1 Scientific RepoRts | 7: 16349 | DOI:10.1038/s41598-017-16259-8 www.nature.com/scientificreports Pseudotrichonympha leei, Pseudotrichonympha lifesoni, and Pseudotrichonympha pearti, new species of parabasalian fagellates and the description of a rotating subcellular structure Javier del Campo 1,2 , erick R. James 1 , Yoshihisa Hirakawa 1 , Rebecca Fiorito 1 , Martin Kolisko 1,3 , Nicholas A. T. Irwin 1 , Varsha Mathur 1 , Vittorio Boscaro 1 , Elisabeth Hehenberger 1 , Anna Karnkowska 1,4 , Rudolf H. Schefrahn 5 & Patrick J. Keeling 1 Pseudotrichonympha is a large and structurally complex genus of parabasalian protists that play a key role in the digestion of lignocellulose in the termite hindgut. Like many termite symbionts, it has a conspicuous body plan that makes genus-level identifcation relatively easy, but species-level diversity of Pseudotrichonympha is understudied. Molecular surveys have suggested the diversity is much greater than the current number of described species, and that many “species” described in multiple hosts are in fact diferent, but gene sequences from formally described species remain a rarity. Here we describe three new species from Coptotermes and Prorhinotermes hosts, including small subunit ribosomal RNA (SSU rRNA) sequences from single cells. Based on host identifcation by morphology and DNA barcoding, as well as the morphology and phylogenetic position of each symbiont, all three represent new Pseudotrichonympha species: P. leei, P. lifesoni, and P. pearti. Pseudotrichonympha leei and P. lifesoni, both from Coptotermes, are closely related to other Coptotermes symbionts including the type species, P. hertwigi. Pseudotrichonympha pearti is the outlier of the trio, more distantly related to P. leei and P. lifesoni than they are to one another, and contains unique features, including an unusual rotating intracellular structure of unknown function. Pseudotrichonympha is a genus of parabasalian protists found exclusively in the hindguts of lower termites, in particular rhinotermitids, where they play a key role in a well-studied symbiotic system in which the microbial community degrades the lignocellulose that makes up most of the animal’s diet 1 . Te ancestral parabasalian body plan was probably a relatively simple cell, characterized by the presence of a hydrogenosome, an anaerobic meta- bolic organelle derived from the mitochondrion, and akaryomastigont system comprising a nucleus, four fagella, and other conserved cytoskeletal elements 2 . But the morphology of parabasalians diversifed greatly within the context of symbiosis with insects: cells expanded in size, and in some lineages cytoskeletal elements were rep- licated to form patterns both so grand and complex that they were considered to form a lineage, the so-called hypermastigotes 3 . Molecular phylogeny and morphology have now shown this elaboration of form actually hap- pened independently in several parabasalian subgroups 4 . Pseudotrichonympha is a member of one such group, the Trichonymphida, which are characterized by very large cells with a single nucleus that are covered by thousands 1 Department of Botany, University of British columbia, Vancouver, British columbia, canada. 2 Department of Marine Biology and Oceanography, institut de ciències del Mar - cSic, Barcelona, catalonia, Spain. 3 institute of Parasitology, Biology Centre, Czech Academy of Sciences, České Budějovice, Czech Republic. 4 Department of Molecular Phylogenetics and Evolution, Faculty of Biology and Biological and Chemical Research Centre, University of Warsaw, Warsaw, Poland. 5 Fort Lauderdale Research & Education Center, University of Florida, Davie, Florida, USA. correspondence and requests for materials should be addressed to P.J.K. (email: pkeeling@mail.ubc.ca) Received: 18 August 2017 Accepted: 3 November 2017 Published: xx xx xxxx opeN