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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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. It is made available under aCC-BY 4.0 International license.

6

102 Table 1. Characterizations of Percolomonas strains; sizes and salinity range supporting growth.

The sizes of cells and long flagellum (n=20) Salinty (‰) Source Strain name Sampling location salinity References length width long (‰) L:W LF:L (μm) (μm) flagellum 225 mean ± mean ± 3 15 30 50 75 100 125150 175200 mean ± mean ± (μm) (+) SD SD SD SD mean ± SD Solar saltern at the LRS Ebre River Delta, 280 this study 7.8±0.8 3.9±0.8 2.0±0.5 17.9±1.6 2.3±0.3 + + + + ++ + + + + Spain Chula Vista solar SD2A 200 this study 6.0±0.9 3.3±0.5 1.9±0.3 17.7±2.7 2.9±0.5 + + ++ + + + + saltern, USA Lake Grassmere, XLG1-P 196 this study 7.6±0.9 4.2±0.8 1.8±0.4 19.1±2.0 2.5±0.5 + + ++ ++ + New Zealand Seosin solar saltern, S4 180 this study 7.6±0.9 3.6±0.6 2.1±0.3 18.2±2.4 1.8±0.4 + + + ++ ++ + + Korea Wielizka Salt Mine, P5-P 73 this study 5.4±0.6 2.9±0.4 1.8±0.5 15.7±2.0 2.4±0.3 + + ++ ++ ++ + Poland Novoe Lake, HLM-6 Orenburg oblast, 36 [22] 6.9±0.6 2.2±0.4 3.3±0.6 16.8±1.5 2.4±0.3 ++ + + + (P. lacustris) Russia ATCC Marine Aquarium, https:/www. 28 4.7±1.0 2.6±0.4 1.8±0.3 14.7±2.6 3.1±0.5 + ++ + 50343 Rockville, MD, USA atcc.org

WS White Sea, Russia 20 [17] 6.0±0.7 3.0±0.3 2.0±0.4 17.9±1.3 3.0±0.4 + + ++ + + +

Lake Turkana, LO 4 this study 9.7±1.1 4.3±0.4 2.2±0.3 16.2±1.3 1.7±0.2 + ++ + + Kenya 103 +: relatively low density of living cells, ++: relatively high density of living cells, blank: no growth, SD: standard deviation, L: length, W:

104 width, LF: long flagellum. *: only morphological data reported. 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. 7

105 Light microscopy

106 Live flagellates mounted on glass slides were observed with phase contrast microscopy or

107 differential interference microscopy using a Leica DM5500B microscope equipped with a DFC550

108 digital camera (Leica, Wetzlar, Germany) or Carl Zeiss AxioScope A.1 microscope equipped with a

109 AVT HORN MC-1009/S analog video camera. To observe the number and shape of the flagella,

110 cultures (1 ml) were centrifuged at ×2,000 g for 10 min, then 900 µL of the supernatant was

111 discarded, and the remaining volumes (i.e. 100 µL) were fixed by addition of 50 µL of 25% v/v

112 glutaraldehyde (electron microscopy grade). The sizes of the live cells (i.e. 20 cells per culture) were

113 measured from digital images.

114 Scanning electron microscopy

115 Cultures (1 ml) were centrifuged at ×1,200 g for 10 min, then 900 µL of the supernatant was

116 discarded, and the remaining volumes (i.e. 100 µL) were fixed by adding 50 µL of 25% v/v

117 glutaraldehyde (electron microscopy grade). Fixed cells were allowed to settle (40 min) on glass

118 coverslips coated with 1% poly-L-lysine. Cells were rinsed with sterile media, and then dehydrated

119 with a graded ethanol series (30–100%). The glass coverslips were then critical-point dried. Fixed

120 cells were coated with gold/platinum using an ion sputter system. Specimens were examined with a

121 SU8220 field emission scanning electron microscope (Hitachi, Tokyo, Japan) or JSM-6510LV

122 scanning electron microscope (JEOL Ltd., Tokyo, Japan).

123 Molecular sequencing and phylogenetic analysis

124 Nucleic acids from the seven new isolates were extracted using a DNeasy Blood and Tissue

125 Kit (Qiagen, Hilden, Germany) or MasterPure Complete DNA and RNA Purification Kit (Epicentre, 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. 8

126 Madison, USA), as described in the supplied protocols.

127 For all strains except LRS and HLM-6, the 18S rRNA gene sequences were obtained by

128 PCR amplification using a combination of the eukaryote primers EukA

129 5′-AACCTGGTTGATCCTGCCAGT-3′ and EukB 5′-TGATCCTTCTGCAGGTTCACCTAC-3′

130 [24]. The 20-µL PCR reactions included 1.5 µL each of 10-µM stocks of the primers, 2 µL of a

131 0.25-mM dNTP-mix, 0.8 µL of 50 mM MgCl2, 0.2 µL of 5 U/µL Taq DNA polymerase (Solgent,

132 Daegeon, Republic of Korea), and 1–3 µL of DNA template. The cycling conditions were as

133 follows: an initial denaturing step at 94 °C for 5 min, followed by 35 cycles of 30 s at 94 °C, 1 min

134 of annealing at 55 °C, and extension at 72 °C for 2 min, with a final extension step for 10 min at 72

135 °C. Amplicons were cloned into a pGEM-T Easy vector, at least five positive clones per sample

136 were partially sequenced, and a positive clone was completely sequenced using various sequencing

137 primers. For strain XLG1-P, the cycling conditions were slightly different: 35 cycles of 20 s at 94 °C,

138 1 min of annealing at 55 °C, and 3 min of extension at 72 °C. Strain LRS was amplified using the

139 different eukaryote primers 82F (5'-GAAACTGCGAATGGCTC-3') and 1498R

140 (5'-CACCTACGGAAACCTTGTTA-3'). The optimized PCR condition was 2 min at 96 °C (an

141 initial denaturation), followed by 35 cycles of 30 s at 96 °C, 1 min at 60 °C, 2 min at 72 °C, with a

142 final extension for 10 min at 72 °C. The PCR products for strains XLG1-P and LRS were directly

143 sequenced by Sanger dideoxy sequencing without cloning.

144 For Strain HLM-6, the primers used were PF1 5′-GCGCTACCTGGTTGATCCTGCC-3′

145 and FAD4 5′-TGATCCTTCTGCAGGTTCACCTAC-3′ [25, 26]. The 25-µL PCR reaction included

146 0.5 µL each of 10-µM primer stocks, 1 µL of DNA template, 10.5 µL PCR-grade water and 12.5 µL

147 EconoTaq® PLUS Green 2x Master Mix (Lucigen, Middleton, USA). The cycling conditions were

148 95 °C for 3 min, followed by 35 cycles of 30 s at 95 °C, 30 s at 50 °C, and 1.5 min at 72 °C for 1.5 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. 9

149 min, with a final extension of 5 min at 72 °C. Amplicons were cloned using Strata Clone PCR

150 Cloning Kit (Agilent, Santa Clara, USA). The 18S rRNA gene sequences from the isolates have

151 been deposited in GenBank under the accession numbers XXXXXXX.

152 The 18S rRNA gene sequences from 62 representative heterolobosean species, plus 16 other

153 Discoba species selected as outgroups, were used for phylogenetic analysis (the seed alignment

154 originated from Jhin and Park [16]). The dataset was aligned and masked by eye, with 1,033

155 unambiguously aligned sites retained for analysis. The alignment is available on request.

156 Phylogenetic trees were inferred by Maximum Likelihood (ML) and Bayesian analyses. The GTR +

157 gamma + I model of sequence evolution was selected for the dataset using MrModeltest 2.2 [27] and

158 was used for both analyses. The ML tree was estimated using RAxML-VI-HPC v.7 [28] with the

159 GTRGAMMAI model setting, 500 random starting taxon addition sequences, and statistical support

160 assessed using bootstrapping with 10,000 replicates. The Bayesian analysis was conducted in

161 MrBAYES 3.2 [29] with two independent runs, each with four chains running for 2 × 107

162 generations with the default heating parameter (0.1) and sampling frequency (0.01). A burn-in of

163 30% was used, by which point convergence had been achieved (the average standard deviation of

164 split frequencies for the last 75% of generations was < 0.05).

165 Salinity ranges for growth

166 To estimate the salinity ranges supporting growth of the seven new isolates and two

167 previously available isolates, we performed an experiment using media with 3‰ to 300‰ salinity,

168 made from artificial seawater stock (Medium V; see above) as reported by Park and Simpson [18,

169 30]. In brief, the medium was supplemented with heat-killed Enterobacter aerogenes at an initial

170 density of 3.46 × 107 cells per ml (20 µL) at 7- to 14-day intervals to support the growth of the

171 protists. All treatments were performed in duplicate. Medium V (0.96 ml) with a range of salinities 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. 10

172 (3‰–300‰) were inoculated with 20 µL of actively growing stock culture (100‰ or 35 ‰ salinity

173 media with autoclaved barley grain) and incubated in the dark at 25 °C for at least 49 days. We

174 confirmed the salinity range supporting growth by transferring a sample of the isolate into fresh

175 media with the same salinity (0.96 ml of media; inoculum size 20 µL), and re-examining the culture

176 for actively moving cells at 7- to 14- day intervals over a period of 35 days.

177 Results

178 General morphology

179 Live cells were usually ovoid-shaped or spindle-shaped with average lengths and widths

180 ranging from 4.7–9.7 µm and 2.2–4.3 µm (Fig 1 and Table 1). The ratio of length and width of the

181 cells was between 1.8 and 3.3 on average (Table 1). The biggest cells (length: 9.7 µm, width: 4.3 µm

182 on average) were from strain LO isolated from Lake Turkana, Kenya, whereas the smallest cells

183 were from strain ATCC 50343 (length: 4.7 µm, width: 2.6 µm on average), but the sizes of different

184 strains represented an overlapping continuum. Cells had four flagella inserted in the sub-anterior

185 part of the cell, at the head of the ventral cytostomal groove (Fig 2). Three flagella were shorter, and

186 one flagellum was longer. The three short flagella were similar in length, similar to that of the

187 cytostomal groove (typically ~4 µm long). The long flagellum averaged 14.7–19.1 µm in length,

188 depending on the strain, which was 1.7–3.1 times longer than the length of the cell body (Table 1).

189 Most cells had an acroneme at the tip of the long flagellum (Fig 2), however, this seems not to be a

190 permanent feature. All species showed jerking motility and sometimes rotated in a counterclockwise

191 direction. The right side of the cytostomal groove was curved, whereas the left side was roughly

192 linear (Fig 2). No amoeboid form was observed during the cultivation of the organisms, and a cyst

193 form was observed occasionally in all cultures except for strain ATCC 50343. We did not observe 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. 11

194 discrete features that distinguished isolates from each other under a light microscope or a scanning

195 electron microscope.

196

197 Fig 1. General light micrographs of Percolomonas cosmopolitus-like strains (ordered by source

198 salinity as in Table 1). (A) New strain LRS isolated from a solar saltern at the Ebre River Delta,

199 Catalonia, Spain (280‰ salinity). (B) New strain SD2A isolated from Chula Vista solar saltern,

200 USA (200‰ salinity). (C) New strain XLG1-P isolated from Lake Grassmere, New Zealand (196‰

201 salinity). (D) New strain S4 isolated from a Korean solar saltern (180‰ salinity). (E) New strain

202 P5-P isolated from Wielizka salt mine, Poland (73‰ salinity). (F) Strain HLM-6 isolated from

203 Novoe Lake, Orenburg oblast, Russia (36‰ salinity); this strain was the type for Percolomonas

204 lacustris as described by A.P. Mylnikov [22]. (G) “Percolomonas cosmopolitus” ATCC 50343. (H)

205 “Percolomonas cosmopolitus” strain WS (White Sea), originally reported by Nikolaev et al. [17]. (I)

206 New strain LO isolated from Lake Turkana, Kenya (4‰ salinity). Note that the right cell image in

207 each panel represents a fixed cell. Groups (A) and (B) indicate halophile clades ‘A’ and ‘B’ in Fig. 3.

208 All scale bars represent 5 μm.

209 Fig 2. Scanning electron micrographs of Percolomonas cosmopolitus-like strains. (A) Strain

210 LRS (B) Strain SD2A. (C) Strain XLG1-P. (D) Strain S4. (E) Strain P5-P. (F) Strain HLM-6 =

211 Percolomonas lacustris. (G) ATCC 50343. (H) Strain WS. (I) Strain LO. The order of strains in Fig.

212 2 is the same as that in Fig. 1 and Table 1. All scale bars represent 5 μm.

213

214 Molecular phylogeny of 18S rRNA gene sequences

215 The 18S rRNA gene sequences from the seven unstudied Percolomonas strains were closest 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. 12

216 by BLASTN search to Percolomonas cosmopolitus strain WS (AF 519443), but with a low identity

217 of 74% to 83%. As in previous studies [14–16, 19, 31, 32], Heterolobosea formed a strong

218 monophyletic group in phylogenetic trees of 18S rRNA gene sequences (Fig 3). The seven new

219 Percolomonas strains and two previously identified sequences branched with the pseudociliate taxon

220 Stephanopogonidae with strong support (100% ML; PP 1; Fig 3) forming the clade Percolatea.

221 Interestingly, Percolomonadidae, including the seven new sequences, formed a monophyletic group,

222 with moderate bootstrap support (ML: 76%) and posterior probability 1. Within Percolomonadidae,

223 ATCC 50343 (USA) and P5-P (Poland) were distinct from each other and the remaining seven

224 strains, which formed a maximally supported clade. This subdivided further into (i) WS (Russia),

225 (ii) HLM-6 (Russia), and (iii) a maximally supported ‘putative halophile clade’ of 5 strains in 2

226 clusters, ‘A’ and ‘B’, for a grand total of 6 distinct clusters or single-strain lineages. The 18S rDNA

227 sequences differences between these 6 clusters range from 15.1 to 42.4%. Group A of the putative

228 halophile clade was composed of strains LRS (Spain), XLG1-P (New Zealand), and S4 (Korea) and

229 formed a maximally supported clade (Fig 3), with low genetic divergence between the strain (98%

230 to 99% identities). All members were isolated from hypersaline waters of 180 to 280‰ salinity

231 (Table 1). Group B consisted of strains LO (Kenya) and SD2A (USA) which showed 98% sequence

232 identity, though formed a weakly supported clade (62% ML; PP 0.75). Strain SD2A was isolated at

233 200‰ salinity, while strain LO was isolated from a low salinity (4‰) sample, though it cannot grow

234 at this salinity (see below and Table 1).

235 Fig 3. Maximum likelihood phylogenetic tree of 18S rRNA gene sequences from Percolomonas

236 strains, other representative heterolobosean species, and outgroups (i.e. Euglenozoa, Jakobida,

237 and Tsukubamonas globosa). Bootstrap support values (> 60%) are shown at the nodes. Solid

238 circles represent a Bayesian posterior probability of 1 (posterior probability < 0.95 not shown). Note 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. 13

239 that ‘Group A’ and ‘Group B’ in Percolomonadidae together represent the putative halophilic

240 Percolomonas clade.

241

242 Salinity tolerance of Percolomonas strains

243 The Percolomonas strains were isolated from a variety of habitats with 4‰–280‰ salinity.

244 Salinity ranges supporting growth of all nine sequenced Percolomonas isolates were determined as

245 described earlier [16, 18, 20, 21]. Percolomonas strain LRS isolated from 280‰ salinity showed the

246 broadest salinity range for growth (15‰–200‰ salinity, Table 1). Strain ATCC 50343 grew in the

247 narrowest salinity range (30‰–75‰ salinity, Table 1). Percolomonas strain LO isolated from a

248 source salinity of 4‰ grew only at 30‰ to 100‰ salinity. This suggests that the strain existed as an

249 alternative life-cycle form (e.g. cyst) in the original sample. The five Percolomonas strains (i.e.

250 LRS, SD2A, XLG1-P, S4, and P5-P) isolated from hypersaline habitats with >70‰ source salinity

251 could grow best at 75‰ to 125‰ salinity (Table 1). In contrast, the four Percolomonas strains (i.e.

252 ATCC 50343, White Sea, HML-6 and LO) isolated from non-hypersaline habitats with < 40‰

253 source salinity appeared to grow best at 30‰ to 50‰ salinity (Table 1).

254 Discussion

255 Morphology of Percolomonas isolates

256 In general, the isolates studied here are morphologically indistinguishable by light and

257 scanning electron microscopy from the seminal modern accounts of Percolomonas cosmopolitus

258 [10, 11]. They all have four flagella, specifically three short flagella (~4 μm long) and one long

259 flagellum (12–20 μm long) directed along a ventral groove. The size of the cell body of P. 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. 14

260 cosmopolitus is 6–12 μm in length, 3–9 μm in width [10], overlapping with all of our isolates. The

261 curved right side of the cytostomal groove differs from the left side with a near-linear shape. All

262 strains move with a jerky gliding motion. Thus, it is appears that all new isolates may be assigned to

263 the morphospecies P. cosmopolitus. The nominal Percolomonas species most similar to P.

264 cosmopolitus include P. denhami, with three flagella, two of which are long [33] and P. similis with

265 two flagella, one short, and the other long [34]. There are no molecular data for either of these

266 species. The third similar species is Percolomonas lacustris, which was introduced based on the

267 description of strain HLM-6. This was distinguished morphologically from P. cosmopolitus

268 primarily by having a small posterior bulbous projection and an acroneme on one short flagellum

269 [22]. However, the bulbous projection appears by light microscopy just as a pointed tip, which was a

270 feature of the original description of P. cosmopolitus [9], and was seen intermittently in several of

271 our isolates (see Fig 1). We confirmed that one short flagellum bearing a proportionately long

272 acroneme can be a feature of strain HLM-6 (compare our Fig 2f to Fig 2g in [22]), but note that

273 other cells of the strain appear to lack it (Fig 2f in [22]), and that (shorter) acronemes can be

274 observed on the short flagella of several of the strains studied here (our Fig 2). Thus, it seems that

275 the morphological distinction between P. lacustris and other P. cosmopolitus-like strains is subtle at

276 best.

277 Genetic structure in Percolomonas

278 The Percolomonas complex includes 6 distinct clades that are separated by genetic distances

279 of 15.1% or more. Considering the similar morphology of P. cosmopolitus-like strains (see above),

280 it is reasonable to regard the Percolomonas complex as a grouping of several (at least) cryptic (or

281 sibling) species. Among protists, it has long been recognized that numerous cryptic species can exist

282 within a single morphospecies [4, 8, 35–38]. Fenchel [36] suggested that the cryptic protists are 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. 15

283 regarded as allopatric speciation, and are distributed in limited geographic regions. In our case,

284 however, no geographic clustering of Percolomonas strains was observed in the two clades

285 represented by multiple isolates, although the numbers of sampling sites and sequencing data are

286 limited. Three Percolomonas strains in ‘Group A’ form a robust clade with low genetic divergences

287 (98% to 99% identities) and are widely dispersed geographically (Korea, New Zealand, and Spain).

288 In addition, two Percolomonas strains in ‘Group B’ (with 98% identity) are from Africa and North

289 America. This pattern is more consistent with extremely widespread dispersal, as inferred for other

290 very small protists (e.g. [39]), and we speculate that environmental selection to different habitats

291 may determine which groups appear where (see below).

292 Halophilicity in Percolomonas

293 Ruinen [9] reported that P. cosmopolitus was detected at salinities ranging from 30‰ to

294 saturated brines. In our study, the nine examined Percolomonas strains derived from a wide range of

295 salinities, up to near-saturation. In general, the halophilicity of these strains was related to the salt

296 concentration of their original habitats. Five Percolomonas strains (i.e. LRS, SD2A, XLG1-P, S4,

297 and P5-P) were isolated from various habitats with a salinity range of 73‰ to 280‰. These all grew

298 best at 75‰–125‰ by our qualitative estimation and most could still grow at 175‰ or even 200‰

299 on our experimental media. This clearly makes them halophiles according to the definition by Oren

300 [40], in which a halophile could grow at 50‰ or higher and tolerated at 100‰ salinity. None were

301 obligate halophiles, however, since all could also grow at 30‰ salinity. By contrast, three

302 Percolomonas strains (i.e. HLM-6, ATCC 50343, and WS) isolated from non-hypersaline habitats

303 (20‰ to 36‰ salinity) grew optimally at 30‰ or 50‰, and failed to grow above 100‰ salinity.

304 Interestingly, Percolmonas strain LO grew optimally at 50‰ salinity, and grew up to 100‰ salinity,

305 although it was isolated from a Lake Turkana, Kenya, with a very low salinity of 4‰. 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. 16

306 Phylogenetically, LO belongs within the putative ‘halophile clade’, and it is possible that it

307 descended from a more halophilic ancestor (see below). Interestingly, the Lake Turkana sample was

308 also the origin for Pharyngomonas turkanensis, a heterolobosean amoeba that grows best at

309 15–30‰ salinity, but is inferred to have descended from halophilic Pharyngomonas ancestor [41].

310 The taxon Heterolobosea includes a substantial proportion of the known halophilic or

311 halotolerant eukaryotes [18–21, 41–44], and is interesting for examining the evolution of halophiles

312 [16, 42, 43]. Recently, Kirby et al. [43] and Jhin and Park [16] suggested that the Tulamoebidae

313 clade (sensu lato) in Heterolobosea were an example of a radiation of morphospecies that stemmed

314 from a common halophilic ancestor. This clade included Pleurostomum flabellatum, Tulamoeba

315 peronaphora, Tulamoeba bucina, and Aurem hypersalina, all with optimal salinities for growth of at

316 least 150‰ [16, 20, 21, 43]. It is possible that the Percolomonas clade consisting of ‘Group A’ and

317 ‘Group B’ may also represent a radiation of halophiles (albeit one of cryptic species within a

318 morphospecies). All of the cultivated Percolomonas in this clade are halophiles, with one borderline

319 case (LO; see above). However, this possibility of an exclusively/predominantly halophile clade

320 could be a biased sampling artefact, and should be tested through additional isolations and study of

321 related strains. This would also be useful to understand the nature of the closest relative of the

322 halophile strain P5-P, which is phylogenetically isolated from others at present.

323 Is Percolomonas monophyletic?

324 For a long time only two 18S rRNA gene sequences of P. cosmopolitus were available, and

325 these were included in many phylogenetic analyses of Heterolobosea [13–21, 45]. Most of these

326 phylogenies showed the two sequences forming a paraphyletic group, with one more closely related

327 to Stephanopogon [45]. This inference may have been affected by the small number of sequences of 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. 17

328 Percolomonas available. In the present study, with seven additional and different 18S rRNA gene

329 sequences, we instead inferred a (moderately supported) Percolomonas clade. Future research will

330 address the cause of this difference, and test the relationships amongst Percolomonas and

331 Stephanopogon using other markers.

332 Conclusions

333 On the basis of light and scanning electron microscopic observations, all Percolomonas

334 strains studied here are morphologically very similar, in spite of the huge genetic diversity they

335 encompass. Percolomonas strains form at least 6 genetically distinct clades in the molecular

336 phylogenetic trees, and could be considered to represent at least as many cryptic species. The

337 speciation of Percolomonas strains could be partially related to salinity preference, rather than

338 spatial distribution. The clusters ‘Group A’ and ‘Group B’, which are specifically related, may

339 collectively represent a halophilic clade.

340 Acknowledgements

341 We thank Dr. Alexander Mylnikov for help with SEM preparations and Dr. Jan Janouškovec for

342 help with SSU rDNA sequencing of strain HLM-6. We thank Dr. Noèlia Carrasco for providing

343 access to the Delta de l’Ebre sampling location, and Golara Sharafi for isolating and obtaining DNA

344 from strain LRS. This research was supported by the Basic Science Research Programs through the

345 National Research Foundation of Korea (NRF), a grant from the National Institute of Biological

346 Resources (NIBR), funded by the Ministry of Education (2016R1A6A1A05011910,

347 2017K2A9A1A06049946, and 2019R1A2C2002379) and the Ministry of Environment (MOE) of

348 the Republic of Korea (NIBR201801208), respectively, to J.S.P., a Russian Foundation for Basic

349 Research grant (No 18-504-51028) to D.V.T., and a Natural Sciences and Engineering Research 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. 18

350 Council of Canada Discovery Grant (298366-2014) to A.G.B.S.

351

352 References

353 1. Bickford D, Lohman DJ, Sodhi NS., Ng PKL, Meier R, Winker K, et al. Cryptic species as a

354 window on diversity and conservation. Trends Ecol Evol. 2007; 22:148–155.

355 2. Ristau K, Steinfartz S, Traunspurger W. First evidence of cryptic species diversity and

356 significant population structure in a widespread freshwater nematode morphospecies (Tobrilus

357 gracilis). Mol Ecol. 2013; 22:4562–4575.

358 3. Trewick SA. Sympatric cryptic species in New Zealand Onychophora. Biol J Linnean Soc.

359 1998; 63:307–329.

360 4. Scheckenbach F, Wylezich C, Mylnikov AP, Weitere M, Arndt H. Molecular comparisons of

361 freshwater and marine isolates of the same morphospecies of heterotrophic flagellates. Appl

362 Environ Microbiol. 2006; 72:6638–6643.

363 5. Finlay BJ. Global dispersal of free-living microbial eukaryote species. Science. 2002;

364 296:1061–1063.

365 6. Škaloud P, Rindi F. Ecological differentiation of cryptic species within an asexual protist

366 morphospecies: a case study of filamentous green alga Klebsormidium (Streptophyta). J

367 Eukaryot Microbiol. 2013; 60:350–362.

368 7. Foissner W. Protist diversity: estimates of the near-imponderable. Protist. 1999; 150:363–368.

369 8. Finlay BJ, Fenchel T. Divergent perspectives on protist species richness. Protist. 1999;

370 150:229–233.

371 9. Ruinen J. Notizen über Salzflagellaten. II. Über die Verbreitung der Salzflagellaten. Arch 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. 19

372 Protistenkd. 1938; 90:210–258.

373 10. Fenchel T. Patterson DJ. Percolomonas cosmopolitus (Ruinen) n. gen., a new type of filter

374 feeding flagellate from marine plankton. J Mar Biol Assoc UK. 1986; 66:465–482.

375 11. Larsen J, Patterson DJ. Some flagellates (Protista) from tropical marine sediments. J Nat

376 History. 1990; 24:801–937.

377 12. Post FJ, Borowitzka LJ, Borowitzka MA, Mackay B, Moulton T. The protozoa of a Western

378 Australian hypersaline lagoon. Hydrobiologia. 1983; 105:95–113.

379 13. Cavalier-Smith T, Nikolaev S. The zooflagellates Stephanopogon and Percolomonas are a clade

380 (Class Percolatea: Phylum Percolozoa). J Eukaryot Microbiol. 2008; 55:501–509.

381 14. Hanousková P, Táborský P, Čepička I. Dactylomonas gen. nov., a novel lineage of

382 heterolobosean flagellates with unique ultrastructure, closely related to the amoeba Selenaion

383 koniopes Park, De Jonckheere & Simpson, 2012. J Eukaryot Microbiol. 2019; 66:120–139.

384 15. Harding T, Brown MW, Plotnikov A, Selivanova E, Park JS, Gunderson JH, et al. Amoeba

385 stages in the deepest branching heteroloboseans, including Pharyngomonas: Evolutionary and

386 systematic implications. Protist. 2013; 164:272–286.

387 16. Jhin SH, Park JS. A new halophilic heterolobosean flagellate, Aurem hypersalina gen. n. et sp.

388 n., closely related to the Pleurostomum-Tulamoeba clade: Implications for adaptive radiation of

389 halophilic eukaryotes. J Eukaryot Microbiol. 2019; 66:221–231.

390 17. Nikolaev SI, Mylnikov AP, Berney C, Fahrni J, Pawlowski J, Aleshin VV, et al. Molecular

391 phylogenetic analysis places Percolomonas cosmopolitus within Heterolobosea: Evolutionary

392 implications. J Eukaryot Microbiol. 2004; 51:575–581.

393 18. Park JS, Simpson AGB. Characterization of Pharyngomonas kirbyi (= “Macropharyngomonas

394 halophila” nomen nudum), a very deep-branching, obligately halophilic heterolobosean 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. 20

395 flagellate. Protist. 2011; 162:691–709.

396 19. Park JS, Simpson AGB. Diversity of heterotrophic protists from extremely hypersaline habitats.

397 Protist. 2015; 166:422–437.

398 20. Park JS, Simpson AGB, Lee WJ, Cho BC. Ultrastructure and phylogenetic placement within

399 Heterolobosea of the previously unclassified, extremely halophilic heterotrophic flagellate

400 Pleurostomum flabellatum (Ruinen 1938). Protist. 2007; 158:397–413.

401 21. Park JS, Simpson AGB, Brown S, Cho BC. Ultrastructure and molecular phylogeny of two

402 heterolobosean amoebae, Euplaesiobystra hypersalinica gen. et sp. nov. and Tulamoeba

403 peronaphora gen. et sp. nov., isolated from an extremely hypersaline habitat. Protist. 2009;

404 160:265–283.

405 22. Mylnikov AP. A new flagellate Percolomonas lacustris sp. n. (Excavata, Percolozoa) from an

406 inland saline lake (Southeastern European Russia). Biol Bull. 2016; 43:587–594.

407 23. Park JS. Effects of different ion compositions on growth of obligately halophilic protozoan

408 Halocafeteria seosinensis. Extremophiles. 2012; 16:161–164.

409 24. Medlin L, Elwood HJ, Stickel S, Sogin ML. The characterization of enzymatically amplified

410 eukaryotic 16S-like rRNA coding regions. Gene. 1988; 71:491–499.

411 25. Keeling PJ. Molecular phylogenetic position of Trichomitopsis termopsidis (Parabasalia) and

412 evidence for the Trichomitopsiinae. Eur J Protistol. 2002; 38:279–286.

413 26. Deane JA, Hill DRA, Brett SJ, McFadden GI. Hanusia phi gen. et. sp. nov. (Cryptophyceae):

414 Characterization of ‘Cryptomonas sp. Φ'’. Eur J Phycol. 1998; 33:149–154.

415 27. Nylander JAA. MrModeltest. Version 2. Program distributed by the author. Evolutionary

416 Biology Centre, Uppsala University, Sweden. 2004.

417 28. Stamatakis A. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with 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. 21

418 thousands of taxa and mixed models. Bioinformatics. 2006; 22:2688–2690.

419 29. Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, et al. MrBAYES 3.2:

420 Efficient Bayesian phylogenetic inference and model choice across a large model space. Syst.

421 Biol. 2012; 61:539–542.

422 30. Park JS, Simpson AGB. Characterization of halotolerant Bicosoecida and Placididea

423 (Stramenopila) that are distinct from marine forms, and the phylogenetic pattern of salinity

424 preference in heterotrophic stramenopiles. Environ Microbiol. 2010; 12:1173–1284.

425 31. Pánek T, Silberman JD, Yubuki N, Leander BS, Čepička I. Diversity, evolution and molecular

426 systematics of the Psalteriomonadidae, the main lineage of anaerobic/microaerophilic

427 heteroloboseans (Excavata: Discoba). Protist. 2012; 163:807–831.

428 32. Pánek T, Simpson AGB, Hampl V, Čepička I. Creneis carolina gen. et sp. nov.

429 (Heterolobosea), a novel marine anaerobic protist with strikingly derived morphology and life

430 cycle. Protist. 2014; 165:542–567.

431 33. Tong SM. Heterotrophic flagellates from the water column in Shark Bay, Western Australia.

432 Mar. Biol. 1997; 128:517–536.

433 34. Lee WJ, Brandt SM, Vørs N, Patterson DJ. Darwin’s heterotrophic flagellates. Ophelia. 2003;

434 57:63–98.

435 35. Beszteri B, Ács É, Medlin LK. Ribosomal DNA sequence variation among sympatric strains of

436 the Cyclotella menghiniana complex (Bacillariophyceae) reveals cryptic diversity. Protist.

437 2005; 156:317–333.

438 36. Fenchel T. Cosmopolitan microbes and their ‘cryptic’ species. Aquat Microb Ecol. 2005;

439 41:49–54.

440 37. Koch TA, Ekelund F. Strains of the heterotrophic flagellate Bodo designis from different 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. 22

441 environments vary considerably with respect to salinity preference and SSU rRNA gene

442 composition. Protist. 2005; 156:97–112.

443 38. Von der Heyden S, Chao EE, Vickerman K, Cavalier-Smith T. Ribosomal RNA phylogeny of

444 Bodonid and Diplonemid flagellates and the evolution of Euglenozoa. J Eukaryot Microbiol.

445 2004; 51:402–416.

446 39. Stoupin D, Kiss AK, Arndt H, Shatilovich AV, Gilichinsky DA, Nitsche F. Cryptic diversity

447 within the choanoflagellate morphospecies complex Codosiga botrytis – Phylogeny and

448 morphology of ancient and modern isolates. Europ J Protistol. 2012; 48:263–273.

449 40. Oren A. Microbial life at high salt concentrations: phylogenetic and metabolic diversity. Saline

450 Syst. 2008; 4:2.

451 41. Park JS, Simpson AGB. Characterization of a deep-branching heterolobosean, Pharyngomonas

452 turkanaensis n. sp., isolated from a non-hypersaline habitat, and ultrastructural comparison of

453 cysts and amoebae among Pharyngomonas strains. J Eukaryot Microbiol. 2016; 63:100–111.

454 42. Harding T, Simpson AGB. Recent advances in halophilic protozoa research. J Eukaryot

455 Microbiol. 2018; 65:556–570.

456 43. Kirby WA, Tikhonenkov DV, Mylnikov AP, Janouškovec J, Lax G, Simpson AGB.

457 Characterization of Tulamoeba bucina n. sp., an extremely halotolerant amoeboflagellate

458 heterolobosean belonging to the Tulamoeba-Pleurostomum clade (Tulamoebidae n. fam.). J

459 Eukaryot Microbiol. 2015; 62:227–238.

460 44. Park JS, De Jonckheere JF, Simpson AGB. Characterization of Selenaion koniopes n. gen., n.

461 sp., an amoeba that represents a new major lineage within Heterolobosea, isolated from the

462 Wieliczka salt mine. J Eukaryot Microbiol. 2012; 59:601–613.

463 45. Pánek T, Čepička I. Diversity of Heterolobosea. In: Caliskan M, editor. Diversity of 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. 23

464 Microorganisms. Rijeka: Intech; 2012. pp. 3–26. 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. 24

466

467 Parts of a Submission 468 Short title: Percolomonas diversity and halophilicity

469 Author contributions 470 Conceptualization: Jong Soo Park, Alastair G.B. Simpson 471 Investigation: Jong Soo Park, Denis V. Tikhonenkov, Soo Hwan Jhin, Yana Eglit, Kai Miller, 472 Andrey Plotnikov 473 Formal analysis: Jong Soo Park, Denis V. Tikhonenkov, Soo Hwan Jhin 474 Visualization: Jong Soo Park, Denis V. Tikhonenkov, Soo Hwan Jhin 475 Supervision: Jong Soo Park, Alastair G.B. Simpson 476 Funding acquisition: Jong Soo Park, Denis V. Tikhonenkov, Alastair G.B. Simpson 477 Writing – original draft preparation: Jong Soo Park, Denis V. Tikhonenkov, Alastair G.B. Simpson 478 Writing – review and editing: all authors. 479

480 481 Keywords: Cryptic species; Heterolobosea; Molecular Phylogeny; Morphospecies; Percolomonas;

482 Protist; Protozoa

483 484 Financial Disclosure Statement 485 This research was supported by the Basic Science Research Programs through the National Research 486 Foundation of Korea (NRF), a grant from the National Institute of Biological Resources (NIBR), 487 funded by the Ministry of Education (2016R1A6A1A05011910, 2017K2A9A1A06049946, and 488 2019R1A2C2002379) and the Ministry of Environment (MOE) of the Republic of Korea 489 (NIBR201801208), respectively, to J.S.P., a Russian Foundation for Basic Research grant (No 490 18-504-51028) to D.V.T., and a Natural Sciences and Engineering Research Council of Canada 491 Discovery Grant (298366-2014) to A.G.B.S.

492 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. 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. 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. 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.