To understand why molecular evolution turned out as it did, we must characterize not only the path that evolution followed across the space of possible molecular sequences but also the many alternative trajectories that could have been taken but were not. A large-scale comparison of real and possible histories would establish whether the outcome of evolution represents an optimal state driven by natural selection or the contingent product of historical chance events; it would also reveal how the underlying distribution of functions across sequence space shaped historical evolution. Here we combine ancestral protein reconstruction with deep mutational scanning to characterize alternative histories in the sequence space around an ancient transcription factor, which evolved a novel biological function through well-characterized mechanisms. We find hundreds of alternative protein sequences that use diverse biochemical mechanisms to perform the derived function at least as well as the historical outcome. These alternatives all require prior permissive substitutions that do not enhance the derived function, but not all require the same permissive changes that occurred during history. We find that if evolution had begun from a different starting point within the network of sequences encoding the ancestral function, outcomes with different genetic and biochemical forms would probably have resulted; this contingency arises from the distribution of functional variants in sequence space and epistasis between residues. Our results illuminate the topology of the vast space of possibilities from which history sampled one path, highlighting how the outcome of evolution depends on a serial chain of compounding chance events.

中文翻译：

---

古代蛋白质序列空间中的替代进化历史

为了理解分子进化为什么会如此，我们不仅必须描述进化在可能的分子序列空间中遵循的路径，而且还必须描述许多本可以采用但没有采用的替代轨迹。对真实历史和可能历史的大规模比较将确定进化的结果是代表自然选择驱动的最佳状态还是历史偶然事件的偶然产物；它还将揭示跨序列空间的潜在功能分布如何影响历史演变。在这里，我们将祖先蛋白质重建与深度突变扫描相结合，以表征古代转录因子周围序列空间中的替代历史，该转录因子通过充分表征的机制进化出一种新的生物学功能。我们发现了数百种替代蛋白质序列，它们使用不同的生化机制来执行衍生功能，至少与历史结果一样。这些替代方案都需要不增强派生功能的先前许可替换，但并非都需要与历史期间发生的相同的许可更改。我们发现，如果进化是从编码祖先功能的序列网络中的不同起点开始的，那么可能会产生具有不同遗传和生化形式的结果；这种偶然性源于序列空间中功能变异的分布和残基之间的上位性。我们的结果阐明了历史从中采样一条路径的广阔可能性空间的拓扑结构，