bioRxiv preprint doi: https://doi.org/10.1101/612531; this version posted April 17, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 4.0 International license. 1 1 Ecological and evolutionary patterns in the enigmatic protist 2 genus Percolomonas (Heterolobosea; Discoba) from diverse 3 habitats 4 Denis V. Tikhonenkov1,2, Soo Hwan Jhin3, Yana Eglit4, Kai Miller4, Andrey Plotnikov5, Alastair 5 G.B. Simpson4,6, Jong Soo Park3* 6 7 1 Papanin Institute for Biology of Inland Waters, Russian Academy of Sciences, Borok, Russia 8 2Zoological Institute, Russian Academy of Sciences, Saint Petersburg, Russia 9 3Department of Oceanography, Research Institute for Dok-do and Ulleung-do Island and 10 Kyungpook Institute of Oceanography, School of Earth System Sciences, Kyungpook National 11 University, Daegu, Korea 12 4Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada 13 5Center of Shared Scientific Equipment “Persistence of Microorganisms”, Institute for Cellular and 14 Intracellular Symbiosis UB RAS, Orenburg, Russia 15 6Canadian Institute for Advanced Research, Program in Integrated Microbial Diversity, Toronto, 16 Ontario, Canada 17 18 * Corresponding author 19 E-mail: [email protected] (JSP) bioRxiv preprint doi: https://doi.org/10.1101/612531; this version posted April 17, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 4.0 International license. 2 20 Abstract 21 The heterotrophic flagellate Percolomonas cosmopolitus (Heterolobosea) is often observed 22 in saline habitats worldwide that range from coastal waters to saturated brines. However, only two 23 cultures assigned to this morphospecies have been examined using molecular methods, and their 24 18S rRNA gene sequences are extremely different. Further the salinity tolerances of individual 25 strains are unknown. Thus, our knowledge on the autecology and diversity in this morphospecies is 26 deficient. Here, we report 18S rRNA gene data on seven strains similar to P. cosmopolitus from 27 seven geographically remote locations (New Zealand, Kenya, Korea, Poland, Russia, Spain, and the 28 USA) with sample salinities ranging from 4‰ to 280‰, and compare morphology and salinity 29 tolerance of the nine available strains. Percolomonas cosmopolitus-like strains show few-to-no 30 consistent morphological differences, and form six clades separated by often extremely large 18S 31 rDNA divergences (up to 42.4%). Some strains grew best at salinities from 75 to 125‰ and 32 represent halophiles. All but one of these belonged to two geographically heterogeneous clusters 33 that formed a robust monophyletic group in phylogenetic trees; this likely represents an ecologically 34 specialized subclade of halophiles. Our results suggest that P. cosmopolitus is a cluster of several 35 cryptic species (at least), which are unlikely to be distinguished by geography. Interestingly, the 9 36 Percolomonas strains formed a clade in 18S rDNA phylogenies, unlike most previous analyses 37 based on two sequences. 38 39 40 bioRxiv preprint doi: https://doi.org/10.1101/612531; this version posted April 17, 2019. 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 4.0 International license. 3 41 Introduction 42 The modern classification of eukaryotic microbes is usually based on molecular sequence 43 information combined with morphological characters obtained using diverse imaging techniques, 44 often including electron microscopy. Many taxonomists have a fundamental question whether small 45 morphological differences among the observed organisms represent multiple species or one variable 46 species [1–3]. Conversely, in protistology, many species (and genera) that were originally proposed 47 based on light microscopy alone have proved to encompass considerable genetic diversity. Also, 48 many morphospecies of protists are cosmopolitan and/or are found across a very wide range of 49 habitats, raising the possibility of recognizing species that are divided more by geography or 50 ecology than morphology [4]. Bickford et al. [1] regarded ‘cryptic species’ as cases where two or 51 more species are distinguished that were previously assigned to a single morphologically defined 52 species (i.e. morphospecies). The concept of cryptic (usually genetically different) species has been 53 examined in a diverse range of protists, e.g. [4, 5, 6]. This concept is widely accepted due to an 54 inconsistency between morphospecies and their DNA sequencing data. Obviously, the estimated 55 number of protist species will vary tremendously depending on the species concept employed [7, 8]. 56 The species Percolomonas cosmopolitus (formerly Tetramitus cosmopolitus Ruinen [9]) is a 57 heteroloboseid flagellate with one long and three shorter flagella at the head of a ventral feeding 58 groove, and no known amoeba stage in its lifecycle. Cell size is given as 6–12 μm long and 3–9 μm 59 wide in the seminal modern accounts [10, 11]; the largest cells reported by Ruinen [9] are somewhat 60 longer. This species is a particularly interesting protist for taxonomists and ecologists for several 61 reasons: Firstly, it is possible that P. cosmopolitus sensu lato may be a remarkably broad assemblage 62 of cryptic species. To date, two strains identified as P. cosmopolitus have been studied using bioRxiv preprint doi: https://doi.org/10.1101/612531; this version posted April 17, 2019. 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 4.0 International license. 4 63 molecular methods, but their 18S rRNA gene sequences share remarkably low sequence identity 64 (58.4%). Secondly, P. cosmopolitus has been reported in samples from an extremely wide range of 65 saline habitats, from coastal waters to saturated brines, throughout the world [9–12]. It is unknown 66 whether there are ecologically or geographically defined subtypes. Thirdly, in addition to the genetic 67 divergence between them (see above) P. cosmopolitus strains are usually not inferred to be sisters in 68 phylogenetic trees, forming instead a paraphyletic group with respect to the pseudociliate 69 Stephanopogon, which has many more flagella and a completely different feeding system [13–21]. 70 Consequently, Percolomonas cosmopolitus and similar organisms (e.g. [22]) are interesting 71 candidates to study the interplay between species distinctions, autecology and evolutionary history 72 among protists. 73 Here, we examined seven Percolomonas cosmopolitus-like organisms, which were isolated 74 from samples with a salinity range from 4‰ to 280‰, from seven different countries (New Zealand, 75 Kenya, Korea, Poland, Russia, Spain, and the USA). The nine Percolomonas strains examined 76 (including the two P. cosmopolitus strains previously sequenced) were morphologically similar to 77 each other, but their 18S rRNA gene sequences showed considerable genetic divergence, and they 78 formed six genetically distinct clusters. The strains isolated from high salinity samples preferred to 79 grow in artificial media with salinity higher than seawater (75‰ or above), indicating ecological 80 specialization. Percolomonas formed a monophyletic group in the 18S rRNA gene phylogeny. 81 82 Materials and methods 83 Isolation and cultivation bioRxiv preprint doi: https://doi.org/10.1101/612531; this version posted April 17, 2019. 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 4.0 International license. 5 84 Seven monoprotistan strains were isolated from brackish to high salinity (4–280‰) 85 water/sediment interface samples collected from New Zealand, Kenya, Korea, Poland, Russia, 86 Spain, and the USA between 2008 and 2015 (Table 1). Isolates from higher salinity waters were 87 LRS, SD2A, XLG1-P, S4, and P5-P; isolates from lower salinity waters were LO, and HLM-6. 88 Strain HLM-6 was examined morphologically by A.P. Mylnikov [22] under the name Percolomonas 89 lacustris. Each monoprotistan culture was established by single-cell isolation (from raw samples or 90 crude cultures) or by serial dilution. High salinity media (~100‰ salinity) was made by dilution of -1 91 Medium V (300‰; 272g NaCl, 7.6g KCl, 17.8g MgCl2, 1.8g MgSO4·7H2O, 1.3g CaCl2 l water, 92 see [23]) with sterile distilled water, whereas normal salinity media (~35‰ salinity) used autoclaved 93 seawater. Luria-Bertani Broth (final concentration of 0.5%, Difco) plus autoclaved barley grains 94 were added into each media to grow indigenous prokaryotes in the culture. For maintenance of each 95 culture, 0.1 ml of inoculum was added into 5 ml of 100‰ salinity liquid media (isolates LRS, 96 SD2A, XLG1-P, S4, and P5-P) or 35‰ salinity liquid media (isolates LO and HLM-6 plus the 97 already available strains ATCC 50343 and White Sea = ‘WS’). Strain HLM-6 was also maintained −1 −1 −1 98 in marine Schmalz-Pratt’s medium (NaCl–28.15 g l , KCl–0.67 g l , MgCl2·6H2O–5.51 g l , −1 −1 −1 −1 99 MgSO4·7H2O–6.92 g l , CaCl2·H2O–1.45 g l , KNO3–0.1 g l , K2HPO4·3H2O–0.01 g l with a 100 final salinity of 35‰) with addition of Pseudomonas fluorescens bacteria as food. All cultures were 101 incubated at 25 °C and subcultured every four weeks. bioRxiv preprint doi: https://doi.org/10.1101/612531; this version posted April 17, 2019. 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.