Multigene Phylogeny of Uncultivable Parabasalia from Single-Cell Transcriptomes

Phylum Parabasalia is a group of anaerobic single-celled eukaryotes that mainly inhabit the guts of terrestrial animals. Most described species are found in termite hindguts as obligate mutualists, some are found in other invertebrate or vertebrate hosts as commensals or parasites, and few are free-living. Despite the medical and agricultural importance of several species, notably Trichomonas vaginalis, a common human STI, the evolutionary history of Parabasalia is not well understood. This is largely due to the difficulty of cultivating Parabasalia, especially termite-associated species, which means sequence data must be acquired using single-cell approaches. Phylogenetic analyses of Parabasalia have, therefore, been limited to just one or a few genes amplified from single cells by PCR. These analyses have lacked resolution and/or conflicted with one another, indicating that more data are needed to resolve the phylogeny of Parabasalia. To address this knowledge gap, we sequenced transcriptomes from 18 individual Parabasalia cells isolated from termite and cockroach hosts. Combining this new data with previously published transcriptomes, we computed a 100-gene phylogeny with 28 ingroup and 4 outgroup taxa. Nearly all nodes received full statistical support from both maximum likelihood bootstrapping and Bayesian posterior probabilities. The root of Parabasalia falls on the branch leading to Trichonymphida and Lophomonadida, a clade we name Blattanymphea to reflect their termite and cockroach hosts. Our phylogeny further suggests that the termite-associated orders Cristamonadida and Spirotrichonymphida are sisters, a clade we name Isoptenymphea. We infer through likelihood-based ancestral state reconstruction that the ancestor of Parabasalia was endobiotic, not free-living, though improved taxon sampling will be necessary to confirm this result.