Protists with Uncertain Phylogenetic Affiliations for Resolving the Deep Tree of Eukaryotes

Resolving the eukaryotic tree of life (eToL) remains a fundamental challenge in biology. Much of eukaryotic phylogenetic diversity is occupied by unicellular microbial eukaryotes (i.e., protists). Among these, the phylogenetic positions of a significant number of lineages remain unresolved due to limited data and ambiguous traits. To address this issue, we introduce the term "PUPAs" (protists with uncertain phylogenetic affiliations) to collectively describe these lineages, instead of using vague or inconsistent labels, such as incertae sedis or orphan taxa. Historically, protists were classified based solely on morphological features, and many with divergent cell structures were left unplaced in the eToL. With the advent of sequence-based approaches, the phylogenetic affiliations of some PUPAs have been clarified using molecular markers, such as small subunit ribosomal DNA. The combination of technological progress and continuous efforts to cultivate diverse protists, including PUPAs and novel protists, now enables phylogenetic analyses based on hundreds of proteins, providing their concrete placements in the eToL. For example, these advances have led to the discovery of new deep-branching lineages (e.g., Hemimastigophora), the resolution of relationships among major groups (e.g., Microheliella, which linked Cryptista and Archaeplastida), and insights into evolutionary innovations within specific clades (e.g., Glissandra). In this review, we summarize current consensus in eukaryotic phylogeny and highlight recent findings on PUPAs whose phylogenetic affiliations have been clarified. We also discuss a few lineages for which the phylogenetic homes remain unsettled, the evolutionary implications of these discoveries, and the remaining challenges in resolving the complete eToL.