Metagenome-assembled genomes (MAGs) of Asgardarchaeota have been recovered from a variety of habitats, broadening their environmental distribution and providing access to the genetic makeup of this archaeal lineage. The recent success in cultivating the first representative of Lokiarchaeia was a breakthrough in science at large and gave rise to new hypotheses about the evolution of eukaryotes. Despite their singular phylogenetic position at the base of the eukaryotic tree of life, the morphology of these bewildering organisms remains a mystery, except for the report of an unusual morphology with long, branching protrusions of the cultivated Lokiarchaeion strain "Candidatus Prometheoarchaeum syntrophicum" MK-D1. In order to visualize this elusive group, we applied a combination of fluorescence in situ hybridization and catalyzed reporter deposition (CARD-FISH) and epifluorescence microscopy on coastal hypersaline sediment samples, using specifically designed CARD-FISH probes for Heimdallarchaeia and Lokiarchaeia lineages, and provide the first visual evidence for Heimdallarchaeia and new images of a lineage of Lokiarchaeia that is different from the cultured representative. Here, we show that while Heimdallarchaeia are characterized by a uniform cellular morphology typified by a centralized DNA localization, Lokiarchaeia display a plethora of shapes and sizes that likely reflect their broad phylogenetic diversity and ecological distribution.IMPORTANCE Asgardarchaeota are considered to be the closest relatives to modern eukaryotes. These enigmatic microbes have been mainly studied using metagenome-assembled genomes (MAGs). Only very recently, a first member of Lokiarchaeia was isolated and characterized in detail; it featured a striking morphology with long, branching protrusions. In order to visualize additional members of the phylum Asgardarchaeota, we applied a fluorescence in situ hybridization technique and epifluorescence microscopy on coastal hypersaline sediment samples, using specifically designed probes for Heimdallarchaeia and Lokiarchaeia lineages. We provide the first visual evidence for Heimdallarchaeia that are characterized by a uniform cellular morphology typified by an apparently centralized DNA localization. Further, we provide new images of a lineage of Lokiarchaeia that is different from the cultured representative and with multiple morphologies, ranging from small ovoid cells to long filaments. This diversity in observed cell shapes is likely owing to the large phylogenetic diversity within Asgardarchaeota, the vast majority of which remain uncultured.

Department of Aquatic Microbial Ecology Institute of Hydrobiology Biology Centre of the Czech Academy of Sciences České Budějovice Czech Republic

Department of Molecular Biology and Biotechnology Faculty of Biology and Geology Babeş Bolyai University Cluj Napoca Romania

Limnological Station Institute of Plant and Microbial Biology University of Zurich Kilchberg Switzerland

Molecular Biology Center Institute for Interdisciplinary Research in Bio Nano Sciences Babeş Bolyai University Cluj Napoca Romania

Zobrazit více v PubMed

Cox CJ, Foster PG, Hirt RP, Harris SR, Embley TM. 2008. The archaebacterial origin of eukaryotes. Proc Natl Acad Sci U S A 105:20356–20361. doi:10.1073/pnas.0810647105. PubMed DOI PMC

Lake JA, Henderson E, Oakes M, Clark MW. 1984. Eocytes: a new ribosome structure indicates a kingdom with a close relationship to eukaryotes. Proc Natl Acad Sci U S A 81:3786–3790. doi:10.1073/pnas.81.12.3786. PubMed DOI PMC

Spang A, Saw JH, Jørgensen SL, Zaremba-Niedzwiedzka K, Martijn J, Lind AE, van Eijk R, Schleper C, Guy L, Ettema T. 2015. Complex archaea that bridge the gap between prokaryotes and eukaryotes. Nature 521:173–179. doi:10.1038/nature14447. PubMed DOI PMC

Zaremba-Niedzwiedzka K, Caceres EF, Saw JH, Bäckström D, Juzokaite L, Vancaester E, Seitz KW, Anantharaman K, Starnawski P, Kjeldsen KU, Stott MB, Nunoura T, Banfield JF, Schramm A, Baker BJ, Spang A, Ettema T. 2017. Asgard archaea illuminate the origin of eukaryotic cellular complexity. Nature 541:353–358. doi:10.1038/nature21031. PubMed DOI

Williams TA, Cox CJ, Foster PG, Szöllősi GJ, Embley TM. 2020. Phylogenomics provides robust support for a two-domains tree of life. Nat Ecol Evol 4:138–147. doi:10.1038/s41559-019-1040-x. PubMed DOI PMC

Bulzu P-A, Andrei A-Ş, Salcher MM, Mehrshad M, Inoue K, Kandori H, Beja O, Ghai R, Banciu HL. 2019. Casting light on Asgardarchaeota metabolism in a sunlit microoxic niche. Nat Microbiol 4:1129–1137. doi:10.1038/s41564-019-0404-y. PubMed DOI

Spang A, Stairs CW, Dombrowski N, Eme L, Lombard J, Caceres EF, Greening C, Baker BJ, Ettema T. 2019. Proposal of the reverse flow model for the origin of the eukaryotic cell based on comparative analyses of Asgard archaeal metabolism. Nat Microbiol 4:1138–1148. doi:10.1038/s41564-019-0406-9. PubMed DOI

Seitz KW, Dombrowski N, Eme L, Spang A, Lombard J, Sieber JR, Teske AP, Ettema TJG, Baker BJ. 2019. Asgard archaea capable of anaerobic hydrocarbon cycling. Nat Commun 10:1822. doi:10.1038/s41467-019-09364-x. PubMed DOI PMC

Liu Y, Zhou Z, Pan J, Baker BJ, Gu J-D, Li M. 2018. Comparative genomic inference suggests mixotrophic lifestyle for Thorarchaeota. ISME J 12:1021–1031. doi:10.1038/s41396-018-0060-x. PubMed DOI PMC

Wong HL, White RA, Visscher PT, Charlesworth JC, Vázquez-Campos X, Burns BP. 2018. Disentangling the drivers of functional complexity at the metagenomic level in Shark Bay microbial mat microbiomes. ISME J 12:2619–2639. doi:10.1038/s41396-018-0208-8. PubMed DOI PMC

Orsi WD, Vuillemin A, Rodriguez P, Coskun ÖK, Gomez-Saez GV, Lavik G, Mohrholz V, Ferdelman TG. 2020. Metabolic activity analyses demonstrate that Lokiarchaeon exhibits homoacetogenesis in sulfidic marine sediments. Nat Microbiol 5:248–255. doi:10.1038/s41564-019-0630-3. PubMed DOI

Seitz KW, Lazar CS, Hinrichs K-U, Teske AP, Baker BJ. 2016. Genomic reconstruction of a novel, deeply branched sediment archaeal phylum with pathways for acetogenesis and sulfur reduction. ISME J 10:1696–1705. doi:10.1038/ismej.2015.233. PubMed DOI PMC

Manoharan L, Kozlowski JA, Murdoch RW, Löffler FE, Sousa FL, Schleper C. 2019. Metagenomes from coastal marine sediments give insights into the ecological role and cellular features of Loki- and Thorarchaeota. mBio 10:e02039-19. doi:10.1128/mBio.02039-19. PubMed DOI PMC

Imachi H, Nobu MK, Nakahara N, Morono Y, Ogawara M, Takaki Y, Takano Y, Uematsu K, Ikuta T, Ito M, Matsui Y, Miyazaki M, Murata K, Saito Y, Sakai S, Song C, Tasumi E, Yamanaka Y, Yamaguchi T, Kamagata Y, Tamaki H, Takai K. 2020. Isolation of an archaeon at the prokaryote–eukaryote interface. Nature 577:519–525. doi:10.1038/s41586-019-1916-6. PubMed DOI PMC

Caceres EF, Lewis WH, Homa F, Martin T, Schramm A, Kjeldsen KU, Ettema T. 2019. Near-complete Lokiarchaeota genomes from complex environmental samples using long and short read metagenomic analyses. bioRxiv 2019.2012.2017.879148.

Spang A, Eme L, Saw JH, Caceres EF, Zaremba-Niedzwiedzka K, Lombard J, Guy L, Ettema T. 2018. Asgard archaea are the closest prokaryotic relatives of eukaryotes. PLoS Genet 14:e1007080. doi:10.1371/journal.pgen.1007080. PubMed DOI PMC

Da Cunha V, Gaia M, Nasir A, Forterre P. 2018. Asgard archaea do not close the debate about the universal tree of life topology. PLoS Genet 14:e1007215. doi:10.1371/journal.pgen.1007215. PubMed DOI PMC

Da Cunha V, Gaia M, Gadelle D, Nasir A, Forterre P. 2017. Lokiarchaea are close relatives of Euryarchaeota, not bridging the gap between prokaryotes and eukaryotes. PLoS Genet 13:e1006810. doi:10.1371/journal.pgen.1006810. PubMed DOI PMC

Andrei A-Ş, Salcher MM, Mehrshad M, Rychtecký P, Znachor P, Ghai R. 2019. Niche-directed evolution modulates genome architecture in freshwater Planctomycetes. ISME J 13:1056–1071. doi:10.1038/s41396-018-0332-5. PubMed DOI PMC

Neuenschwander SM, Ghai R, Pernthaler J, Salcher MM. 2018. Microdiversification in genome-streamlined ubiquitous freshwater Actinobacteria. ISME J 12:185–198. doi:10.1038/ismej.2017.156. PubMed DOI PMC

Amann R, Fuchs BM. 2008. Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques. Nat Rev Microbiol 6:339–348. doi:10.1038/nrmicro1888. PubMed DOI

Mussmann M, Brito I, Pitcher A, Sinninghe Damsté JS, Hatzenpichler R, Richter A, Nielsen JL, Nielsen PH, Müller A, Daims H, Wagner M, Head IM. 2011. Thaumarchaeotes abundant in refinery nitrifying sludges express amoA but are not obligate autotrophic ammonia oxidizers. Proc Natl Acad Sci U S A 108:16771–16776. doi:10.1073/pnas.1106427108. PubMed DOI PMC

Salcher MM, Andrei A-Ş, Bulzu P-A, Keresztes ZG, Banciu HL, Ghai R. 2019. Visualization of Loki- and Heimdallarchaeia (Asgardarchaeota) by fluorescence in situ hybridization and catalyzed reporter deposition (CARD-FISH). bioRxiv 580431. doi:10.1101/580431 PubMed DOI PMC

Stahl DA, Amann R. 1991. Development and application of nucleic acid probes, p 205–248. In Stackebrandt E, Goodfellow M (ed), Nucleic acid techniques in bacterial systematics. John Wiley & Sons Ltd, Chichester, United Kingdom.

Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, Peplies J, Glöckner FO. 2013. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res 41:D590–D596. doi:10.1093/nar/gks1219. PubMed DOI PMC

Knittel K, Lösekann T, Boetius A, Kort R, Amann R. 2005. Diversity and distribution of methanotrophic archaea at cold seeps. Appl Environ Microbiol 71:467–479. doi:10.1128/AEM.71.1.467-479.2005. PubMed DOI PMC

Jørgensen SL, Thorseth I, Pedersen RB, Baumberger T, Schleper C. 2013. Quantitative and phylogenetic study of the Deep Sea Archaeal Group in sediments of the Arctic mid-ocean spreading ridge. Front Microbiol 4:299. doi:10.3389/fmicb.2013.00299. PubMed DOI PMC

Karst SM, Dueholm MS, McIlroy SJ, Kirkegaard RH, Nielsen PH, Albertsen M. 2018. Retrieval of a million high-quality, full-length microbial 16S and 18S rRNA gene sequences without primer bias. Nat Biotechnol 36:190–195. doi:10.1038/nbt.4045. PubMed DOI

Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, Tiedje JM. 2007. DNA–DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol 57:81–91. doi:10.1099/ijs.0.64483-0. PubMed DOI

Ashelford K, Chuzhanova N, Fry J, Jones A, Weightman A. 2005. At least 1 in 20 rRNA sequence records currently held in public repositories is estimated to contain substantial anomalies. Appl Environ Microbiol 71:7724–7736. doi:10.1128/AEM.71.12.7724-7736.2005. PubMed DOI PMC

Fuchs B, Glöckner F, Wulf J, Amann R. 2000. Unlabeled helper oligonucleotides increase the in situ accessibility to 16S rRNA of fluorescently labeled oligonucleotide probes. Appl Environ Microbiol 66:3603–3607. doi:10.1128/aem.66.8.3603-3607.2000. PubMed DOI PMC

Yilmaz LS, Parnerkar S, Noguera DR. 2011. mathFISH, a web tool that uses thermodynamics-based mathematical models for in silico evaluation of oligonucleotide probes for fluorescence in situ hybridization. Appl Environ Microbiol 77:1118–1122. doi:10.1128/AEM.01733-10. PubMed DOI PMC

Teeling H, Fuchs BM, Becher D, Klockow C, Gardebrecht A, Bennke CM, Kassabgy M, Huang S, Mann AJ, Waldmann J, Weber M, Klindworth A, Otto A, Lange J, Bernhardt J, Reinsch C, Hecker M, Peplies J, Bockelmann FD, Callies U, Gerdts G, Wichels A, Wiltshire KH, Glöckner FO, Schweder T, Amann R. 2012. Substrate-controlled succession of marine bacterioplankton populations induced by a phytoplankton bloom. Science 336:608–611. doi:10.1126/science.1218344. PubMed DOI

Salcher MM, Posch T, Pernthaler J. 2013. In situ substrate preferences of abundant bacterioplankton populations in a prealpine freshwater lake. ISME J 7:896–907. doi:10.1038/ismej.2012.162. PubMed DOI PMC

Wallner G, Amann R, Beisker W. 1993. Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms. Cytometry 14:136–143. doi:10.1002/cyto.990140205. PubMed DOI

Fuerst JA, Sagulenko E. 2011. Beyond the bacterium: planctomycetes challenge our concepts of microbial structure and function. Nat Rev Microbiol 9:403–413. doi:10.1038/nrmicro2578. PubMed DOI

Ludwig W, Strunk O, Westram R, Richter L, Meier H, Yadhukumar, Buchner A, Lai S, Steppi S, Jobb G, Förster W, Brettske I, Gerber S, Ginhart A, Gross O, Grumann S, Hermann S, Jost R, König A, Liss T, Lüßmann R, May M, Nonhoff B, Reichel B, Strehlow R, Stamatakis A, Stuckmann N, Vilbig A, Lenke M, Ludwig T, Bode A, Schleifer K. 2004. ARB: a software environment for sequence data. Nucleic Acids Res 32:1363–1371. doi:10.1093/nar/gkh293. PubMed DOI PMC

Stamatakis A, Ludwig T, Meier H. 2005. RAxML-II: a program for sequential, parallel and distributed inference of large phylogenetic trees. Concurr Comput 17:1705–1723. doi:10.1002/cpe.954. DOI

Yılmaz LŞ, Noguera DR. 2007. Development of thermodynamic models for simulating probe dissociation profiles in fluorescence in situ hybridization. Biotechnol Bioeng 96:349–363. doi:10.1002/bit.21114. PubMed DOI

Yilmaz LS, Bergsven LI, Noguera DR. 2008. Systematic evaluation of single mismatch stability predictors for fluorescence in situ hybridization. Environ Microbiol 10:2872–2885. doi:10.1111/j.1462-2920.2008.01719.x. PubMed DOI

Hoshino T, Yilmaz LS, Noguera DR, Daims H, Wagner M. 2008. Quantification of target molecules needed to by fluorescence in situ hybridization (FISH) and catalyzed reporter deposition-FISH. Appl Environ Microbiol 74:5068–5077. doi:10.1128/AEM.00208-08. PubMed DOI PMC

Gastescu P, Bretcan P, Teodorescu DC. 2016. The lakes of the Romanian Black Sea coast. man-induced changes, water regime, present state. Rom J Geogr 60:27–42.

Ishii K, Mussmann M, MacGregor BJ, Amann R. 2004. An improved fluorescence in situ hybridization protocol for the identification of bacteria and archaea in marine sediments. FEMS Microbiol Ecol 50:203–213. doi:10.1016/j.femsec.2004.06.015. PubMed DOI

Ecogenomics sheds light on diverse lifestyle strategies in freshwater CPR

Visualization of Lokiarchaeia and Heimdallarchaeia (Asgardarchaeota) by Fluorescence In Situ Hybridization and Catalyzed Reporter Deposition (CARD-FISH)