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Patchy-distributed ciliate (Protozoa) diversity of eight polar communities as determined by 454 amplicon pyrosequencing Patchy-distributed ciliate (Protozoa) diversity of eight polar communities as determined by 454...
Jung, Jae-Ho; Park, Kyung-Min; Yang, Eun Jin; Joo, Hyoung Min; Jeon, Misa; Kang, Sung-Ho; Choi, Han-Gu; Park, Mi-Hyun; Min, Gi-Sik; Kim, Sanghee
2015-09-03 00:00:00
ECOLOGY, POPULATION BIOLOGY & ANIMAL BEHAVIOR Animal Cells and Systems, 2015 Vol. 19, No. 5, 339–349, http://dx.doi.org/10.1080/19768354.2015.1082931 Patchy-distributed ciliate (Protozoa) diversity of eight polar communities as determined by 454 amplicon pyrosequencing a,b,d a,b c c c c b Jae-Ho Jung , Kyung-Min Park , Eun Jin Yang , Hyoung Min Joo , Misa Jeon , Sung-Ho Kang , Han-Gu Choi , a a b Mi-Hyun Park , Gi-Sik Min and Sanghee Kim * a b Department of Biological Sciences, Inha University, Incheon 402-751, South Korea; Division of Life Sciences, Korea Polar Research Institute (KOPRI), 26 Songdomirae-ro, Yeonsu-gu, Incheon 406-840, South Korea; Division of Polar Ocean Sciences, Korea Polar Research Institute (KOPRI), 26 Songdomirae-ro, Yeonsu-gu, Incheon 406-840, South Korea; Department of Biology, Gangneung-Wonju National University, Gangneung 210-702, South Korea (Received 30 April 2015; received in revised form 3 July 2015; accepted 18 August 2015) To determine ciliate diversity and community structure in the polar ecosystem efficiently, we applied the pyrosequencing technique to the polar samples. To select the appropriate sequencing depth using a ciliate-specific primer, we evaluated different pyrosequencing depths, ranging 4149–112,306 reads. At a 3% distance cutoff for clustering, 750 operational taxonomic units (OTUs) were identified, and 332 were composed of a single read (singletons). The singletons showed a 1.8-fold increase in OTU richness, although their beta diversity showed no significant changes. The ratio of singletons in each sequencing depth was sharply decreased after reaching the sequencing depth of approximately 10,000 reads, and the singletons did not completely disappear even at 73,435 qualified reads. The data set without singletons showed saturated trends in rarefaction curves. In addition, we built a normalized data set without the singletons (1227 reads × eight samples). Among the samples, one brackish water having a broad range of salinity (3–23 PSU) at the Arctic coast presented the highest OTU richness (103), while a temporal pool at the Antarctic coast with a high salinity of 53.4 PSU, showed a relatively lower OTUs (8). Each normalized sample showed a distinct community structure. Interestingly, a freshwater lake on King George Island shared relatively higher OTUs with salt-water samples (72.1%), suggesting a higher inter-relationship with closely located coastal water environment. Keywords: community structure; eukaryotic microbial ecology; polar ciliate; pyrosequencing; V4 region Introduction and are associated with higher trophic levels. For High-throughput next-generation sequencing (NGS) tech- example, ciliates are consumed by metazoans and play nology is a cost-effective method and has replaced clone an important role in the ecosystem, as a part of nutrient library method for community analysis (DeLong 2009). cycling (Sherr & Sherr 1988). In addition, ciliates can Among the various NGS platforms, 454 pyrosequencing be used as bioindicators for assessing environmental is applicable to metagenome, biodiversity, and transcrip- quality by their relatively prompt response to environ- tome analyses because of the longer read lengths com- mental contaminants (Chen et al. 2008; Xu et al. 2013). pared to that of other NGS platforms (Glenn 2011). However, traditional taxonomic approaches to identify Generally, longer reads contain more reliable information ciliates, such as microscopic examination with taxon- so that each read can be assigned as a species without any specific impregnation methods (Foissner 2014), are often assembly. To date, the 454 platform has been the pre- difficult and time-consuming (Dopheide et al. 2008). ferred approach for investigations of community According to Foissner et al. (2008), approximately 4500 structures. free-living ciliate morphospecies, as valid species, have Previous studies have attempted to examine large been described, and it is estimated that approximately diverse groups such as meiofauna (Creer et al. 2010), 27,000–40,000 ciliates might exist on the earth. Thus, metazoans (Fonseca et al. 2010), and protists/picoeukaryotes 83–89% of extant ciliates remains and is waiting for our (Amaral-Zettler et al. 2009;Stoecket al. 2009;Cheung etal. discovery. 2010; Nolte et al. 2010; Edgcomb et al. 2011;Kilias etal. The high diversity of ciliates with undiscovered species 2014). To achieve a more thorough understanding of including endemic ones hampers non-taxonomists in iden- specific ecological groups, we focused on the phylum Cilio- tifying ciliates. However, rapid climate change (e.g. global phora, a monophyletic unicellular microorganism. warming) and human activities have changed the polar eco- Ciliates are globally distributed in various diverse system; thus, the inherent ciliate diversity might have been habitats, ranging from the oceans to terrestrial habitats, altered. To assess the ciliate diversity and community *Corresponding author. Email: sangheekim@kopri.re.kr © 2015 Korean Society for Integrative Biology 340 Jae-Ho Jung et al. structure in the polar ecosystem efficiently, we applied the Environmental DNA preparation and pyrosequencing massive pyrosequencing technique to eight polar samples Genomic DNA (gDNA) from the samples was extracted collected from five coastal, two freshwater, and one using the PowerSoil DNA Isolation Kit (Mo Bio Co., Carls- pelagic environment. Our aims are as follows: (1) to evalu- bad, CA), because the humic acid in the samples prevents ate the specificity of a ciliate-specific primer in the polar direct amplification of the target gene fragment. A pair of samples, (2) to determine an appropriate pyrosequencing ciliate-specific primers, Pyro-F (5’-AGC AGC CGC GGT depth that could be utilized for the efficient monitoring AAY HCC-3’) and CiliPyro-R (5’-TAS GAC GGT ATC of a single community/sample, (3) to evaluate the TGA TCG TCT AT-3’), was used to amplify the hypervari- effect of singletons in data processing, and (4) to able V4 region of the small subunit ribosomal RNA (SSU examine the relationship among the polar ciliate rRNA) gene from the environmental gDNA. The reverse communities. primer specifically binds to the V4 region of ciliates based on a single-nucleotide polymorphism (SNP)-based phylum-specific polymerase chain reaction (PCR) amplifi- Materials and methods cation technique (SPAT) (Jung et al. 2012). PCR was per- Sample collection formed in a total volume of 35 μL, which contained 3.5 Eight samples were collected from Arctic and Antarctic μL of 10 × AccuPrime PCR Buffer II, 10 pmol of each environments (Figure 1). Detailed information on the primer, and 0.5 units (0.1 μL) AccuPrime Taq DNA Poly- samples is presented in Table 1. To compare ciliate diversity merase High Fidelity (Invitrogen, Carlsbad, CA) by using in bipolar ecosystems, we collected three salt-water samples the following cycle conditions: one cycle of 180 s at 92°C and one freshwater sample from each polar environment. (initial denaturation), 30–35 cycles of denaturation, primer Most of the salt-water samples were collected from coastal annealing, and elongation (96°C for 10 s, 52°C for 20 s, regions, with the exception of one pelagic sample collected and 68°C for 60 s), and a subsequent 7-min final extension during a research activity on ARAON, a Korean icebreaking step at 72°C. Twelve PCRs were performed for each gDNA, research vessel. The bottom of the sampling station was agi- and the PCR products were pooled together to reduce biased tated to collect both benthic and planktonic ciliates using a amplification of specific taxa (Lahr & Katz 2009). These planktonic net (20-µm mesh size). One of the six salt-water PCR products were purified using the QIAquick PCR Puri- samples was collected from the coastal area around King fication Kit (Qiagen Co., Hilden, Germany). The purified George Island (the Antarctic) using a polyurethane foam products were then concentrated up to 100 ng/µL by using unit (PFU). All samples were preserved in ethanol (70% a Centricon YM-3 (EMD Millipore, Billerica, MA) and final concentration) and maintained in a freezer at −80°C were pooled together. The pooled purified product was before the further analysis. sequenced using a 454 FLX Titanium sequencer. The Figure 1. Map of eight sampling stations. Scar bars: km. Animal Cells and Systems 341 Table 1. Summary of sample localities and analyses conducted on samples from the Svalbard (Arctic), Arctic Ocean, and King George Island (Antarctic). Antarctic Arctic Coastal area, Coastal area, King George King George Coastal area, King Freshwater, King Coastal area, Coastal area, ARAON, Freshwater, Island Island George Island George Island Svalbard Svalbard Arctic Ocean Svalbard Sample name ANtCoast1 ANtCoast2 ANtCoastPFU ANtFresh ArCoast1 ArCoast2 ArOcean ArFresh Location 62°14′S, 62°14′S, 62°13′S, 62°20′S, 78°54′N, 78°55′N, 73°07′N, 78°54′N, 58°46′W 58°46′W 58°47′W 58°47′W 11°57′E 11°55′E 168°56′W 11°52′E Date 21 January 2010 21 January 2010 20–30 January 21 January 2010 18 July 2010 15 July 2010 20 July 2010 14 July 2010 Temperature (°C) 5.6 1.5 0.5–2 1.4 9.5–12.2 8.2 −1.5 11 Salinity (PSU) 53.4 34.3 33–34.5 0.1 3–23 28.4 31 0.1 No. of raw reads 30,159 7502 112,306 4877 4149 8307 5071 6270 No. of high-quality reads 21,393 2367 80,633 2787 3598 7342 4522 5551 No. of high-quality reads in 19,690 2339 73,435 2774 3541 7312 4481 5413 Ciliophora (singleton) (1351) (1112) (7058) (728) (1026) (1001) (936) (1061) % of ciliate specificity 92 98.8 91.1 99.5 98.4 99.6 99.1 97.5 No. of ciliate OTUs (3% cutoff) 43 128 301 121 219 69 102 66 No. of ciliate OTUs (3% cutoff 17 70 123 66 116 30 78 51 without singletons) No. of OTUs in normalized 8 (1.09) 69 (2.81) 48 (1.75) 61 (2.27) 103 (3.17) 26 (1.95) 67 (2.44) 39 (2.51) data without singleton (Shannon diversity) Polyurethane foam unit (PFU) sample soaked in the coastal water for ten days Normalized data set composed of 1227 high-quality ciliate reads per a sample 342 Jae-Ho Jung et al. amplicons from each environmental sample contained its own multiplex identifier (MID) sequence at the 5′ end of both forward and reverse primers. Operational taxonomic unit determination and taxonomic assignment To isolate high-quality reads, we preliminarily filtered the raw reads with the following criteria: complete MID sequences at both ends, minimum sequence length of 300 bp (including PCR primers), no ambiguous nucleotide (N), ≥25 average quality score, no chimera, and no poor quality of alignments with the SILVA eukaryotes, using Mothur software (Schloss et al. 2009). Both ends of the fil- tered reads were trimmed to extract the hypervariable V4 Figure 2. Percentage of singletons in reads with exponential region. Finally, the V4 region was examined to define oper- decay fit. ational taxonomic units (OTUs) based on p-distance by using Mothur version 1.33.3 and JAGUC version 2.1 (Nebel et al. 2011). USEARCH version 7.0.1090 (Edgar Singletons 2010) was implemented to assign the V4 region to SILVA The ratio of singletons varied among the samples and SSU rRNA database release 119 (Pruesse et al. 2007). showed a range of 6.9–47.5%. ANtCoast2 consisted of According to the assigned results, these reads were classi- 2339 qualified-ciliate reads and showed the highest single- fied at higher taxonomic levels (kingdom to order level). ton ratio of 47.5%. The singleton ratio was negatively The output data of the USEARCH were transferred to exponentially correlated with the number of reads MEGAN version 5.5.3, and all communities were combined (Figure 2). ANtCoastPFU, the biggest data set, was com- and visualized together (Huson et al. 2009). A Perl script posed of 73,435 qualified-ciliate reads with a singleton was used to transform these data to the preferred formats ratio of 9.6%. The lowest singleton ratio was 6.9% from of the programs mentioned above and was applied for 19,690 qualified-ciliate reads of ANtCoast1. simple repetitive procedures. Additionally, we generated several data sets to identify the influences of the singletons. Here, we applied two similarity thresholds (97% and Richness and diversity of operational taxonomic units 99%). Reads showing ≥99% similarity with the reference The number of raw reads in each sample ranged from were classified as species level (i.e. taxonomic assign- 4149 to 112,306, and qualified-ciliate reads showed 43– ment), and the species list was used to interpret the environ- 301 OTUs at a 3% cutoff (750 OTUs in this study). The mental features based on taxonomic literatures (e.g. singletons accounted for 11.8% of the total ciliate reads occurrence, ecology). Based on the OTU (3% distance covering 44.3% of the OTUs (332 out of 750). OTU rich- cutoff), we calculated the Bray–Curtis similarity, and ness was highly influenced by two factors: non-ciliate these values were used to construct dendrograms and to sequences and singletons (Figure 3). In particular, single- implement non-metric multidimensional scaling (NMDS) tons highly affected and increased the number of OTUs in Primer 6.1.16 (Clarke & Gorley 2006) to analyze (1.8-fold). Elimination of both non-ciliate reads and sin- relationships among polar ciliate communities. gletons displayed a saturated trend in the rarefaction curves. Therefore, we excluded the non-ciliate reads and Results singletons for reliable data analysis of ciliate community. From the qualified reads, we built normalized data sets. Ciliate specificity of SPAT primers Each sample of the normalized data set was A total of 178,641 raw reads were composed of 4149– composed of 1227 ciliate reads without a singleton. 112,306 reads from each of the eight samples. From the OTU richness ranged from 8 to 103 OTUs and Shannon raw data set of 178,641 reads, 128,193 reads satisfied our diversity was in the range of 1.09–3.17 (Figure 4, Table criteria for high quality. Using USEARCH (Edgar 2010), 1). ArCoast1 showed the highest diversity among the we assigned the high-quality reads to SILVA SSU rRNA eight samples. database release 119 (Pruesse et al. 2007). A total of 118,985 reads from the high-quality reads were identified Community structure as ciliate. The ciliate-specific primer presented high speci- ficity for ciliates, ranging from 91.1% to 99.6% (92.8% on In the normalized data sets, a total of 258 OTUs was ident- average) (Table 1). ified, and Antarctic and Arctic samples were composed of Animal Cells and Systems 343 Figure 3. Rarefaction curves of three different data sets from eight samples. The raw reads are high-quality reads that satisfied the filter criteria using Mothur (Schloss et al. 2009). Based on USEARCH results (Edgar 2010), the ciliate reads were separated from the raw reads. Additionally, we constructed one additional data set that excluded the singletons from the ciliate reads, and these three data sets were ana- lyzed for their rarefaction curves by using JAGUC (Nebel et al. 2011). The number of OTUs differed slightly from that generated using Mothur (Schloss et al. 2009), which may be attributable to variations in algorithms in each software. 344 Jae-Ho Jung et al. similarity without read abundance, 0–38.3%; Figure 7). One additional normalized data set was constructed to evaluate the effect of singletons on beta diversity. The community similarity values were not significantly affected by the singletons (Figure 6). The normalized data set was employed for taxonomic assignments using USEARCH (Edgar 2010) and reads showing ≥99% sequence similarity with the reference SILVA were assigned to species level. Each normalized data set was composed of 1227 reads and the number of assigned reads was in the range of 8–819 (0.7–66.7%, Table 2). The ratio of reads showing ≥99% similarity was loosely positively correlated with number of OTUs (y = 32.1065 + 0.9364x, r = 0.5190, p < 0.05, n = 8). As anticipated, the assigned taxa usually showed its occur- Figure 4. Venn diagram showing co-occurring OTUs using a nor- rence at appropriate environments, such as terrestrial, malized data set without singletons. freshwater, and salt-water. However, ANtFresh contained many marine species such as Apokeronopsis bergeri, 152 and 179 OTUs, respectively. Seventy-three OTUs Cyclotrichium cyclokaryon, Euplotes nobilii, E. sinicus, (28.3%) co-occurred in both polar regions. The normalized Loxophyllum rostratum, Psammomitra retractilis, Pseu- eight samples were categorized as salt-water or freshwater, doamphisiella alveolata, Spathidiopsis buddenbrocki, and each category of Antarctic and Arctic samples was Tintinnopsis rapa, and T. spp. composed of 3681 (1227 reads × 3 salt-waters) and 1227 reads (1227 reads × 1 freshwater), respectively (Figure 4). Discussion The number of co-occurring OTUs between the Antarctic Appropriate sequencing depth with SPAT primer and Arctic regions was 50 OTUs (22.2% in salt-water cat- egory) and 15 OTUs (17.6% in freshwater category), We analyzed the ciliate community structure of eight respectively. Six OTUs co-occurred in all categories from polar environments using the ciliate-specific primer we bipolar regions, and their top hit taxa were as follows: previously designed, and the SPAT primer had showed OTU050—Hypotrichia, with a similarity of 85.8–91.6%; high specificities of 94.5% for cloning and 99.2% for OTU054—Hypotrichia, with a similarity of 98.2–100%; the pyrosequencing method (Jung et al. 2012). OTU067—Oligotrichia, with a similarity of 86.1–89.2%; Although several primers targeting partial ciliate groups OTU105—Tintinnopsis sp., with a similarity of 99.1– have been reported (Costas et al. 2007; Bachy et al. 100%; OTU155—Euplotes sp., with a similarity of 99.6– 2012a; Liu & Gong 2012), the SPAT primer encom- 100%; and OTU197—Euplotes balteatus, with a similarity passes the majority of the phylum Ciliophora both in of 94.9–96%. silico and experimentally, thus indicating its applicability The most abundant classes in the normalized data set to mass pyrosequencing of diverse polar ciliate were Spirotrichea (43.6%), Litostomatea (33.1%), and Oli- communities. gohymenophorea (10.6%) (Figure 5). However, the ratios The singleton was mainly responsible for increasing the of the abundant groups varied among the samples. For number of OTUs. Even though we increased the sequencing example, the most abundant class Spirotrichea showed a depth up to a maximum of 112,306 reads to recover rare minimum of 5.3% (ANtCoast1) and a maximum of 86% species, singletons were not completely removed, and the (ANtFresh) (Figure 5). The most abundant orders were rarefaction curve with singletons showed a non-saturated Pleurostomatida (Litostomatea, 19.6%), Tintinnida (Spiro- trend (Figure 3). If we exclude singletons from the data trichea, 10.4%), and Haptorida (Litostomatea, 10%). Spir- set, the curves showed more saturated trends at a range of otrichea showed more diverse orders and greater evenness 1000–20,000 reads. The OTUs showing low abundance than did the other orders. For example, the class Litostoma- (or consisting of a single read) could be rare species tea was mainly composed of three orders, Pleurostomatida, (Sogin et al. 2006), while Behnke et al. (2011) reported Haptorida, and Cyclotrichida, while Spirotrichea was com- that singletons should be eliminated for reliable data posed of nine orders. interpretation. Thus, removal of singletons from data sets The normalized data set was used to analyze beta could be an appropriate way to avoid overestimation of diversity among the eight samples (Figures 6 and 7). ciliate diversity in despite of loss of few rare species. Even Each community showed a distinct community structure though the singletons were not completely disappeared and low similarity between the samples (Bray–Curtis with the increased reads, the singleton ratio sharply similarity with read abundance, 0–24.3%; Bray–Curtis decreased at approximately 10,000 reads (Figure 2). Animal Cells and Systems 345 Figure 5. Megan tree showing assigned reads at higher taxonomic level with their abundances (circles). Based on these results, we propose that the appropriate The number of OTUs significantly varied depending on sequencing depth using the SPAT primer could be 10,000 the data processing criteria, cutoff threshold, and target reads per sample, because this number of reads showed a region (Schloss 2010; Behnke et al. 2011; Bachy et al. very low singleton ratio and could recover most of the 2013). Recently, Meng et al. (2012) investigated ciliate species in a sample. Similarly, Lundin et al. (2012) proposed diversity by broad sampling of offshore sediments from that 5000 denoised reads are effective in describing trends the Yellow Sea with silver impregnation, and the number for bacterial communities. Because ciliate biodiversity is of morphospecies (ranging from eight to 65 in each usually lower than bacterial biodiversity, a lower number station; a total of 198 morphospecies) was higher than that of reads than the 10,000 reads would be adequate for from the intertidal sediments, as reported by Hamels et al. ciliate monitoring to capture existing trends of ciliate (2005). Meng et al. (2012) reported that the observed diversity. higher morphospecies richness might have been attributable 346 Jae-Ho Jung et al. Figure 6. Dendrogram of two normalized data sets based on the Bray–Curtis similarity at 3% cutoff. These dendrograms consist of 18,712 reads and 9816 reads, respectively. The topology of these dendrograms is nearly identical whether the singletons are included or excluded. Figure 7. Multidimensional scaling with cluster analysis of Bray–Curtis similarity matrix by using abundance (a) and presence/absence (b) data. to their broad sampling approach. In terms of species rich- (Foissner 1999, 2004; Foissner et al. 2008). Petz also ness, the polar ciliate diversity does not deviate from the reported that some ciliates show a restricted geographic microscopic approach and slightly higher. However, some distribution (Petz 2003; Petz et al. 2007). Based on the mol- closely related ciliate species could have identical 18S ecular approach of terminal restriction fragment length sequences (some congeners in Tetrahymena; see Lynn & polymorphism (T-RFLP) (Engel et al. 2012), the intertidal Strüder-Kypke 2006). Here we should note that the ciliate community, collected from the German island of number of OTUs could be underestimated because of the Sylt in the Wadden Sea showed a highly patchy structure. inherent nature of the 18S rRNA gene. Our results show trends similar to those of these previous studies and emphasize the need for a broader sampling to understand polar environments better. In addition, we Community structure of bipolar regions should be aware that cell size (Zhu et al. 2005) and 18S rDNA copy number (Gong et al. 2013) could affect the Each sample showed a distinct community structure (Bray– read abundance in an OTU. With the distinctness of the Curtis similarity with abundance data, 0–24.3%), and the structures, we found some interesting results from the number of co-occurring OTUs between the eight polar polar samples. samples was 0–33 OTUs (Bray–Curtis similarity of pres- The two freshwater samples ANtFresh and ArFresh ence/absence data, 0–38.3%). The singletons increased contained and shared some OTUs with salt-water the number of OTUs while they did not significantly samples, and ANtFresh shared more OTUs than did affect the beta diversity among the eight samples because ArFresh. Assigned taxa in Table 2 indicate that the of their low abundance. The distinct structure of each shared OTUs were typically marine ciliates, and ANtFresh, sample made it difficult to understand their relationships in particular, showed 12 marine species (vs. 4 in ArFresh). (e.g. indigenous species). Previously, Foissner (1996) The sampling station of ANtFresh is located about 50 m reported a patchy distribution of soil ciliates from Antarc- away from the coastline and salt-water can easily penetrate tica and proposed the “moderate endemicity model” Animal Cells and Systems 347 Table 2. Ciliate species showing high similarity (≥99%) with pyrosequencing data, compiled with SILVA release 119. Ant Ant Ant Ant Ar Ar Ar Ar Species (≥99% with Ref seq) Coast1 Coast2 CoastPFU Fresh Coast1 Coast2 Ocean Fresh Habitat Amphisiella annulata GU170843 0 4 0 0 0 0 0 0 S/B Apokeronopsis bergeri DQ777742 0 0 0 18 3 0 13 0 S/B Arcuseries scutellum FJ156105 0 0 0 0 1 0 0 0 S/B Aspidisca steini AF305625 0 0 0 0 0 0 1 0 S/B Cyclotrichium cyclokaryon FJ876971 0 0 0 8 0 87 127 0 S/P Diophrys scutum DQ353851 0 0 0 0 3 0 0 0 S/B Diophrys sp. HE664174 8 0 0 0 0 0 0 0 S/B Euplotes focardii EF094960 0 0 2 0 0 0 0 0 S/B Euplotes nobilii GU479383 0 0 0 2 4 0 0 0 S/B Euplotes nobilii GU479391 0 3 0 2 1 0 0 0 S/B Euplotes rariseta JX437134 0 3 0 0 1 0 0 0 S/B Euplotes sinicus FJ423448 0 8 0 2 4 0 0 2 S/B Euplotes sp. EF193242 0 12 0 0 0 0 0 0 S/B Gastrostyla steinii AF508758 0 0 0 0 0 0 0 1 F/B Halteria grandinella AF508759 0 2 0 151 0 0 0 0 F/P Laboea strobila AF399154 0 0 0 0 0 3 0 0 S/P Loxophyllum rostratum DQ190465 0 3 5 8 5 0 1 0 S/B Metaurostylopsis antarctica JF906730 0 0 0 0 4 0 0 0 S/B Metaurostylopsis cheni GU170204 0 2 0 0 0 0 4 0 S/B Orthoamphisiella sp. JQ723974 0 0 0 204 0 0 0 0 F?/B Oxytricha trifallax AMCR01020474 0 0 0 0 0 0 0 29 F/B Parauronema longum AY212807 0 0 16 0 0 0 0 0 S/B Pleuronema setigerum JX310015 0 6 0 0 0 0 3 0 S/B Psammomitra retractilis EF486865 0 0 1 3 2 0 0 0 S/B Pseudoamphisiella alveolata DQ503583 0 0 0 6 2 0 0 0 S/B Pseudotontonia sp. JX178817 0 0 0 0 0 0 1 0 NA/P Spathidiopsis buddenbrocki HM051056 0 2 0 4 2 0 0 0 S/P Stentor multiformis FN659821 0 0 0 0 0 0 1 62 F/B Strombidinopsis sp. JQ028734 0 2 0 0 0 0 0 0 S/P Strombidinopsis sp. AM412524 0 0 0 0 0 0 1 0 S/P Strombidium biarmatum AY541684 0 0 0 0 3 0 5 0 S/P Strombidium rassoulzadegani AY257125 0 0 0 0 47 0 0 0 S/P Strombidium sp. AY143564 0 17 10 0 2 0 0 0 S/P Tintinnopsis cylindrica JN831811 0 0 0 0 0 2 18 0 S/P Tintinnopsis rapa JN831833 0 0 0 23 7 164 5 1 S/P Tintinnopsis sp. JX178854 0 0 0 0 0 2 0 0 S/P Tintinnopsis sp. JX178862 0 0 0 0 0 3 0 0 S/P Tintinnopsis sp. JX178881 0 0 0 2 0 1 0 0 S/P Tintinnopsis sp. JX178900 0 3 0 90 0 557 0 1 S/P Uroleptus gallina AF508779 0 0 0 0 0 0 6 185 F/B Uroleptus willii EU399543 0 0 0 0 0 0 0 15 F/P Uronema marinum GQ465466 0 0 3 0 0 0 3 0 S/B Uronychia setigera HQ380023 0 22 0 0 1 0 3 0 S/B Uronychia setigera JF694042 0 3 0 0 83 0 0 0 S/B Vorticella microstoma DQ868347 0 6 0 0 0 0 0 0 F/B Zoothamnium sp. JX178767 0 0 0 0 0 0 0 3 NA/B Total 8 98 37 523 175 819 192 299 Notes: NA, data not available; B, benthic; F, freshwater; P, planktonic; S, salt-water. Soil environment was classified as freshwater in this study. the island and reach to the freshwater lake. However, most species. However, we can assume that these samples OTUs were not assigned to the species level because of low shared some OTUs and they were likely euryhaline similarity with SILVA DB. Because of this limitation of the species or were in their resting stage (cyst) or were cell reference sequence, we could not ascertain whether the debris. All sampling stations of each polar region were other co-occurring OTU is a marine or freshwater closely situated along the coastline, except for one 348 Jae-Ho Jung et al. pelagic sample. Thus, these environments might easily taxonomic assignment (Pawlowski et al. 2012), and (iii) the influence each other by physical forces such as wind and low resolution of 18S rRNA for closely related species waves. Additionally, we need to consider a dispersal by (Lynn & Strüder-Kypke 2006). resting cysts of terrestrial ciliates (including freshwater species) to coastal waters because live cells are very Funding fragile for the dispersal as an active form (Foissner This study was supported by the Inha University research grant, 2011). Moreover, the two freshwater samples share only the Graduate Program for the Undiscovered Taxa of Korea 15 OTUs (17.6% of total freshwater OTUs) that might be (1834-302), the Ministry of Environment, Korea, and the Polar related with the limited distribution of limnetic ciliates as Academic Program (PAP, PE15020) of KOPRI. reported by Petz et al. (2007). Among the eight polar samples of the normalized data set, ArCoast1 showed the highest number of OTUs (103). ORCID The sample ArCoast1 includes ciliate species collected Jae-Ho Jung http://orcid.org/0000-0001-5497-8678 from a sandy sediment. According to Hamel et al. 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