Reticulate evolution in eukaryotes: Origin and evolution of the nitrate assimilation pathway

Genes and genomes can evolve through interchanging genetic material, this leading to reticular evolutionary patterns. However, the importance of reticulate evolutionin eukaryotes, and in particular of horizontal gene transfer(HGT), remains controversial. Given that metabolic pathways with taxonomically-patchy distributions can be indicative of HGT events, the eukaryoticnitrate assimilation pathway is an ideal object of investigation, as previous results revealed a patchy distribution and suggested that the nitrate assimilation cluster of dikaryotic fungi (Opisthokonta) could have been originatedand transferredfrom a lineage leading to Oomycota (Stramenopiles). We studied the origin and evolution of this pathway through both multi-scale bioinformatic and experimental approaches. Our taxon-rich genomic screening shows that nitrate assimilation is present in more lineages than previously reported, although being restricted to autotrophs and osmotrophs. The phylogenies indicate a pervasive role of HGT, with three bacterial transfers contributing to the pathway origin, and at least seven well-supported transfers between eukaryotes. In particular, we propose a distinct and more complex HGT path between Opisthokonta and Stramenopiles than the one previously suggested, involving at least two transfers of a nitrate assimilation gene cluster. We also found that gene fusion played an essential role in this evolutionary history, underlying the origin of the canonical eukaryotic nitrate reductase, and of a chimeric nitrate reductasein Ichthyosporea (Opisthokonta). We show that the ichthyosporean pathway, including this novel nitrate reductase, is physiologically active and transcriptionally co-regulated, responding to different nitrogen sources; similarly to distant eukaryoteswith independent HGT-acquisitions of the pathway. This indicates that this pattern of transcriptional control evolved convergently in eukaryotes, favoring the proper integration of the pathway in the metabolic landscape. Our results highlight the importance of reticulate evolutionin eukaryotes, by showing the crucial contribution of HGT and gene fusion in the evolutionary history of the nitrate assimilation pathway.