定，可能解释了非同义替换率远低于同义替换率的常见观察结果。大多数同义突变的强烈非中性，如果它适用于其他基因和其他生物体，将需要重新检查依赖于假设的关于突变、选择、有效种群大小、分歧时间和疾病机制的众多生物学结论同义突变是中性的。