Exploring the Phylogenetic Diversity of Phagotrophic Euglenids

Abstract

Euglenids are a widespread, complex group of flagellated, single-celled eukaryotes. The majority of described species are phototrophic forms, yet a large portion of the phylogenetic diversity is composed of phagotrophs, from which phototrophs arose through secondary endosymbiosis. To understand euglenid evolution it is necessary to understand phylogenetic relationships among phagotrophic euglenids. Yet despite their diversity and evolutionary relevance, phagotrophs are underrepresented in molecular sequence data that is crucial in reconstructing evolutionary relationships. Unfortunately they are difficult to culture, which complicates molecular data collection for most species, as standard methods of nucleic acid extraction cannot be used. I used a culture-independent single-cell approach to increase sampling of the SSU-rDNA gene for phagotrophs almost five-fold (now 141 sequences). Phylogenetic trees show that ploeotids, an assemblage of rigid phagotrophs, make up much of the basal phylogenetic diversity. Several morphotypes that were previously lumped together into a single genus Ploeotia are not monophyletic and belong to multiple separate genera, including Olkasia nov. gen. Two species of ‘spirocute’ Anisonema proved to be ploeotids and were transferred to new genera Hemiolia and Liburna. More previously unsampled phagotrophs belong within Spirocuta (which includes phototrophs and osmostrophs), and phylogenetic analyses revealed several morphologically-defined genera are likely not monophyletic. To better resolve the tree of euglenids, I generated 24 single-cell transcriptomes to provide data for a 20-gene phylogenetic analysis. This divided Spirocuta into phototrophs, a robust ‘Anisonemids plus’, and a weakly supported ‘Peranemids’ clade. Ploeotids are paraphyletic, with Olkasia robustly inferred as sister to Spirocuta, whereas petalomonads are placed basal to all other euglenids with high support. The multigene analyses suggest that symbiontids are not euglenids, but may be more closely related to diplonemids and kinetoplastids. Hemimastigophora are a group of enigmatic multiflagellated cells that have long evaded molecular sequencing, and were inferred at one point to be related to euglenids based on electron-microscopy data. A single-cell approach generated transcriptomes of group members Spironema and Hemimastix, enabling them to be included in eukaryote-wide phylogenomic analyses. Remarkably, Hemimastigophora do not fall into any recognised supergroup of eukaryotes, but form their own independent group that branches outside both Diaphoretickes and Amorphea.