A cnidarian parasite of salmon (Myxozoa: Henneguya) lacks a mitochondrial genome Dayana Yahalomia,1, Stephen D. Atkinsonb,1, Moran Neuhofc, E. Sally Changd,e, Hervé Philippef,g, Paulyn Cartwrightd, Jerri L. Bartholomewb, and Dorothée Huchona,h,2 aSchool of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University, 6997801 Tel Aviv, Israel; bDepartment of Microbiology, Oregon State University, Corvallis, OR 97331; cSchool of Neurobiology, Biochemistry & Biophysics, George S. Wise Faculty of Life Sciences, Tel-Aviv University, 6997801 Tel- Aviv, Israel; dDepartment of Ecology and Evolutionary Biology, University of Kansas, Lawrence, KS 66045; eComputational and Statistical Genomics Branch, Division of Intramural Research, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892; fCNRS, Station d’Ecologie Expérimentale du CNRS, 09200 Moulis, France; gDépartement de Biochimie, Centre Robert-Cedergren, Universitéde Montréal, Montréal, QC H3C 3J7, Canada; and hThe Steinhardt Museum of Natural History and National Research Center, Tel Aviv University, 6997801 Tel Aviv, Israel Edited by W. Ford Doolittle, Dalhousie University, Halifax, Canada, and approved January 10, 2020 (received for review June 25, 2019) Although aerobic respiration is a hallmark of eukaryotes, a few uni- have highly divergent genome structures, with large multipartite cellular lineages, growing in hypoxic environments, have second- circular mt chromosomes and unusually high evolutionary rates (8, arily lost this ability. In the absence of oxygen, the mitochondria of 9). To gain further insight into the evolution of the myxozoan these organisms have lost all or parts of their genomes and evolved mt genome, we studied two closely related freshwater species, into mitochondria-related organelles (MROs). There has been debate regarding the presence of MROs in animals. Using deep sequencing approaches, we discovered that a member of the Significance Cnidaria, the myxozoan Henneguya salminicola, has no mitochon- drial genome, and thus has lost the ability to perform aerobic Mitochondrial respiration is an ancient characteristic of eukary- cellular respiration. This indicates that these core eukaryotic fea- otes. However, it was lost independently in multiple eukaryotic tures are not ubiquitous among animals. Our analyses suggest lineages as part of adaptations to an anaerobic lifestyle. We that H. salminicola lost not only its mitochondrial genome but also show that a similar adaptation occurred in a member of the nearly all nuclear genes involved in transcription and replication of Myxozoa, a large group of microscopic parasitic animals that are the mitochondrial genome. In contrast, we identified many genes closely related to jellyfish and hydroids. Using deep sequencing that encode proteins involved in other mitochondrial pathways approaches supported by microscopic observations, we present and determined that genes involved in aerobic respiration or mi- evidence that an animal has lost its mitochondrial genome. The tochondrial DNA replication were either absent or present only as myxozoan cells retain structures deemed mitochondrion-related pseudogenes. As a control, we used the same sequencing and organelles, but have lost genes related to aerobic respiration annotation methods to show that a closely related myxozoan, and mitochondrial genome replication. Our discovery shows Myxobolus squamalis, has a mitochondrial genome. The molecular that aerobic respiration, one of the most important metabolic results are supported by fluorescence micrographs, which show pathways, is not ubiquitous among animals. the presence of mitochondrial DNA in M. squamalis, but not in H. salminicola. Our discovery confirms that adaptation to an anaerobic Author contributions: P.C., J.L.B., and D.H. designed research; D.Y., S.D.A., and D.H. per- environment is not unique to single-celled eukaryotes, but has also formed research; D.Y., J.L.B., and D.H. contributed new reagents/analytic tools; D.Y., M.N., E.S.C., H.P., and D.H. analyzed data; and D.Y., S.D.A., and D.H. wrote the paper. evolved in a multicellular, parasitic animal. Hence, H. salminicola provides an opportunity for understanding the evolutionary transi- The authors declare no competing interest. tion from an aerobic to an exclusive anaerobic metabolism. This article is a PNAS Direct Submission. Published under the PNAS license. Cnidaria | mitochondrial evolution | mitochondria-related organelle | Data deposition: Voucher paratype material was deposited at the US National Parasite MRO | cristae Collection, Smithsonian Institution, Washington, DC (https://collections.nmnh.si.edu/ search/iz/) under the following accession numbers: Henneguya salminicola myxospores: USNM1611578 (from genome sample) and USNM1611579 (from transcriptome sample); he acquisition of the mitochondrion was a fundamental event Myxobolus squamalis myxospores: USNM1611580 (from genome sample); M. squamalis Tin the evolution of eukaryotes, and most extant eukaryotes myxospores and developmental stages: USNM1611581 (from transcriptome sample). All sequence data have been deposited in the National Center for Biotechnology Information cannot survive without oxygen. Interestingly, the loss of aerobic (NCBI) database (https://www.ncbi.nlm.nih.gov/). The H. salminicola data are available respiration has occurred independently in several eukaryotic under the BioProject accession number PRJNA485580. The SSU rDNA sequence was de- lineages that adapted to low-oxygen environments and replaced posited under MK480607. The raw transcriptome and genome reads are available under accession numbers SRR7754566 and SRR7754567, respectively. The transcriptome and the standard mitochondrial (mt) oxidative phosphorylation path- genome shotgun assembly projects were deposited at under the accession GHBP00000000 way with novel anaerobic metabolic mechanisms (Fig. 1) (1, 2). and SGJC00000000, respectively. The M. squamalis data are available under the BioProject Such anaerobic metabolism occurs within mitochondria-related accession number PRJNA485581. The SSU rDNA sequence was deposited under organelles (MROs), which have often lost their cristae, and in- MK480606. The raw transcriptome and genome reads are available under the accession numbers SRR7760054 and SRR7760053, respectively. The transcriptome and genome shot- clude hydrogenosomes and mitosomes (1, 2). There is debate gun assembly projects were deposited under accession numbers GHBR00000000 and regarding the existence of exclusively anaerobic animals and QWKW00000000, respectively. The mt genome of M. squamalis was deposited under accompanying MROs (3). Although it was reported that some the accession number MK087050. These accession numbers are provided in Dataset S6. loriciferans found in anoxic conditions possess hydrogenosomes The Bayesian trees and all alignments were deposited in the Dryad Digital Repository (https://doi.org/10.5061/dryad.v15dv41sm). Finally, the uncropped images underlying (4, 5), genomic data are not yet available for these organisms, and Fig. 2 and SI Appendix,Figs.S7andS8were deposited in the Figshare repository (https:// alternative explanations have been proposed (3). Here, we show doi.org/10.6084/m9.figshare.8300003, https://doi.org/10.6084/m9.figshare.9897284, that a myxozoan parasite (Cnidaria) has lost both its mt genome https://doi.org/10.6084/m9.figshare.9897320). and aerobic metabolic pathways, and has a novel type of anaerobic 1D.Y. and S.D.A. contributed equally to this work. MRO. Myxozoans are a large group of enigmatic, parasitic, cni- 2To whom correspondence may be addressed. Email: [email protected]. darians with complex life cycles that require two hosts, usually a This article contains supporting information online at https://www.pnas.org/lookup/suppl/ fish and an annelid (6). They have a substantial negative economic doi:10.1073/pnas.1909907117/-/DCSupplemental. impact on fisheries and aquaculture (7). Myxozoan mitochondria First published February 24, 2020. 5358–5363 | PNAS | March 10, 2020 | vol. 117 | no. 10 www.pnas.org/cgi/doi/10.1073/pnas.1909907117 Downloaded by guest on September 25, 2021 EVOLUTION Fig. 1. Eukaryote phylogenetic relationships infer- red from a supermatrix of 9490 amino acid positions for 78 species. Bayesian majority-rule consensus tree reconstructed using the CAT + Γ model from two in- dependent Markov-chain Monte Carlo chains. Branches with low node support (posterior probabili- ties PP < 0.7) were collapsed. Most nodes were highly supported (PP > 0.98), and PP are only indicated for nodes with PP < 0.98. The eukaryote classification is based on Adl et al. (47). Species known to have lost their mt genome are indicated in bold with an aster- isk. Myxozoan species form a well-supported group (PP = 1.0) and our reconstructions agree with previous studies (14), which show monophyly of the fresh-wa- ter/oligochaete host lineage (10). Henneguya salminicola and Myxobolus squamalis (SI Appendix, (15). This view is supported by calculations using only the most Fig. S1), both of which are parasites of salmonid fish (10–12). conserved CEGMA genes, which have higher recovery in both H. salminicola and M. squamalis (76.9% and 56.9%, respectively). Results Assembly of the mt genomes revealed striking differences We assembled transcriptomes and genomes from both species between the two parasites. For M. squamalis, we successfully using identical protocols and computational pipelines. Our phylo- recovered a circular