Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Dinoflag- in the deeper 3 Ciliates and , and M. R. Landry 1 Dinoflagellates , H. Liu 1 13484 13483 Costa Rica by 454 ff , X. Xia 1 , L. Kong 1 700 m. A clear shift of protist composition from photosynthetic ∼ , E. Rocke 1,2 in the surface to potential parasitic This discussion paper is/has beenPlease under refer review to for the the corresponding journal final Biogeosciences paper (BG). in BG if available. Division of Life Science, The Hong Kong University of ScienceSanya and Institute Technology, Clear of Water Deep-Sea Science and Engineering, Chinese Academy ofScripps Sciences, Institution of Oceanography, University of California, San Diego, La Jolla, CA, USA ellates Abstract Marine planktonic protists, includingtant microalgae contributor and to protistan global primarylittle grazers, is production are and known an carbon about impor- column. and their We mineral population used cycles, shifts however, 454 along pyrosequencingstudy the of the oxic-anoxic the community gradient 18S composition in rRNA ofumn the in gene whole water the and and Costa active gene Rica protists transcriptsdepth Dome, throughout of to where 400 a a water stable col- oxygen minimum zone (OMZ) exists at a water was revealed alongprotist groups the recovered only vertical at profile theing rDNA at from level represent both the either upper rRNA lyseddemonstrated aggregates that waters and sink- total or rDNA and potential active levels.tinct protists hosts Those from in for those the anoxic in parasitic core otherof groups. of water OMZ UPGMA a depths. (550 The clustering m) parasitic/symbiotic reduced were trophic communitygests dis- diversity that lifestyle and OMZs in presence can the exertin a OMZ, community selective structure especially pressure and on the a protistmicrobial anoxic shift communities. loop in core, Such and trophic changes sug- associated lifestyle biogeochemical could cycling. result in a modulation of the 1 Introduction Protists, the unicellular eukaryotes, have aare ubiquitous distribution important in contributors oceanic to waters global and They primary are production a and very carbon and complextially mineral group, in cycles. spanning size, all shape and eukaryotic motility,are kingdoms and the but produce basis varied upwards of of substan- aquatic halfin food of webs various the (Thomas world’s ecosystems et oxygen al., have and cal 2012). been classification Protist remarkably previously community (Bachy diversities underestimated et based al., 2013). on Until morphologi- last decade, with the application pyrosequencing H. Jing Protist communities in a marineminimum oxygen zone o Biogeosciences Discuss., 12, 13483–13509, 2015 www.biogeosciences-discuss.net/12/13483/2015/ doi:10.5194/bgd-12-13483-2015 © Author(s) 2015. CC Attribution 3.0 License. 1 Bay, Kowloon, Hong Kong2 SAR, China Sanya 572000, China 3 Received: 8 July 2015 – Accepted: 8Correspondence July to: 2015 H. – Liu Published: ([email protected]) 20 August 2015 Published by Copernicus Publications on behalf of the European Geosciences Union. 5 10 15 20 25 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | erent natural erent depths ff ff 20 µM) occur in the in- < 13485 13486 erent DO concentrations from six di ects of oxygen on various biological assemblages ff ff ect of OMZ formation on the communities of marine microbial ff ect the ecological functions of various groups. As a special niche, ff erent DO concentrations are still largely unknown. In our study, a typical marine OMZ in the Costa Rica Dome (CRD) was selected Low oxygen conditions as a putative stress can reshape local microbial community Protists as an important component of marine ecosystems consist of phototrophs, ff in order toThe characterize CRD the located composition inproximately of the 300 Eastern km, protists Tropical is along North oneFiedler, Pacific the of 2002; (ETNP) oxic-anoxic the Helly with eastern gradients. and a boundarytween Levin, diameter upwelling 400 2004), of to systems and ap- 700 (Wyrtki, generates m 1984; tems, (Wyrtki, a 1984). stable the Compared OMZ with CRD-OMZ usually othermuch found is marine be- thicker oxygen characterized deficient OMZ by sys- layerdeeper its (300–400 m). oxic permanent, water This much layers specialtion and deeper habitat to the containing location investigate intermediate the and theTherefore, anoxic upper water impact water and samples of could with be DO a di on perfect the loca- protist communities and their functions. spanning the aerobic photic(400 zone m) (20 and m), lower the (700 m)as secondary oxic-anoxic the DO boundary oxygen-rich deep and peak water the (200 (2000put m), core m) pyrosequencing. were of the For collected OMZ upper a and (550 studied comparative m), using study,oxygen as the high gradient well active through- were protist communities investigated alongsights at this into rRNA the level ecological aswaters. significance well. of Our marine study protist provides communities in new suboxic/anoxic in- and processes. The e termediate waters of some partsan of ideal the oceans place (Ulloa for and Pantoja, studying 2009) the representing e eukaryotes (Orsi et al.,bic 2012; Ciliates Parris and et Dinoflagellates al., werespatial 2014) found variations have as of been the protists, investigated dominant especially anddi groups. the anaero- Nevertheless, active the portion, along the water column with anoxic waters has caused(Wylezich significant and changes Jürgens, of 2011; the OrsiKong community et et structures al., al., 2013). of 2012) Increased and protists diversityto of prokaryotes those picoeuaryotes (Stewart in in oxic et the waters hypoxic al., waters hasing 2012; compared been to observed, and parasitism a had shift been inlow-oxygen microbial proposed environments, food (Rocke webs marine et from graz- al., oxygenstably 2013). minimum depleted Among dissolved zones the oxygen di (OMZs) (DO) characterized concentrations by (e.g. structure and a upper photic zones represent theComparatively, main heterotrophic protists primary provide producers a in vitalthe surface link waters prokaryotic for (Li, the fraction 1994). cycling to of higherSherr, nutrients trophic 1994), from levels and in have the amixotrophic marine much microbial protists, wider loop they distribution (Sherr either throughout and other harbor the eukaryotes endosymbionts water (Harada (Not column. et et As al.,Considering al., for 2007), the 2007) playing diverse or a metabolic diverse parasitize andtribution and trophic and complex capabilities responses ecological of to role. protists, variations their in distinct environmental dis- conditions would be expected. heterotrophs and mixotrophs. The phototrophic protists with confined distribution to the of various molecular techniquessity based of on protists 18S haveet rRNA been al., genes, revealed 2001; unexpected (López-García NotMore high et et recently, diver- next al., al., generation 2001; 2007; 454most pyrosequencing Moon-van Guillou abundant technology der et and has Staay al., rare detectedrevealed protist 2012; both a species the Massana vast (Edgcomb diversity and et up Pedrós-Aliò,based al., to assessment 2008). several 2011; (Bachy orders Oris et of al., etpicture magnitude 2013), al., of of therefore 2012) the those enabling protist and from a community morphology- more in realistic/exhaustive natural environments. 5 5 10 15 20 25 20 15 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | N, ◦ 40 m ∼ ) (López- 0 C for 40 s; final ◦ C for 20 min to re- ◦ ) (Díez et al., 2001), 0 C for 30 s, 72 ◦ the Costa Rica coast (8.9845 ff -GAAACTGCGAATGGCTC-3 0 C for 30 s, 55 ◦ 13488 13487 -ACCAGACTTGCCCTCC-3 0 erent parts of the water column described above ff er. rDNA and rRNA concentrations were measured on ff PCR DNA and Gel Band Purification kit (GE Healthcare). An ampli- TM C for 7 min. ◦ GFX C for 2 min; 35 cycles of 95 ◦ W) during the FLUZiE cruise in June 2010 as described previously (Kong ◦ TM C. Total RNA was extracted from 0.22 µm polycarbonate filters with the TRIzol ◦ C until further analysis. C isotherm depth) (Fiedler, 2002). Seawater hydrographical data was collected by ◦ 80 ◦ erent MID sequences for pyrosequencing were synthesized together with the for- − TriplicatePCR products for each sample were combined and subsequently purified Before cDNA synthesis, purified total RNA was treated with DNase I to eliminate For DNA/RNA sample collection, 1 L seawater from 6 depths, 20, 200, 400, 550, ff by illustra con library was constructed andof emPCR sequences was linked conducted to to each generateration bead, millions according (Roche, of to copies 454 the instructions LifePicoTiterPlate of and Science). Rapid sequenced library DNA on prepa- a beads GS were Junior system successfully (Roche,2.4 deposited 454 Life onto Science). the Post-run sequence analysis All forward-oriented reads generated fromspecific this study multiplex were identifiers separated (MIDs), accordingrosequencing to and pipeline, their for quality example, control readsless were was flagged than checked as 300 in low bp quality thesequence in when and RDP they length, when were py- the one or start(Cole more et of ambiguous al., the bases 2009). (N) sequence Chimeras were did were present detected not in with exactly the the sequence match Chimera a Slayer primer algorithm imple- (20 90.50911 et al., 2013). The sampling station has an annual mean thermocline depth of 35 designed to amplify the completeDi V2 and V3 domains ofward all primer. eukaryote PCR 18S was rDNA carried gene. cycles: out 95 with Peltier Thermal Cyclerextension at (Bio-Rad) 72 with following Garcia et al., 2003) and Euk-516R (5 using the SuperScript III first strand cDNA synthesis kit (Invitrogen, Carlsbad, CA). DNA contamination. Total RNA (up to 200 ng) was then reverse transcribed to cDNA a NanoDrop 1000 Spectrophotometer (Thermo Scientific). move RNA residue and used foralways subsequent used PCR as amplification. a Non-RT negative samples control. were 2.3 PCR amplification and 454 pyrosequencing Both rDNAs and(Roche) cDNAs with were amplified universal using primers FastStart Euk-82F High (5 Fidelity PCR system was removed before thenally preparation eluted with in TRIzol 50 µL Reagent elution and bu extracted RNA was fi- A parallel reaction without SuperScriptSynthesized III RT cDNA was was used as further an digested RT-PCR negative control. with 2 U RNase H at 37 2.2 DNA and RNA extraction andGenomic cDNA DNA synthesis was extracted fromnomic 0.22 µm DNA Kits polycarbonate (Invitrogen, filters Carlsbad, withat CA) the and PureLink eluted Ge- into 100 µLplus TE RNA butter and purification stored kit (Invitrogen, Carlsbad, CA). RNA later immersing the filters 700 and 2000 m,was representing filtered di through aa low 0.22 µm vacuum pressure. pore Filterssolution for size (Ambion) RNA polycarbonate sample immediately collection filter after were filtration.–80 (47 immersed mm, All in filters Millipore) RNA were later with flash frozen and stored at a conductivity-temperature-depth (CTD) rosette systemtached (Sea-Bird Niskin Electronics) bottles. with The at- were concentrations also of measured dissolved (Kong oxygen, et nitrite al., 2013). and ammonium 2 Materials and methods 2.1 Sample collection and environmental conditions Water samples were collected from the OMZ o 5 5 10 15 20 25 25 20 15 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | , orts in Fungi ff , rarefac- value of ff was detected at level of 3 % using erent DO concen- ff ff Cryptophyta , 0.97) than for rRNA reads , were identified from all the Cryptophyta ∼ Alveolata value at the rRNA level (Fig. 1a) ff Viridiplanctae 1.0 µM). and 13490 13489 ≤ routine. Rarefaction curves were calculated using Stramenopiles , erent samples was generating using the R-package. OTU species af- ff Rhizaria value. Comparatively, richness (Chao1) and diversity (Shannon) at the , 40 m as described earlier (Kong et al., 2013). Very di ff ∼ with 10 000 iterations. Taxonomic identification of each read was carried out summary.single 0.81). The highest coverage was obtained from the core (550 m) of the OMZ, ∼ Pyrosequencing generated a total of 26 411 rRNA reads and 41 895 rDNA reads after Higher coverages were shown for rDNA reads (0.87 The estimators for richness (Chao1), diversity (Shannon index), coverage and oper- samples at the rRNA level, contrasting to the fact that no filtering out low-quality reads accordingology to (Table the 1). applied On criteria average, much describedrDNA more in quality level the reads (6983 method- per reads sample perwhilst were sample) the obtained at than latter the at waslarity the classified rRNA into as level much cuto (4402 more reads total per OTUs sample), using 97 % sequence simi- rRNA level were remarkablyvertical higher profiles, than the those highest number at ofrDNA the total and OTUs, rDNA Chao1 rRNA level and levels (Table Shannonindices 1). were indices (Chao1 at Along shown both the and at the Shannon)rRNA level depth were and of found the 200 surface from m; water the whilst (2 m) the core at lowest the of rDNA diversity the level. OMZ (550 m) at the Haptophyceae while the lowest onesrespectively were (Table 1).This shown was at consistentefaction 700 curves with with and the 97 % patterns 400 sequence m similarity demonstrated at as by cuto the the rRNA rar- and rDNA levels, (0.70 and at the DNAthose level from (Fig. other 1b): depths; samples theorder at to 550 the m adequately rRNA assess sample level community became still composition. flat need more earlier sampling3.2 compared e with Microbial community structure In total, seven protistan phylogenetic groups, including trations were detected atconcentration the dropped sharply six from typicaling the depths surface at along water around with the 200 m a700 vertical m and secondary with water decreased peak a profiles. again appear- core to DO at generate around a 550 m clear (DO OMZ zone from 400 to depth of 30 60. To estimate similarity amongbased samples, on hierarchical a cluster matrix analysis of wasMothur OTUs conducted in and each a sample dendrogram usingmetic inferred Bray-Curtis with means similarity calculated the (UPGMA). from unweighted AOTUs pair-group among heatmap method di plot with depicting arith- the abundance and distribution of 3 Results 3.1 OMZ conditions and sequencing statistics A stable thermocline formedevents as in indicated the by sampling increased station salinity and due decreased to temperature the occurred persistent at upwelling the Mothur’s Bray distance and the complete clustering method. 2.5 Accession number All the sequences obtained fromfor this Biotechnology study Information have (NCBI) been Sequence deposited Readnumber in Archive the of (SRA) National SAMN03839054-SAMN03839065. under Center BioSample filiations to the reddatabase (Guillou portions et of al., the 2012). heatmap The were relationship acquired among manually samples using was determined the by PR2 tion.single ational taxonomic unit (OTU) numbers were calculated at the cuto with Mothur against the Silva bacteria no-gap reference database at a cuto mented in the Mothur softwareAfter package above using filtration, the 18Spackage the rDNA for remaining alignment, data distance sets reads calculation as and were references. classification analyzed (Schloss et with al., the 2009). Mothur software 5 5 10 15 10 20 20 15 25 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | and and was were Dino- Rhizaria , Perkinsea , were re- Phyllopha- Suessiales erent water Peridiniales , which was Eustigmato- ff Rhizaria Dinophyceae was generally Stramenopiles and was always the which was unde- , particularly occurred in the sur- Raphidophycea 7.0 %) levels (Fig. 2). Raphidophyceae , Pinguiophyceae ∼ Spirotrichea Haptophyceae Eustigmatales were both found at 400 m , were two minor groups and was more numerous at the was only found at 200, 550 Alveolata Dinophyceae , seven phylogenetic groups were two major groups more Bacillariophyta 91.7 %) levels. and Fungi ∼ Gymnodiniales Phyllopharyngea was the most abundant group, and had broad distribution in each sam- Prorocentrales Perkinsea Haptophyceae reads was more pronounced at the , predominant in all samples. The dom- , PX-clade and Alveolata Ciliophora and was only detected at the rRNA level with Litostomata Pyrocystales Fungi were found from our samples. Chattonellales and 13492 13491 , presented at 200 and 400 m (Fig. 3b). On the Eumastigochyceae , were predominant at the rDNA level (Fig. 5). In Gymnodiniales 29.8 %) than the rDNA (4.5 Ciliophora Donophyceae was always the predominant group in di ∼ Cryptophyta and Gymnodiniales were recovered from the deep (2000 m) and surface Chrysophyceae 75.1 %) and rDNA (82.6 Eustigmatophyceae ∼ Stramenopiles as the most abundant group. in total Blastodiniales was almost undetectable at the rDNA level except very little accounted for less than 1 % in all samples except at 550 m at was the predominant ciliate class at the rRNA level, and all Stramenopiles Alveolata Apicomplexa , the dominant group of Spirotrichea , four groups were identified from rRNA reads, but no and Oligohymenophorea were much less abundant at both levels. For example, Prorocentrales in addition to was only recovered from 200 m at the rRNA level. occupied less than 5 % of the protist community. Dinophyceae predominated in all twelve samples and comprised of four phylogentic and Spirotrichea Apicomplexa Litostomatea Gymnodiniales Ciliophora Dinophyceae occupied around 50 % of this phylum (Fig. 3d). Cilophora was recovered for the rDNA reads (Fig. 3b). At the rDNA level, surface water liation and abundance of the top 10 most abundant OTUs at the rRNA and rDNA , ffi more highly distributed in the surface water (2 m) at both rDNA and rRNA levels, Viridiplantae In total, eight groups of Within As for Alveolata Consistent with the pattern of OTUs, major protist orders recovered from the rRNA was the second most abundant phylatist in all community samples, at contributing the more to rRNA the (17.0 whole pro- at the rDNA level and 2000 m at the rRNA level, respectively. inance of four groups were revealed in all samples except for For the remaining minor groups, was ryngea (2 m) and deep wateralso (2000 m) predominant were in composed the exclusively of other water depths; other hand, tected at 700 m. were successfully identified with rRNA level than at the rDNA level (Fig. 3c). At both rRNA and rDNA levels, less than 1 % of the total community in each sample; the rDNA level (Fig. 3a). On the verticalwhereas the profile, highest the abundance predominant of group rRNA level; and 700 m; whilst and were more abundant at the rDNA level, whilst only found at 200deeper m waters (0.53 (700 %) and at 2000 m) the at rRNA the rDNA level, level. butgroups undetectable with in the surface and Viridiplantea and rDNA levels also varied with samples. Although vealed from the rRNA level, contrasting to the fact that Gonyaulacales more abundant in the surface waters than in other water depths. dominating order, more phycea had its lowest presencePeridiniales at the core(2 m) of waters, OMZ respectively. (550 m); whilst the highest presence of phyceae abundant at the rRNA level. At both rRNA and rDNA levels, the least of Synurophyceae ple, contrasting to theundetectable fact in that the surfacelevel. (2 m) and deeper (700 and3.3 2000 m) waters Community at comparison the rDNA The a levels varied with samples (Fig.was 4). more abundant at rDNAthe surface than waters. rRNA levels with the highest abundance presentappeared in at the coreface of water. OMZ and the highest of water of 2000 m. The remaining groups of on the surface water; its highest abundance at the rRNA level appeared in the deep the rDNA level (Fig. 2). depths at both the rRNA (65 5 5 25 10 15 20 20 25 15 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Di- . A few Gymno- erent OTUs ff ect the diversity ff Prasinophytes and were only present at the , dominated in all depths was the dominant order at the (Fig. 6a). At the core of 550 m, Prorocentrum dinoflagellates Prasinophytes Ciliates northern Chile, which could be caused by in 700 and 2000 m; 400 m was composed in our study are mainly composed of Gymnodiniales ff 13494 13493 occurred only at 200 and 400 m, but were not erent samples (Fig. 6). The density of OTUs was and ff Ciliates erent samples. At the rRNA level, high number of pho- , particularly Alveolata ff Radiolaria (gp I, II, III) and appeared at 2 and 200 m and their abundance decreased Alveolata erent samples at both rRNA and rDNA levels (Fig. 7). Surface appeared exclusively in the deep waters of 700, 2000 m and the Eustigmatales ff axis) among di erent from other water depths and the core of the OMZ formed ff y Ciliates were the most abundant group throughout the water column, but shifted Dinoflagellates Dinoflagellates Protists have diverse trophic life styles, and suboxic/anoxic waters acting as a stress The heatmap presented in a color-matrix illustrates the distribution of di UPGMA demonstrated a generally similar clustering pattern regarding to the protist 4 Discussion Naturally formed oxygen-depleted OMZ ecosystems representextreme one environments of and the are largely ocean’s most unexplored byproposed far. as Suboxic/oxicwaters have an been environmental stress leadingenergy to diversion habitat into compression, microbial loss of pathwaysberg, fauna in 2008). and Protists the are marine a ecosystemskaryotic very (Diaz diversified organisms, group, and and comprising Rosen- their all distributionResults major in single from the celled our eu- OMZ study regions showgeneral that, remain are like largely metazoan not unknown. zooplankton, the suboxic/anoxicdiversity favorable watersin and habitats total for number protists,thy of indicated that OTUs by the in the highest the diversity lowestbeen anoxic of community reported microeukaryotes core from of from the OMZ the 0.2–1.6 (550 µm core m). size It of fraction is has OMZ notewor- o could induce thegroups outgrowth due to of elevated certainFenchel bacterial abundance bacterivorous and at Finlay, mixotrophic ora 2008; below and shift the Edgcomb in heterotrophic oxycline protist and (Fenchel, communitythroughout 1990; Pachiadaki, the composition. water Distinct 2014), column distribution as a subsequently patternsexhibited result of of causing in adapting protist to our groups the study. verticalacross oxygen gradient oxygen were gradients, inregions agreement (Parris with et al., their 2014). The predominance in other oxic/suboxic a contamination of lysedever, cells the from leakage the of largerestimation lysed size at cells fraction the (Parris from rRNA etfor level larger as al., metazoans eukaryotes conducted 2014). (Levin, would in 2003) How- not ourhas and study. a been protists Reduced reported (Oris community diversity et previously.low Together al., DO with concentration 2012) our acts under findings, asspecies low an assemblages it DO to environmental can conditions sustain stress be the that ecosystem summarized selectively balance support that and certain processes in OMZs. the core of the OMZ, 48 % of the mostI, abundant OTUs II at and the rRNA III,ciliate level or or were nanoflagellate parasitic identified grazers. as dinoflagellates. At parasitic the The groups rDNA remaining level, as 52 % the consisted red block exclusively demonstrated, of from photosynthetic species inin the deeper surface waters. waters Phototrophic to diatoms parasitic and species gpI, II and III rRNA and rDNA levels, respectively. (clustering on the noflagellates diatoms also presented here, most likely silica detritus driftingcommunity from surface among waters. di water was verya di distinct branch apparentlysamples separating fell it into from adeeper the water rest single (700 and of clade, 2000 the m) withincommunity formed samples. structure two which The of separate shallower clusters 700 rest m at water400 four was the m) rRNA (200 more rather level; similar than and but to to the 400 the those m) deepest of and water shallower (2000 waters m) (200 at and the rDNA level. with depth and was replaced by scaled by color andcells) was the highly variable phylogenetic with di compositiontosynthetic of the most abundant OTUs (red surface, while core of the OMZ (Fig. 6b). of parasitic detected at the rRNA level.consisted At of the many hypoxic mixotrophic 550 protists m, such the as most abundant rDNA sequences 5 5 10 25 20 15 10 20 25 15 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Litosto- Rhizari- together was found and Prorocentrales only occurred in Rhizaria , a special group are generally surface- . Both had been proposed as Rhizaria Stramenopiles northern Chile (Parris et al., ff with diverse trophic lifestyles Apicomplexa erent suboxic/anoxic conditions ff Oligohymenophorea Alevolata seem more abundant in the suboxic Gymnodiniales Gymnodiniales and liations. This highly diverse supergroup ffi Stramenopiles 13496 13495 Perkinsea and , dominated at the upper and lower boundary of the ect was more apparent in the upper boundary of the , belonging to respective of dinoflagellate orders and Stramenopiles ff was most abundant in the surface water in our study. had the highest abundance in the core of OMZ, where (Wylezich and Jürgens, 2011). The latter group possibly acted Ciliophora Ciliophora Spirotrichea have known mutual symbiotic associations with planktonic had its confined distribution, may be a proof of the global distribution of and Bacillariophyta Synurophyceae liated with metazoans were frequently detected with high abundance due to , occurring mainly in the core of the OMZ, as this unicellular and spore- ffi ect of sustained oxygen depletion on protists was the most obvious in the ff Stramenopiles , which is an order of mixotrophic non-thecate dinoflagellates with the potential , two groups of erent geographical locations (Blackwell and Powell, 2000). Like anywhere else, Spirotrichea (Spero and Angel, 1991). It is interesting to find ff Alveolata In addition to the core of OMZ, the upper and lower boundary of the OMZ acts as an Most eukaryote community studies by far have been based on DNA-derived analysis, The e in both the upperwith and the lower dominant boundary mixotrophic of OMZ. The heterotrophic the upper boundary, and a higher abundance of PX-clade of a SAR supergroup with close phylogenetic a (Lynn, 2008) including OMZs (Parris et al., 2014). In addition, has been found to predominate in2014). all The depths relatively of high the nutrient OMZ andlower o low boundaries oxygen-deficiency of tensions the in theprovide OMZ upper more represent and harboring broader niches transition for zones, protist and communities. therefore might which cannot distinguish intact livingmaterials cells of from other species. inactive It or isquences lysed problematic a especially cells for and protist extracellular studies, sincethe many se- high copy numbers ofRNA-based RNA investigations genes have in revealed the a metazoan cell complex (Prescott, and 1994). metabolically Previous active marine matea OMZ, respectively. Some members ofsess these hydrogenosomes two groups belonging have tofacultative been anaerobes obligatory suggested and to anaerobes, have pos- been while reported other from di members are OMZ (400 m) as the oxic-suboxic transition interface. interface with a rich supply ofatively organic moderate matter ecological/physiological but threshold less for oxygenous certain stress, studies microbial representing have a groups. demonstrated rel- Previ- thatof the zooplankton edges (Morrison of et OMZ al., 1999) typicallyhad and support occurred that a apparent in high shifts the of density 2011). microbial oxic/anoxic communities interface In (Vetriani our et study, al., the 2003; edge Wylezich e and Jürgens, tion as isolated habitatscommunity contributing adaptive to to the this development stress. of a high degree of endemic as the host of some parasitic/symbiotic groups particularly harbored in the anoxic core. of forming protist group is comprisedprotists. almost Heterotrophic entirely of obligate endoparasitic pathogenic hypoxic diniales of forming blooms (Tester and Steidinger, 1997). Chrysophyceae anoxic core of thefrom OMZ, grazing as food it web not toProrocentrales only a led symbiosis/parabiosis-based to lifestyle a asciliate well. reduced classes, For microbial are instance, diversity, butby two a major the shift groups most in abundant the group core of of photosynthetic the OMZ in our study, followed dwelling, and their appearancecation in of the resting cysts, deep whichthrough waters adverse act is conditions, as i.e. likely an the resulted adaptive lowjor DO strategy from group in for the our of dinoflagellates case amplifi- eukaryotic to (Anderson,generally survive 1984). microorganisms, occur Another predominantly ma- in surfacein waters di in spite ofphotosynthetic their ubiquitous distribution The fact that and ans waters and the lattersymbiotic/parasitic is protist a groups parasite mentioned of aboveprevious bivalve in reports mollusks. our that study The the is detectionOMZs presence in of (Levin, of agreement 2003) these an and to distinct further endosymbiotic the proves lifestyle that seems anoxic conditions widespread at in the core of OMZs func- 5 5 15 25 20 10 15 10 25 20 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ects ff ering opportu- ff and heterotrophic Chytridiumlagenaria for o Melville Cryptophyta erent trophic processes and integrated ff 13498 13497 ect the microbial populations. A clear shift from photosynthetic erence between the total and active proportions of eukaryotes ff ff We thank the captain and crew of the R/V were exclusively found at the rRNA level but undetected at the rDNA erent from the surface water which harbors a large amount of photosyn- ff erent trophic lifestyles, such as predation, parasitism and symbioses. These ff DO concentrations primarily drive the nutrient and energy flow in marine ecosys- in: Seafood Toxins, edited by:USA, Ragelis, 125–138, E. 1984. P., American Chemical Society, Washington, DC, diversity assessments: morphology compared with cloningrRNA and genes direct and ITS pyrosequencing of regions using 18S 7, the 244–255,2013. conspicuous tintinnid ciliates as a case study, ISME J., Schenk, Inoculum, Mycologia, 51, 19–20, 2000. Mohideen, A. S., McGarrell, D. M.,database Marsh, project: T., Garrity, improved G. alignments M.,37, and and D141–D145, Tiedje, new 2009. J. tools M.: The for ribosomal rRNA analysis, Nucl. Acids Res., Bachy, C., Dolan, J. R., Lòpez-García, P., Deschamps, P., and Moreira, D.: Accuracy of protist Blackwell, W. H. and Powell, M. J.: Internal sporangial proliferation in nity for sampling during661813) the and FLUZiEcruise. Supports the from Strategic(Grant Hong No. Priority Kong XDB06010202) Research RGC and Program (GRF66191241406180)are the also and of National acknowledged. the Natural Chinese Science Foundation Academy of of China Sciences (Grant No. References Anderson, D. M.: The roles of dormant cysts in toxic dinoflagellate blooms and shellfish toxicity, Cole, J. R., Wang, Q., Cardenas, E., Fish, J., Chai, B., Farris, R. J., Kulam-Syed- are possible survival strategiesture that and regulate in the endemic turnthe microbial mediate whole community and the active struc- biogeochemical protistare community cycling needed suggests in that to OMZs. analysis reflect of Discrepancyvironmental the both between real microbial rRNA ecological and studies. rDNA rolesharbor The of a marine vast diverse microbial assemblage and of communitiestion relatively protists in and and unexplored en- represent specialization. OMZ a Therefore, source ecosystems help studies for focusing to genetic on diversifica- understand this theand special ecological metabolic environment processes and could that evolutionary prevail in strategy oxygen-deficient of marineAcknowledgements. microbial environments. adaptations protist community in anoxic/sulfidic2011), waters however, the in di thewas Black not Sea investigated. In (Wylezich our and study, both Jürgens, photosynthetic Phyllopharyngea our study. It is notellates surprising in to the find surface waters, much while moreciliates the abundance (rRNA abundance of of level) photosynthetic parasitic increased dinoflag- dinoflagellatesbe with and a water the few depth potential especially hostsence in for of the the phototrophic OMZ. Dinoflagellates parasitic Ciliatessinking in Dinoflagellates. could down the On to deep the the watersand other water was rDNA column. hand, possibly levels In the dead demonstrates our skeletons the pres- a study, oxic relative the waters homogeneity UPGMA below of clusteringOMZ, the suggesting the at euphotic that zone, microbial both the distinct rRNA community oxygenon from in gradient those both presented in in total the thestrikingly and OMZ anoxic exerts di metabolically core similar of active e the protistthetic protists. communities. The high All abundances theproved of active their water protists depths importance uncovered from are conditions as the OMZ (Stoeck a further et majorparasitic/symbiotic al., trophic trophic 2007). link lifestyles in The in thethe reduced the suboxic/anoxic waters oxygen community microbial of deficiency OMZ diversity loop occurring suggestsbial in and under that communities the presence hypoxic and OMZ leads of exerts tobiogeochemical a modulation selective of cycles pressure di as on a themany micro- di result of community adaptation. Protists are capable of level. This highlights the necessityfor of a integrated comprehensive rRNA- community and study, since rDNA-basedmunity the investigations and latter might targets not the be whole sensitive complex enough com- to detect speciestems with and low selectively abundance. a groups in the surface to parasitic protist groups in the deeper waters was observed in 5 5 10 15 20 25 15 20 25 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | are Costa Rica, ff Duboscquella nities to other eukary- ffi ect of oxygen minimum zone formation ff northern Chile, Front. Microbiol., 5, 543, ff ects of the Costa Rica Dome, Deep-Sea Res. ff 13500 13499 ) gene in the Oxygen Minimum Zone o hao of small eukaryotes in deep-sea Antarctic plankton, Nature, 409,hydrothermal 603–607, sediment 2001. and experimentalAcad. microcolonizers Sci. at USA, the 100, mid-Atlantic 697–702, Ridge, 2003. P. Natl. 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L. and J.: Jürgens, K.: Fingerprinting Protist microbial diversity in assemblages suboxicWyrtki, from and K.: sulfidic the Upwelling in waters the of Costa the Rica Black Dome, Sea, Fish Bull., 63, 355–372, 1984. Spero, H. J. and Angel, D. L.: Planktonic sarcodines: microhobitat forStewart, oceanic F. J., dinoflagellates, Ulloa, J. O., and Delong, E. F.: Microbial metatranscriptomics in aStoeck, permanent marine T., Stoeck, T., Zuendorf, A., Breiner, H. W., and Behnke, A.: ATester, P. A. molecular and approach Steidinger, to K. A.: 5 10 20 15 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | sequences (b) and rDNA (a) sequences achieved by pyrosequenc- (b) 13504 13503 and rDNA liation of all the rRNA (a) ffi erent water samples collected in the Costa Rica Dome. ff Relative abundance and a Rarefaction curves for rRNA erent water samples collected in the Costa Rica Dome. OTU was defined with 3 % as ff value. ff achieved by pyrosequencing for di Figure 2. Figure 1. ing for di cuto Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Stra- , (c) Dinophyceae , (b) erent water samples collected in the ff Ciliophora , (a) 13506 13505 Alveolata liation of the 10 most abundanct OTUs based on rRNA ffi erent water samples collected in the Costa Rica Dome. ff asseblages based on rRNA (left panel) and rDNA (right panel) sequences (d) Phylogenetic compositions of Relative abundance and a achieved by pyrosequencing for di menopiles Costa Rica Dome. Figure 4. and rDNA sequences achieved by pyrosequencing for di Figure 3. Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | levels. Red blocks represent the most (b) 13508 13507 and rDNA (a) erent water samples collected in the Costa Rica Dome. ff erent phylogenetic compositions (right panel). Relative abundance in ff score ((each value–mean of row)/standard deviation) is indicated by color as z Hierarchical dendrogram illustration for the abundance and distribution of OTUs erent samples at rRNA Comparison of major ten orders of protists based on rRNA and rDNA sequences ff among di shown in the legend (bottom right). the form of abundant OTUs with di Figure 6. Figure 5. achieved by pyrosequencing for di Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | erent ff erent samples based ff 13509 UPGMA demonstating protistcommunity similarities among di water samples collected in the Costa Rica Dome. Figure 7. on rRNA (left panel)and rDNA sequences (right panel) achieved by pyrosequencing for di