ORIGINAL RESEARCH Microbial eukaryotic diversity and distribution in a river plume and cyclonic eddy-influenced ecosystem in the South China Sea Wenxue Wu1,2,3, Lei Wang1,2, Yu Liao1,2 & Bangqin Huang1,2 1Key Laboratory of Coastal and Wetland Ecosystems, Xiamen University, Xiamen, China 2State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen, China 3Institute of Oceanography, National Taiwan University, Taipei, Taiwan.

Keywords Abstract 18S rDNA, mesoscale process, microbial , South China Sea. To evaluate microbial eukaryotic diversity and distribution in mesoscale pro- cesses, we investigated 18S rDNA diversity in a river plume and cyclonic eddy- Correspondence influenced ecosystem in the southwestern South China Sea (SCS). Restriction Bangqin Huang, College of the Environment fragment length polymorphism analysis was carried out using multiple primer and Ecology, Xiamen University, South sets. Relative to a wide range of previous similar studies, we observed a signifi- Xiang’an Road, Xiamen 361102, China. cantly higher proportion of sequences of pigmented taxa. Among the photosyn- Tel: +86 592 2187783; thetic groups, Haptophyta accounted for 27.7% of the sequenced clones, which Fax: +86 592 2180655; E-mail: [email protected] belonged primarily to . Unexpectedly, five operational taxo- nomic units of Cryptophyta were closely related to freshwater species. The Chlorophyta mostly fell within the Prasinophyceae, which was comprised of six Funding Information clades, including Clade III, which is detected in the SCS for the first time in This work was supported by the Natural this study. Among the photosynthetic stramenopiles, Chrysophyceae was the Science Foundation of China (Nos. 41330961 most diverse taxon, which included seven clades. The majority of 18S rDNA and 41176112), the Doctoral Fund of the sequences affiliated with the Dictyochophyceae, Eustigmatophyceae, and Pelago- Ministry of Education of China (No. phyceae were closely related to those of pure cultures. The results of redun- 20130121110031) and the Special Fund of the State Oceanic Administration of the dancy analysis and the permutation Mantel test based on unweighted UniFrac People’s Republic of China (No. 530-03-02- distances, conducted for spatial analyses of the Haptophyta subclades suggested 02-03). that the Mekong River plume and cyclonic eddy play important roles in regu- lating microbial eukaryotic diversity and distribution in the southwestern SCS. Received: 21 March 2015; Revised: 9 July 2015; Accepted: 14 July 2015

MicrobiologyOpen 2015; 4(5): 826–840 doi: 10.1002/mbo3.282

Introduction lar have been widely demonstrated to enhance nutrient inputs to the surface ocean and increase new production Microbial , pivotal components of marine (Falkowski et al., 1991; Moran et al., 2001). However, ecosystems, are ubiquitous in the world ocean (de Vargas although numerous studies have been conducted to deter- et al., 2015). Mesoscale features, such as river plumes and mine particle flux in cyclonic eddies, and only a few cyclonic eddies, are widespread in the global ocean and focused on the phylogenetic diversity of planktonic organ- are critical components that enhance the efficiency of the isms. Baltar et al. (2010) investigated prokaryotic assem- biological pump (Oschlies and Garcßon, 1998; Chelton blage structure and activity in mesoscale eddies in the et al., 2011; Kang et al., 2013). Cyclonic eddies in particu- Canary Current system, revealing that these eddies had

826 ª 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. W. Wu et al. Microbial Eukaryotes in Mesoscale Processes distinct community compositions. In the Sargasso Sea, a (Sea-Bird) were used to obtain in situ vertical profiles of distinguished phylogenetic difference in bacterioplankton temperature, salinity and fluorescence. Irradiances were has been clearly demonstrated by community profiling obtained from Moderate Resolution Imaging Spectrora- according to relative abundances of pyrosequenced ampli- diometer (MODIS) observation and an empirical formula cons (Nelson et al., 2014). Despite their important (Morel, 1988). Mixed layer depth was defined as the first ecological roles (Massana, 2011; Caron et al., 2012), depth where the temperature was 0.2°C lower than that microbial eukaryotic diversity and distribution have been at surface (Steinhoff et al., 2010). rarely reported in the cyclonic eddy-influenced ecosystem. For molecular analyses, two samples were collected Among marine microbial eukaryotes, the pigmented from surface water and the deep chlorophyll maximum taxa are extremely diverse and are recognized as important (DCM) layer at each of the nine sampling stations. primary producers in open oceans (Li, 1994; Vaulot et al., Briefly, after being filtered through a 10-lm pore size 2008). However, the results of molecular surveys are com- mesh, 8–10-L seawater samples were prefiltered through monly dominated by heterotrophs when polymerase chain 5-lm pore size polycarbonate filters (Millipore, Ireland). reaction (PCR)-based approaches are used (Dıez et al., Subsequently, the penetrated seawater samples were fil- 2001; Moon-van der Staay et al., 2001; Not et al., 2007; tered through GF/F filters (Whatman, USA). Samples Stoeck et al., 2009). Methodological bias was even were immediately frozen in liquid nitrogen, brought back observed using novel flow cytometry sorting approaches to the laboratory and stored at 80°C until analyses. (Marie et al., 2010), with the diversity of pigmented For pigment analyses, six samples were collected at var- microbial eukaryotes typically being dominated by several ious depths (surface, 25, 50, 75, 100, and 150 m). One of taxa, including the Prymnesiophyceae, Mamiellophyceae, the six selected layers was replaced by the DCM at each and , in sorted samples. The utility of station based on real-time hydrological and fluorescence molecular methods (e.g., 18S rDNA clone library construc- profiles obtained from CTD sensors. Seawater samples of tion and next-generation sequencing) is considered to be 4–16 L were directly filtered onto GF/F filters at a pres- severely limited due to the use of universal primers to sure of lower than 150 mm Hg. Samples were immedi- address photosynthetic microbial eukaryotic diversity. ately frozen in liquid nitrogen on board and protected From August to September 2007, a cyclonic eddy in from light. Filters were then stored at 80°C in the labo- the southwestern South China Sea (SCS) was well ratory until further processing. depicted (Hu et al., 2011), and its ecological characteris- Nutrient samples and pigment samples were collected tics were discussed (Chen et al., 2009; Zhang et al., 2011). together. Seawater samples were filtered through 0.45-lm Chen et al. (2010) reported the biogeochemical effects pore size acetate-fiber filters, and they were analyzed associated with the Mekong River plume in this area. within 24 h on board. Here, we further characterize the genetic diversity of microbial eukaryotes in these mesoscale process-influ- DNA extraction, cloning, and sequencing enced environments. We performed 18S rDNA cloning and sequencing associated with restriction fragment Filters were thawed at room temperature, and DNA was length polymorphisms (RFLPs), revealing an abundance extracted according to Countway et al. (2005) using the of sequences affiliated with pigmented taxa in these phenol:chloroform:isoamylalcohol (Sigma-Aldrich, USA) mesoscale-influenced ecosystems for the first time. Fur- method. PCR and cloning were performed in two steps. thermore, using redundancy analysis (RDA) and the First, four universal eukaryotic primer sets (Table 1 and permutation Mantel test, we analyzed the effects of Fig. S1) were selected to amplify the near-full-length 18S mesoscale processes on the regulation of diversity and dis- rDNA fragments of the DNA mixture containing the 18 tribution of the major pigmented group Haptophyta. This samples. PCR amplification was carried out using a Go-Taq study significantly expands upon the current knowledge Flexi DNA Polymerase Kit (Promega, USA). Thermal of microbial eukaryotic diversity and distribution in these cycling was performed with a thermocycler (Bio-Rad, Mex- mesoscale process-influenced environments. ico) using the following protocol: one cycle at 95°C for 5 min, 30 cycles at 94°C for 40 sec, 55°C for 40 sec, 72°C ° Materials and Methods for 1.5 min, and a final extension at 72 C for 10 min, followed by a hold at 4°C. For each of the four primer sets, three separate reactions were performed and the PCR Sample collection products were pooled for construction of the following Sampling was conducted at nine stations in the south- four clone libraries: WS071 (Euk328f+1498R), WS072 western SCS on board from September 2 through 8, 2007 (EukA+1498R), WS073 (82F+1498R), and WS074 (Fig. 1). CTD casts equipped with an SBE-911 instrument (Euk328f+Euk329r). Clone libraries were constructed using

ª 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. 827 Microbial Eukaryotes in Mesoscale Processes W. Wu et al.

Depth 25˚ (m) 14˚ 30 N 50 N 100 250 29.75 500 20˚ 750 13˚ Y90 Y93 Y96 29.5 na Sea 1000 1250

1500 Vietnam 29.25 South Chi 2000 12˚ Y10 Y13 15˚ 2500 Y16 3000 29 3500 4000 28.75 10˚ 4500 11˚ Y30 Y33 Y36 5000 5500 28.5 6000 Mekong River plume 6500 5˚ 10˚ Ocean data view 28.25 105˚ 110˚ 115˚ 120˚E 108˚ 109˚ 110˚ 111˚ 112˚ 113˚E

Figure 1. Sampling locations in the southwestern South China Sea (Schlitzer, 2014). Dots on the map correspond to the sampled stations for molecular analyses and pigment measurements. The colored background depicts the real-time surface water temperatures (°C) at all stations investigated during the cruise in 2007.

Table 1. List of primers used in this study. showed the highest diversity among the four primer sets. Direction Primer Sequence (50–30) Source Eighteen clone libraries were constructed following the above-mentioned procedures. For each clone library, 100 Forward EukA AACCTGGTTGATCCTGCCAGT Medlin et al. (1988) positive clones were randomly selected and partially Euk328f ACCTGGTTGATCCTGCCAG Moon-van sequenced in one sequencing reaction. der Staay et al. Nucleotide sequences identified in this study were (2001) deposited in GenBank database under the accession num- 82F GAAACTGCGAATGGCTC Lopez-Garc ıa bers KP404633–KP404914 (ca. 1800 bp) and KP404915– et al. (2003) KP405223 (ca. 900 bp). Reverse EukB TGATCCTTCTGCAGGTTCACCTAC Medlin et al. (1988) Euk329r TGATCCTTCYGCAGGTTCAC Moon-van der Phylogenetic analyses Staay et al. (2001) Sequences were analyzed with KeyDNAtools (www.keyd- 1498R CACCTACGGAAACCTTGTTA Lopez-Garc ıa natools.com) for detecting chimeras. Partial fragments of et al. (2003) putative chimera were then individually examined using BLAST (Zhang et al., 2000). Good-quality, near-full- a TA cloning kit (TaKaRa, China), following the length sequences identified with the four primer sets were manufacturer’s instructions. For each clone library, PCR used for the following phylogenetic analyses. Sequences amplification products of 400 positive clones were digested aligned with MAFFT 7 (Katoh and Standley, 2013) were with the restriction enzymes HaeIII and HhaI (TaKaRa) and grouped into OTUs, as defined using Mothur v1.30.0 run on a 3% agarose gel for RFLP analysis. For highly abun- (Schloss et al., 2009), at 98% similarity. Venn diagrams dant RFLP patterns, 1–3 clones were sequenced with an ABI were generated to compare the number of overlapping 3730xl system (Applied BioSystems, USA) to address the OTUs among the four clone libraries. possibility that the patterns were present in different species Subsets of sequences at different taxonomic levels were (Lekunberri et al., 2014). Two sequencing reactions were chosen based on assignment using PR2 database (Guillou performed for each selected clone to obtain an approxi- et al., 2013). Sequences from each subset, together with mately 1800-bp fragment. For phylogenetic analyses, these their closest relatives, were realigned using MAFFT 7. sequences were re-grouped into operational taxonomic Poorly aligned and divergent regions were eliminated units (OTUs) based on 98% similarity. using Gblocks v0.91b (Castresana, 2000), with a mini- Second, PCR amplification was separately performed mum block of five and allowed gap positions equal to for each of the 18 samples using the primer set that half. Nest models of DNA substitution and associated

828 ª 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. W. Wu et al. Microbial Eukaryotes in Mesoscale Processes parameters were estimated using jModelTest 2 (Darriba Pigment and nutrient analyses et al., 2012), and maximum likelihood (ML) phylogenetic trees were constructed using PhyML 3.1 (Guindon et al., Pigment concentrations were determined using high-per- 2010), with bootstrap analysis of 1000 replicates. Bayesian formance liquid chromatography (HPLC), according to interferences were calculated with MrBayes v3.2.1 (Ron- previously decribed methods (Zapata et al., 2000; Mendes quist et al., 2012) using the GTR + G + I model and two et al., 2007). Phytoplankton pigments were extracted runs of 1 million generations. The first 25% of the using N,N-dimethylformamide (Furuya et al., 1998), and samples were discarded as ‘burn-ins’. the extracts were then passed through GF/F filters of 13 mm in diameter to remove debris. For each sample, 600 lL of extract was mixed with a 1 mol L 1 ammo- Distribution patterns of Haptophyta nium acetate solution of an equal volume. Pigment con- subclades centrations were determined with an Agilent series 1100

Unique Haptophyta sequences (100% sequence similarity) HPLC system and a 3.5-lm Eclipse XDB C8 column (Agi- obtained from the 18 separate clone libraries were used lent Technologies, USA). The analytical methods based for spatial analyses. Relative contributions of different on a gradient elution procedure were performed with a Haptophyta groups to the eukaryotic communities were methanol:ammonium acetate (1 mol L 1) mixture calculated to identify distribution variations in the differ- (80:20) and a 100% methanol solution (solvents A and B, ent environments. According to the relative abundances respectively). Standards supplied by DHI Water and Envi- of Haptophyta phylotypes, the distribution patterns were ronment (Denmark) were used to identify and quantify illustrated by a heatmap coupled with an unweighted of the following pigments: 190-butanoyloxyfucoxanthin, pair-group method with arithmetic mean (UPGMA) den- 190-hexanoyloxyfucoxanthin, alloxanthin, a-carotene, b- drogram of the 18 stations based on Bray-Curtis dis- carotene, chlorophyll a, chlorophyllide a, chlorophyll b, tances. RDA, combined with the ‘envfit’ function in vegan chlorophyll c1 + chlorophyll c2, chlorophyll c3, diadinox- packages (Oksanen et al., 2013) was performed using R anthin, diatoxanthin, divinyl chlorophyll a, fucoxanthin, program (R Core Team 2014). The permutation Mantel lutein, neoxanthin, peridinin, prasinoxanthin, violaxan- test was used to estimate the correlations between the thin, and zeaxanthin. The relative contributions of the environmental distances and unweighted UniFrac dis- different phytoplankton groups to the total chlorophyll a tances (Lozupone and Knight, 2005) based on a neigh- (Chl a) biomass were estimated using CHEMTAX matrix bor-joining tree. factorization (Mackey et al., 1996). Nine phytoplankton

3 (A)Potential density anomaly σ0 (kg/m ) (B) 0 200 400 6001900 1950 2000 2050 26 0 Chlorophyll a (ng/L 21 Y36 19 30 Y33 Y16 25 Y90 Y30 Y13 Y10 20 Y93 Y96 22 24

(°C) 50 θ

25 23

23 Depth (m) 22

100 Y90 Y93

Potential temperature 20 21 Y96 Y10 Y13 Y16 25 20 Y30 Y33 Y36 150 24 15 Ocean data view 19 32 33 34 Salinity

Figure 2. (A) T-S diagram of hydrographic stations and (B) vertical profiles of chlorophyll a.

ª 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. 829 Microbial Eukaryotes in Mesoscale Processes W. Wu et al.

86 WS071.041 0.1 92 Haptolina brevifilum Kawachi AM490995 parkeae Kawachi AM490994 Euk328f+1498R 100 WS074.031 EukA+1498R 82F+1498R Chrysocampanula spinifera AM491022 Euk328f+Euk329r 100 WS072.030

99 chiton PLY146A AM491029 86 WS072.024 Prymnesium minor PLY304 AM491010 97 100 WS073.052 Prymnesiales B1 Prymnesium patelliferum L34671 100 84 WS074.036 Haptolina hirta UIO030 AJ246272 64 99 WS072.025

65 Chrysochromulina scutellum UIO046 AJ246274 WS073.122

99 ST3500.080 KF129663 WS073.125 54 Chrysochromulina simplex UIO047 AM491021 100 WS072.028 99 94 Chrysochromulina rotalis UIOP16 AM491025 WS072.029 99 Prymnesiales B2

Chrysochromulina parva CCMP291 AM491019 Prymnesiophyceae 100 WS072.026 ST3500.121 KF129696 99 WS073.105 pelagicus PLY182g AJ246261 100 74 WS073.046 Cocco Biosope T123.034 FJ537301

100 WS074.038 Isoch Biosope T33.008 FJ537311 E 100 WS074.041 Biosope T33.039 FJ537312 99 D 100 WS074.039

100 Phaeocystis antarctica MM E2C1 JN381495 WS074.033 78 99 Phaeocystis sp. PLY559 AF180940 100 WS074.035 Phaeocystales

97 Pavlova salina AF106059 WS073.048 100 A 100 Pavlova salina L34669 Pavlovo 100 WS073.049 Biosope T65.100 FJ537342

100 WS073.051 Hap-2 Micromonas sp. RCC299 HM191693 100 Micromonas pusilla CCMP1545 AY954994

Figure 3. Phylogenetic tree for Haptophyta based on maximum likelihood analysis of 18S rDNA sequences (1696 positions) retrieved from the southwestern South China Sea (bold). The tree is based on a TrNef + G + I model of DNA substitution with a gamma distribution shape parameter of 0.495 (I = 0.477), and R(b)[A–G] = 1.9121, R(e)[C–T] = 4.0299 and 1.0 for the other substitution rates. Bootstrap values of >50% (1000 replicates) are shown, and the filled circles at the nodes indicate Bayesian posterior probabilities of >0.9. The tree is rooted by two Micromonas sequences. The multi-value bar charts correspond to the number of clones represented by each operational taxonomic unit. The scale bars indicate 0.1 substitutions per base (black line) and one clone (color bar chart). Cocco, ; Isoch, ; Pavlovo, Pavlovophyceae.

830 ª 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. W. Wu et al. Microbial Eukaryotes in Mesoscale Processes groups, including dinoflagellates, diatoms, haptophytes_8, 23.4% to the total recovered sequences. Sequences belong- haptophytes_6, chlorophytes, cryptophytes, prasinophytes, ing to heterotrophs accounted for only 17.4% of the total Synechococcus, and Prochlorococcus, were identified to be sequenced clones. Notably, only 6 OTUs were shared by responsible for the total Chl a biomass (Goericke and the four clone libraries (Fig. S3). Primer bias is widely Repeta, 1993; Latasa, 2007). The major differences known as one of the main shortcomings of PCR-based between hyptophytes_8 and hyptophytes_6 were the diversity surveys (Potvin and Lovejoy, 2009). The use of distinct concentrations of 190-butanoyloxyfucoxanthin multiple primer sets has been suggested as an approach for (abundant in haptophytes_8) and monovinyl chlorophyll improving the recovery of protistan sequences (Stoeck c3 (present in haptophytes_6; Zapata et al., 2004). Nutri- et al., 2006). This method also appears to be essential for ent concentrations were analyzed using a Technicon AA3 photosynthetic microbial eukaryotes, according to the Auto-Analyzer (Bran-Luebbe, Germany). results of this study. For example, the primer set 82F and 1498R amplified all recovered Cryptophyta sequences, but Results and Discussion Dictyochophyceae sequences were only detected in the clone library using Euk328f and Euk329r. To elucidate how mesoscale processes regulate the major microbial eukary- Hydrographic characteristics otic groups, particular focus was placed on the Hapto- A well-developed cyclonic eddy was identified in the phyta. The primer set 82F+1498R, which detected the southwestern SCS during the survey period (Fig. 1), and greatest number of OTUs (97 OTUs), was selected for the its three-dimensional structure and physical properties following PCR amplifications of the 18 separate samples. were thoroughly characterized by Hu et al. (2011). Satel- lite altimeter data showed that the core of this eddy was Photosynthetic microbial eukaryotic stably located at 110.8°E, 12.2°N and that the horizontal diversity measurements were approximately 60–90 km for 2 weeks, from August 26th to September 10th, 2007 (Hu et al., Sequences affiliated with the Haptophyta belonged pri- 2011). Accordingly, we found that all investigated stations marily to Prymnesiophyceae (Fig. 3). Of the seven in this study were influenced by the moving cyclonic eddy detected Prymnesiophyceae clades, Prymnsiales B1 and B2 to some extent. The T-S diagram revealed vertical and were characterized by higher diversity. Previously, we horizontal variations in hydrography among the nine sam- conducted a relatively complete survey of the SCS, rang- pled stations (Fig. 2A). Due to the strong influences of the ing from the southernmost Nansha Islands to the north- plume, the highest Chl a concentration of 2026.9 ng L 1 ernmost coast (Wu et al., 2014a), and the Haptophyta was observed at a 28 m depth at station Y30 (Fig. 2B). At was found to be the most diverse group of photosynthetic the three stations (Y10, Y13, and Y90) characterized by microbial eukaryotes. Surprisingly, sequences affiliated lower surface temperatures, all of the DCM depths were with the order Prymnesiales B1 that were frequently shallower than 50 m, indicating stronger influences of the detected in this area have been rarely reported in previous cyclonic eddy. However, Y33, Y36, and Y96 possessed surveys of the SCS (Yuan et al., 2004; Cheung et al., deeper DCMs at 85 m (527.8 ng L 1) and 75 m 2008). Coccolithales and Clade E, represented by the (551.1 ng L 1, Y36; 478.6 ng L 1, Y96). The highest Chl a clones WS073.046 and WS074.041, respectively, were concentrations of Y93 and Y16 were 410.6 ng L 1 at 25 m detected for the first time in the SCS in this study. Nota- and 267.9 ng L 1 at 50 m, respectively; however, we bly, clones of Clade E, Clade D (WS074.039) and obtained samples at 50 m (Y93) and 80 m (Y16) with Chl Isochrysidales (WS074.038) were closely related to envi- a concentrations of 164.6 and 320.2 ng L 1. ronmental sequences detected from the oligotrophic southeastern Pacific Ocean, with sequence similarities of 100%. One OTU affiliated with the Phaeocystales pos- Community structures sessed the greatest number of clones, totaling 14, and In total, 282 near-full-length 18S rDNA sequences were was represented by WS074.033. Clone WS073.051 was obtained from the four clone libraries using mixed DNA affiliated with the recently established Hap-2, sharing templates and different primer sets. Remarkably, the 100% sequence similarity with Biosope T65.100, support- majority of the sequences identified were associated with ing the existence of deep-branching novel lineages and photosynthetic groups (Fig. S2). The Haptophyta was the high diversity of the Haptophyta in the subtropical–tropi- most abundant taxon, accounting for 27.7% of the cal environment (Egge et al., 2015a; Egge et al., 2015b). sequenced clones on average. Photosynthetic stramenopiles Thirty-one Cryptophyta sequences were identified and (including the Bacillariophyceae, Pelagophyceae, Chryso- were grouped into 15 OTUs. Surprisingly, 5 OTUs were phyceae, and Dictyochophyceae) contributed an average of closely related to freshwater species (Fig. 4). Specifically,

ª 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. 831 Microbial Eukaryotes in Mesoscale Processes W. Wu et al.

0.1 99 Cryptophyta sp. CR-MAL11 FJ884690 WS073.036 Euk328f+1498R 58 EukA+1498R 100 Teleaulax amphioxeia KF734087 82F+1498R 100 WS073.037 Euk328f+Euk329r nannoplanctica FM876311 Teleaulax 78 100 WS074.028

99 Cryptophyceae sp. CCMP2293 GQ375265 WS073.031 73 Falcomonas 98 sp. AF508273 WS073.038 baltica AB241128 61 100 sp. CCMP1868 AF508276 Rhodomonas 100 WS073.035

100 nordstedtii AF508269 WS073.023 98 95 Chroomonas mesostigmatica AF508268

100 WS073.022 Chroomonas

65 marssonii EU163586 WS073.019 100 paramecium L28811

99 WS073.021 Cryptomonas 100 100 Biosope T39.105 FJ537320 WS073.020 93 PFF1AU2004 DQ244012 51 100

WS073.039 Unknown clade sp. AY705740 100 Goniomonas sp. AY360459 53 100 Goniomonas sp. ATCC-50108 AY360454 WS073.030 Goniomonadales 99 99 Uncultured AJ536451

WS072.009 Goniomonas 100 Goniomonas pacifica AF508277 100 WS073.029

100 Micromonas sp. HM191693 Micromonas pusilla CCMP1545 AY954994

832 ª 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. W. Wu et al. Microbial Eukaryotes in Mesoscale Processes

Figure 4. Phylogenetic tree for Cryptophyta based on maximum likelihood analysis of 18S rDNA sequences (1615 positions) retrieved from the southwestern South China Sea (bold). The tree is based on a TrN + G + I model of DNA substitution with a gamma distribution shape parameter of 0.67 (I = 0.49), and R(b)[A–G] = 2.2363, R(e)[C–T] = 4.557 and 1.0 for the other substitution rates. Bootstrap values of >50% (1000 replicates) are shown, and the filled circles at the nodes indicate Bayesian posterior probabilities of >0.9. The tree is rooted by two Micromonas sequences, and sequences that originated from freshwater are marked in cyan. The multi-value bar charts correspond to the number of clones represented by each operational taxonomic unit. The scale bars indicate 0.1 substitutions per base (black line) and one clone (color bar chart). three of the five detected Cryptomonadales genera 2009). Our frequent identification of Clade IX sequences (Teleaulax, Chroomonas and Cryptomonas) contained in filtered samples from the SCS using universal primers sequences that shared 100% similarity with 18S rDNA was unexpected (Wu et al., 2014a; Wu et al., 2014b). The sequences from freshwater organisms. Such a large number limited knowledge of Clade IX highlights the need for of 18S rDNA sequences related to freshwater species has increased culture isolation efforts to characterize common not been previously reported in marine environments. species in molecular studies (Pedros-Ali o, 2006). Cryptophyta is well known to be extremely diverse in fresh- The photosynthetic stramenopiles were the most water environments, and some taxa, such as Cryptomonas diverse groups, containing 66 sequences grouped into 39 are widespread in lakes (Eiler et al., 2013). Goes et al. OTUs (Fig. 6). Among the Chrysophyceae, sequences of (2014) have reported the sporadic presence of Cryptophyta Clades G and I shared high similarities of above 99% with in the Amazon River estuary and in a plume in the western environmental references detected in the southeastern tropical North Atlantic. High abundances and biomass Pacific Ocean, except for WS073.092. A number of Bacil- have been frequently reported in the Amazon River estuary; lariophyceae sequences were detected in these size-frac- however, strikingly small biomass contributions of the tioned samples. The clone WS073.081 clustered with Cryptophyta have been observed in the river plume far Biosope T123.033 as a novel group sharing 100% similar- from the Amazon River estuary. Similarly, higher biomass ity. Notably, the similarity of WS073.081 to the second contributions were observed in the DCMs of the stations most closely related sequence in GenBank database with lower salinity levels in this study (Fig. S4), and dropped sharply to 91%. The majority of sequences affili- the contributions of the Cryptophyta were decreased ated with the Dictyochophyceae, Eustigmatophyceae, and accordingly with increasing distances from the Mekong Pelagophyceae were the most closely matched to River estuary (e.g., 3.31, 3.29 and 1.87% in the DCMs of sequences from cultures. One clone was recovered for Y30, Y33 and Y36, respectively). It is reasonable to hypothe- each of Xanthophyceae, Synurophyceae, and Bolido- size that the Cryptophyta sequences that we detected origi- phyceae. Generally, Dictyochophyceae, Eustigmatophyceae nated from species transported by the Mekong River plume. Xanthophyceae, Synurophyceae, and Bolidophyceae are Furthermore, it could also be inferred from our results that rarely identified in 18S rDNA clone libraries and seem to these Cryptophyta species survive not only in freshwater be ‘rare species’ in molecular surveys. In fact, numerous systems but also in marine environments. Among the order 18S rDNA sequences from pure cultures have proven dif- Goniomonadales, nine sequences (3 OTUs) affiliated with ficult to detect in molecular surveys, and the reason for the Goniomonas were closely related to seawater spe- this difficulty has been primarily attributed to culture bias cies, with sequence similarities of above 99%. Additionally, (del Campo et al., 2014). Our results demonstrate the two OTUs represented by WS073.020 and WS073.039 were recovery of numerous environmental sequences from unknown clades that shared 100% similarity with their these taxa in these mesoscale process-influenced systems, references, Biosope T39.105 and PFF1AU2004, respectively. providing new insights into the diversity and distribution The Chlorophyta fell primarily within the Prasino- of these organisms. phyceae, which was comprised of six clades, Clades I, III, VI, VII, and IX and Mamiellophyceae (Fig. 5). Clade III Haptophyta communities in relation to identified in this study is a novel clade that has not been environmental variables previously reported in the SCS (Yuan et al., 2004; Cheung et al., 2008; Wu et al., 2014a). Among the Chlorophyta, The separate clone libraries using the primer set sequences affiliated with Micromonas, Ostreococcus, and 82F+1498R were found to contain abundant Haptophyta Bathycoccus are ubiquitous and are commonly detected in lineages and they were extremely similar to the taxa various habitats. Surprisingly, no sequences of these three detected using the four different primer sets (Fig. 7). A genera of Mamiellophyceae were recovered here. In previ- total of 309 clones affiliated with the Haptophyta were ous studies of other seas, Clade IX has been identified detected and included 28 unique phylotypes (100% only in flow cytometry-sorted samples or using a phy- sequence similarity). One OTU (OTU 10) affiliated with lum-biased approach (Viprey et al., 2008; Shi et al., the Phaeocystales (Phaeocystis antarctica) was detected in

ª 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. 833 Microbial Eukaryotes in Mesoscale Processes W. Wu et al.

0.1 95 Bathycoccus prasino CCMP1898 JX625115 Ostreococcus tauri Y15814 100 Euk328f+1498R Prasinophyceae sp. SL-175 EF432530 II EukA+1498R 100 82F+1498R WS073.062 100 Euk328f+Euk329r Micromonas pusilla CCMP1545 AY954994

100 Biosope T65.141 FJ537349 100 WS073.065 Biosope T41.155 FJ537332 IX 100 WS071.038 100 Biosope T41.151 FJ537330 100 WS071.036 WS073.124 79 SGYX420 KJ761285 100 Biosope T65.119 FJ537346 100 VII-A WS073.066 99 Biosope T65.109 FJ537344 100 WS073.064

100 Biosope T123.014 FJ537298 WS073.069 VII-B 100 Biosope T88.015 FJ537357 100 WS071.040 Chloroidium saccharophilum FR865677 100 Trebo WS073.063 100 Chlorophyceae sp. CCMP1189 AF203398 Chlor 63 100 WS073.053

100 Nephroselmis spinosa AB158375 100 WS073.056 Nephroselmis sp. MBIC11149 AB214975 III 100 100 WS073.055 100 Nephroselmis astigmatica NIES-252 FN562433 100 WS073.054 Pyramimonas gelidicola AnM0046 EU141942 I 100 WS073.060 Prasinoderma coloniale CCMP1413 FN562437 VI 100 WS073.057

100 Phaeocystis globosa CCMP1528 AF182110 Phaeocystis globosa CCMP1524 AF182109

Figure 5. Phylogenetic tree for Chlorophyta based on maximum likelihood analysis of 18S rDNA sequences (1680 positions) retrieved from the southwestern South China Sea (bold). The tree is based on a TrNef + G + I model of DNA substitution with a gamma distribution shape parameter of 0.534 (I = 0.313), and R(b)[A–G] = 2.5138, R(e)[C–T] = 4.4253 and 1.0 for the other substitution rates. Bootstrap values of >50% (1000 replicates) are shown, and the filled circles at the nodes indicate Bayesian posterior probabilities of >0.9. The tree is rooted by two Phaeocystis sequences. The multivalue bar charts correspond to the number of clones represented by each operational taxonomic unit. The scale bars indicate 0.1 substitutions per base (black line) and one clone (color bar chart). Trebo, Trebouxiophyceae; Chlor, Chlorophyceae. each clone library with a high relative contribution of a by the Mekong River plume (Y30S, Y33S and Y36S) were maximum of 6%. Based on Bray-Curtis distances, the separated from the others. Surface (Y10S, Y13S, and samples collected from locations significantly influenced Y90S) and DCM samples (Y10D, Y13D, and Y90D) col-

834 ª 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. W. Wu et al. Microbial Eukaryotes in Mesoscale Processes

Bacillariophyceae

Bolidophyceae

Cylindrothec

Novel group 6

6

a fusiformis Synurophyceae

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9

2 CCM

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80 999 15 AY788937 WS0 p. CC - f 9 A 17 ica CCW50 AY180025 8 WS071.068 s AY4854 Y1800 Y Pelagophyceae CCMP1866 HQ912 s

072 NIES 72.042 A le WS 79995 96 039 1 Biosope T123.033 F 72.03 9 ula 072.03 9 00 AY WS073.127 S0 7 57 S073. vic

W a Mic CCA67 S072. 14 W 0021 ro N 072.040 W 8 AY1 monas WS072.031 7 S CCI 2 U14389 W a sp. RCC2 CI thodiscus luteus Micromonas pusilla 557 C J Olis WS073.081537 CW34 AY18 C 007 99 HM1 300 097 EU247837 WS073.126 monas calceolat WS074. AY95499491693 sp. CCMP2 Pelago S073.076yceae W ph lago Pe U14385 Biosope culum T60.030 FJ537340 WS074.003 Dictyocha spe 9 AB21762 WS073.078 WS074.016 Biosope T60.01 Pseudochattonella verruculosa 1 FJ537338 WS074.009 WS073.079 Rhizochromulina cf. marina U14388 Dictyochophyceae Biosope T84.071 FJ537356 WS074.008 WS074.013 Pseudopedinella elastica WS074.001 U14387 Apedi Biosope T39.013WS074.010 FJ537315 nella radians WS074.014 U14384 WS073.092 Uncultured WS074.004 Pedinellales FN2630 LG34-12 AY91980 Gon WS072iochl o LG44-09 AY919813 EF165139 Eustigm ris sculpta W .033 29 WS073.073 S074 W a CCMP1149 73.074 S tos F 0 Monodopsis0 .005 J8 sp. W 72. magn 58970 P1278 WS0U42382 WI S 03 onas 519 36 W 071.015 AB474963 0 RS M 4 a CCM W S073.087 U4 hrom Bioso WS c Biosope T39.040 FJ537317 1 O sp. heri WS073.07 41 S . 03 1051 33 12 71 0 s

EF432 0 7 ubterranea 2.0 f AM1148 ta 74. 4.011 .10 pe T3 WS072. 651 3.0 07 1 ton visc WS072.032 01 m.00 chromonas S 07 S Eustigmatophyceae O W 2 9.09

W 10 163 99 EF U CCI40 AY179989 4105 Chromophy 8 F P18 KC onas imperfora J5 4 5 CM 3 82

73

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m

o Clade G r

Clade C Och Clade E Xanthophyceae 0.1

Clade F1 Euk328f+1498R Clade D EukA+1498R Clade I 82F+1498R Clade H Euk328f+Euk329r

Figure 6. Phylogenetic tree for photosynthetic stramenopiles based on maximum likelihood analysis of 18S rDNA sequences (1657 positions) retrieved from the southwestern South China Sea (bold). The tree is based on a TrN + G + I model of DNA substitution with a gamma distribution shape parameter of 0.502 (I = 0.359), and R(b)[A–G] = 2.3457, R(e)[C–T] = 4.3974 and 1.0 for the other substitution rates. The red dots at the nodes indicate a bootstrap value of 100% (1000 replicates) and a Bayesian posterior probability of 1.0. The tree is rooted by two Micromonas sequences. The multivalue bar charts correspond to the number of clones represented by each operational taxonomic unit. The scale bars indicate 0.1 substitutions per base (black line) and one clone (color bar chart). lected from the cyclonic eddy-influenced station were also An RDA plot was generated based on the environmental clustered together, suggesting the important role of parameters and relative abundances of Haptophyta phylo- mesoscale processes in regulating Haptophyta population types, showing that 27.5% of the variability could be structures. explained by the environmental parameters (Fig. 8). In

ª 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. 835 Microbial Eukaryotes in Mesoscale Processes W. Wu et al.

Relative abundance (%)

0246

-OTU 01 Pavlovophyceae -OTU 02 -OTU 03 Hap-2 -OTU 04 -OTU 05 -OTU 06 Zygodiscales -OTU 07 -OTU 08 -OTU 09 -OTU 10 Phaeocystales -OTU 11 -OTU 12 Isochrysidales -OTU 13 Clade D -OTU 14 Coccolithales -OTU 15 Clade E -OTU 16 Phylotypes -OTU 17 -OTU 18 -OTU 19 -OTU 20 -OTU 21 Prymnesiales -OTU 22 -OTU 23 -OTU 24 -OTU 25 -OTU 26 -OTU 27 -OTU 28 Y90S Y10S Y13S Y16S Y93S Y96S Y30S Y33S Y36S Y30D Y16D Y93D Y96D Y13D Y10D Y90D Y36D Y33D Samples

Figure 7. Hierarchical heatmap showing distribution patterns based on the relative abundances of unique Haptophyta phylotypes (100% sequence similarity), coupled with a unweighted pair-group method with arithmetic mean dendrogram of 18 samples constructed based on Bray- Curtis distances. addition, the Mantel test based on unweighted UniFrac salina (L34669), which has undergone adaptation to distances suggested that environmental variables played brackish environments (Bendif et al., 2011). Clade D, important roles in shaping the communities of Hapto- Clade E, Hap-2, and the Isochrysidales appeared to be pre- phyta subclades (P < 0.05, permutations = 999). RDA fur- dominant in the DCM samples. The Coccolithales and ther suggested that changes in community composition Zygodiscales were located in the corner and center of the were positively correlated with salinity, nutrients and Chl RDA plot, indicating that there were no significantly close a concentrations in surface samples, in contrast with the relationships between their distributions and the environ- DCMs, in which community shifts were positively corre- mental variables. No single environmental variable was lated with temperature and irradiance. Negative clustering found to be responsible for the distribution of the Prym- by temperature indicated that Y10S, Y13S, and Y90S were nesiales due to their widespread dispersal. Special traits of more strongly influenced by the cyclonic eddy in the shap- the Prymnesiales, such as mixotrophy might contribute to ing of the Haptophyta community. The distribution of their adaptability to diverse environments (Unrein et al., Pavlovophyceae showed strong correlations with the nutri- 2014). Pigment analyses showed that haptophytes_8 were ent concentrations and the sample Y30S, which was the most important eukaryotic group, contributing an affected by the Mekong River plume. As revealed in Fig. 3, average of 9.3% to the Chl a biomass (Fig. S4). Particu- the Pavlovophyceae sequences were affiliated with Pavlova larly in the DCMs, haptophytes_8 was the dominant

836 ª 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. W. Wu et al. Microbial Eukaryotes in Mesoscale Processes

Y33S

Y36S Phae

Y16S MLD Y16D ClaE

Y30S Pavl Y36D Y90S Y90D

Y13D Y10S Y93D Isoc S N Y10D ClaD Y13S Irr Y96D Hap2T RDA2 (12.8%) Figure 8. Ordination diagram of the first two Zygo Y96S axes of redundancy analysis, showing the Y33D relationships between the relative abundances Y93S of Haptophyta subclades and environmental Y30D Prym variables. T, temperature; S, salinity; Irr, −1.0 −0.5 0.0 0.5 1.0 irradiance; MLD, mixed layer depth; N, –1 0 + NO2 NO3; Cocc, Coccolithales; Isoc, Cocc Isochrysidales; ClaD, Clade D; ClaE, Clade E; Pavl, Pavlovophyceae; Phae, Phaeocystales; −1.0 −0.5 0.0 0.5 1.0 1.5 Prym, Prymnesiales; Hap2, Hap-2; Zygo, Zygodiscales. RDA1 (14.7%) group, with Chl a biomass contributions ranging from 9% Acknowledgments (Y30) to 67.1% (Y10). Haptophytes_8 primarily included species affiliated with the order Phaeocystales (e.g., Phaeo- We sincerely thank X. Wang for the wet-laboratory work cystis antarctica, Phaeocystis pouchetii and Phaeocystis glo- and C. Hsieh for the useful comments. We also thank the bosa) and was characterized by an abundance of 190- captain and crew of R/V Dongfanghong 2 for their help butanoyloxyfucoxanthin (Zapata et al., 2004). This finding with sampling. This work was supported by the Natural was confirmed by molecular analysis, which revealed an Science Foundation of China (Nos. 41330961, and abundance of Phaeocystales sequences (Figs. 3 and 7). 41176112), the Doctoral Fund of the Ministry of Educa- Due to the frequent recovery of Phaeocystales in distinct tion of China (No. 20130121110031) and the Special environments, no parameter was determined to be respon- Fund of the State Oceanic Administration of the People’s sible for their distribution patterns in the RDA plot. Republic of China (No. 530-03-02-02-03).

Conclusions Conflict of Interest In summary, our study expands upon previous similar stud- None declared. ies, reporting microbial eukaryotic communities character- ized by abundant pigmented taxa in a river plume and cold eddy-influenced ecosystem. Undoubtedly, phylogenetic References analyses based on numerous sequences provide important Baltar, F., J. Arı´stegui, J. M. Gasol, I. Lekunberri, and G. J. background information on these vital primary producers Herndl. 2010. Mesoscale eddies: hotspots of prokaryotic that have been missed in previous environmental surveys. activity and differential community structure in the ocean. Our study also has revealed that mesoscale processes have ISME J. 4:975–988. substantial effects on the diversity and distribution of Bendif, E. M., I. Probert, A. Herve´, C. Billard, D. Goux, C. microbial eukaryotes. To identify how mesoscale processes Lelong, et al. 2011. Integrative of the shape microbial eukaryotic communities in detail and how Pavlovophyceae (Haptophyta): a reassessment. Protist microbial eukaryotic diversity responds to mesoscale pro- 162:738–761. cesses, more systematical studies are needed in the future.

ª 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. 837 Microbial Eukaryotes in Mesoscale Processes W. Wu et al.

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species (65 strains) of Haptophyta: implications for Figure S1. Localization of the primers used in this study. oceanography and chemotaxonomy. Mar. Ecol. Prog. Ser. The environmental sequence OLI11115 (GenBank acces- 270:83–102. sion number AJ402326) is used as a reference. Zapata, M., F. Rodrıguez, and J. L. Garrido. 2000. Separation Figure S2. Relative abundances of microbial eukaryotic of chlorophylls and carotenoids from marine phytoplankton: groups in the four clone libraries using mixed DNA and a new HPLC method using a reversed phase C8 column and different primer sets (Euk328f+1498R, EukA+1498R, pyridine-containing mobile phases. Mar. Ecol. Prog. Ser. 82F+1498R, and Euk328f+Euk329r). 195:29–45. Figure S3. Venn diagram illustrating the operational tax- Zhang, Y., Z. Zhao, J. Sun, and N. Jiao. 2011. Diversity and onomic units for the clone libraries determined using distribution of diazotrophic communities in the South four primer sets. China Sea deep basin with mesoscale cyclonic eddy Figure S4. The phytoplankton communities at each sta- – perturbations. FEMS Microbiol. Ecol. 78:417 427. tion with the percent contributions of the nine main Zhang, Z., S. Schwartz, L. Wagner, and W. Miller. 2000. A groups to the total chlorophyll a biomass at the surface greedy algorithm for aligning DNA sequences. J. Comput. and the deep chlorophyll maximum (DCM). Biol. 7:203–214.

Supporting Information Additional Supporting Information may be found in the online version of this article:

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