The origin of plastids was a major evolutionary event that paved the way for an astonishing diversification of photosynthetic eukaryotes. Plastids originated by endosymbiosis between a heterotrophic eukaryotic host and a cyanobacterium, presumably in a common ancestor of all primary photosynthetic eukaryotes (Archaeplastida). A single origin of primary plastids is well supported by plastid evidence but not by nuclear phylogenomic analyses, which have consistently failed to recover the monophyly

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... of Archaeplastida hosts. Importantly, the monophyly of both plastid and host (nuclear) genomes is required to support a single ancestral endosymbiosis, whereas non-monophyletic hosts could be explained under scenarios of independent or serial eukaryote-to-eukaryote endosymbioses. Here, we assessed the strength of the signal for the Archaeplastida host monophyly in four available phylogenomic datasets. The effect of phylogenetic methodology, data quality, alignment trimming strategy, gene and taxon sampling, and the presence of outlier genes loci were investigated. Our analyses revealed a general lack of support for host monophyly in the shorter individual datasets. However, when analyzed together under rigorous data curation and complex mixture evolutionary models, the combined dataset consistently recovered the monophyly of Archaeplastida hosts. This study represents an important step towards better understanding the eukaryotic evolution and the origin of plastids.