bioRxiv preprint doi: https://doi.org/10.1101/2020.05.14.095992; this version posted May 15, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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.
1 A freshwater radiation of diplonemids 2 3 4 Indranil Mukherjee1#, Michaela M Salcher1, Adrian-Ștefan Andrei1*, Vinicius 5 Silva Kavagutti1,2, Tanja Shabarova1, Vesna Grujčić3, Markus Haber1, Paul 6 Layoun1,2, Yoshikuni Hodoki4, Shin-ichi Nakano4, Karel Šimek1,2, Rohit 7 Ghai1# 8 9 1Department of Aquatic Microbial Ecology, Institute of Hydrobiology,Biology Centre of 10 the Czech Academy of Sciences, , Na Sadkach 7, 37005, České Budějovice, Czech 11 Republic 12 13 2Department of Ecosystem Biology, Faculty of Science, University of South Bohemia, 14 Branišovská 1760, 37005, České Budějovice, Czech Republic 15 16 3KTH Royal Institute of Technology, Science for Life Laboratory, Department of Gene 17 Technology, School of Engineering Sciences in Chemistry, Biotechnology and Health, 18 Stockholm, Sweden 19 20 4Center for Ecological Research, Kyoto University, Otsu, Shiga, Japan 21 22 *Current address: Limnological Station, Institute of Plant and Microbial Biology, 23 University of Zurich, Seestrasse 187, 8802, Kilchberg, Switzerland 24 25 26 27 Keywords: 18S amplicon sequencing/ CARD-FISH/ diplonemids/ freshwater lakes/
28 protists/ metagenomic 18S rRNA assembly
29 Running Title: Diplonemids in freshwater lakes 30 31 32 #Authors to whom correspondence should be addressed
33 Indranil Mukherjee ([email protected])
34 Rohit Ghai ([email protected])
35
36
37
38
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39 Abstract
40 Diplonemids are considered marine protists and have been reported among the most
41 abundant and diverse eukaryotes in the world oceans. Recently we detected the presence
42 of freshwater diplonemids in Lake Biwa, Japan. However, their distribution and
43 abundances in freshwater ecosystems remain unknown. We assessed abundance and
44 diversity of diplonemids from several geographically distant deep freshwater lakes of the
45 world by amplicon-sequencing, shotgun metagenomics and CARD-FISH. We found
46 diplonemids in all the studied lakes, albeit with low abundances and diversity. We
47 assembled long 18S rRNA sequences from freshwater diplonemids and showed that they
48 form a new lineage distinct from the diverse marine clades. Freshwater diplonemids are a
49 sister-group to marine isolates from coastal and bay areas, suggesting a recent habitat
50 transition from marine to freshwater habitats. Images of CARD-FISH targeted freshwater
51 diplonemids suggest they feed on bacteria. Our analyses of 18S rRNA sequences retrieved
52 from single cell genomes of marine diplonemids shows they encode multiple rRNA copies
53 that may be very divergent from each other, suggesting that marine diplonemid abundance
54 and diversity both have been overestimated. These results have wider implications on
55 assessing eukaryotic abundances in natural habitats by using amplicon-sequencing alone.
56
57 Introduction
58 Diplonemids appear as one of the more intriguing findings in amplicon-sequencing
59 surveys of the global oceans microbiota [1]. While the abundance and distribution of these
60 protists were nearly unknown till the last decade, they are now considered (based mainly
61 on metabarcoding approaches) as one of the most abundant and diverse microbial
62 eukaryotic group in the world oceans [2,3]. Marine diplonemid 18S rRNA sequences
63 exhibit high diversity and are the sixth most frequently recovered eukaryotic signatures in
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64 the world’s oceans in amplicon-based surveys [3]. They appear to be more abundant in the
65 mesopelagic zone of the oceans [1,4]. These discoveries have ignited interest in
66 understanding the importance and distribution of these protists in other aquatic non-
67 marine ecosystems. Diplonemids are considered marine protists [5] and were till recently
68 never reported from freshwater ecosystems. Our interest in studying the distribution of
69 free-living kinetoplastids (a sister group of diplonemids) from freshwater lakes using
70 kinetoplastid-specific primers accidentally led us to the discovery of freshwater
71 diplonemids in deep freshwater lakes of Japan [6]. Similar to kinetoplastids, diplonemids
72 are generally not targeted by commonly used eukaryotic V4 primers due to their divergent
73 18S rRNA genes [7,8] and this explains their exclusion from most diversity studies in
74 freshwater environments. This finding suggested that diplonemids are not limited to
75 marine ecosystems and are likely present in other freshwater lakes of the world. To
76 examine the distribution of diplonemids in freshwater habitats, we sampled seven deep
77 freshwater lakes (different depths, both monthly time series and isolated samples) of
78 various trophic statuses from Japan, Czech Republic, and Switzerland. The diversity and
79 abundance of diplonemids were analyzed using PCR based amplicon sequencing, shotgun
80 metagenomic sequencing and CARD-FISH analyses. Our results suggest a broad
81 distribution (albeit with low abundance) of diplonemids in freshwater ecosystems, and the
82 presence of one widespread freshwater clade that appears to be a radiation of known
83 marine isolates.
84
85 Results and Discussions
86 Freshwater diplonemid 18S rRNA gene sequences
87 A single diplonemid sequence (OTU_4) was described recently from the lakes Biwa and
88 Ikeda using 18S rRNA amplicon sequencing [6]. This sequence provided the first
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89 intriguing evidence for the existence of freshwater diplonemids, which have been until
90 now considered as marine eukaryotes [5]. However, more conclusive evidence for the
91 presence of these eukaryotes and their distribution in other freshwater habitats is lacking.
92 As the first diplonemid sequence was described from Lake Biwa, in an effort to more
93 clearly examine the depth- or seasonal-related abundance of freshwater diplonemids, we
94 carried out multiple sampling campaigns with different sampling frequencies at this site.
95 The first campaign in Lake Biwa lasted for three months in spring/summer 2016 and
96 samples were collected from three different depths (Table 1, Supplementary Figure S3).
97 The second campaign lasted throughout the year 2017 with a monthly sampling frequency
98 (Table 1, Supplementary Figure S4). A single depth profile was also collected from Lake
99 Ikeda to examine the vertical distribution of diplonemids (Table 1, Supplementary Figure
100 S5). To expand the known distribution of diplonemids outside Japan, we also collected
101 samples from five European lakes (Řimov, Medard, Zurich, Constance and Lugano). A
102 complete list of sampling sites and their locations, depths and analyses performed with the
103 samples from each site is given in Table 1.
104 Amplicon-sequencing was performed for the sampling campaigns from Lake Biwa
105 and Lake Ikeda (Figure 1A, B, C and Supplementary Figure S3, S4, S5). Relative
106 abundance of diplonemid sequences in these samples was always below 1%. A total of 18
107 sequences were recovered, somewhat more frequently from the hypolimnion. The highest
108 relative abundance was observed in the hypolimnion of Lake Ikeda (100 m), contributing
109 up to 0.96 % of total sequence reads (Figure 1C). Although rare, they were present in
110 nearly all the seasons in Lake Biwa (Figure 1) in both epilimnion (5 m) and metalimnion
111 (10-20 m) but only occasionally in the hypolimnion (50-60 m). The highest amplicon read
112 abundance in Lake Biwa was observed in the hypolimnion of May (0.5% of total
113 sequences, Figure 1A). Diplonemid sequences were more abundant in the epilimnion and
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114 metalimnion during the early part of the spring and as the stratification developed their
115 numbers increased in the hypolimnion (Figure 1A). However, data from the entire year
116 showed the absence of diplonemid sequences in the hypolimnion during summer and
117 autumn (Figure 1B). This discrepancy could be due to the sample collection in different
118 years (spring sampling in 2016 and monthly sampling in 2017) and depths (50 vs. 60 m).
119 However, the low numbers of recovered sequences preclude definitive conclusions
120 regarding seasonal or depth related preferences for diplonemids. Similar results were
121 obtained upon examining the abundance of freshwater diplonemids using an existing
122 shotgun metagenomic timeline from the Řimov reservoir (Figure 1D). Here, diplonemid
123 sequences in the epilimnion were found only during spring or early summer with low
124 abundance (maximum 0.04% of total eukaryotic 18S rRNA reads) (Figure 1D). In contrast
125 to Lake Biwa, diplonemid sequences were found at all seasons in the hypolimnion of the
126 Řimov reservoir with relatively higher proportions (up to 0.6% of total metagenomic
127 reads) (Supplementary Table S2) although these samples were pre-filtered with 5 μm
128 filters, which may have reduced the number of diplonemids. Moreover, the differences in
129 trophic status and sample collection from different depths (Table 1) might be responsible
130 for the observed variation. It is also relevant to mention that dynamics of diplonemids
131 inferred from raw sequence reads is not fully quantitative, as we observed multiple copies
132 of 18S rRNA in single cell genomes from marine diplonemids (Supplementary Table S1).
133 To further examine the distribution of diplonemids at other locations, we sampled
134 several European lakes for shotgun metagenomics (Table 1). As the sequences obtained
135 by amplicon-sequencing were short (300 bp), we wondered if it was possible to identify
136 longer assembled 18S rRNA sequences from freshwater diplonemids in assembled
137 metagenomic contigs. Additionally, we also used previously available metagenomic
138 samples from the Řimov reservoir [17] and other published metagenomic datasets from
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139 the deep Lake Baikal [18,19]. Multiple long sequences were retrieved from all samples
140 except for Lake Baikal and all of them were closely related to the amplicon-derived
141 sequences (See Figure 2). The longest assembled 18S rRNA sequence was 2029 bp
142 suggesting the less examined potential for assembled metagenomic sequence data for
143 recovery of long eukaryotic 18S rRNA sequences.
144 Long sequences (>300 bp) of diplonemids from the metagenomic datasets allowed
145 us to construct a robust maximum-likelihood phylogenetic tree including all closely
146 related sequences of diplonemids from public databases (Figure 2). We observed that the
147 sequences recovered from geographically distant freshwater lakes were highly similar and
148 appeared to be a sister clade of several marine isolates. High similarity of diplonemid
149 sequences from freshwater lakes of the world reiterates what has been suggested already
150 [32] that geographic distance does not restrict the distribution of protists in freshwater
151 lakes around the world. The freshwater sequences formed a separate freshwater cluster
152 that was separated from the marine cultured and environmental sequences with high
153 bootstrap values, suggesting that freshwater diplonemids diverged from the marine ones
154 and are phylogenetically different. The highly congruent freshwater clade provides
155 conclusive evidence for an evolutionary radiation of diplonemids in freshwater
156 ecosystems. Intriguingly, a single diplonema isolate (accession MF422200) that was
157 obtained from Tokyo Bay also appeared to be part of this freshwater clade. This might
158 suggest that even though this isolate was obtained in the bay of Tokyo (Supplementary
159 Table S3), it is possible that it is actually derived from the freshwater inflow into the bay.
160 Moreover, the freshwater cluster is closely related to the marine isolates (Figure 2) and
161 majority of those isolates were from bay, estuarine and near-shore regions (Supplementary
162 Table S3) further suggesting a habitat transition of diplonemids from marine to
163 freshwaters.
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164
165 Occurrence, morphology, and potential feeding mode of diplonemids
166 We conducted CARD-FISH with the diplonemid-specific probe ‘Diplo1590R’ [31]
167 targeting all freshwater and marine sequences, to enumerate their abundance in freshwater
168 lakes. However, the number of CARD-FISH stained cells was too low to confidently
169 assess their abundance and dynamics in all the studied lakes. Their abundance was low
170 even during spring, when 18S rRNA read numbers were relatively high. In total, we
171 detected 22 CARD-FISH stained cells from all lakes, again pointing at their rarity in
172 freshwater habitats. However, images of these cells provide some insights about cell size,
173 morphology and potential trophic strategies. All detected diplonemids were ovoid cells in
174 the range of 5-10 μm in length (Figure 3, Supplementary Figure S6). Thus, the freshwater
175 diplonemids observed in the present study are smaller when compared to the cultured
176 marine diplonemids (which are around 20 μm in size), and also the ones observed from
177 marine waters by single cell sorting, which are much diverse in size (7-20 μm) and shape
178 [20].
179 There is little information on the feeding behaviour of diplonemids apart from the
180 consensus that they are heterotrophic. Some studies have also reported diplonemids as
181 mainly benthic dwellers feeding by phagotrophy on bacteria, osmotrophy or by detrivory
182 [33,34]. Protists of size ranges observed here are also known to be efficient grazers of
183 bacteria in aquatic ecosystems [35–39]. All CARD-FISH positive cells observed here also
184 appeared to contain several bacteria (Figure 3). However, it is impossible to conclude
185 from these images whether or not these microbes are inside the cells (suggesting
186 phagotrophic behavior or symbiosis) or outside (epibionts, commensals, etc.) (Figure 3).
187 Although there is no direct evidence of their feeding behavior from environmental
188 samples, diplonemid sequences were detected from the larger planktonic fractions (180-
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189 2000 µm) in TARA Ocean data and suggested to possess a parasitic or symbiotic lifestyle
190 [40]. Moreover, a recent study reported the presence of endosymbiotic bacteria inside the
191 cells of some cultured diplonemids [41], which might mimic bacterivory. Direct evidence
192 using FLB (fluorescently labelled bacteria) uptake experiments [42,43] are necessary to
193 confirm their phagotrophy on bacteria.
194
195 Copy number of 18S rRNA genes in diplonemids
196 Diplonemids have divergent rRNA genes and due to the large number of mismatches, they
197 are generally not targeted in diversity studies using universal eukaryotic primers
198 (especially V4 primers) [7,8]). This explains why they remained undetected in a majority
199 of eukaryotic diversity studies in the world oceans until the TARA expedition. TARA
200 strategically used primers targeting the V9 region [3], which is a short region (~150 bp)
201 considered best to capture a large diversity [10]. Although sequence abundance of
202 diplonemids was high in the global oceanic waters, direct quantitative evidence of their
203 actual abundances is scarce. Only a single study from the Atlantic Ocean quantified the
204 abundance of diplonemids from various depths using CARD-FISH and found their
205 abundance to be <1% of total planktonic eukaryotes [31], similar to our observations from
206 freshwaters. In the microscopic analysis we could barely observe CARD-FISH positive
207 cells even in those samples, which had relatively higher sequence numbers assigned to
208 diplonemids. These observations suggest that diplonemids (especially marine ones)
209 possess high copy numbers of divergent 18S rRNA genes, that could contribute to their
210 amplified sequence abundance [44]. In order to test this, we analyzed ten previously
211 available single cell genomes from marine diplonemids [20]. These genomes are very
212 incomplete (most complete being 9%), so the number of actual 18S rRNAs detected in
213 these is likely to be severely underestimated. Even so, we detected multiple 18S rRNA
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214 gene sequences in these incomplete genomes (up to 21 sequences >300bp in cell_13,
215 Supplementary Table S4). Moreover, the sequences from the same cell did not cluster
216 with each other in a phylogenetic tree in several cases (e.g. cell-13, cell-27 and cell-4sb),
217 suggesting a high intra-genomic diversity of this frequently used gene for assessing protist
218 diversity [44] (Supplementary Figure S2). As the raw sequence data for the single cell
219 genomes are not available, we used the coverage of the contigs to obtain an estimate of
220 18S rRNA copy number (Supplementary Table S4). A conservative estimate, using
221 median coverage of an 18S rRNA containing contig vs coverage of the remaining contigs
222 (>3kb) suggests multiple copies of 18S rRNA genes in one single cell (up to 141 copies,
223 Supplementary Table S1 and Supplementary Table S4). Only a single, related isolate
224 genome is available (Diplonema papillatum sp.) with an estimated genome completeness
225 of ca. 30% (Supplementary Figure S1). We could detect 6 copies of 18S rRNAs, which
226 appears to be somewhat low [22]. However, we could not estimate the putative number of
227 rRNAs, as coverage information for contigs was not available. Given such wide variations
228 in the number of 18S rRNA copies and sequence divergence in the presently available
229 incomplete genomic data, accurate quantifications of protist diversity or abundance by
230 sequencing data alone are expected to remain a pressing issue until improved genomes are
231 available.
232
233 Conclusions
234 We describe a clade of freshwater diplonemids from multiple lakes around the world.
235 Freshwater diplonemids are related to cultured marine diplonemids and distinct from the
236 as yet uncultured marine diplonemids. Direct observations using CARD-FISH also
237 suggest that freshwater diplonemids are consistently smaller in size in comparison to those
238 from the marine habitat. Although diplonemid 18S rRNA gene sequences were found to
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239 be highly abundant in marine waters [3], a prior quantification of marine diplonemids
240 using CARD-FISH suggests that they are rare components of the marine habitat [31]. It
241 has also been recently suggested that estimates from amplicon sequencing are far from
242 quantitative [44,45]. Taken together with the highly divergent and multiple 18S sequences
243 derived from single diplonemid cells, it appears the actual abundances of marine
244 diplonemids using sequence data might have been overestimated. Direct counts by
245 CARD-FISH using an identical probe that targets both freshwater and marine clades
246 suggest that diplonemid abundances in both marine and freshwater ecosystems are very
247 low. However, amplicon-sequencing results from both habitats are drastically different,
248 while a large diversity of diplonemid sequences have been obtained from the marine
249 habitat [3], very limited diversity was observed in freshwaters. This suggests that
250 freshwater diplonemids are less diverse than their marine counterparts and their close
251 relationship indicates a recent habitat transition from marine to freshwaters. Nevertheless,
252 it is important to note that this contradictory result of diversity and abundance might not
253 be restricted to diplonemids alone and may also extend to other protistan groups as well
254 [7,43,45]. Further studies using single-cells, cultures, genome sequencing and
255 experimental approaches will be necessary for an improved understanding of the
256 ecological role and evolutionary history of these poorly understood groups of protists.
257
258 Materials and Methods
259 Sampling: Samples were collected from seven freshwater lakes, in Japan (Biwa- 35°12′
260 N 135°59′ E and Ikeda- 31°14′ N 130°33′ E), Czech Republic (Řimov- 48°51′ N 14°29′ E,
261 and Medard- 50°10′ N 12°35′ E), and Switzerland (Zurich- 47°18’N 8°34’E, Lugano-
262 45°59'N 8°58'E, and Constance- 47°32'N 9°31'E) (Table 1). All these lakes are >100m
263 deep with the exception of the Řimov reservoir and Medard, which are 43 and 50m deep,
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264 respectively. Samples from Lake Biwa, Japan were collected from three depths
265 representing epilimnion, metalimnion and hypolimnion from station Ie-1, a long-term
266 limnological survey station of Kyoto University, Japan. These samples were collected
267 monthly for one year in 2017 (from depths 5, 20 and 60 m) and also from a high
268 frequency weekly sampling, which was conducted for three months during the spring of
269 2016 (from depths 5, 10 and 50 m) (Table 1). Samples from the Řimov Reservoir, Czech
270 Republic were collected seasonally for two years (2016 and 2017) from two depths (0.5
271 and 30 m), representing epilimnion and hypolimnion (Table 1) from a station located at
272 the dam (deepest part of the reservoir). For other lakes, samples were collected once or
273 twice during the thermal stratification period from depths representing the epilimnion and
274 hypolimnion from stations located at the deepest part of the lakes. Samples were collected
275 with a 5-liter Niskin sampler (General Oceanics, Miami, USA) or with a Freidinger
276 sampler. Temperature, concentration of chlorophyll a and dissolved oxygen was
277 determined with a multiparameter probe (911 plus, Sea Bird Electronics, Inc., USA; or
278 YSI 6600 multiparameter sonde V2, Yellow Springs Instruments, USA) (Figure 1).
279
280 DNA extraction, amplicon sequencing and data processing: Amplicon sequencing was
281 conducted only from samples collected from Biwa and Ikeda lakes. Water from the
282 different sampling depths was pre-filtered just after collection using a 20-μm mesh
283 plankton net to exclude larger organisms. One to two liters were filtered onto 0.8 μm
284 polycarbonate filters (47 mm diameter, Costar) at low vacuum (7.5 cm Hg) and
285 immediately frozen at -30°C until analysis. DNA was extracted with the Power Soil DNA
286 isolation and purification kit (MoBio laboratories, Carlsbad, CA, USA) and quantified
287 using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc.,
288 Wilmington, DE, USA). Polymerase chain reactions (PCR) were conducted using
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289 universal eukaryotic primers Euk1391f and EukBr to amplify the V9 region of 18S rRNA
290 genes of all eukaryotes [9,10]. PCRs were performed in 25 µl of reaction volume
291 according to the 18S Illumina amplicon protocol of the Earth Microbiome Project
292 (http://www.earthmicrobiome.org/protocols-and-standards/18s/). Amplicons were
293 examined using agarose gel electrophoresis and DNA was purified using magnetic beads.
294 Samples were quantified using Qubit. The samples were pooled to have uniform DNA
295 concentration in each and were sequenced using the Illumina MiSeq platform. The
296 processing and quality control of the sequencing data were conducted using DADA2 [11].
297 Chimera check was conducted with DADA2 and also with the reference-based chimera
298 searches against the PR2 database [12]. Amplicons with quality score more than Q30 were
299 retained and amplicons were trimmed to have 300 bp using the ‘Filter and Trim’ function
300 of DADA2. A total of 656,427 reads were obtained after processing. Classification of
301 unique sequences was conducted against the SILVA 132 reference database [13]. The
302 unclassified sequences were additionally examined for their closest relatives with BLAST
303 searches against the NCBI NT database [14].
304
305 Filtration and DNA extraction for shotgun metagenomic analysis: Shotgun
306 metagenomic sequencing was conducted for all lakes except for the Japanese ones (Biwa
307 and Ikeda). Water samples (ca. 10 L each) from lakes Řimov (Czech Republic, depths 0.5
308 m and 30 m), Medard (Czech Republic, depths 5 m and 20 m), Zurich (Switzerland,
309 depths 5 m and 80 m), Constance (Switzerland/Germany, depths 5 m and 200 m) and
310 Lugano (Switzerland/Italy, depths 5 m and 50 m) were progressively filtered through 20
311 μm, 5 μm, and 0.22 μm polycarbonate or polysulfone membrane filters (Sterlitech, USA).
312 DNA extraction was performed from the 0.22 μm filters (containing the 5–0.22 μm
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313 microbial size fraction) using the ZR Soil Microbe DNA MiniPrep kit (Zymo Research,
314 Irvine, CA, USA), following the manufacturer’s instructions.
315
316 Preprocessing of metagenomic datasets: Shotgun metagenome sequencing was
317 performed with Novaseq 6000 (2 × 151 bp) (Novogene, HongKong, China). Low quality
318 bases/reads and adaptor sequences were removed from the raw Illumina reads using the
319 bbmap package [15]. Briefly, the reads were quality trimmed by bbduk.sh (using a Phred
320 quality score of 18). Subsequently, bbduk.sh was used for adapter trimming and
321 identification/removal of possible PhiX and p-Fosil2 contamination. Additional checks
322 (i.e., de novo adapter identification with bbmerge.sh) were performed in order to ensure
323 that the datasets meet the quality threshold necessary for assembly. Each metagenomic
324 dataset (i.e. each sample) was assembled independently with MEGAHIT (v1.1.5) [16]
325 using the k-mer sizes: 49, 69, 89, 109, 129, 149, and default settings. Previously available
326 metagenomes and assemblies from Řimov reservoir [17] and Lake Baikal were included
327 in the analysis [18,19].
328
329 Genomic data processing: Assembled sequence data for single cell derived genomes
330 from 10 published marine diplonemids were downloaded [20]. Genome completeness
331 estimates performed by us (and similar to those reported earlier) for these were found to
332 be very low with the best assembly missing 91% of conserved single copy orthologs (as
333 reported by BUSCO [21]). The assembled Diplonema papillatum genome [22] was
334 downloaded from NCBI (accession LMZG00000000). Only the assembled genome is
335 available and raw data was not available although it was mentioned to be 21.5Gb. The
336 genome assembly size is ca. 107Mb (shortest contig 1000bp, longest contig 100kb). The
337 authors estimated the genome size to be 175Mb, but no details were provided on how this
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338 estimate was obtained [22]. Genome completeness estimates for all available genomes
339 (Diplonema papillatum genome and the 10 marine diplonemid SAGs) were obtained using
340 BUSCO [21] with the euglenozoa lineage (euglenozoa_odb10 version 2019-11-21) (busco
341 -m genome -i genome.fna -o output --lineage_dataset euglenozoa_odb10) (Supplementary
342 Figure S1). Eukaryotic small ribosomal subunit sequences were detected in these genomic
343 assemblies using ssu-align [23]. Only those sequences that were >300bp were retained for
344 further analysis. The results are summarized in Supplementary Table S1.
345
346 Retrieval of 18S rRNA sequences from assembled shotgun metagenomic datasets: All
347 diplonemid sequences were extracted from the SILVA database version 138 [13]. These
348 sequences along with the freshwater diplonemid sequence (OTU_4) described previously
349 [6], were used as queries for BLASTN against the metagenomic datasets. Hits with e-
350 value <1e-5, alignment length >200 bp and percentage identity >90% to the queries were
351 extracted. These candidate sequences were aligned to the global SILVA alignment for
352 rRNA genes using the SINA web aligner [24] and subsequently inserted into the guide
353 tree of SILVA SSU database 138 RefNR 99 in ARB [25] using maximum parsimony and
354 only those sequences that affiliated to diplonemids and of length >300 bp were retained.
355
356 Quantification of 18S rRNA sequences of freshwater diplonemids in a shotgun
357 metagenomic time-series from Řimov reservoir: All assembled freshwater diplonemid
358 sequences (from previous section) along with OTU_4 from Lake Biwa [6] were added to
359 the existing SILVA 138 database of 18S sequences. At least half the reads from each
360 metagenomic dataset (minimum 94 million reads, maximum 277 million reads) were
361 scanned by ssu-align to extract potential eukaryotic 18S reads (minimum ca. 65,000,
362 maximum 180,000 found) and these were used as queries against the SILVA138 database
14 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.14.095992; this version posted May 15, 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.
363 supplemented with freshwater diplonemid sequences using mmseqs2 [26]. Metagenomic
364 query sequences matching the freshwater diplonemid sequences with >95% identity,
365 evalue <1e-5 and alignment length >50bp were considered to originate from freshwater
366 diplonemids (Supplementary Table S2).
367
368 18S rRNA phylogenetic tree: Assembled 18S rRNA sequences identified to be from
369 freshwater diplonemids were aligned to all known clades of Euglenids using PASTA [27]
370 and a maximum likelihood phylogenetic tree (Figure 2) was made using IQ-TREE 2 using
371 automatic model selection (TIM3e+R10 model), ultrafast bootstraps and SH-aLRT tests (-
372 bb 1000 -alrt 1000) [28–30]. Additionally, all 18S sequences (>300 bp) from the single
373 cell diplonemid genomes and the Diplonema papillatum genome were aligned to the same
374 set of sequences and a phylogenetic tree (Supplementary Figure S2) was created as
375 described above.
376
377 Catalyzed Reporter Deposition-Fluorescent in situ Hybridization (CARD-FISH):
378 Samples for CARD-FISH were collected from the same depths as the samples used for
379 DNA extractions (Table 1). Water samples were fixed in a 2% final concentration of
380 formaldehyde (freshly prepared by filtering through 0.2 µm syringe filter) for at least 3-4
381 hours before filtration. 50 ml of epilimnion/metalimnion and 100 ml of hypolimnion
382 samples were filtered through polycarbonate filters (pore size 0.8 µm, Advantec), rinsed
383 twice with 1X PBS and twice with MilliQ water, air dried and frozen at -20°C until further
384 processing. CARD-FISH was performed according to the method described previously
385 [7]. The filters were hybridized at 35°C for 12 h with a 0.5 µg ml-1 probe concentration
386 and a 30% concentration of formamide. The probe targeting diplonemids ‘DiploR1792’
387 [31] was purchased from Biomer, Germany. Counting was performed using an Olympus
15 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.14.095992; this version posted May 15, 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.
388 BX 53 epifluorescence microscope under 1000 × magnification at blue/UV excitation.
389 Total microbial eukaryotes were counted simultaneously by DAPI staining under UV
390 excitation. Images were taken and processed using a semiautomatic image analysis system
391 (NIS-Elements 3.0, Laboratory Imaging, Prague, Czech Republic).
392
393 Data Accessibility
394 The sequence data of Japanese lakes generated from amplicon sequencing were submitted
395 to European Nucleotide Archive (ENA) and are available under the BioProject:
396 PRJEB37597, BioSamples ERS4413153–226. Sequence data for all metagenomes
397 generated in this work are archived at EBI European Nucleotide Archive and can be
398 accessed under the BioProject PRJEB35770, BioSamples ERS4146214-50 (Řimov
399 reservoir), BioProject PRJEB35640, BioSamples ERS4065084-85 (Lake Medard),
400 ERS4065070-71 (Lake Lugano), BioProject PRJEB35770, BioSamples ERS4409418–21
401 (Lake Constance), BioSamples ERS4409422–23 (Lake Zurich). All 18S rRNA sequences,
402 alignments and trees are available at figshare
403 (https://doi.org/10.6084/m9.figshare.12091749.v1). This study did not generate any code.
404
405 Author’s Contributions
406 IM and RG conceived the study. IM performed sampling, amplicon analyses and CARD-
407 FISH. RG performed metagenomic and phylogenetic analyses. MMS, A-ŞA and VSK
408 performed sampling and phylogenetic analyses. A-ŞA, TS, VG, MH, PL, YH, SN, KS
409 performed sampling and DNA extractions. IM and RG drafted the manuscript with the
410 input of all other authors.
411
412
16 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.14.095992; this version posted May 15, 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.
413 Competing interests
414 The authors declare that they have no competing interests.
415
416 Funding
417 This study was supported by the JSPS Bilateral Japanese-Czech Joint Research Project
418 No. JSPS-17-17, funding the collaboration of KS, SN, IM, VG and YH. IM was supported
419 by the research grants CZ.02.1.01/0.0/ 0.0/16_025/0007417 (ERDF/ESF) and 20-12496X
420 (Grant Agency of the Czech Republic). RG was supported by the research grants 17-
421 04828S, 20-12496X (Grant Agency of the Czech Republic) and CZ.02.1.01/0.0/
422 0.0/16_025/0007417 (ERDF/ESF). MMS was supported by the research grants 20-
423 12496X (Grant Agency of the Czech Republic) and 310030_185108 (Swiss National
424 Science Foundation). A-ŞA was supported by the research grants 17-04828S (Grant
425 Agency of the Czech Republic) and MSM200961801 (Academy of Sciences of the Czech
426 Republic). VSK was supported by the research grants 17-04828S, 20-12496X (Grant
427 Agency of the Czech Republic) and 116/2019/P (Grant Agency of the University of South
428 Bohemia in České Budějovice 2019-2021). MH was supported by the research grant
429 310030_185108 (Swiss National Science Foundation). PL was supported by the research
430 grant 19-23469S (Grant Agency of the Czech Republic) and 022/2019/P (Grant Agency of
431 the University of South Bohemia in České Budějovice 2019-2021).
432
433 Acknowledgements
434 We are thankful to the captains of the research vessel ‘Hasu’, Y. Goda and T. Akatsuka
435 for their help in sample collection in Lake Biwa. We are also thankful to Shohei Fujinaga
436 for his help in sample collection and data analysis. P. Znachor, P. Rychtecky and J.
437 Nedoma are acknowledged for help in sampling of Řimov Reservoir and Lake Medard. T.
17 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.14.095992; this version posted May 15, 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.
438 Posch, E. Loher and V. Lanta are thanked for help in the sample collection of Lake
439 Zurich. S. Dirren and the crew of the research vessel ‘Kormoran’ are thanked for help in
440 sampling of Lake Constance, and F. Leporelli and V. Lanta for help in sampling of Lake
441 Lugano.
442
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608 Table 1. Details of sampling sites Lake, Trophic Surface Z max Sampling depths Sampling date Analyses country status area (km2) (m) (m) performed
Biwa, JP Mesotrophic 674.0 104 5, 10, 50 (2016) Mar-May 2016 (weekly) AS, CF 5, 20, 60 (2017) 2017 (monthly) AS, CF
Ikeda, JP Mesotrophic 11.0 200 5, 50, 100, 200 25.11.2017 AS, CF
Řimov, CZ Eutrophic 2.1 43 0.5, 30 Apr, Aug, Nov 2016, MG, CF 2017 (monthly) MG, CF
Medard, CZ Oligotrophic 4.9 50 5, 20 09.07.2019 MG
Constance, Mesotrophic 536 252 5, 200 03.07.2018 MG, CF CH/DE/AT
Zurich, CH Oligo- 88.7 136 5, 80 11.07.2018 MG, CF mesotrophic 13.11.2019 CF
Lugano, Eutrophic 48.7 288 5, 50 02.04.2019 MG CH/IT 05.11.2019 CF
609 Abbreviations: JP, Japan; CZ, Czech Republic; CH, Switzerland; DE, Germany; AT, Austria; IT, 610 Italy; AS, 18S amplicon sequencing, MG, shotgun metagenomics; CF, CARD-FISH
611
612
613
25 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.14.095992; this version posted May 15, 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.
A B C Proportion of diplonemid 18S rRNA reads (%) 0 0.2 0.4 0.6 0.8 1.0 1.2 0.5 30 6 0.5 30 6 0 5m 5m Chl. (μg/L) 25 5 25 5 0.4 Chl. (μg/L) 0.4 50 Temp. (°C) Temp. (°C) 20 4 20 4 0.3 0.3 15 3 15 3 100 0.2 0.2 Temp. (°C) Chl. (μg/L) Depth (m) 10 2 10 2 150 Lake Ikeda 0.1 5 1 0.1 5 1 Profile 2017 200 0 0 0 0 0 0 J F M A M J J A S O N D 0 5 10 15 20 25 30 March April May
Proportion of diplonemid 18S rRNA reads (%) Proportion of diplonemid 18S rRNA 0 1 2 3 4 D 0.5 30 6 0.5 30 6 0.6 25 50 10m 20m 0.5m 0.4 25 5 0.4 25 5 0.5 20 40 Chl. (μg/L) 20 4 20 4 0.4 0.3 0.3 15 30 Temp. (°C) 15 3 Temp. (°C) 15 3 0.3 Chl. (μg/L) 0.2 Temp. (°C) 0.2 10 20 10 2 10 2 0.2 Chl. (μg/L)
0.1 5 1 0.1 5 1 0.1 5 10
0 0 0 0 0 0 0 0 0 March May April Apr Aug Nov Apr May Jun Jul Aug Proportion of diplonemid 18S rRNA reads (%) Proportion of diplonemid 18S rRNA 2016 2017
0.5 30 6 0.5 30 6 0.6 25 50 50m 60m 30m 0.4 25 5 0.4 25 5 0.5 20 40 20 4 20 4 0.4 0.3 0.3 15 30 15 3 15 3 0.3 0.2 0.2 10 20 Temp. (°C) Temp. (°C) 10 2 10 2 0.2 Temp. (°C) 0.1 Chl. (μg/L) 5 1 0.1 5 1 0.1 5 10 Chl. (μg/L) Chl. (μg/L) 0 0 0 0 0 0 0 0 0 March May J F M A M J J A S O N D April Apr Aug Nov Apr May Jun Jul Aug Proportion of diplonemid 18S rRNA reads (%) Proportion of diplonemid 18S rRNA 2016 2017 Lake Biwa - Spring 2016 Lake Biwa - Timeline 2017 Rimov reservoir 2016-2017
Figure 1. Dynamics of freshwater diplonemids (18S rRNA gene sequence abundance) by A) amplicon sequencing in a weekly spring/summer sampling campaign from Lake Biwa, Japan (depths 5, 10 and 50m), B) amplicon sequencing in a one year sampling campaign from Lake Biwa (depths 5, 20 and 60m) C) shotgun metagenomic time series of the Rimov reservoir, Czech Republic (depths 0.5 and 30m) and D) in a single depth profile from Lake Ikeda (depths 0, 50, 100, 150 and 200m). bioRxiv preprint doi: https://doi.org/10.1101/2020.05.14.095992; this version posted May 15, 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.
FN598299 Uncultured marine diplonemid clone BIO9 D4 (1345) EU635597 Uncultured marine diplonemid clone Ti11 D1 20 (1199) EU635596 Uncultured marine diplonemid clone Bi2 D1 21 (1198) FJ000259 Uncultured eukaryote clone SW1F01 (1616) FJ000076 Uncultured eukaryote clone BB2F06 (1610) FN598233 Uncultured marine diplonemid clone BIO1 H12 (1319) FN598252 Uncultured marine diplonemid clone BIO3 A2 (1343) FN598397 Uncultured marine diplonemid clone BS23 B2 (1325) KX189135 Uncultured marine diplonemid clone Tara 960 6 (1585) EU635593 Uncultured marine diplonemid clone Ma126 D1 21 (1198) FN598248 Uncultured marine diplonemid clone BIO2 F3 (1348) FN598420 Uncultured marine diplonemid clone BS25 H3 (1336) FN598413 Uncultured marine diplonemid clone BS25 B2 (1341) KX532973 Uncultured marine eukaryote clone o10 B 77 (1466) KX533083 Uncultured marine eukaryote clone G3 93 (1466) FN598250 Uncultured marine diplonemid clone BIO2 H4 (1348) KJ757576 Uncultured eukaryote clone SGYY747 (2061) KX189168 Uncultured marine diplonemid clone Tara 1645 11 (1606) KX532691 Uncultured marine eukaryote clone o10 800 118 (1335) FN598441 Uncultured marine diplonemid clone BS3 G10 (1348) FN598287 Uncultured marine diplonemid clone BIO7 E10 (1349) FJ000090 Uncultured eukaryote clone 571C11 (1614) Marine EU635618 Uncultured marine diplonemid clone DH117 D1 19 (1200) FN598436 Uncultured marine diplonemid clone BS3 D8 (1349) Diplonemids FJ000260 uncultured eukaryote (1351) FJ000261 Uncultured eukaryote clone 452D08 (1610) DQ504319 Uncultured diplonemid clone LC22 5EP 10 (1098) FJ000071 Uncultured eukaryote clone BB2A02 (1612) EU635651 Uncultured marine diplonemid clone Ma121 D1 21 (1198) KY947153 Diplonemida sp 27 RG-2016 (2068) EU635652 Uncultured marine diplonemid clone Ma131 D1 06 (1198) EU635654 Uncultured marine diplonemid clone DH136 D1 57 (1198) EU635630 Uncultured marine diplonemid clone R26 D1 33 (1199) FJ000255 Uncultured eukaryote clone SW1D09 (1565) EU635606 Uncultured marine diplonemid clone BS16 D1 14 (1195) FN598347 Uncultured marine diplonemid clone BS13 H8 (1331) KX533087 Uncultured marine eukaryote clone G3 97 (1239) EU635672 Uncultured marine diplonemid clone Ma126 D1 39 (1198) 100/100 KY947154 Diplonemida sp 37 RG-2016 (2063) EU635605 Uncultured marine diplonemid clone DH117 D1 41 (1199) AY665088 Uncultured eukaryote clone SCM38C39 (2005) GU820768 Uncultured euglenozoan clone BCA3F14RJ1A09 (1610) 72/94 KJ757308 Uncultured eukaryote clone SGYY1477 (2055) KX189132 Uncultured marine diplonemid clone Tara 960 3 (1907) KJ762701 Hemistasia uncultured eukaryote (2064) 97/100 AB948221 Hemistasia phaeocysticola (2020) MF422202 Hemistasiidae sp (1995) Hemistasia MF422203 Hemistasiidae sp (2064) LH-02apr19-C1499987 Lugano hypolimnion (542) ZE-Oct18-C228045 Zurich hypolimnion (1223) RH-26jul17-C234517 Rimov hypolimnion (1093) MH-09jul19-C1664111 Medard hypolimnion (307) RH-14apr17-C3362255 Rimov hypolimnion (331) RH-27jun17-C1571205 Rimov hypolimnion (491) MH-09jul19-C523660 Medard hypolimnion (762) RH-27jun17-C1179729 Rimov hypolimnion (590) CE-Jul18-C700394 Constance hypolimnion (770) RH-18aug17-C423291 Rimov hypolimnion (994) Freshwater RH-26jul17-C462493 Rimov hypolimnion (487) RH-23may17-C110669 Rimov hypolimnion (2029) Diplonemids RH-27jun17-C1910473 Rimov hypolimnion (417) MH-09jul19-C873310 Medard hypolimnion (518) 100/100 RH-20APR16-C630432 Rimov hypolimnion (642) RH-18aug17-C497411 Rimov hypolimnion (939) CE-Jul18-C713090 Constance epilimnion (440) RH-15aug16-C835002 Rimov hypolimnion (437) CE-Oct18-C2726209 Constance epilimnion (387) 100/100 CH-Jul18-C1818609 Constance hypolimnion (380) OTU 4 Lake Biwa amplicon (293) MF422200 Diplonemida sp (2007) DQ504350 uncultured diplonemid Rhynchopus (1917) AY490209 Rhynchopus sp SH-2004-I (1995) MF422197 Rhynchopus sp (1990) AY425013 Rhynchopus sp ATCC 50226 (2032) AF380997 Rhynchopus sp ATCC 50226 (2012) GAOU01013166 Rhynchopus (1352) AY490210 Rhynchopus sp SH-2004-II (2008) AY180037 uncultured euglenid (1743) Marine AY425012 Diplonema sp ATCC 50232 (2148) AF119811 Diplonema papillatum (1981) Isolates FN598401 uncultured marine diplonemid (1330) AY425009 Diplonema ambulator (2062) AY425011 Diplonema sp ATCC 50225 (2067) MF422192 Diplonema sp (2021) MF422190 Diplonema sp (2023) 88/96 AY425010 Diplonema sp ATCC 50224 (2046) Mf422201 Diplonemida gen 2 sp 1 AH-2017 (2010)
100/100 Kinetoplastids
0.4
Figure 2. Maximum-likelihood tree of 18S rRNA of diplonemids. Marine diplonemids, Hemistasia, freshwater diplonemids, sequences from isolates and kinteoplastids are shown. Other euglenids (e.g. EUglena, Phacus) were used as outgroup (not shown and indicated by an arrow). Ultra-fast bootstrap values and SH branch supports are indicated for selected nodes. Scale bar indicates the number of substitutions per site. bioRxiv preprint doi: https://doi.org/10.1101/2020.05.14.095992; this version posted May 15, 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.
Lake Biwa Řimov Reservoir Lake Zurich A A A
10 µm 10 µm 10 µm
B B B 10 µm
Figure 3. Freshwater diplonemids under fluorescent microscope. A - DAPI, B - FISH, bacteria inside the cells are shown by arrows. bioRxiv preprint doi: https://doi.org/10.1101/2020.05.14.095992; this version posted May 15, 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.
cell_13 C:1 [S:1, D:0], F:0, M:129, n:130
cell_1sb C:0 [S:0, D:0], F:0, M:130, n:130
cell_21 C:1 [S:1, D:0], F:4, M:125, n:130
cell_21sb C:0 [S:0, D:0], F:0, M:130, n:130
cell_27 C:0 [S:0, D:0], F:0, M:130, n:130
cell_3 C:1 [S:1, D:0], F:0, M:129, n:130
cell_37 C:0 [S:0, D:0], F:0, M:130, n:130
cell_47 C:1 [S:0, D:1], F:0, M:129, n:130
cell_4sb C:0 [S:0, D:0], F:2, M:128, n:130
dpap C:30 [S:23, D:7], F:12, M:88, n:130
0 20 40 60 80 100
%BUSCOs
Complete (C) and single-copy (S) Complete (C) and duplicated (D)
Fragmented (F) Missing (M)
Supplementary Figure S1. Genome completeness estimation for all available diplonemid genomes using BUSCO (Seppey et al. 2019). Single cell genomes are prefixed with "cell", Diplonema papillatum isolate genome: dpap. bioRxiv preprint doi: https://doi.org/10.1101/2020.05.14.095992; this version posted May 15, 2020. The copyright holder for this preprint cell-27-c31026 (333) (which was not certifiedcell-27-c26100 by peer review)(594) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is cell-27-c602 (382) cell-27-c39174 (411)made available under aCC-BY-NC-ND 4.0 International license. cell-27-c41645 (501) KY947153 Diplonemida sp 27 RG-2016 (2068) FJ000071 Uncultured eukaryote clone BB2A02 (1612) EU635651 Uncultured marine diplonemid clone Ma121 D1 21 (1198) cell-27-c28331 (428) cell-27-c35771 (341) EU635652 Uncultured marine diplonemid clone Ma131 D1 06 (1198) EU635654 Uncultured marine diplonemid clone DH136 D1 57 (1198) cell-9sb-c18102 (386) EU635630 Uncultured marine diplonemid clone R26 D1 33 (1199) cell-37-c56087 (847) cell-4sb-c77108 (810) cell-13-c30360 (745) cell-27-c9571 (320) FJ000255 Uncultured eukaryote clone SW1D09 (1565) EU635606 Uncultured marine diplonemid clone BS16 D1 14 (1195) FN598413 Uncultured marine diplonemid clone BS25 B2 (1341) FN598347 Uncultured marine diplonemid clone BS13 H8 (1331) cell-13-c25882 (377) cell-13-c30273 (673) cell-13-c31185 (492) cell-13-c1523 (1355) cell-13-c16512 (781) cell-13-c7866 (525) cell-13-c11377 (399) cell-13-c9716 (1044) cell-13-c11965 (344) cell-13-c31257 (338) cell-13-c10157 (1421) cell-13-c25288 (302) cell-13-c15957 (717) cell-13-c20800 (379) cell-13-c18566 (379) cell-13-c24568 (732) cell-13-c28805 (972) cell-13-c15517) (300 FJ000259 Uncultured eukaryote clone SW1F01 (1616) FJ000076 Uncultured eukaryote clone BB2F06 (1610) FN598299 Uncultured marine diplonemid clone BIO9 D4 (1345) EU635597 Uncultured marine diplonemid clone Ti11 D1 20 (1199) EU635596 Uncultured marine diplonemid clone Bi2 D1 21 (1198) FN598233 Uncultured marine diplonemid clone BIO1 H12 (1319) FN598252 Uncultured marine diplonemid clone BIO3 A2 (1343) FN598397 Uncultured marine diplonemid clone BS23 B2 (1325) cell-4sb-c42147 (417) EU635593 Uncultured marine diplonemid clone Ma126 D1 21 (1198) KX189135 Uncultured marine diplonemid clone Tara 960 6 (1585) Marine cell-27-c10228 (360) FN598248 Uncultured marine diplonemid BIO2 F3 (1348 Diplonemids cell-47-c14652 (682) cell-47-c7122 (682) cell-47-c11972 (682) cell-47-c12257 (682) cell-47-c22866 (682) cell-47-c14510 (682) cell-4sb-c52750 (593) FN598420 Uncultured marine diplonemid clone BS25 H3 (1336) KX532973 Uncultured marine eukaryote clone o10 B 77 (1466) KX533083 Uncultured marine eukaryote clone G3 93 (1466) FN598250 Uncultured marine diplonemid clone BIO2 H4 (1348) KJ757576 Uncultured eukaryote clone SGYY747 (2061) KX189168 Uncultured marine diplonemid clone Tara 1645 11 (1606) KX532691 Uncultured marine eukaryote clone o10 800 118 (1335) cell-21-c159800 (1279) cell-21-c173570 (395) cell-21-c133830 (568) cell-3-c4076 (2066) FN598287 Uncultured marine diplonemid clone BIO7 E10 (1349) FN598441 Uncultured marine diplonemid clone BS3 G10 (1348) cell-21sb-c5277 (1548) FJ000090 Uncultured eukaryote clone 571C11 (1614) EU635618 Uncultured marine diplonemid clone DH117 D1 19 (1200) 0.8 FN598436 Uncultured marine diplonemid clone BS3 D8 (1349) cell-1sb-c28866 (936) cell-1sb-c37040 (682) KX533087 Uncultured marine eukaryote clone G3 97 (1239) FJ000260.1.1351 Uncultured eukaryote (1351) FJ000261 Uncultured eukaryote clone 452D08 (1610) cell-1sb-c19113 (1194) DQ504319 Uncultured diplonemid clone LC22 5EP 10 (1098) cell-4sb-c100636 (314) cell-4sb-c10234 (707) cell-47-c13440 (1206) cell-37-c68542 (494) cell-37-c110782 (365) cell-4sb-c20910 (302) cell-37-c16423 (445) cell-37-c12122 (902) cell-37-c68032 (597) cell-37-c50818 (786) cell-37-c88864 (561) KY947154 Diplonemida sp 37 RG-2016 (2063) EU635672 Uncultured marine diplonemid clone Ma126 D1 39 (1198) EU635605 Uncultured marine diplonemid clone DH117 D1 41 (1199) AY665088 Uncultured eukaryote clone SCM38C39 (2005) GU820768 Uncultured euglenozoan clone BCA3F14RJ1A09 (1610) KJ757308 Uncultured eukaryote clone SGYY1477 (2055) KX189132 Uncultured marine diplonemid clone Tara 960 3 (1907) 89.8/98 cell-13-c31734 (306) cell-13-c25200 (306) KJ762701 Hemistasia Uncultured eukaryote (2064) Ab948221 Hemistasia phaeocysticola (2020) Mf422202 Hemistasiidae sp (1995) Hemistasia Mf422203 Hemistasiidae sp (2064) LH-02apr19-C1499987 (542) ZE-Oct18-C228045 (1223) RH-26jul17-C234517 (1093) RH-27jun17-C1571205 (491) MH-09jul19-C1664111 (307) RH-23may17-C110669 (2029) CE-Jul18-C713090 (440) RH-27jun17-C1910473 (417) MH-09jul19-C873310 (518) RH-20APR16-C630432 (642) Freshwater RH-15aug16-C835002 (437) CE-Oct18-C2726209 (387) Diplonemids RH-18aug17-C497411 (939) MH-09jul19-C523660 (762) RH-14apr17-C3362255 (331) CH-Jul18-C1818609 (380) RH-26jul17-C462493 (487) 100/100 RH-18aug17-C423291 (994) CE-Jul18-C700394 (770) 100/99 RH-27jun17-C1179729 (590) OTU 4 (293) MF422200 Diplonemida sp (2007) DQ504350 Uncultured diplonemid (1917) AY490209 Rhynchopus sp SH-2004-I (1995) Mf422197 Rhynchopus sp (1990) AF380997 Rhynchopus sp ATCC 50226 (2012) AY425013 Rhynchopus sp ATCC 50226 (2032) GAOU01013166 Rhynchopus sp(1352) AY490210 Rhynchopus sp SH-2004-II (2008) LMZG01024242 Diplonema papillatum (414) LMZG01012232 Diplonema papillatum (358) LMZG01005445 Diplonema papillatum (423) AF119811 Diplonema papillatum (1981) LMZG01015819 Diplonema papillatum (301) Marine 100/100 LMZG01013342 Diplonema papillatum (302) Isolates 99.9/ LMZG01022859 Diplonema papillatum (323) 100 AY180037 Uncultured euglenid (1743) AY425012 Diplonema sp ATCC 50232 (2148) AY425009 Diplonema ambulator (2062) AY425011 Diplonema sp (2067) 88.5/99 MF422192 Diplonema sp (2021) MF422190 Diplonema sp (2023) AY425010 Diplonema sp (2046) FN598401 Uncultured marine diplonemid (1330) MF422201 Diplonemida gen 2 sp 1 AH-2017 (2010)
100/100 Kinetoplastids Supplementary Figure S2. Maximum-likelihood tree of 18S rRNA gene sequences of diplonemids highlighting the copy numbers in single cells. 18S rRNA sequences from the genome of single cells were obtained from public databases and are highlighted with same color. D. papillatum group is highlighted in red in marine isolates. Marine diplonemids, Hemistasia, freshwater diplonemids, sequences from isolates and kinteoplastids are also shown. Other euglenids (e.g. Euglena, Phacus) were used as outgroup (not shown and indicated by an arrow). Ultra-fast bootstrap values and SH branch supports are indicated for selected nodes. The scale bar indicates the number of substitutions per site. bioRxiv preprint doi: https://doi.org/10.1101/2020.05.14.095992; this version posted May 15, 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. A 100 Bacillariophyta Cercozoa Chlorophyta 75 Chrysophyta Ciliophora Cryptophyta Dinophyta 50 Fungi Kathablepharis 18S Abundance (%) 18S Oomycota 25 Others Telonema Unclassified Uroglena 0 Mar Apr May
100 B
Bacillariophyta Cercozoa Chlorophyta 75 Chrysophyta Ciliophora Cryptophyta 50 Dinophyta Fungi Kathablepharis 18S Abundance (%) 18S Others 25 Synurophyta Telonema Unclassified
0 Uroglena
Mar Apr May C 100
Bacillariophyta Bicosoecida Cercozoa 75 Chlorophyta Choanoflagellata Chrysophyta 50 Ciliophora Cryptophyta Dinophyta 18S Abundance (%) 18S Fungi 25 Mesomyceta Others Telonema
0 Unclassified
Mar Apr May
Supplementary Figure S3. Community composition of microbial eukaryotes in Lake Biwa during weekly spring/summer sampling campaign by 18S amplicon sequencing from A) Epilimnion (5m), B) Metalimnion (10m) and C) Hypolimnion (50m). bioRxiv preprint doi: https://doi.org/10.1101/2020.05.14.095992; this version posted May 15, 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 A made available under aCC-BY-NC-ND 4.0 International license. Bacillariophyta 100 Cercozoa Chlorophyta Choanoflagellida
75 Chrysophyta Ciliophora Cryptophyta Diatom 50 Dinophyta Fungi
18S Abundance (%) 18S Kathablepharis Others 25 Prymnesiophyta Stramenopile Telonema Unclassified 0 Uroglena J F M A M J J A S O N D
B Bacillariophyta 100 Bicosoecida Cercozoa Chlorophyta Choanoflagellida 75 Chrysophyta Ciliophora Cryptophyta Diatom 50 Dinophyta Fungi
18S Abundance (%) 18S Kathablepharis Others 25 Prymnesiophyta Stramenopile Telonema Unclassified 0 J F M A M J J A S O N D Uroglena
C Bacillariophyta 100 Bicosoecida Cercozoa Choanoflagellida Chrysophyta 75 Ciliophora Cryptophyta Diatom Dinophyta 50 Fungi Heliozoa
18S Abundance (%) 18S Kathablepharis Others 25 Pedinella Perkinsozoa Stramenopile Telonema 0 J F M A M J J A S O N D Unclassified Supplementary Figure S4. Community composition of microbial eukaryotes in Lake Biwa in 2017 by 18S amplicon sequencing from (A) epilimnion (5 m), (B) metalimnion (20 m) and (C) hypolimnion (60 m) bioRxiv preprint doi: https://doi.org/10.1101/2020.05.14.095992; this version posted May 15, 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.
100
Cercozoa Chlorophyta 75 Chrysophyta Ciliophora Cryptophyta Diatom 50 Dinophyta Fungi Kathablepharis 18S Abundance (%) 18S Kinetoplastida 25 Others Telonema Unclassified Uroglena 0 5 50 100 150 200 Depth (m)
Supplementary Figure S5. Community composition of microbial eukaryotes in various depths of Lake Ikeda by 18S amplicon sequencing. bioRxiv preprint doi: https://doi.org/10.1101/2020.05.14.095992; this version posted May 15, 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.
A1 B1 C1
10µm 10µm 10µm A2 B2 C2
10µm 10µm 10µm
D1 E1 F1
10µm 10µm 10µm D2 E2 F2
10µm 10µm 10µm
Supplementary Figure S6. Fluorescent microscopic images of freshwater diplonemids from lake Biwa (A-D), Římov (E) and lake Zurich (F). 1 and 2 refers to the same cells visualized by DAPI (under UV excitation) and CARD-FISH (under blue excitation) respectively.