Lack of evidence for the presence of plastids in the evolutionary history of kinetoplastid protists.

The discovery of multiple metabolic enzymes encoded by genes of apparent plant or cyanobacterial origin in trypanosomatids led to the influential hypothesis that the common ancestor of Euglenozoa harboured a plastid that was subsequently lost in kinetoplastids. Here, we critically re-evaluate this hypothesis using an expanded, phylogenetically balanced dataset comprising 299 eukaryotic and 102 bacterial species. We reassess the evolutionary histories of 16 genes encoding proteins previously interpreted as evidence for an ancestral euglenozoan plastid using state-of-the-art maximum-likelihood and Bayesian phylogenetic approaches, supplemented by topology tests. Our analyses reveal that none of the examined genes provides compelling support for a plastid-bearing ancestor of Euglenozoa. Instead, these enzymes display heterogeneous evolutionary origins consistent with multiple independent horizontal gene transfer events between euglenozoans and diverse bacterial (distinct from cyanobacteria) and eukaryotic donors. Only the gene encoding a vacuolar H⁺-pyrophosphatase, an electrogenic proton pump, shows limited affinity to chloroplast-bearing lineages, and this signal alone is insufficient to infer plastid ancestry. Taken together, our results strongly suggest a horizontal gene transfer from various non-plastid bearing lineages over the hypothesis of plastid presence in the euglenozoan common ancestor with subsequent loss in kinetoplastids, diplonemids, and non-photosynthetic euglenids.