The Zooflagellates Stephanopogon and Percolomonas are a Clade (Class Percolatea: Phylum Percolozoa)

ABSTRACT. The enigmatic marine protozoan Stephanopogon was first classified with ciliate protozoa because its pellicle also has rows of cilia. As ciliates have nuclear dimorphism with separate germline and somatic nuclei, Stephanopogon with several identical nuclei was regarded as a model for a hypothetical homokaryotic ancestor of ciliates. When electron microscopy revealed radical differences from ciliates this idea was abandoned, but its evolutionary position remains controversial, affinities with three other phyla being suggested. We sequenced 18S rDNA from Stephanopogon aff. minuta and actin genes from it and Stephanopogon apogon to clarify their evolutionary position. Phylogenetic analyses of 18S rRNA nest S. aff. minuta and Stephanopogon minuta securely within the protozoan phylum Percolozoa with zooflagellates of the genus Percolomonas, their closest relatives, comprising the clade Percolatea. This supports a previous grouping of Stephanopogon (order Pseudociliatida) with Percolomonas (order Percolomonadida) as a purely zooflagellate class Percolatea within Percolozoa, in contrast to the fundamentally amoeboid Heterolobosea, which are probably ancestral to Percolatea. Stephanopogon actins evolve exceptionally fast: actin trees place them as a long branch within bikont eukaryotes without revealing their sisters. We establish Percolomonadidae fam. n. for Percolomonas, excluding Pharyngomonas kirbyi g., sp. n. and Pharyngomonas (=Tetramastix=Percolomonas) salina comb. n., which unlike Percolomonas have two anterior and two posterior cilia and a pocket‐like pharynx, like “Macropharyngomonas”, now grouped with Pharyngomonas as a new purely zooflagellate class Pharyngomonadea, within a new subphylum Pharyngomonada; this contrasts them with the revised ancestrally amoeboflagellate subphylum Tetramitia. We discuss evolution of the percolozoan cytoskeleton and different body forms.     

Characterization of Selenaion koniopes n. gen., n. sp., an Amoeba that Represents a New Major Lineage within Heterolobosea,Isolated from the Wieliczka Salt Mine

A new heterolobosean amoeba, Selenaion koniopes n. gen., n. sp., was isolated from 73‰ saline water in the Wieliczka salt mine, Poland. The amoeba had eruptive pseudopodia, a prominent uroid, and a nucleus without central nucleolus. Cysts had multiple crater‐like pore plugs. No flagellates were observed. Transmission electron microscopy revealed several typical heterolobosean features: flattened mitochondrial cristae, mitochondria associated with endoplasmic reticulum, and an absence of obvious Golgi dictyosomes. Two types of larger and smaller granules were sometimes abundant in the cytoplasm—these may be involved in cyst formation. Mature cysts had a fibrous endocyst that could be thick, plus an ectocyst that was covered with small granules. Pore plugs had a flattened dome shape, were bipartite, and penetrated only the endocyst. Phylogenies based on the 18S rRNA gene and the presence of 18S rRNA helix 17_1 strongly confirmed assignment to Heterolobosea. The organism was not closely related to any described genus, and instead formed the deepest branch within the Heterolobosea clade after Pharyngomonas, with support for this deep‐branching position being moderate (i.e. maximum likelihood bootstrap support—67%; posterior probability—0.98). Cells grew at 15–150‰ salinity. Thus, S. koniopes is a halotolerant, probably moderately halophilic heterolobosean, with a potentially pivotal evolutionary position within this large eukaryote group.     

Neovahlkampfia nana n. sp. Reinforcing an Underrepresented Subclade of Tetramitia,Heterolobosea

The study provides robust genetic evidence that a newly isolated naked ameba with morphological and ultrastructural features indicative of Heterolobosea is a new species. Neovahlkampfia nana n. sp. associates with the yet underrepresented subclade of Tetramitia I. Considerable differences found in 18S rRNA gene sequences of individual molecular clones derived from DNA of five clonal cultures, using a low fidelity DNA polymerase, raised the issue of intragenomic sequence variation, a phenomenon that has not been previously studied in Heterolobosea. However, as proved using a higher fidelity DNA polymerase, the sequence variability observed was introduced by PCR mediated by the low fidelity polymerase and fixed by molecular cloning. This points to the potentially dubious validity of some current nominal species of Heterolobosea that differ from one another in just one or two base positions.     

Principles of protein and lipid targeting in secondary symbiogenesis: euglenoid, dinoflagellate, and sporozoan plastid origins and the eukaryote family tree

The biggest unsolved problems in chloroplast evolution are the origins of dinoflagellate and euglenoid chloroplasts,which have envelopes of three membranes not two like plants and chromists, and of the sporozoan plastid, bounded by four smooth membranes. I review evidence that all three of these protozoan plastid types originated by secondary symbiogenesis from eukaryotic symbionts. Instead of separate symbiogenetic events, I argue that dinoflagellate and sporozoan plastids are directly related and that the common ancestor of dinoflagellates and Sporozoa was photosynthetic. I suggest that the last common ancestor of all Alveolata was photosynthetic and acquired its chlorophyll c-containing plastids in the same endosymbiogenetic event as those of Chromista. Chromistaand Alveolata are postulated to be a clade designated chrornalveolates. I propose that euglenoids obtained their plastids from the same(possibly ulvophycean) green alga as chlorarachneans and that Discicristata (Euglenozoa plus Percolozoa) and Cercozoa (the group including chlorarachneans) form a clade designated cabozoa (protozoa with chlorophyll a + b). If both theories are correct, there were only two secondary symbiogenetic events (witnessed by the chlorarachnean and cryptomonad nucleormorphs) in the history of life, not seven as commonly assumed. This greatly reduces the postulated number of independent origins of chloroplast protein-targeting machinery and of gene transfers from endosymbiont to host nuclei. I discuss the membrane and plastid losses and innovations in protein targeting implied by these theories, the comparative evidence for them, and their implications for eukaryote megaphylogeny. The principle of evolutionary conservatism leads to a novel theory for the function of periplastid vesicles in membrane biogenesis ofchlorarachneans and chromists and of the key steps in secondary symbiogenesis. Protozoan classification is also slightly revised by abandoning the probably polyphyletic infrakingdom Actinopoda, grouping Foraminifera and Radiolaria as a new infrakingdom Retaria,placing Heliozoa within a revised infrakingdom Sarcomastigota, establishing a new flagellate phylum Loukozoa for Jakobea plus Anaeromonadea within an emended subkingdom Eozoa, and ranking Archezoa as an infrakingdom within Eozoa.