ORIGINAL RESEARCH

published: 14 June 2017

doi: 10.3389/fmicb.2017.01121

Microbial Eukaryote Diversity and

Activity in the Water Column of the
South China Sea Based on DNA and
RNA High Throughput Sequencing
Dapeng Xu 1*, Ran Li 1 , Chen Hu 1 , Ping Sun 2 , Nianzhi Jiao 1 and Alan Warren 3
1
State Key Laboratory of Marine Environmental Science, Institute of Marine Microbes and Ecospheres, Xiamen University,
Xiamen, China, 2 Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystem, College of the Environment
and Ecology, Xiamen University, Xiamen, China, 3 Department of Life Sciences, Natural History Museum, London, United
Kingdom

To study the diversity and metabolic activity of microbial eukaryotes in the water column
of the South China Sea, genomic DNA and RNA were co-extracted from samples
collected down to bathyal depth at two sites. V9 regions of both SSU rRNA gene and its
transcript (cDNA) were amplified and sequenced using high throughput sequencing. Our
study revealed: (1) DNA and RNA datasets showed significant differences in microbial
Edited by:
Télesphore Sime-Ngando,
eukaryote community composition, with the variability between the two datasets for
Centre National de la Recherche the same sample exceeding that between samples within each dataset, indicating that
Scientifique (CNRS), France nucleic acid source overrode environmental factors in determining the composition of
Reviewed by: microeukaryotes; (2) despite the differences in community composition between the
Zhenfeng Liu,
University of Southern California, two datasets, both DNA and RNA revealed similar depth-related distribution patterns
United States of microbial eukaryotes; (3) using the ratio of RNA: DNA as a proxy of relative metabolic
Sébastien Monchy,
Université du Littoral Côte d’Opale,
activity, a depth-related pattern was found for the relative metabolic activity of some
France but not all groups of microbial eukaryotes, with the highest activity for the groups with
*Correspondence: depth-related pattern usually found in the middle water layers; and (4) the presence of
Dapeng Xu live and active photoautotrophic microbial eukaryotes in the deep ocean was confirmed,
[email protected]
indicating that they play an important role in controlling the deep-sea organic carbon
Specialty section: pool. Overall, our study sheds light on the diversity and activity of microbial eukaryotes
This article was submitted to in the water column of a tropical oligotrophic ocean and their potential contributions in
Aquatic Microbiology,
a section of the journal the downward transportation of organic material from the surface ocean to the deep via
Frontiers in Microbiology the biological pump.
Received: 25 January 2017
Keywords: biological pump, deep sea, metabolic activity, protist, RNA/DNA ratio
Accepted: 01 June 2017
Published: 14 June 2017

Citation: INTRODUCTION
Xu D, Li R, Hu C, Sun P, Jiao N and
Warren A (2017) Microbial Eukaryote
Single-celled eukaryotes (protists) comprise a wide range of morphologically, genetically, and
Diversity and Activity in the Water
Column of the South China Sea Based
functionally diverse lineages that act as primary producers, consumers, decomposers, parasites,
on DNA and RNA High Throughput and/or trophic links in microbial food webs. They thus play key roles in global biogeochemical
Sequencing. Front. Microbiol. 8:1121. cycles of numerous elements including carbon, nitrogen, and silica (Azam et al., 1983; Jiao et al.,
doi: 10.3389/fmicb.2017.01121 2010; Caron et al., 2012; Worden et al., 2015). Studies on the taxonomy, diversity, and phylogeny

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Xu et al. Microbial Eukaryotes Diversity and Activity

of protists based on conventional methods, e.g., observations active microbial eukaryotes in the water column of the South
by microscopy of specimens in vivo or following fixation China Sea? (II) do DNA and RNA reveal similar alpha and
and staining, have been carried out for centuries and have beta diversities of microbial eukaryotes and to what extent do
laid a strong foundation for our understanding of their they differ? (III) what is the relative metabolic activity of major
biology and diversity (Whittaker, 1969). In recent decades, the eukaryotic assemblages in the water column at the two locations?
application of culture-independent methods (especially DNA-
based sequencing) to natural samples collected from a wide range MATERIALS AND METHODS
of aquatic and terrestrial ecosystems has played a central role
in the discovery of novel microbial eukaryote lineages (López- Sample Collection
García et al., 2001a,b; Stoeck et al., 2003; Massana et al., 2004; Sampling was conducted on board of R/V Dongfanghong 2 in
Not et al., 2007; Dunthorn et al., 2014) as well as revealing their summer of 2014. Two sites, H7 (116◦ E, 14’N) and F3 (118◦ E,
patterns of distribution (Rodriguez-Martinez et al., 2013; Logares 16’N) located in the mid-region of the South China Sea, were
et al., 2014; de Vargas et al., 2015; Grattepanche et al., 2016; sampled on 11 and 13 July 2014, respectively (Figure S1).
Santoferrara et al., 2016; Sun et al., 2016). Seawater was collected from eight discrete depths from the
Environmental DNA samples usually consist of nucleic acids surface to the bathyal zone at both sites: F3 (25, 75, 200, 300,
derived from living, dormant (e.g., cysts) and dead microbial 500, 1,000, 1,500, and 2,000 m) and H7 (5, 25, 75, 200, 500, 1,000,
cells (Josephson et al., 1993), as well as extracellular free DNA 1,500, and 3,900 m).
(Dell’Anno and Danovaro, 2005). Recent studies have shown
the importance of differentiating the active from the total DNA/RNA Co-extraction, PCR
communities (Jones and Lennon, 2010; Campbell et al., 2011). Amplification, and Sequencing
Moreover, the SSU rRNA gene copy number varies from one to Total DNA and RNA were extracted simultaneously from each
thousands in single eukaryotic genomes (Zhu et al., 2005; Godhe cryopreserved filter membrane using AllPrep DNA/RNA kit
et al., 2008; Gong et al., 2013) making it difficult to directly link (Qiagen, USA) based on an initial mechanical cell disruption
read numbers to abundances of individual organisms. Compared employing bead beating (Stoeck et al., 2007) followed by
with DNA, extracellular RNA molecules are much less stable and chemical disruption using lysis buffer provided in the kit. Total
survive for much shorter time periods (Karl and Bailiff, 1989). DNA and RNA were quantified using a Nanodrop ND-2000
By targeting RNA, the problems associated with interference Spectrophotometer (Labtech International) and the quality was
by nucleic acids from inactive/dead cells, extracellular nucleic assessed by gel electrophoresis. After treatment with DNase
acids, and differences in rRNA gene copy number, can be (Qiagen, Hilden, Germany) to remove the carry-over genomic
avoided. Furthermore, the community diversity revealed by RNA DNA, RNA was then transcribed into cDNA using QuantiTect R
responds more sensitively to environmental conditions than that Reverse Transcription kit (Qiagen, USA) according to the
revealed by DNA (Charvet et al., 2014). There are thus several manufacturer’s instructions. The universal forward and reverse
advantages of including both DNA and RNA when analyzing primers, 1389F (5′ -TTG TAC ACA CCG CCC-3′ ) and 1510R (5′ -
environmental nucleic acids. This approach has been proven to CCT TCY GCA GGT TCA CCT AC-3′ ), were used to amplify
be effective for prokaryotes (Mills et al., 2005; Moeseneder et al., the hyper-variable loop V9 of the DNA and RNA (Amaral-
2005; Gentile et al., 2006), but has only recently been applied to Zettler et al., 2009). Six individual PCR reactions for each sample
protistan community surveys (Stoeck et al., 2007; Not et al., 2009; were run and later pooled. The resulting PCR amplicons (ca.
Orsi et al., 2013; Logares et al., 2014; de Vargas et al., 2015). In 130 bp) were excised from the gel using Gel extraction kit
addition, only a few studies have applied RNA: DNA ratios to (Promega, Shanghai, China). Bridge amplification and paired-
infer the metabolic activity of microbial eukaryotes assemblages, end sequencing of the amplicons were performed with an
and these were either restricted to protist communities above Illumina MiSeq sequencer by a commercial sequencing company.
the euphotic/disphotic zones (Charvet et al., 2014; Logares et al., All sequence data generated in this study have been submitted to
2014; Massana et al., 2015; Hu et al., 2016) or focused on the NCBI Sequence Read Archive and are accessible under the
communities in shallow coastal water sediments (Massana et al., accession number SRP104547.
2015). The activity of microbial eukaryote assemblages at bathyal
depths in oligotrophic waters remains largely unknown. Data Processing
In this study, we investigate the diversity and activity of Overlapping reads were merged using the program FLASH
microbial eukaryotes in the water column, from the surface to the with default parameters (Magoc and Salzberg, 2011). Barcoded
bathyal zone, of the South China Sea which is one of the world’s datasets were de-multiplexed and filtered to remove low quality
largest marginal seas and represents typical tropical oligotrophic sequences using QIIME (Caporaso et al., 2010). Chimeras were
waters (Gong et al., 1992). Microbial eukaryotes were sampled at detected and removed with UCHIME (Edgar et al., 2011) using
discrete depths down the water column at two separate locations. both de novo and reference-based chimera searches against PR2
Small subunit (SSU) rRNA gene and cDNA (reverse transcribed (de Vargas et al., 2015). For each sample, clean reads were
from extracted RNA, hereafter referred to as RNA) tags were dereplicated. Reads present as a single copy (singleton) were also
sequenced by high-throughput sequencing based on total RNA removed. Reads were then clustered into Operational Taxonomic
and DNA co-extracts of 32 samples. This study aimed to address Units (OTUs) at 95% sequence similarity using UPARSE (Edgar,
the following questions: (I) what is the community structure of 2013). The taxonomy assignment of OTUs was achieved by

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Xu et al. Microbial Eukaryotes Diversity and Activity

UCLUST against the PR2 database to detect and remove taxa 75 m; middle: 200, 300, and 500 m; deep: 1,000, 1,500, 2,000, and
that are not affiliated with eukaryotes (e.g., Bacteria, Archaea, 3,900 m) were pooled and the differences were evaluated using
and plastidial sequences) (de Vargas et al., 2015). The OTUs analysis of variance (ANOVA) (P < 0.05).
affiliated with Metazoa or Unassigned were removed from the
dataset before downstream analysis because the former would
distort the relative abundance of DNA sequences of microbial
eukaryotes and the latter are taxonomically uninformative. RESULTS
To normalize sampling effort, OTU counts were rarefied at Sampling Sites and Environmental Factors
a uniform sequencing depth based on the lowest sequence Two sites were chosen to represent the central part of the
count (20,204 sequences) from the 75 m depth of site F3 South China Sea (Figure S1). The physical properties of the
retrieved from RNA samples (F3.75R). Following removal of oceanic waters at these two sites exhibited similar environmental
low quality reads, potential chimeras, reads that were not conditions at each of the depths sampled (Figure S2). A well-
assigned as eukaryotes (including Unassigned) and singletons, defined pycnocline was found with the steepest gradients in
there were 2,309,424 sequences representing both protistan and salinity (33.1 to 36.4 psu) and temperature (ca 30.2 to 2.4◦ C)
metazoan taxa, ranging 22,481 to 174,306 reads per sample between 20 and 100 m (Figure S2). The bacterial abundance
(Table 1). After removing sequences affiliated with Metazoa, decreased with increasing depth, from ca. 7.4 × 105 to 8.4 × 104
there was a total of 2,067,712 microbial eukaryote sequences, cells ml−1 between 25 and 2,000 m water depths at site F3 and
ranging 20,204 to 169,189 sequences per sample. Using a from ca. 6.7 × 105 to 4.3 × 104 cells ml−1 between 5 and 3,900 m
95% sequence similarity threshold, sequences were further at site H7.
clustered into 2,799 protistan OTUs. The number of protistan
OTUs ranged 918–1,504 per sample (Table 1). Alpha diversity
estimations (Chao1, Shannon, ACE, Inverse of Simpson, and
Phylogenetic Diversity) were calculated using QIIME (Caporaso
Alpha and Beta Diversity of Microbial
et al., 2010). Eukaryotes in the Water Column of the
An OTU table generated in QIIME was used to study the South China Sea
similarities among microbial eukaryote communities of the 32 Metazoa represented approximately 2.9–42.1% of total eukaryote
samples using the Bray-Curtis coefficient within PRIMER v.6 reads in each sample. The proportions of metazoan sequences
(Clarke and Warwick, 2001). OTU abundances were normalized generally decreased with increasing water depth at both sites
to relative abundances within each library using a square- (Table 1). For the same sample for which DNA and RNA were co-
root transformation to prevent a few well-represented OTUs extracted and sequenced, the proportions of metazoan sequences
from driving the similarity analysis (Clarke and Warwick, were smaller in RNA than in DNA amplicons in the majority of
2001). ANOSIM was conducted to establish the significance of the samples. One exception was the sample collected at 25 m at
dendrogram nodes resulting from cluster analysis. To check the site H7, for which the proportion of metazoan sequences was
robustness of dissimilarity among communities, beta diversity higher in the RNA extract (ca. 42.1%) than in the DNA extract
was calculated using the unweighted Unifrac metric, which (32.7%). Other exceptions were the samples collected at 1,000 m
compares samples based on the phylogenetic relatedness of at site F3 and 75 m at site H7, where the proportions of metazoan
OTUs in a community without taking into account relative sequences were only slightly higher in the RNA than in the DNA
OTU abundance (Lozupone and Knight, 2005). Visualization extracts (4.8 vs. 4.7% and 11.6 vs. 11.1%, respectively).
of community dissimilarity based on the unweighted Unifrac To calculate alpha diversity and evaluate differences in
metric was made using a two-dimensional Principal Coordinate microbial eukaryote community composition between samples,
Analysis (PCoA). Simple Mantel tests were conducted in R with OTU counts were rarefied at a uniform sequencing depth based
the Vegan package to assess correlations between environmental on the lowest sequence count from sample F3.75R, i.e., RNA
variables and community variability (Legendre and Legendre, extract from sample collected at 75 m at site F3 (n = 20,204
1998) sequences). As a general trend, the richness decreased with
increasing depth but increased again at the greatest depths
sampled (2,000 m at F3 and 3,900 m at H7, respectively). In the
Relative Metabolic Activity of Major RNA extracts, the highest richness of microbial eukaryotes was at
Microbial Eukaryote Assemblages 25 m (1,092 OTUs) and the lowest was at 1,500 m (1,037 OTUs).
To reveal the relative metabolic activity of major microbial At site H7, the highest richness was at 25 m and the lowest was
eukaryote assemblages for samples collected at each water depth, at 1,500 m, both in DNA (1,272 and 664 OTUs, respectively)
OTUs occurring only in the DNA or only in the RNA dataset and RNA (1,027 and 579 OTUs, respectively) extracts. Site F3
were not included in the analysis (Hu et al., 2016). RNA: DNA showed a similar trend with the lowest richness at 2,500 m,
ratios for each OTU were calculated, then average ratios for each however the highest was at 200 m (1,097 OTUs) in the DNA
major microbial eukaryote group were used as a proxy for relative extracts and at 25 m (1,092 OTUs) in RNA extracts. The four
activity of the group (Hu et al., 2016). To reveal the differences diversity estimators, i.e., Chao1, ACE, Shannon, and Simpson,
in RNA: DNA ratios of different groups of microbial eukaryotes, showed the same trend (Table 1). Phylogenetic diversity (PD),
OTUs from the same group and water layers (shallow: 5, 25, and which measures the total branch length connecting all OTUs

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 TABLE 1 | Diversity parameters of South China Sea samples.
Xu et al.

Sample Clean reads* Metazoa reads (%) Clean reads (metazoa Metazoan OTUs OTU0.05 (metazoan OTU0.05 ** Chao1** ACE** Shannon** Simpson ** PD**
reads excluded) OTUs excluded)

F3.25D 54,581 13.0 47,484 64 1,371 1,065 1,418 1,417 6.34 0.015 163.15
F3.75D 34,495 18.5 28,117 67 1,125 1,031 1,346 1,331 7.25 0.039 159.29
F3.200D 35,586 16.2 29,805 50 1,262 1,097 1,491 1,456 7.09 0.031 164.15
F3.300D 77,840 5.0 73,968 75 1,459 952 1,429 1,398 5.54 0.012 150.66
F3.500D 99,944 9.1 90,828 76 1,385 836 1,201 1,218 5.26 0.013 129.84
F3.1KD 150,687 4.7 143,668 70 1,323 633 918 968 4.47 0.013 111.00
F3.15KD 148,897 5.1 141,365 78 1,268 599 914 1,022 3.40 0.009 110.85

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F3.2KD 174,306 2.9 169,189 67 1,381 669 1,083 1,151 4.16 0.009 120.26
F3.25R 29,060 8.7 26,522 49 1,172 1,092 1,380 1,401 8.06 0.105 173.86
F3.75R 22,481 10.1 20,204 43 918 918 1,239 1,196 7.67 0.093 148.53
F3.200R 24,836 12.3 21,772 39 1,032 1,007 1,288 1,246 7.12 0.022 157.56
F3.300R 31,288 4.2 29,968 45 1,147 1,011 1,413 1,337 7.00 0.031 158.61
F3.500R 40,374 7.5 37,332 50 1,129 942 1,215 1,213 7.03 0.042 154.67
F3.1KR 52,331 4.8 49,802 52 1,161 867 1,120 1,118 5.92 0.016 141.83
F3.15KR 67,120 3.6 64,716 53 1,026 665 1,037 1,081 3.91 0.005 119.70
F3.2KR 63,444 2.8 61,696 53 1,111 771 1,048 1,100 5.89 0.029 134.28
H7.5D 38,986 41.5 22,815 65 1,119 1,082 1,426 1,421 7.62 0.052 162.61

4
H7.25D 35,680 32.7 24,013 60 1,320 1,272 1,702 1,647 7.88 0.052 180.16
H7.75D 52,050 11.1 46,285 62 1,451 1,098 1,526 1,497 6.84 0.028 166.21
H7.200D 70,027 17.9 57,510 85 1,504 1,078 1,509 1,486 6.49 0.021 164.96
H7.500D 31,488 11.2 27,946 59 1,155 1,051 1,420 1,450 6.80 0.030 161.60
H7.1KD 122,774 23.4 94,077 83 1,398 836 1,269 1,335 5.11 0.012 133.54
H7.15KD 134,465 12.1 118,161 80 1,330 664 999 1,065 4.26 0.011 115.79
H7.39KD 118,192 7.1 109,746 94 1,498 810 1,329 1,295 5.13 0.013 140.79
H7.5R 40,821 21.8 31,924 66 1,003 901 1,230 1,199 7.48 0.081 147.53
H7.25R 55,457 42.1 32,131 70 1,155 1,027 1,429 1,404 7.48 0.061 162.22
H7.75R 56,374 11.6 49,842 68 1,300 994 1,352 1,377 6.81 0.029 161.07
H7.200R 50,270 17.2 41,628 80 1,238 985 1,398 1,377 6.42 0.022 160.56
H7.500R 37,634 5.0 35,734 61 965 789 1,194 1,134 6.34 0.029 133.99
H7.1KR 114,111 4.9 108,558 66 1,187 679 1,021 1,087 4.93 0.019 121.41
H7.15KR 133,136 7.3 123,380 67 1,127 579 976 1,016 4.02 0.010 111.64

OTU0.05 , Operational taxonomic unit at 95% SSU rRNA V9 gene sequence identity. *Reads affiliated with protists only; **Standardized numbers based on subsampling of 22,875 sequences without replacement.
Microbial Eukaryotes Diversity and Activity

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Xu et al. Microbial Eukaryotes Diversity and Activity

in the SSU rRNA gene phylogeny, also showed the same trend by the principle component analysis of community taxonomic
(Table 1). relatedness quantified by the Unweighted Unifrac metric (Figure
Four supergroups dominated the microbial eukaryote S4). Statistical analyses showed the composition of the two
assemblages at the two sites regardless of the water depth: clusters was significantly different (ANOSIM, R = 0.5506,
Alveolata, Rhizaria, Stramenopiles, and Excavata (Figure S3). p = 0.0001). Within the RNA cluster, the samples were generally
In terms of OTU richness, the pattern of distribution of these grouped by water depth. Samples collected from deep waters
supergroups was similar at the two sites retrieved either with (over 1,000 m) formed a group which then clustered with samples
DNA or RNA. The proportions of each supergroup did not from the middle layers (200, 300, and 500 m) plus the sample
change significantly with increasing depth. In the DNA survey, from 75 m at site H7, and these two groups formed a sister
these four supergroups represented 86–90% of the total richness group to the samples retrieved from shallow waters (5, 25, and
(Figures S3A,B). In the RNA survey, the same four supergroups 75 m). The samples within the DNA cluster were also grouped
dominated with comparable, but slightly smaller, contributions, by water depth, except that samples from middle layers clustered
i.e., 80–85% of total richness (Figures S3C,D). first with those of shallow waters rather than with samples from
To reveal the differences of alpha-diversity along the depth deep layers.
gradients, the samples were categorized into three groups:
shallow (5, 25, and 75 m), middle (200, 300, and 500 m), and
deep (1,000, 1,500, 2,000, and 3,900 m). The nonparametric test
Community Composition of Microbial
(Monte Carlo permutations) based on the Chao1 and Observed Eukaryotes Revealed by DNA and RNA
species showed that the alpha-diversity of the shallow water Surveys
group was not significantly different from the middle group Pooling all data from different depths and sites gave the first
in both the DNA and RNA surveys. By contrast, there were insight of the microbial eukaryote community compositions
significant differences in the alpha-diversity between the shallow at different sites and depths in the South China Sea revealed
and the deep groups and between the middle and the deep groups by DNA and RNA datasets. The reads affiliated with Rhizaria
(Figure 1). (mainly Cercozoa and Radiolaria) represented over half of the
The samples were clustered into two groups, first by total DNA reads followed by Alveolata (21%), Opisthokonta
nucleic acid sources based on the Bray-Curtis similarity, i.e., (9.7%) (represented mainly by Fungi and Choanoflagellida),
samples retrieved from DNA and samples retrieved from RNA, and Stramenopiles (5.8%). The other supergroups, i.e., Excavata,
respectively (Figure 2). This grouping pattern was also supported Amoebozoa, Apusozoa, Archaeplastida, Hacrobia, and Picozoa,

FIGURE 1 | Alpha-diversity measures (Chao1 and Observed species) for the pooled samples from the two sampling sites grouped by water depths, shallow (5, 25,
and 75 m), middle (200, 300, and 500 m), and deep (1,000, 1,500, 2,000, and 3,900 m), revealed by DNA and RNA, respectively. The line in each box plot indicates
the median, the box delimits the 25th and 75th percentile, and the whisker is the range.

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Xu et al. Microbial Eukaryotes Diversity and Activity

FIGURE 2 | Cluster diagram of Bray-Curtis similarities calculated from square-root transformed relative OTU abundances for each sample. The asterisk at the node in
the dendrogram separating DNA and RNA surveys indicates significant compositional differences between these two groups (R = 0.5506, p = 0.0001) determined by
the ANOSIM.

collectively contributed only ca. 10.8% of total reads (Figure 3A). small across all water depths but their relative abundance slightly
Compared with the DNA dataset, reads in the RNA survey were increased with increasing depth. The other groups (Picozoa,
relatively evenly distributed among the different supergroups. Hacrobia, Excavata, Archaeplastida and Apusozoa) made only
Stramenopiles was the dominant supergroup, representing minor contributions, generally less than 10% of the total reads
24.7% of total microbial eukaryote reads. Rhizaria was the individually (Figures 4A,B).
second most dominant with 17.1% of total reads, followed Similar to the DNA survey, the active microbial eukaryotic
by Opisthokonta (15.6%), Alveolata (14.9%), and Amoebozoa assemblages as revealed by RNA also showed depth-related
(10.5%) (Figure 3B). However, in terms of OTU richness, trends in the water column (Figures 4C,D). The contributions of
DNA and RNA showed similar patterns in the three most Rhizaria reads were generally higher in deep than shallow waters,
dominant supergroups, i.e., Alveolata, Stramenopiles, and accounting for 7.4 to 15.8% at site F3 and 7 to 42.2% at site H7,
Rhizaria, accounting for 47, 12.8, and 13.4% in DNA and 43, peaking at 500 m and 200 m, respectively. The contributions of
16.2, and 12.9% in RNA surveys, respectively. Excavata (mainly Stramenopiles reads ranged from 8.8 to 45% of the total reads at
Euglenozoa and other unidentified Discoba) represented 14% site F3, peaking at 200 m and 5.7 to 35.8% at H7 with a peak at
of total DNA OTUs and 11% of total RNA OTUs, respectively 500 m. In contrast to these two groups, Alveolata reads generally
(Figures 3C,D). decreased with increasing water depth, ranging from 5.3 to 41.7%
Several supergroups followed clear depth-specific trends in the at site F3 and 2.4 to 25.9% at site H7. Opisthokonta reads reached
DNA survey (Figure 4). Alveolata usually dominated the shallow their highest contribution (up to 20.3% of total reads) at 2,000 m
waters but its relative abundance decreased with increasing at site F3 and 54.7% at 1,500 m at site H7. The contribution of
depth, ranging from 41.7 to 5.3% at site F3 and from 53.1 Amoebozoa reads did not vary much across different depths,
to 8.3% at site H7 (Figures 4A,B). The relative contribution peaking at 2,000 m at site F3 (30.5%). The other groups (Picozoa,
of Rhizaria showed a reverse trend, with relative abundance Hacrobia, Archaeplastida, Excavata, and Apusozoa) were minor
generally increasing with the increase of depth, especially at site constituents of the total microbial eukaryote assemblages except
F3 where Rhizaria contributed more than 60% of all microbial at 1,500 m at site F3 where the contribution of Apusozoa was
eukaryote reads in waters below 300 m. The contribution 53.2% of the total reads.
of Opisthokonta generally increased with increasing depth, The four most consistently abundant groups within Alveolata
accounting for 14.4% of total reads at 1,000 m at site F3 and up were Ciliophora, Dinophyceae, MALV-I, and MALV-II, each
to 43% at 1,500 m at site H7. The contribution of Stramenopiles showing various relative contributions to both DNA and
was small across all water depths but generally higher in shallow RNA datasets (Figure S5). Reads affiliated with Dinophyceae
than deep waters. The contribution of Amoebozoa was also dominated the alveolate assemblages in the DNA dataset,

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Xu et al. Microbial Eukaryotes Diversity and Activity

FIGURE 3 | Overview of assemblages of microbial eukaryotes in the South China Sea water column at the supergroup taxonomic level. Relative sequence number in
DNA survey (A) and RNA survey (B); relative OTU number in DNA survey (C) and RNA survey (D).

whereas those affiliated with Ciliophora, most of which in the was almost undetectable in DNA survey, contributed more in
euphotic zone of open oceans are active grazers of prokaryotes the RNA survey (Figure S6A). Opisthokonta was dominated
and pico-/nano-sized eukaryotes (Sherr and Sherr, 1987), by Fungi both in DNA and RNA surveys; Choanoflagellida
contributed more in the RNA dataset and were the dominant contributed more in the RNA than the DNA survey and its
alveolates at some water depths (e.g., 2,000 m at F3; 5, 25, 75, and contribution to total sequences was generally larger in the deep
500 m at H7; Figure S5A). Within Stramenopiles, MAST reads than shallow waters (Figure S6B).
dominated the shallow water and showed substantial depth-
specific variation in both the DNA and RNA surveys (Figure Relative Activity of Major Groups of
S5B). In deep waters, Bicoecea replaced MAST as the dominant Microbial Eukaryotes
Stramenopiles group with up to 85% of total stramenopile reads. Analyses of the relative metabolic activity for major microbial
The relative contributions of photosynthetic Stramenopiles eukaryote assemblages were conducted separately by employing
(Dictyochophyceae, Pelagophyceae, Pinguiophyceae, RNA: DNA ratio as a proxy of relative activity. This was achieved
Chrysophyceae, Synurophyceae, and Bacillariophyta) were by combing samples into three groups according to the water
larger in the RNA than the DNA survey. Surprisingly, their depth from which samples were collected: shallow (5, 25, and
contributions in the deep water were sometimes comparable 75 m), middle (200, 300, and 500 m), and deep (1,000, 1,500,
to those in shallow waters, even in the RNA dataset (Figure 2,000, and 3,900 m) waters. Most but not all major groups of
S5B). Within Rhizaria, the most dominant group in both DNA microbial eukaryotes showed depth-related distribution patterns
and RNA surveys was Spumellarida, the contribution of which for their relative activities. The activities of Cercozoa, Acantharea,
generally increased with increasing water depth (Figure S5C). and RAD-B, which are all members of Rhizaria, showed similar
Within Hacrobia, Haptophyta was the most dominant group in depth-related patterns of relative activity throughout the water
both DNA and RNA surveys, its contribution to total sequences column. Their activities were significantly higher in middle
being larger in the RNA than in the DNA survey (Figure (average RNA: DNA ratio of 4.0, 2.4, and 1.3, respectively),
S6A). Another photoautotrophic group, Cryptophyta, which followed by deep (average RNA: DNA ratios of 3.7, 2.1, and 1.2,

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Xu et al. Microbial Eukaryotes Diversity and Activity

FIGURE 4 | Relative sequence abundance of microbial eukaryotes in the water column at the two sampling sites, as revealed by DNA (A,B) and RNA (C,D) surveys.

respectively) and shallow (average RNA: DNA ratio of 2.4, 1.5, photoautotrophic groups of Stramenopiles (Chrysophyceae,
and 0.9, respectively) waters (Table 2; Figure 5, Figure S7). The Synurophyceae, Dictyochophyceae, and Pelagophyceae) in the
activity of Polycystinea, another major group of Rhizaria, was water column. For the other groups of microbial eukaryotes,
the highest in shallow (1.4) followed by middle (0.9) and deep Amoebozoa, Archaeplastida (mainly represented by Chlorophyta
(0.5) waters. (Table 2; Figure S7). Ciliophora, mainly represented and Streptophyta), Choanoflagellatea, and Hacrobia (mainly
by pelagic groups such as Choreotrichia, Oligotrichia, and represented by Cryptophyta, Haptophyta, and Telonemia), the
Scuticociliatia, were overrepresented in RNA surveys and showed average RNA: DNA ratios were above 1:1 regardless of the water
no significant differences of activity in different depth groups depth and their activity did not show any significant difference
(on average of 5.7, 5.7, and 8.2, respectively) (Table 2; Figure 5). between shallow, middle and deep water groups (Table 2). The
Dinophyta, represented primarily by Dinophyceae, MALV-I, highest activity of Fungi and Excavata (mainly represented by
and MALV-II, were overrepresented in DNA surveys (Figure 5, Discoba) was found in middle waters, followed by deep and
Figures S7, S8). The activities of Dinophyceae and MALV-I shallow waters (Table 2; Figure S8).
were the highest in middle followed by deep and shallow
waters. For MALV-II, the highest activity was found in middle
followed by shallow and deep waters. Stramenopiles represented DISCUSSION
mainly by Bacillariophyta, MAST, and other photoautotrophic
groups (Chrysophyceae, Synurophyceae, Dictyochophyceae, and Analysis of RNA Reduces the Interference
Pelagophyceae) were overrepresented in the RNA compared to of Metazoan Sequences in Protistan
the DNA survey even in deep waters (Table 2). The highest Surveys
activities of Bacillariophyta and MAST were found in deep In the present study, metazoan sequences were often
followed by middle and shallow waters (Figure 5, Figure S8). encountered, especially in the DNA dataset, even after pre-
No significant difference of activity was found in the other main filtration using different sizes of mesh. The presence of metazoan

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Xu et al. Microbial Eukaryotes Diversity and Activity

TABLE 2 | Average RNA: DNA ratios for major groups of microbial eukaryotes in shallow (5, 25, and 75 m), middle (200, 300, and 500 m), and deep (1,000, 1,500, 2,000,
and 3,900 m) waters.

Average RNA: DNA ratios Pairwise comparisons

Shallow Middle Deep Shallow-Middle Shallow-Deep Middle-Deep

Rhizaria Cercozoa 2.38 4.00 3.68 0.030 0.215 0.495

Acantharea 1.47 2.43 2.08 0.017 0.096 0.573
Polycystinea 1.42 0.92 0.46 0.044 0.001 0.066
RAD-B 0.87 1.26 1.15 0.036 0.156 0.539
Alveolata Ciliophora 5.67 5.74 8.17 0.944 0.075 0.089
Dinophyceae 0.68 1.33 0.93 0.000 0.077 0.006
MALV-I 0.47 0.84 0.71 0.000 0.012 0.180
MALV-II 0.79 0.92 0.74 0.056 0.575 0.037
Stramenopiles Bacillariophyta 2.44 5.62 8.93 0.000 0.000 0.005
Photosynthetic Stramenopiles* 5.07 5.58 5.52 0.555 0.419 0.807
MAST 3.94 5.26 5.91 0.014 0.001 0.252
Amoebozoa 7.02 4.92 5.33 0.208 0.285 0.799
Archaeplastida 7.91 9.17 3.98 0.341 0.545 0.138
Opisthokonta Choanoflagellatea 5.20 9.34 8.91 0.065 0.174 0.881
Fungi 1.37 3.28 2.76 0.004 0.035 0.372
Excavata 1.02 1.43 1.27 0.002 0.069 0.180
Hacrobia 5.41 7.00 5.79 0.092 0.738 0.333

Difference in RNA: DNA ratios between shallow, middle, and deep waters were evaluated by analysis of variance (ANOVA) for significantly difference (P < 0.05). Bolded values are
significant. *Phototrophic Stramenopiles included Chrysophyceae, Synurophyceae, Dictyochophyceae, and Pelagophyceae.

sequences probably came from extracellular metazoan DNA after shaping factors (Countway et al., 2007; Schnetzer et al., 2011;
death or leakage from damaged metazoans during the filtration Xu et al., 2017). In the present study, depth-related changes
process. In the shallow waters (5, 25, 75 m), their contributions in the protist community structure based on the DNA dataset
were over 10% in most samples. In 5 m and 25 m waters at site were consistent with those based on the RNA dataset (Figures 2,
H7, metazoan sequences accounted for up to 41.7 and 32.7% of 4, Figure S4). Therefore, the “total” (as seen in DNA dataset)
the total eukaryote sequences, respectively. In the RNA dataset, and the “active” (as seen in RNA dataset) communities of
however, the contributions of metazoan sequences were notably microbial eukaryote were both shaped by water depth. Several
smaller in most samples, the only exception being 25 m depth at studies based on analyses of DNA from samples collected at
site H7 in which the contribution of metazoan sequences was local or regional scales have shown Rhizaria to be the dominant
higher in the RNA than in the DNA dataset. In the middle and micoeukaryote group in the deep sea (Countway et al., 2007;
deep water samples, the contributions of metazoan sequences Not et al., 2007; Orsi et al., 2011; Schnetzer et al., 2011; Xu
were comparably smaller than in the shallow waters both in et al., 2017). Recently, a large-scale survey on the diversity of
DNA and RNA surveys. Although the contributions of metazoan bathypelagic microbial eukaryotes also revealed Rhizaria being
sequences revealed by DNA did not differ as dramatically as a dominant group in the world’s oceans (Pernice et al., 2015).
those revealed by RNA in middle and deep waters, they still However, whether Rhizaria is the dominant functional group
contributed less in the RNA than in the DNA surveys. A recent in deep ocean water was still unclear as most previous studies
study that analyzed samples collected seasonally at several sites are based on analyses of DNA. Compared with DNA, the RNA
in the eastern North Pacific also found that the contributions of molecule is much less stable and consequently has much a
metazoan sequences were lower in RNA than DNA surveys (Hu shorter lifetime outside the cell (Karl and Bailiff, 1989). Thus,
et al., 2016). Thus, the application of RNA sequencing can serve RNA can serve as an indicator of metabolic activity in microbial
as an alternative approach to reduce the interference of metazoan communities (Stoeck et al., 2007). Nevertheless, few studies
sequences in protistan surveys even for deep water samples. have been performed that include simultaneous DNA and RNA
analyses from the same sample source (Logares et al., 2014;
Massana et al., 2015). Not et al. (2009) compared the community
Alpha and Beta Diversity of Microbial composition of pico-sized microeukaryotes in the euphotic zone
Eukaryotes in the Bathyal Zone of the of the Mediterranean Sea based on both DNA and RNA data
South China Sea and found that the contribution of radiolarian sequences, which
Several studies on the diversity of microbial eukaryotes in the overwhelmingly dominated DNA libraries, was significantly less
water column have been carried out in the world’s oceans and in the RNA libraries. In the present study, the proportion
depth has been identified to be one of the major environmental of Rhizaria-affiliated reads in deep layers reached up to ca.

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Xu et al. Microbial Eukaryotes Diversity and Activity

FIGURE 5 | Relative abundance of RNA (y-axis) and DNA (x-axis) reads for each OTU in representative microbial eukaryotes assemblages, Acantharea, Dinophyceae,
Ciliophora, and MAST, in the water column of the South China Sea.

75% in the DNA dataset. However, in the RNA extracts, the and a depth-related distribution pattern. Previous studies have
proportion of rhizarian reads was significantly lower throughout shown the presence of new radiolarian clades in the deep sea
the water column (Figure S9), indicating that some of the deep (Orsi et al., 2011; Xu et al., 2017). Our study provides evidence
rhizarian sequences may come from dead or dormant, rather for the presence of active assemblages of radiolarians in the
than metabolically active, cells. However, the RNA approach deep sea, so these new clades might represent active radiolarian
did reveal the presence of Radiolaria sequences in the deep sea assemblages.

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Xu et al. Microbial Eukaryotes Diversity and Activity

In the cluster analysis, samples were first clustered into m). RNA-DNA comparisons of the major components of the
two groups by nucleic acid source, i.e., the RNA group and microeukaryote communities in the water column of the South
the DNA group (ANOSIM, R = 0.5506, p = 0.0001). Within China Sea revealed depth-related pattern for their relative
each group, the samples generally clustered into subgroups by activity: the highest activity of most groups (e.g., Cercozoa,
water depth (Figure 2). A previous study on benthic prokaryotes Acantharea, RAD-B, Dinophyceae, MALV-I, MALV-II, Fungi,
from an active mud volcano in the Gulf of Mexico also found and Excavata) was found in middle waters. The highest
that the communities revealed by DNA differed significantly activity of Bacillariophyta and MAST was found in deep
from those revealed by RNA and this difference was more waters and that of Polycystinea was found in shallow waters.
significant than that caused by the sediment depth (Martinez Unexpectedly, however, we did not, find any depth-related
et al., 2006). Lanzén et al. (2011) explored prokaryotes at trends in the relative activity of some heterotrophic groups,
the Jan Mayen hydrothermal vent field and found that the e.g., Ciliophora, Amoebozoa, Choanoflagellatea, and even some
composition and diversity predictions differed systematically photoautotrophic groups such as Stramenopiles (represented
between extracted DNA and RNA samples. An investigation by Chrysophyceae, Synurophyceae, Dictyochophyceae, and
of bacterial communities in Antarctic coastal waters using Pelagophyceae), Archaeplastida, and Hacrobia, and their relative
both 16S RNA and 16S DNA showed similar results (Gentile activities did not change significantly along depth gradients.
et al., 2006). In the present study, however, although DNA Ciliates have long been known as the major grazers of
and RNA showed a distinctly different community composition prokaryotes and pico-/nano-sized eukaryotes in the euphotic
for the same sample, the two approaches revealed similar zone (Sherr and Sherr, 1987). However, data on the abundance
or comparable community structure (depth-related gradients) and community structure of ciliates in the deep sea are scarce
in each sample. Combined with the above evidence, our compared with prokaryotes (Nagata et al., 2010). The roles of
results support the notion that nucleic acid source has a ciliates in deep-sea microbial food webs thus remain enigmatic,
greater influence than depth (as seen in the present study), or largely because of the lack of metabolic data (Nagata et al., 2010).
other environmental factors (as seen in the above references), One study that surveyed protistan assemblages along European
in determining eukaryote microbial community composition. coasts showed ciliates were overrepresented in RNA compared to
The differences of community composition revealed by DNA DNA datasets, both in pico- and nano-sized fractions of waters
and RNA were likely caused by the differences between the and sediments (Massana et al., 2015). A study at a coastal ocean
activity levels of taxa because the former generally reflects site in the eastern North Pacific showed higher ciliate activity
activity and the latter usually includes active, dormant, and in April compared to other months and higher activity below
dead cells (Lanzén et al., 2011). In addition, the techniques the euphotic zone compared to shallower depths (Hu et al.,
applied, e.g., the bias introduced during the extraction of 2016). In the present study, relative metabolic activity of deep-
DNA/RNA and reverse transcription, may also contribute to sea ciliates was comparable to that of those in the euphotic zone
these differences. Thus, environmental RNA sequencing can (Figure 5), which indicates: (1) the presence of active ciliates in
serve as a reliable approach to reveal the diversity and community the bathyal zone of the South China Sea, and; (2) ciliate activity
structure of active microorganisms and provide supplementary did not decrease with increasing depth. Previous studies showed
perspectives of microbial eukaryotes. Care should be taken, that ciliate abundance varies in the range of <0.1–52 cells L−1
however, when comparing communities, especially if data is in deep (>1,000 m) waters (Nagata et al., 2010 and references
derived from different nucleic acid sources, i.e., DNA and therein). A parallel study on ciliates in the water column at
RNA. the same two sites in the South China Sea using quantative
protargol staining (QPS) method showed that their abundances
were <1–7 cells L−1 in the waters below 1,000 m depth (data not
Metabolic Activities of Major Microbial shown), which is similar to previous studies (Nagata et al., 2010
Eukaryotic Groups in the Bathyal Zone of and references therein). Although the abundance of ciliates in
the South China Sea the deep sea is much lower than prokaryotes and heterotrophic
Comparisons of RNA and DNA sequence abundance (RNA: nanoflagellates, the discovery of high ciliate metabolic activity
DNA ratios) have been widely used as proxies of in situ in the bathyal zone of the South China Sea indicates that they
microbial metabolic activity (Poulsen et al., 1993; Logares are probably grazing on such prey and are thus likely playing
et al., 2014; Massana et al., 2015). However, this approach an important role in controlling biogeochemical processes in the
has only recently been applied to the study of microbial deep sea. Unfortunately, the present study did not employ time-
eukaryotes (Charvet et al., 2014; Massana et al., 2015; Hu series sampling so temporal variations in the activity of deep-sea
et al., 2016). Each of these studies focused on microbial ciliates remain unknown.
eukaryote communities either above the euphotic/disphotic Rhizaria and Dinophyta were predominant in the DNA
zones or in shallow coastal water sediments; the metabolic dataset and their RNA: DNA ratios were on average below
activity of microeukaryotes in deep sea (>1,000 m) pelagic 1:1. (Table 2; Figure 5, Figure S7). The highest activity of
waters remains largely unknown. Therefore, to our best Dinophyceae and MALV-I were found in middle water followed
knowledge, the present study is the first investigation of by deep and shallow waters in the present study. The highest
the relative metabolic activity of microbial eukaryotes in the activity of MALV-II was also found in middle water but
water column from the surface to bathypelagic depths (>3,500 followed by shallow then deep waters. The recent San Pedro

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Xu et al. Microbial Eukaryotes Diversity and Activity

Ocean Time-series (SPOT) study of protist diversity and activity Bacillariophyta, Boliodphyta, Dictyochophyta, Prasinophyceae,
at a coastal site in the eastern North Pacific showed high Prymnesiophyceae, Raphidophyta, and Eustigmatophyceae,
RNA: DNA ratios for Radiolaria at the subsurface chlorophyll which represented 0.14% of the pyrotaps and 2% of the OTUs
maximum layer, 150 and 890 m (Hu et al., 2016). Similarly, in the sequence analyses (Pernice et al., 2015). This work was
generally high RNA: DNA ratios for dinoflagellates were found generated based on DNA sources therefore it remains unclear
in all euphotic zone samples in April (Hu et al., 2016). whether the photosynthetic signals were from live cells, dead
By contrast, Massana et al. (2015) reported that: (1) most cells, dormant cells or extracellular DNA. The DNA survey in the
lineages of Radiolaria were equally well represented in DNA present study likewise recovered photoautotrophic groups which
and RNA surveys of pico-, nano-, micro/meso-sized plankton represented ca. 0.1–0.4% of the total microbial eukaryote reads
and sediment samples collected from European coastal waters; in the samples ≥1,000 m. Furthermore, the RNA survey revealed
(2) Acantharea was overrepresented in RNA surveys of the that photoautotrophic groups accounted for ca. 0.9–4.3% of the
micro/meso-sized plankton; and (3) RAD-B was overrepresented total reads which is even higher than their contribution in the
in DNA surveys of sediments. Therefore, it seems likely that the DNA dataset. The photoautotrophic groups were still present
activities of Radiolaria and Dinophyta might be influenced by even after applying much stricter screening processes, i.e.,
geographical location, water depth, and season. In the present considering only the OTUs represented in both RNA and DNA
study, another major lineage of microeukaryotes, Stramenopiles, datasets (Figure S8). Furthermore, contrary to expectations,
also showed high RNA: DNA ratios in the water column we did not find any decrease in the metabolic activity of some
(Table 2), which is consistent with the findings of Massana et al. photoautotrophic groups (photoautotrophic Stramenopiles
(2015). such as Chrysophyceae, Synurophyceae, Dictyochophyceae,
Overall, we did find depth-related distribution patterns for Pelagophyceae, and members of Archaeplastida and Hacrobia)
most of the major groups of microbial eukaryotes. Surprisingly, with increasing depth (Figure S8). Thus, the present study
the highest activity for most groups was usually found in middle confirmed the presence of not only live but also active
(200, 300, and 500 m) rather than shallow (5, 25, and 75 m) or photoautotrophic microbial eukaryotes in the deep sea. Agusti
deep (1,000, 1,500, 2,000, and 3,900 m) waters. More than 90% et al. (2015) hypothesized that fast-sinking is the main reason
of the organic carbon annually exported from surface waters is for the presence of photoautotrophic cells in the dark ocean
respired back to CO2 in the mesopelagic waters (generally the and that when these cells die they serve as fresh organic carbon
depth range from 200 to 1,000 m) by prokaryotes (Arístegui input fueling the deep-sea ecosystem. If fast-sinking is the reason
et al., 2009). Although still being controversial, grazing of for the presence of photoautotrophic cells in the deep sea, their
prokaryotes by small eukaryotes such as small heterotrophic sinking rates must either be much faster than expected (some
flagellates (HF) are proposed to remove most of the prokaryotic of these cells were within the pico-sized fraction), or they can
production in the mesopelagic waters (Arístegui et al., 2009). survive in the dark for much longer than previously realized.
HF is a phylogenetically diverse group and include members Furthermore, we cannot rule out the possibility that some of
from a variety of microbial eukaryotes such as Stramenopiles these photoautotrophic microbial eukaryotes are facultative
and Rhizaria. The high metabolic activity of some groups heterotrophs because mixotrophy in protists is well documented
of microbial eukaryotes, such as Cercozoa, Acantharea, and and such forms are thought to contribute significantly to
Excavata, observed in the present study may help explain the biogeochemical cycles in the oceans globally (Mitra et al., 2014;
active grazing impact of HF on prokaryotes in the mesopelagic Worden et al., 2015; Stoecker et al., 2016). If the latter is the case,
waters (Arístegui et al., 2009). However, the present study did there are at least two possibilities that need to be investigated in
not include temporal sampling and the two water columns are further studies: (1) that these cells come from the surface layers
represent only a tiny fraction of the global oceans. A strategy sink to the deep sea and survive by phagotrophy (e.g., grazing on
that integrates both temporal and large-scale spatial sampling is prokaryotes), or; (2) they are endemic in the deep sea and act as
therefore needed in order to shed more light on the functions of grazers in the microbial loop.
microbial eukaryotes in marine biogeochemical cycling and their
responses to environmental change.
AUTHOR CONTRIBUTIONS

The Presence of Photoautotrophic DX and PS conceived and designed the experiments; DX, PS,
CH, RL, NJ, and AW performed onboard ample processing
Microbial Eukaryotes in the Dark Ocean and analyzed data; DX, PS, AW, NJ, RL, and CH wrote the
Previous studies based on direct microscopy have reported
paper.
the presence of well-preserved phytoplankton cells in the
deep sea (Kimball et al., 1963; Smayda, 1971; Wiebe et al.,
1974). Healthy photoautotrophic cells, especially diatoms, FUNDING
were recently reported down to 4,000 m in a global survey of
the dark ocean (Agusti et al., 2015). A parallel study on the This work was supported by the National Basic Research
diversity of bathypelagic microbial eukaryotes from the same Program of China (2013CB955700), SOA grant (GASI-03-01-
circumnavigation expedition using pyrosequencing also showed 02-05), the National Natural Science Foundation of China
the presence of photoautotrophic groups in the deep sea, e.g. (41306125, 31372167), the Fundamental Research Funds for

Frontiers in Microbiology | www.frontiersin.org 12 June 2017 | Volume 8 | Article 1121

Xu et al. Microbial Eukaryotes Diversity and Activity

the Central Universities (20720140504), and the State Key sampling. Special thanks are given to the two reviewers for their
Laboratory of Marine Environmental Science of Xiamen valuable comments.
University (MELRI1303).
SUPPLEMENTARY MATERIAL
ACKNOWLEDGMENTS
The Supplementary Material for this article can be found
We thank the captain, crew, and marine technicians of the online at: http://journal.frontiersin.org/article/10.3389/fmicb.
R/V Dongfanghong 2 during the cruise for ensuring smooth 2017.01121/full#supplementary-material

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Pernice, M., Giner, C. R., Logares, R., Perera-Bel, J., Acinas, S. G., Conflict of Interest Statement: The authors declare that the research was
Duarte, C. M., et al. (2015). Large variability of bathypelagic microbial conducted in the absence of any commercial or financial relationships that could
eukaryotic communities across the world’s oceans. ISME J. 10, 945–958. be construed as a potential conflict of interest.
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Rodriguez-Martinez, R., Rocap, G., Salazar, G., and Massana, R. (2013). original author(s) or licensor are credited and that the original publication in this
Biogeography of the uncultured marine picoeukaryote MAST-4: temperature- journal is cited, in accordance with accepted academic practice. No use, distribution
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