A Deeply Branching Lineage in Heterolobosea (Discoba) With Multiflagellated and Multigrooved Life Stages Prokina, Kristina I. Yubuki, Naoji Galindo, Luis Javier Torruella, Guifré Inagaki, Yuji Iwamoto, Koji López-García, Purificación Moreira, David Electron microscopy Heterolobosea Karyomastigont Several distantly related, deep-branching lineages within the eukaryotic tree of life share what is known as the “typical excavate” morphology. This cell architecture includes a suspension-feeding ventral groove, supported by complex and specific structures of the cytoskeleton, and a vane-bearing posterior flagellum. Within Discoba, such characteristics were found only in the deepest branch, Jakobida, also famous for having the most gene-rich and bacterial-like mitogenomes. Some members of the discoban lineage Heterolobosea also possess a ventral groove. However, they lost many other typical excavate characteristics, including the flagellar vane and some flagellar root elements. We have established a clonal culture of a free-living freshwater flagellate from Malaysia, strain NY0229, described here under the provisional name Multisulcus malaysiensis nom. prov. Electron microscopy observations show a conspicuous ventral groove reinforced with microtubules of the flagellar apparatus and a vane on the posterior flagellum. Strain NY0229 has typical excavate biflagellated forms but also exhibits very atypical multiflagellated and multigrooved forms. The kinetid consists of two orthogonally oriented kinetosomes; both flagella of biflagellate cells display proximal and distal projections. Molecular phylogenetic analyses support a deep-branching position of the new strain within Heterolobosea, as the sister lineage to all previously known Eutetramitia. 2025-09-24T10:26:33Z 2025-09-24T10:26:33Z 2025-08-06 journal article Prokina, K. I., N. Yubuki, L. J. Galindo, et al. 2025. “ A Deeply Branching Lineage in Heterolobosea (Discoba) With Multiflagellated and Multigrooved Life Stages.” Journal of Eukaryotic Microbiology 72, no. 5: e70037. https://doi.org/10.1111/jeu.70037 https://hdl.handle.net/10481/106594 10.1111/jeu.70037 eng info:eu-repo/grantAgreement/ERC/H2020/787904 info:eu-repo/grantAgreement/ERC/H2020/101141745 http://creativecommons.org/licenses/by-nc-nd/4.0/ open access Attribution-NonCommercial-NoDerivatives 4.0 Internacional Wiley Periodicals LLC