The Windblown: Possible Explanations for Dinophyte DNA

Home , Alexandrium fundyense, Alexandrium ostenfeldii, Ceratiaceae, Ceratocorys horrida, Ellobiopsis, Gymnodiniaceae

bioRxiv preprint doi: https://doi.org/10.1101/2020.08.07.242388; this version posted August 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

The windblown: possible explanations for dinophyte DNA

in forest soils

Marc Gottschlinga, Lucas Czechb,c, Frédéric Mahéd,e, Sina Adlf,

Micah Dunthorng,h,*

a Department Biologie, Systematische Botanik und Mykologie, GeoBio-Center, Ludwig- Maximilians-Universität München, D-80638 Munich, Germany

b Computational Molecular Evolution Group, Heidelberg Institute for Theoretical Studies, D- 69118 Heidelberg, Germany

c Department of Plant Biology, Carnegie Institution for Science, Stanford, CA 94305, USA

d CIRAD, UMR BGPI, F-34398, Montpellier, France

e BGPI, Université de Montpellier, CIRAD, IRD, Montpellier SupAgro, Montpellier, France

f Department of Soil Sciences, College of Agriculture and Bioresources, University of Saskatchewan, Saskatoon, S7N 5A8, SK, Canada

g Eukaryotic Microbiology, Faculty of Biology, Universität Duisburg-Essen, D-45141 Essen, Germany

h Centre for Water and Environmental Research (ZWU), Universität Duisburg-Essen, D- 45141 Essen, Germany

Running title: Dinophytes in soils

Correspondence M. Dunthorn, Eukaryotic Microbiology, Faculty of Biology, Universität Duisburg-Essen, Universitätsstrasse 5, D-45141 Essen, Germany Telephone number: +49-(0)-201-183-2453; email: [email protected] bioRxiv preprint doi: https://doi.org/10.1101/2020.08.07.242388; this version posted August 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

ABSTRACT

Dinophytes are widely distributed in marine- and fresh-waters, but have yet to be conclusively documented in terrestrial environments. Here we evaluated the presence of these protists from an environmental DNA metabarcoding dataset of Neotropical rainforest soils. Using a phylogenetic placement approach with a reference alignment and tree, we showed that the numerous sequencing reads that were assigned to the dinophytes did not associate with taxonomy, environmental preference, nutritional mode, or dormancy. All the dinophytes in the soils are most likely windblown dispersal units of aquatic species, and are not biologically active residents of terrestrial environments.

Keywords dinoflagellates; distribution; phylogenetic placement; operational taxonomic unit; rRNA. bioRxiv preprint doi: https://doi.org/10.1101/2020.08.07.242388; this version posted August 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Environmental high-throughput sequencing (HTS) studies of protists have now been performed for over a decade (Santoferrara et al. 2020). During that time, a large diversity of dinophyte DNA sequences has also been uncovered. Dinophytes are an ecologically and economically important group of protists that exhibit many types of life styles and nutritional modes, including phototrophic, mixotrophic and heterotrophic forms as well as some being parasitic (Saldarriaga and Taylor 2017). All known dinophytes are from marine or freshwater environments (Adl et al. 2019). As they constitute a considerable fraction of the plankton and play an important role in the global aquatic ecosystem, HTS studies have detected dinophytes from waters sampled from the polar regions through to the tropics (de Vargas et al. 2015; Le Bescot et al. 2016; Elferink et al. 2017; Decelle et al. 2018; Lentendu et al. 2018; Annenkova et al. 2020; Giner et al. 2020; Gottschling et al. 2020). HTS studies have also detected DNA of dinophytes in terrestrial environments (Bates et al. 2013; Geisen et al. 2015; Mahé et al. 2017; Venter et al. 2017; Voss et al. 2019), although they are not expected to be there.

Aquatic protists can sometimes be detected in terrestrial environments, notably riparian soil, such as foraminifera (Meisterfeld et al. 2001; Lejzerowicz et al. 2010) and possibly haptophytes (Mahé et al. 2017). However, that does not mean the normally aquatic protists are biologically active in soils or other drier environments (Geisen et al. 2018). In the absence of observing putative soil dinophytes using direct microscopic observations, here we used Mahé et al.’s (2017) metabarcoding data from three lowland Neotropical rainforest soils to ask if the presence of dinophytes in those soils associate with taxonomy, environmental preference, nutritional mode, or dormancy. bioRxiv preprint doi: https://doi.org/10.1101/2020.08.07.242388; this version posted August 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

MATERIALS AND METHODS

Environmental sampling and data Sampling and sequencing of tropical soils originally took place in lowland rainforest in Costa Rica, Panama, and Ecuador (Mahé et al. 2017). The extracted soils DNAs were amplified for the hyper-variable V4 region of the SSU-rRNA locus using general eukaryotic primers (Stoeck et al. 2010); this short region has relatively strong phylogenetic signal, although it is not as strong as the full-length SSU-rRNA (Dunthorn et al. 2014; Gottschling et al. 2020). Illumina sequencing reads were clustered into OTUs using Swarm v2 (Mahé et al. 2015) and taxonomically assigned to the Protist Ribosomal Reference database (Guillou et al. 2013) using VSEARCH (Rognes et al. 2016). The 269 OTUs that were assigned to the dinophytes by Mahé et al. (2017), were extracted and used here for phylogenetic placements (File S1).

Reference tree From GenBank, 228 ingroup dinophytes, plus 10 outgroups, were downloaded, then aligned with MAFFT v6.624b (Katoh and Standley 2013) using the –auto option. Based on previous analyses (Gottschling et al. 2012, 2020; Žerdoner Čalasan et al. 2019), the full sequences of each species were used without excluding ambiguously aligned positions sites. Phylogenetic inferences of the reference alignment were carried out by using Maximum Likelihood (ML) as described in detail by Gottschling et al. (2012, 2020), using RAxML v8.2.10 (Stamatakis 2014) with the GTR+G substitution model. To determine the best fitted ML tree, we executed 10-tree searches from distinct random stepwise addition sequence Maximum Parsimony starting trees and performed 1,000 non-parametric bootstrap replicates. Reference alignment and tree available upon request.

Phylogenetic placement of environmental OTUs The OTU representative sequences obtained from Swarm were aligned against the reference alignment using PaPaRa v2.0 (Berger and Stamatakis 2011), and phylogenetically placed onto the ML reference tree using the Evolutionary Placement Algorithm (EPA) of RAxML. Next, all OTUs that were placed with at least 95% probability (combined likelihood weight ratios) in the dinophyte clade were extracted and visualized, using Gappa (Czech et al. 2020). For details on the extraction, see Czech et al. (2018); details of the workflow are published in a GitHub code repository (https://github.com/lczech/dinoflagellate-paper).

RESULTS AND DISCUSSION

For the dinophyte reference alignment and tree, we included a broad representative taxon sample covering the known DNA sequence diversity with comprehensive sequence information. The alignment was 7,270 bp long and had 3,753 parsimony informative sites (52%, 15.7 per terminal taxon). The ML tree had many bipartitions that had high if not maximal bootstrap values. The Dinophyceae was inferred to be monophyletic, and it contained well-known subclades: Dinophysales, Gonyaulacales, Gymnodiniales, Peridiniales, Prorocentrales, †Suessiales as well as Amphidomataceae, Brachydiniaceae, and Tovelliaceae. Only 207 Neotropical soil OTUs from from the Mahé et al.’s (2017) that were assigned to the dinophytes, phylogenetically placed across the reference tree with high likelihood weight scores (Fig. 1, File S2).

There were no exclusive associations with taxonomy. Some of the dinophyte OTUs formed distinct clades of sequences that were unknown until the present study. However, the amount of such undescribed diversity is low compared to other microbial lineages such as the Fungi (Jones et al. 2011; Rosling et al. 2011). They placed onto early branches, which comprise heterotrophic, mainly parasitic species (Saldarriaga et al. 2003; Gómez et al. 2009; Bachvaroff et al. 2012; Gu et al. 2013), but also within the Peridiniales. Most of the OTUs, though, placed within already known lineages of the Gymnodiniaceae, Peridiniales and †Suessiales, a truly heterogeneous set of dinophytes including unarmored and thecate algae as well. There is no morphological trait that the OTUs would therefore necessarily share and that would unite them with these different taxa. Some thecate groups such as Dinophysales, Gonyaulacales, Prorocentrales and Protoperidiniaceae (Peridiniales) did not include any of the OTUs obtained from the environmental samples.

There were no exclusive associations with habitat preference. Some of the dinophyte OTUs placed onto branches that contain freshwater species, such as the Gymnodiniaceae (Kretschmann et al. 2015; Romeikat et al. 2020), Tovelliaceae (Lindberg et al. 2005), and peridinialean Naiadinium comprising a freshwater lineage within otherwise marine Scrippsiella s.l. (Kretschmann et al. 2014; Luo et al. 2016). This placement of OTUs within freshwater clades was similar to what was shown for the haptophyte OTUs from the same rainforest soils (Mahé et al. 2017). However, none of the OTUs placed with the Peridiniaceae, which is one of the most prominent freshwater dinophyte lineages (Moestrup bioRxiv preprint doi: https://doi.org/10.1101/2020.08.07.242388; this version posted August 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

and Calado 2018), but some OTUs placed on branches that contain just marine species, such as the Amphidomataceae (Tillmann et al. 2014) and Brachydiniaceae (Bergholtz et al. 2006; Henrichs et al. 2011). Groups such as Dinophysales, Gonyaulacales, Prorocentrales and Protoperidiniaceae (Peridiniales) are primarily marine and did not include any of the OTUs obtained from the environmental samples.

There were no exclusive associations with nutritional mode. Some of the dinophyte OTUs placed onto branches that predominantly contain phototrophic species, such as Gymnodiniaceae, Peridiniales and †Suessiales. Other OTUs placed with the heterotrophic species such as in Thoracosphaeraceae (i.e., Pfiesteria and relatives), but not in the consistently heterotrophic Dinophysales and Protoperidiniaceae (Peridiniales). Transitions between phototrophic and heterotrophic modes are thought to occur in the dinophytes (Jeong et al. 2012; Fawcett and Parrow 2014), but there is no phylogenetic signal for this trait at high taxonomic levels. Additionally, some OTUs placed onto early branches that include many parasitic species (Saldarriaga et al. 2003; Gómez et al. 2009; Bachvaroff et al. 2012; Gu et al. 2013), and they placed onto younger branches that also include parasites, such as the Gymnodiniaceae (Gómez et al. 2009; Kretschmann et al. 2015; Romeikat et al. 2020) and Peridiniales (Coats et al. 2010; Gottschling and Söhner 2013).

There were no exclusive associations with dormancy practice. In addition to flagellated trophic cells, coccoid stages are integral part in the life-history of many dinophytes from marine and freshwater environments. Coccoid cells are particularly abundant in the Gonyaulacales where no OTUs placed, or in the Peridiniales where many OTUs placed (Dale 1983; Evitt and Davidson 1964; Fensome et al. 1993; Wall 1971). Exact functions of coccoid cells are not worked out rigorously for more than a handful of dinophyte species, but may frequently correspond to resting and/or dormancy stages (Fensome et al. 1993). Deposited in sediments, coccoid cells have the potential to preserve the local biodiversity like diaspores in a seed bank (Dale 1983). They can be considered either as old remnants from former aquatic and today terrestrial habitats (Boere et al. 2011; Sønstebø et al. 2010) or the result of random dispersal (Foissner 2006, 2011) and subsequent loss in terrestrial habitats. However, the difference between the taxonomic assignments of the rainforest soil samples (see above) may indicate that the ability to form coccoid cells during life-history is not decisive for their terrestrial occurrence. An ecological group that has been receiving more interest in the past years and that may also be considered for the evaluation of bioRxiv preprint doi: https://doi.org/10.1101/2020.08.07.242388; this version posted August 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

the terrestrial samples are benthic dinophytes living in the intertidal (Hoppenrath et al. 2014); phylogenetically, it is a heterogeneous assemblage recruiting members particularly from Gonyaulacales and Peridiniales. But the bigger question remains, as to whether there are any soil dinophytes at all, or we are simply detecting windblown cells or dormant cells.

Conclusion The presence of dinophyte DNA sequences in the Neotropical rainforest soils—as an exemplar of a terrestrial environment—did not associate with taxonomy, environmental preference, dormancy practice during life history, or nutrition mode/organismal interaction. The reason for the presence thus remains to be identified. We have to keep in mind that presence of DNA does not necessarily indicate biological activity. The environmental DNA sequences identified here are scattered unevenly across the classification, but it may represent more easily dispersed surface algae. To completely resolve this paradox, microscopy of soil samples with dinophyte DNA needs to be performed to verify their presence; however, in two centuries of looking at soil samples, dinophytes have never been observed. The most likely explanation for dinophyte DNA in forest soils is that they were passively dispersed there— they are the windblown.

ACKNOWLEDGMENTS We thank Alexandros Stamatakis for computational support. Funding came from the Deutsche Forschungsgemeinschaft (grant DU1319/5-1) to MD. bioRxiv preprint doi: https://doi.org/10.1101/2020.08.07.242388; this version posted August 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

LITERATURE CITED

Adl S. M., Bass D., Lane C. E., Lukeš J., Schoch C. L., Smirnov A., Agatha S., Berney C., Brown M. W., Burki F., Cárdenas P., Čepička I., Chistyakova L., del Campo J., Dunthorn M., Edvardsen B., Eglit Y., Guillou L., Hampl V., Heiss A. A., Hoppenrath M., James T. Y., Karpov S., Kim E., Kolisko M., Kudryavtsev A., Lahr D. J. G., Lara E., Le Gall L., Lynn D. H., Mann D. G., Massana i Molera R., Mitchell E. A. D., Morrow C., Park J. S., Pawlowski J. W., Powell M. J., Richter D. J., Rueckert S., Shadwick L., Shimano S., Spiegel F. W., Torruella i Cortes G., Youssef N., Zlatogursky V. & Zhang Q. 2019. Revisions to the classification, nomenclature, and diversity of eukaryotes. J. Eukaryot. Microbiol., 66:4–119.

Annenkova N. V., Giner C. R. & Logares R. 2020. Tracing the origin of planktonic protists in an ancient lake. Microorganisms, 8:543.

Bachvaroff T. R., Kim S., Guillou L., Delwiche C. F. & Coats D. W. 2012. Molecular diversity of the syndinean genus Euduboscquella based on single-cell PCR analysis. Appl. Environ. Microbiol., 78:334–345.

Bates S. T., Clemente J. C., Flores G. E., Walters W. A., Parfrey L. W., Knight R. & Fierer N. 2013. Global biogeography of highly diverse protistan communities in soil. ISME J., 7:652–659.

Berger S. A. & Stamatakis A. 2011. Aligning short reads to reference alignments and trees. Bioinformatics, 27:2068–2075.

Bergholtz T., Daugbjerg N., Moestrup Ø. & Fernández-Tejedor M. 2006. On the identity of Karlodinium veneficum and description of Karlodinium armiger sp. nov. (Dinophyceae), based on light and electron microscopy, nuclear-encoded LSU rDNA, and pigment composition. J. Phycol., 42:170–193.

Boere A. C., Rijpstra W. I. C., de Lange G. J., Malinverno E., Sinninghe Damsté J. S. & Coolen M. J. L. 2011. Exploring preserved fossil dinoflagellate and haptophyte DNA signatures to infer ecological and environmental changes during deposition of sapropel S1 in the eastern Mediterranean. Paleoceanography, 26:PA2204.

Coats D. W., Kim S., Bachvaroff T. R., Handy S. M. & Delwiche C. F. 2010. Tintinnophagus acutus n. g., n. sp. (Phylum Dinoflagellata), an ectoparasite of the ciliate Tintinnopsis cylindrica Daday 1887, and its relationship to Duboscquodinium collini Grassé 1952. J. Eukaryot. Microbiol., 57:468–482.

Czech L., Barbera P. & Stamatakis A. 2018. Methods for automatic reference trees and multilevel phylogenetic placement. Bioinformatics, 35:1151–1158.

Czech L., Barbera P. & Stamatakis A. 2020. Genesis and Gappa: processing, analyzing and visualizing phylogenetic (placement) data. Bioinformatics, 36:3263–3265.

Dale B. 1983. Dinoflagellate resting cysts: “Benthic plankton.” In: Fryxell G. A. (ed.), Survival Strategies of the Algae. Cambridge, Cambridge University Press. p. 69–136.

Decelle J., Carradec Q., Pochon X., Henry N., Romac S., Mahé F., Dunthorn M., Kourlaiev A., Voolstra C. R., Wincker P. & de Vargas C. 2018. Worldwide occurrence and activity of bioRxiv preprint doi: https://doi.org/10.1101/2020.08.07.242388; this version posted August 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

the reef-building coral symbiont Symbiodinium in the open ocean. Current Biology, 28:3625– 3633.

de Vargas C., Audic S., Henry N., Decelle J., Mahé F., Logares R., Lara E., Berney C., Le Bescot N., Probert I., Carmichael M., Poulain J., Romac S., Colin S., Aury J.-M., Bittner L., Chaffron S., Dunthorn M., Engelen S., Flegontova O., Guidi L., Horák A., Jaillon O., Lima- Mendez G., Lukeš J., Malviya S., Morard R., Mulot M., Scalco E., Siano R., Vincent F., Zingone A., Dimier C., Picheral M., Searson S., Kandels-Lewis S., Tara Oceans Coordinators, Acinas S. G., Bork P., Bowler C., Gorsky G., Grimsley N., Hingamp P., Iudicone D., Not F., Ogata H., Pesant S., Raes J., Sieracki M. E., Speich S., Stemmann L., Sunagawa S., Weissenbach J., Wincker P. & Karsenti E. 2015. Eukaryotic plankton diversity in the sunlit ocean. Science, 348:1261605.

Dunthorn M., Otto J., Berger S. A., Stamatakis A., Mahé F., Romac S., de Vargas C., Audic S., BioMarKs Consortium, Stock A., Kauff F. & Stoeck T. 2014. Placing environmental next- generation sequencing amplicons from microbial eukaryotes into a phylogenetic context. Mol. Biol. Evol., 31:993–1009.

Elferink S., Neuhaus S., Wohlrab S., Toebe K., Voß D., Gottschling M., Lundholm N., Krock B., Koch B. P., Zielinski O., Cembella A. & John U. 2017. Molecular diversity patterns among various phytoplankton size-fractions in West Greenland in late summer. Deep Sea Research Part I: Oceanographic Research Papers, 121:54–69.

Evitt W. R. & Davidson S. E. 1964. Dinoflagellate studies. I. Dinoflagellate cysts and thecae. In: Harbaugh J. W. (ed.), Stanford University Publications in the Geological Sciences. Stanford, Stanford University Press. p. 1–12.

Fawcett R. C. & Parrow M. W. 2014. Mixotrophy and loss of phototrophy among geographic isolates of freshwater Esoptrodinium/Bernardinium sp. (Dinophyceae). J. Phycol., 50:55–70.

Fensome R. A., Taylor F. J. R., Norris G., Sarjeant W. A. S., Wharton D. I. & Williams G. L. 1993. A classification of living and fossil dinoflagellates. Micropaleontology, Special Publication Number 7:1–245.

Foissner W. 2006. Biogeography and dispersal of micro-organisms: a review emphasizing protists. Acta Protozool., 45:111–136.

Foissner W. 2011. Dispersal of protists: the role of cysts and human introductions. In: Fontaneto D. (ed.), Biogeography of microscopic organisms: is everything small everywhere? Cambridge, U.K., Cambridge University Press. p. 61–87.

Geisen S., Mitchell E. A. D., Adl S., Bonkowski M., Dunthorn M., Ekelund F., Fernández L. D., Jousset A., Krashevska V., Singer D., Spiegel F. W., Walochnik J. & Lara E. 2018. Soil protists: a fertile frontier in soil biology research. FEMS Microbiology Reviews, 42:293–323.

Geisen S., Tveit A. T., Clark I. M., Richter A., Svenning M. M., Bonkowski M. & Urich T. 2015. Metatranscriptomic census of active protists in soils. ISME J., 9:2178–2190.

Giner C. R., Pernice M. C., Balagué V., Duarte C. M., Gasol J. M., Logares R. & Massana R. 2020. Marked changes in diversity and relative activity of picoeukaryotes with depth in the world ocean. ISME J., 14:437–449. bioRxiv preprint doi: https://doi.org/10.1101/2020.08.07.242388; this version posted August 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Gómez F., López-García P., Nowaczyk A. & Moreira D. 2009. The crustacean parasites Ellobiopsis Caullery, 1910 and Thalassomyces Niezabitowski, 1913 form a monophyletic divergent clade within the Alveolata. Syst. Parasitol., 74:65–74.

Gottschling M., Chacón J., Žerdoner Čalasan A., Neuhaus S., Kretschmann J., Stibor H. & John U. 2020. Phylogenetic placement of environmental sequences using taxonomically reliable databases helps to rigorously assess dinophyte biodiversity in Bavarian lakes (Germany). Freshw. Biol., 65:193–208.

Gottschling M., Soehner S., Zinssmeister C., John U., Plötner J., Schweikert M., Aligizaki K. & Elbrächter M. 2012. Delimitation of the Thoracosphaeraceae (Dinophyceae), including the calcareous dinoflagellates, based on large amounts of ribosomal RNA sequence data. Protist, 163:15–24.

Gottschling M. & Söhner S. 2013. An updated list of generic names in the Thoracosphaeraceae. Microorganisms, 1:122–136.

Gu H., Kirsch M., Zinssmeister C., Soehner S., Meier K. J. S., Liu T. & Gottschling M. 2013. Waking the dead: morphological and molecular characterization of extant †Posoniella tricarinelloides (Thoracosphaeraceae, Dinophyceae). Protist, 164:583–597.

Guillou L., Bachar D., Audic S., Bass D., Berney C., Bittner L., Boutte C., Burgaud G., de Vargas C., Decelle J., Del Campo J., Dolan J. R., Dunthorn M., Edvardsen B., Holzmann M., Kooistra W. H., Lara E., Le Bescot N., Logares R., Mahé F., Massana R., Montresor M., Morard R., Not F., Pawlowski J., Probert I., Sauvadet A. L., Siano R., Stoeck T., Vaulot D., Zimmermann P. & Christen R. 2013. The Protist Ribosomal Reference database (PR2): a catalog of unicellular eukaryote small sub-unit rRNA sequences with curated taxonomy. Nucleic Acids Research, 41:D597-604.

Henrichs D. W., Sosik H. M., Olson R. J. & Campbell L. 2011. Phylogenetic analysis of Brachidinium capitatum (Dinophyceae) from the Gulf of Mexico indicates membership in the Kareniaceae. J. Phycol., 47:366–374.

Hoppenrath M., Murray S. A. & Chomérat N. 2014. Marine benthic dinoflagellates – Unveiling their worldwide biodiversity. Stuttgart, Schweizerbart Science Publishers.

Jeong H. J., Yoo Y. D., Kang N. S., Lim A. S., Seong K. A., Lee S. Y., Lee M. J., Lee K. H., Kim H. S., Shin W., Nam S. W., Yih W. & Lee K. 2012. Heterotrophic feeding as a newly identified survival strategy of the dinoflagellate Symbiodinium. Proc. Natl. Acad. Sci. USA, 109:12604–12609.

Jones M. D. M., Forn I., Gadelha C., Egan M. J., Bass D., Massana R. & Richards T. A. 2011. Discovery of novel intermediate forms redefines the fungal tree of life. Nature, 474:200–203.

Katoh K. & Standley D. M. 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular biology and evolution, 30:772–780.

Kretschmann J., Filipowicz N. H., Owsianny P. M., Zinssmeister C. & Gottschling M. 2015. Taxonomic clarification of the unusual dinophyte Gymnodinium limneticum Wołosz. (Gymnodiniaceae) from the Tatra Mountains. Protist, 166:621–637. bioRxiv preprint doi: https://doi.org/10.1101/2020.08.07.242388; this version posted August 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Kretschmann J., Zinssmeister C. & Gottschling M. 2014. Taxonomic clarification of the dinophyte Rhabdosphaera erinaceus Kamptner, ≡ Scrippsiella erinaceus comb. nov. (Thoracosphaeraceae, Peridiniales). Syst. Biodivers., 12:393–404.

Le Bescot N., Mahé F., Audic S., Dimier C., Garet M.-J., Poulain J., Wincker P., de Vargas C. & Siano R. 2016. Global patterns of pelagic dinoflagellate diversity across protist size classes unveiled by metabarcoding. Environ. Microbiol., 18:609–626.

Lejzerowicz F., Pawlowski J., Fraissinet-Tachet L. & Marmeisse R. 2010. Molecular evidence for widespread occurrence of Foraminifera in soils. Environ. Microbiol., 12:2518– 2526.

Lentendu G., Buosi P. R. B., Cabral A. F., Segovia B. T., de Meira B. R., Lansac-Toha F. M., Velho L. F. M., Ritter C. & Dunthorn M. 2018. Planktonic protist biodiversity and biogeography in lakes from four brazilian river-floodplain systems. J. Eukaryot. Microbiol., 66:592–599.

Lindberg K., Moestrup Ø. & Daugbjerg N. 2005. Studies on woloszynskioid dinoflagellates I: Woloszynskia coronata re-examined using light and electron microscopy and partial LSU rDNA sequences, with description of Tovellia gen. nov. and Jadwigia gen. nov. (Tovelliaceae fam. nov.). Phycologia, 44:416–440.

Luo Z., Mertens K. N., Bagheri S., Aydin H., Takano Y., Matsuoka K., McCarthy F. M. G. & Gu H. 2016. Cyst-theca relationship and phylogenetic positions of Scrippsiella plana sp. nov. and S. spinifera (Peridiniales, Dinophyceae). Eur. J. Protistol., 51:188–202.

Mahé F., Rognes T., Quince C., de Vargas C. & Dunthorn M. 2015. Swarm v2: highly- scalable and high-resolution amplicon clustering. PeerJ, 3:e1761.

Mahé F., de Vargas C., Bass D., Czech L., Stamatakis A., Lara E., Singer D., Mayor J., Bunge J., Sernaker S., Siemensmeyer T., Trautmann I., Romac S., Berney C., Kozlov A., Mitchell E. A. D., Seppey C. V. W., Egge E., Lentendu G., Wirth R., Trueba G. & Dunthorn M. 2017. Parasites dominate hyperdiverse soil protist communities in Neotropical rainforests. Nat. Ecol. Evol., 1:0091.

Meisterfeld R., Holzmann M. & Pawlowski J. 2001. Morphological and molecular characterization of a new terrestrial allogromiid species: Edaphoallogromia australica gen. et spec. nov. (Foraminifera) from Northern Queensland (Australia). Protist, 152:185–192.

Moestrup Ø. & Calado A. J. 2018. Süßwasserflora von Mitteleuropa, Bd. 6 - Freshwater Flora of Central Europe, Vol. 6: Dinophyceae. Berlin, Springer Spektrum.

Rognes T., Flouri T., Nichols B., Quince C. & Mahé F. 2016. VSEARCH: a versatile open source tool for metagenomics. PeerJ PrePrints, 4:e2584.

Romeikat C., Knechtel J. & Gottschling M. 2020. Clarifying the taxonomy of Gymnodinium fuscum var. rubrum from Bavaria (Germany) and placing it in a molecular phylogeny of the Gymnodiniaceae (Dinophyceae). Syst. Biodivers., 18:102–115.

Rosling A., Cox F., Cruz-Martinez K., Ihrmark K., Grelet G.-A., Lindahl B. D., Menkis A. & James T. Y. 2011. Archaeorhizomycetes: unearthing an ancient class of ubiquitous soil fungi. Science, 333:876–879. bioRxiv preprint doi: https://doi.org/10.1101/2020.08.07.242388; this version posted August 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Saldarriaga J. F., McEwan M. L., Fast N. M., Taylor F. J. R. & Keeling P. J. 2003. Multiple protein phylogenies show that Oxyrrhis marina and Perkinsus marinus are early branches of the dinoflagellate lineage. Int. J. Syst. Evol. Microbiol., 53:355–365.

Saldarriaga J. F. & Taylor F. J. R. 2017. Dinoflagellata. In: Handbook of the Protists, second edition. Cham, Springer International Publishing AG. p. 625–678.

Santoferrara L., Burki F., Filker S., Logares R., Dunthorn M. & McManus G. B. 2020. Perspectives from ten years of protist studies by high-throughput metabarcoding. J. Eukaryot. Microbiol., doi:10.1111/jeu.12813.

Sønstebø J. H., Gielly L., Brysting A. K., Elven R., Edwards M., Haile J., Willerslev E., Coissac E., Rioux D., Sannier J., Taberlet P. & Brochmann C. 2010. Using next-generation sequencing for molecular reconstruction of past Arctic vegetation and climate. Mol. Ecol. Resour., 10:1009–1018.

Stamatakis A. 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics, 30:1312–1313.

Stoeck T., Bass D., Nebel M., Christen R., Jones M. D. M., Breiner H. W. & Richards T. A. 2010. Multiple marker parallel tag environmental DNA sequencing reveals a highly complex eukaryotic community in marine anoxic water. Mol. Ecol., 19:21–31.

Tillmann U., Elbrächter M., Gottschling M., Gu H., Jeong H. J., Krock B., Nézan E., Potvin E., Salas R. & Soehner S. 2014. The dinophycean genus Azadinium and related species – morphological and molecular characterization, biogeography, and toxins. In: Kim H. G., Reguera B., Hallegraeff G. M., Lee C. K., Han H. S. & Choi J. K. (eds.), Harmful Algae 2012 (Proceedings of the 15th International Conference on Harmful Algae). International Society for the Study of Harmful Algae. p. 149–152. Available from: http://hdl.handle.net/10793/1247

Venter P. C., Nitsche F., Domonell A., Heger P. & Arndt H. 2017. The protistan microbiome of grassland soil: diversity in the mesoscale. Protist, 168:546–564.

Voss C., Fiore-Donno A. M., Guerreiro M. A., Peršoh D. & Bonkowski M. 2019. Metatranscriptomics reveals unsuspected protistan diversity in leaf litter across temperate beech forests, with Amoebozoa the dominating lineage. FEMS Microbiol. Ecol., 95:fiz142.

Wall D. 1971. Biological problems concerning fossilizable dinoflagellates. Geoscience and Man, 3:1–15.

Žerdoner Čalasan A., Kretschmann J. & Gottschling M. 2019. They are young, and they are many: dating freshwater lineages in unicellular dinophytes. Environ. Microbiol., 21:4125– 4135.

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5 Gymnodiniales Gonyaulacaceae Ceratocoriaceae

Amphidiniaceae Torodiniaceae Tovelliaceae

Figure 1: A molecular reference tree recognising major groups of dinophytes. Maximum likelihood (ML) tree of 228 representative dinophyte sequences (with strain number information), plus 10 outgroups, as inferred from a SSUrRNA nucleotide alignment (3,767 parsimony informative positions). Numbers on branches are ML bootstrap (above) and Bayesian support values (below) for the clusters (* = maximal support values; values <50 not shown).