Barthelonids represent a deep-branching metamonad clade with mitochondrion-related organelles predicted to generate no ATP

We here report the phylogenetic position of barthelonids, small anaerobic
flagellates previously examined using light microscopy alone. Barthelona
spp. were isolated from geographically distinct regions and we established
five laboratory strains. Transcriptomic data generated from one Barthelona
strain (PAP020) were used for large-scale, multi-gene phylogenetic
(phylogenomic) analyses. Our analyses robustly placed strain PAP020 at the
base of the Fornicata clade, indicating that barthelonids represent a
deep-branching Metamonad clade. Considering the anaerobic/microaerophilic
nature of barthelonids and preliminary electron microscopy observations on
strain PAP020, we suspected that barthelonids possess functionally and
structurally reduced mitochondria (i.e. mitochondrion-related organelles
or MROs). The metabolic pathways localized in the MRO of strain PAP020
were predicted based on its transcriptomic data and compared with those in
the MROs of fornicates. We here propose that strain PAP020 is incapable of
generating ATP in the MRO, as no mitochondrial/MRO enzymes involved in
substratelevel phosphorylation were detected. Instead, we detected the
putative cytosolic ATP-generating enzyme (acetyl-CoA synthetase),
suggesting that strain PAP020 depends on ATP generated in the cytosol. We
propose two separate losses of substrate-level phosphorylation from the
MRO in the clade containing barthelonids and (other) fornicates.