The successful colonization of new habitats has played a fundamental role during the evolution of life. Salinity is one of the strongest barriers for organisms to cross, which has resulted in the evolution of distinct marine and non-marine (including both freshwater and soil) communities. Although microbes represent by far the vast majority of eukaryote diversity, the role of the salt barrier in shaping the diversity across the eukaryotic tree is poorly known. Traditional views suggest rare and ancient marine/non-marine transitions but this view is being challenged by the discovery of several recently transitioned lineages. Here, we investigate habitat evolution across the tree of eukaryotes using a unique set of taxon-rich phylogenies inferred from a combination of long-read and short-read environmental metabarcoding data spanning the ribosomal DNA operon. Our results show that, overall, marine and non-marine microbial communities are phylogenetically distinct but transitions have occurred in both directions in almost all major eukaryotic lineages, with hundreds of transition events detected. Some groups have experienced relatively high rates of transitions, most notably fungi for which crossing the salt barrier has probably been an important aspect of their successful diversification. At the deepest phylogenetic levels, ancestral habitat reconstruction analyses suggest that eukaryotes may have first evolved in non-marine habitats and that the two largest known eukaryotic assemblages (TSAR and Amorphea) arose in different habitats. Overall, our findings indicate that the salt barrier has played an important role during eukaryote evolution and provide a global perspective on habitat transitions in this domain of life.

新栖息地的成功殖民在生命的进化过程中发挥了基础性作用。盐度是生物体跨越的最强障碍之一，导致了独特的海洋和非海洋（包括淡水和土壤）群落的进化。尽管迄今为止，微生物代表了真核生物多样性的绝大多数，但盐屏障在塑造真核生物树多样性中的作用却鲜为人知。传统观点认为罕见且古老的海洋/非海洋转变，但这种观点受到了几个最近转变谱系的发现的挑战。在这里，我们使用一组独特的富含分类单元的系统发育研究整个真核生物树的栖息地进化，这些系统发育是从跨越核糖体 DNA 操纵子的长读和短读环境元条形码数据的组合推断出来的。我们的结果表明，总体而言，海洋和非海洋微生物群落在系统发育上是不同的，但几乎所有主要真核谱系都在两个方向上发生了转变，检测到了数百个转变事件。一些群体经历了相对较高的转变率，尤其是真菌，跨越盐屏障可能是其成功多样化的一个重要方面。在最深的系统发育水平上，祖先栖息地重建分析表明，真核生物可能首先在非海洋栖息地中进化，并且已知最大的两个真核生物组合（TSAR 和 Amorphea）出现在不同的栖息地。总的来说，我们的研究结果表明盐屏障在真核生物进化过程中发挥了重要作用，并为这一生命领域的栖息地转变提供了全球视角。