Microb Ecol (2013) 66:823–830 DOI 10.1007/s00248-013-0275-3

ENVIRONMENTAL MICROBIOLOGY

Abundance and Novel Lineages of Thraustochytrids in Hawaiian Waters

Qian Li & Xin Wang & Xianhua Liu & Nianzhi Jiao & Guangyi Wang

Received: 15 April 2013 /Accepted: 30 July 2013 /Published online: 14 August 2013 # Springer Science+Business Media New York 2013

Abstract Thraustochydrids has been known for their ubiqui- cluster and the others aligned with uncultured clones or none, tous distribution in the ocean. However, a few efforts have thus appeared to be undescribed. This study indicates the been made to investigate their ecology. In this study, we have presence of new thraustochytrids lineages and their quantita- applied molecular method, acriflavine direct detection, and tive importance in the marine water column. classical oceanographic methods to investigate the abundance and diversity of thraustochytrids in the North Pacific subtrop- ical gyre. Our results revealed interesting temporal and spatial Introduction variations of their population. Out of three seasons (spring, summer, and fall), cruise Hawaii Ocean Time-series (HOT)- As a dominant and obligate osmo-heterotrophic group of 216 during November 2009 obtained the highest abundance of marine , thraustochytrids have been reported to thraustochytrids ranging from 1,890 (Station S1C1, 45 m) to occupy their own spatial and trophic niches in the ocean. 630,000 (Station S2C12, 100 m)cells L−1 of seawater, which These fungal-like protists have broad ability to reside on accounted for a 0.79 to 281.0 % biomass ratio to that of multiple substrates [1–4]. More recently, there has been an bacteria in terms of gram carbon per liter. A patchy distribution increasing body of evidence regarding the role of of these organisms was widely observed in the water column thraustochytrids in marine environments [3, 5]. They can and they were somehow related to the maximum chlorophyll produce an array of unique degradative enzymes for highly layers. A total of 25 operational taxonomic units (OTUs) from refractory substrates such as cell wall material and phenolic cruise HOT-216 formed four phylogroups in the specific compounds [6, 7]. More importantly, their apparent season- 18S rRNA-based phylogenetic tree, with ally high population (varying from undetectable to a few the largest group of 20 OTUs fell into the Aplanochytrium hundred thousand cells per liter seawater) has drawn attention and been documented in oceanic environments in- Electronic supplementary material The online version of this article cluding the Indian Ocean [8], the coastal Mediterranean [9], (doi:10.1007/s00248-013-0275-3) contains supplementary material, and coastal Sea of Japan [10]. However, they currently which is available to authorized users. remain one of the most understudied marine microbial : Q. Li N. Jiao groups. State Key Laboratory of Marine Environmental Science, In previous decades, unraveling the evolutionary Xiamen University, Xiamen 361005, China of thraustochytrids had posed an ambiguous problem for – X. Liu (*) : G. Wang (*) researchers of this group [4, 11 14]. In said studies, routine School of Environmental Science and Engineering, culture methods were applied to provide us the general picture Tianjin University, Tianjin 300072, China of diversity: thraustochytrids comprise five genera with about e-mail: [email protected] 30 species [15], combined with related labyrinthulids (represent- e-mail: [email protected] edbyasinglegenusofLabyrinthula Cienk) and aplanochytrids : X. Wang G. Wang (represented by a single genus of Aplanochytrium Bahnweg et Department of Microbiology, University of Hawaii at Manoa, Sparrow), mainly constitute the Labyrinthulomycetes [16]ofthe Honolulu, HI 86822, USA Stramenopila. However, cultured diversity represents G. Wang only a small fraction of their actual diversity [5] and increasing e-mail: [email protected] evidence for the presence of novel Labyrinthulomycetes 824 Q. Li et al. lineages [17–19] emphasizes the need for both novel culture methods and uncultured diversity study on thraustochytrids. This study site, the Hawaii Ocean Time-series (HOT) station ALOHA (22°45′N, 158°W), represents the expansive North Pacific Subtropical Gyre (NPSG). This habitat has been able to provide tremendous data including both physicochemical and biological oceanographic parameters and, thus, is a good eco- logical habitat for locating microbial distribution profiles and community composition into appropriate oceanographic con- text [20–22]. In order to answer the following fundamental questions: First, does the abundance profile of thraustochytrids in former studies happens habitats- or seasons-specific? Second, how many of them exist in extremely oligotrophic oceans and what are their biomasses? Third, is there a living preference between coastal and open oceans? Finally, what is the potential of discovering their novel lineages? We designed our experiments with one coastal (S1C1) and a few oceanic sampling stations (S2C6, S2C7, S2C12, and S52C1 for HOT- 216; S2C6 and S52C1 for HOT-219 and HOT-223) in three different seasons, with the application of microscopic enumer- ation and phylogenetic analysis.

Materials and Methods

Collection of Samples

Samples were collected during three Hawaii Ocean Time- series cruises aboard R/V K-O-K, namely HOT-216 Fig. 1 Location of sampling sites in the Hawaiian waters during the three (November 2009), HOT-219 (March 2010), and HOT-223 Hawaii Ocean time-series (HOT) cruises (July 2010). Five stations including one coastal station (S1C1) and four oceanic stations (S2C6, S2C7, S2C12, and S52C1), and three stations (S1C1, S2C6, S52C1) were sam- microscope. A minimum of 100 microscope fields of pled during HOT-216 and HOT-219 and HOT-223, respec- thraustochytrid cells were counted based on their characteristic tively (Fig. 1). Depths from the upper euphotic zone (5, 45, red cell walls and yellow–green cell contents. Whenever cells and 75 m), the base of the chlorophyll maximum (100 and were not detected within 100 microscope fields, up to 125 125 m), and below the base of the euphotic zone (200 m) were fields were scanned (corresponding to a detection limit of sampled with 12-L polyvinyl chloride bottles attached to a Sea 6,166 cells/L), and values below this were designated as Bird CTD rosette sampler. For thraustochytrid counting, we zero in this study. The microscopic counting method based preserved 15 mL seawater (10 ml for bacteria) with 20 % on the use of brighter and/or more specific DNA fluoro- paraformaldehyde at a final concentration of 1 %. Afterwards, chromes DAPI was used to record bacterial abundance [23]. the samples were filtered over a 25-mm diameter, 0.8 μm Further calibration was made to the flow cytometry data from (0.2 μm for bacteria) pore size blackened polycarbonate mem- the HOT database. A cell biomass value of 20.6×10–12 g brane. Another 2 L water samples were vacuumed through carbon per cell of 5 μm diameter was used for determining 47-mm diameter, 0.2-μm pore-sized filters for the DNA ex- the thraustochytrid biomass [24, 25]. Bacterial biomass was traction. All samples were stored at −4 °C for subsequent estimated based on a value of 10×10–15 g per bacterial cell in experiments. Hawaiian waters [26].

Abundance and Biomass of Thraustochytrid and Bacteria Correlation Analysis

Cell counting of thraustochytrid was carried out using the All oceanographic data including temperature, salinity, acriflavine direct detection method [1]. Cell enumeration was dissolved oxygen, chloropigments, nitrate, and partial recorded with an OLYMPUS BX51 epifluorescence dissolved organic carbon used in this study were obtained Ecology of Planktonic Thraustochytrids 825 from HOT database (http://www.soest.hawaii.edu/HOT_ observed at 75 m of station S2C6 and 200 m of station S1C1, WOCE/ftp.html). Vertical thermosalinographs were drawn respectively (Fig. 2 and Table 1). Clearly, thraustochytrids based on these data. Pearson's correlation coefficients (r)and revealed a different vertical distribution pattern from that of probability values (p) were calculated using the SPSS PASW bacteria in the water column. A rough vertical pattern of two statistics 18.0.0 to analyze the relationship between peaks of thraustochytrid density was found at each station, one thraustochytrid abundance with bacterial abundance and above the depth of the chlorophyll maximum layer (DCML) environmental parameters of water samples. of 100 m, and the other one below it (Fig. 2). Thraustochytrids were present less frequently and in lower abundance during Clone Library Construction and Phylogenetic Analysis cruise HOT-219 and HOT-223. They were detected only in 7 out of 18 samples collected for both cruises, with the highest The total genomic DNA of seawater samples from cruise density observed at station S52C1. The value is 2.95×103 at HOT-216 was extracted using the FastDNA kit (Qbiogene, 75 m for HOT-219 and 1.75×103 at 100 m for HOT-223 Irvine, CA, USA), followed by PCR using the specific (Fig. 2 and Table 1). Correspondingly, the highest abundance labyrinthulomycetes 18S rRNA gene primer pairs LABY-A of bacterioplankton was detected at 45 m (6.09×108) for and LABY-Y (∼450 bp product) [19, 27]. Fifty microliters of HOT-219 and 125 m (6.81×108) for HOT-223 of station PCR reaction mixture was run with the Expand High Fidelity S52C1. Furthermore, thraustochytrids displayed a less patchy PCR System (Bio-rad, Hercules, CA, USA). The PCR prod- distribution pattern in the water column during these two ucts of correct size were purified using a QIAquick PCR cruises in comparison with HOT-216. Their maximum values Purification Kit (Qiagen, Valencia, CA, USA), cloned into usually occurred concomitantly at the DCML and displayed a pGEM-T Easy vector (Promega, Madison, Wisconsin, USA), similar vertical profile to that of chlorophyll at station S1C1, and finally transformed into Escherichia coli cells. Plasmids S2C12 of HOT-216, and S52C1 of both HOT-219 and HOT- were isolated from positive white colonies and sequenced with 223 (Fig. 2). the T7 or SP6 primers, at the University of Hawaii DNA Core Sequencing Facility on an Applied Biosystems 3730 automat- Biomass of Thraustochytrids and Bacterioplankton ed DNA sequencer. A similarity cutoff of 98 % [28]was in the Water Column applied to determine the operational taxonomic units (OTUs) obtained by software package DOTUR [29]. Thraustochytrid According to the calculation method mentioned above (see 18S rRNA sequences of this study and the matched reference “Materials and Methods”), thraustochytrids yield a biomass of sequences from NCBI GenBank were edited and aligned using 3.9×10−8 to 1.30×10−5 gcarbonL−1 during HOT-216. Its DNAMAN 5.1.0.0 (Lynnon Biosoft), ClustalX version 1.83 relative percentage contribution ranged from 0.79 to 365.1 % [30, 31], and BioEdit with a structure-based 18S rDNA align- to that of bacteria whose biomass varied from 1.45×10−6 to ment [32]. The aligned sequences were ultimately imported 6.94×10−6 (Table 1). A total of seven water samples mainly into PhyML 3.0 [33] to generate the maximum likelihood (ML) from depths below the DCML (125 m of station S2C7; 45, phylogenetic tree, based on the Markov (HKY 85) model of 100, and 200 m of station S2C12; and 100, 125, and 200 m of substitution. Sequences obtained in this study were submitted station S52C1) revealed higher thraustochytrid biomass than to GenBank under accession numbers JN935777-JN935807. that of bacterioplankton (Fig. 4). Whereas, relatively small values of 0–13.49 and 0–8.36 % were obtained from HOT- 219 and HOT-223, respectively (Table 1). Results Correlation Analysis of Thraustochytrids Vertical Distribution of Thraustochytrids in the Water Column with Bacterioplankton and Oceanographic Parameters

Compared to cruise HOT-219 and HOT-223, high numbers Correlation between thraustochytrids and bacterioplankton (103–105 cells/L) of thraustochytrids were detected at all along with other oceanographic parameters was summarized depths of the sampled stations during HOT-216. Four oceanic in Table 2. With all the collected samples during three season- stations (S1C1, S2C6, S2C12, and S52C1) contained compa- al cruises (n=66), one significant positive correlation of rable numbers (5.25–66.30×104 cells/L) of thraustochytrids, thraustochytrids was found with nitrate (p=0.551, r=0.000). 1 order of magnitude higher than the coastal station (S1C1). To better understand the possible mechanisms controlling the Their maximum population (6.30×105 cells/L) was found at distribution of thraustochytrids, separate correlation analysis 100 m of station S2C12, and the minimum (1.89×103 cells/L) of thraustochytrids with both bacterioplankton and environ- detected at 45 m of station S1C1 (Fig. 2 and Table 1). mental parameters was also carried out, using samples from Correspondingly, the maximum bacterial abundance of each cruise (n=30, 18, 18) and each station (n=6).A summa- 6.94×108 cells/L and the minimum of 1.45×108 cells/L were ry of all the Pearson “r” and “p” values are summarized in 826 Q. Li et al.

Fig. 2 Vertical distributions of thraustochytrid and bacterial abundance and chlorophyll at each station during cruise HOT-216 (November 2009), HOT-219 (March 2010), and HOT-223 (July 2010)

Table 2. Interestingly, thraustochytrids collected at two sta- Diversity and Phylogenetic Analysis tions (S1C1 and S52C1) of HOT-216 were negatively (r = −0.837/−0.945, p =0.038/0.005) correlated with Out of the 97 sequences analyzed, 25 OTUs were identified bacterioplankton. The abundance of thraustochytrids collected using the method described by Gao et al. [34]. The vast at all the stations during cruise HOT-223 have positive majority (n=20) of them aligned with Aplanochytrium sp. relationships with salinity (r=0.523, p=0.026) and oxygen (EU851170) recovered from the Indian Ocean [35]in (r=0.673, p=0.002). the ML phylogenetic tree (Fig. 5), with relatively high identity Ecology of Planktonic Thraustochytrids 827

Table 1 Extreme values occurrence of thraustochytrids abundance and biomass percentage during all three cruises (each cell displayed in value and location)

Features of Bacterioplankton and thraustochytrids HOT-216 (November 2009) HOT-219 (March 2010) HOT-223 (July 2010)

Maximum thraustochytrid abundance (cells/L) 6.3×105, 100 m, S2C12 2.95×103, 75 m, S52C1 1.75×103, 100 m, S52C1 Minimum thraustochytrid abundance (cells/L) 1.89×103,45m,S1C1 UD UD Maximum bacterioplankton abundance (cells/L) 6.94×108,75m,S2C6 6.09×108, 45 m, S52C1 6.81×108, 125 m, S52C1 Minimum bacterioplankton abundance (cells/L) 1.45×108, 200 m, S1C1 2.41×108, 200 m, S1C1 1.41×108,200m,S1C1 Maximum thraustochytrid biomass percentage 365.1 %, 200 m, S52C1 13.49 %, 100 m, S2C12 8.36 %, 100 m, S2C12 against bacterioplankton Minimum thraustochytrid biomass percentage 0.79 %, 45 m, S1C1 UD UD against bacterioplankton

UD undetectable

(97–99 %). The other 5 OTUs appeared not to have close Discussion affiliation with cultured species. One OTU from the coastal station S1C1 fell into the phylogroup of Oblongichytrium,with Environmental factors controlling thraustochytrids population a 98 % similarity to uncultured clone (GU825121) dynamics are one of the most fascinating subjects of study in retrieved from the Cariaco Basin of the Caribbean Sea and a marine eukaryotic microbial ecology. Some studies have sug- 96 % identity to the closest known Oblongichytrium sp. from gested that thraustochytrids biomass is related to riverine inputs Long Island, NY of USA. Two OTUs from station S1C1 and of organic material [24, 25] while other studies have showed S52C1 fell into the family of thraustochytrid and clustered with that it is often associated with autochthonous oceanic material Ulkenia. Both of them had a 99 % identity with uncultured such as chlorophyll in the Arabian Sea [36] and POC in the marine eukaryote (HM369554) recovered from Boothbay equatorial Indian Ocean [8]. In the NPSG area, a large abun- Harbor in the Gulf of Maine, USA, and a 95–97 % identity to dance of these protists were observed at both coastal (S1C1) a Ulkenia profunda isolate from fallen mangrove leaves along and oceanic stations (S2C6, S2C7, S2C12, and S52C1), with the Eastern coast of the Gulf of Thailand. Finally, an unknown their unexpected higher numbers (one order of magnitude) group with no associated references consists of 2 OTUs from observed at the oceanic stations during cruise HOT-216. This station S2C6 displayed at the base of the tree (Fig. 5). This indicated their potential role of utilizing both allochthonous and unknown group revealed a fairly low similarity of 94 % to the autochthonous organic matters in the ocean, even with a likely Aplanochytrium sp. S2125 (EU851170). preference to oceanic substrates in this case. Another point that

Table 2 Collection of significant correlations (Pearson coefficient index) dissolved oxygen (Oxy), chloropigments (CHLPIG), nitrate (Nit), for thraustochytrids abundance against that of bacterioplankton (Bac) and among cruises and stations oceanographic factors, including temperature (Tem), salinity (Sal),

Pearson Correlation Bac Tem Sal Oxy CHLPIG Nit

All three cruises 0.059 0.038 0.048 −0.022 0.089 0.551a (n=66) 0.640 0.763 0.704 0.860 0.476 0.000 HOT-216 0.083 −0.063 0.069 0.124 0.157 0.496b (n=30) 0.664 0.739 0.718 0.513 0.407 0.014 HOT-223 0.357 0.103 0.523b 0.673a −0.091 −0.310 (n=18) 0.146 0.684 0.026 0.002 0.720 0.327 216-S1C1 −0.837b −0.872b −0.875b −0.424 −0.016 0.792 (n=6) 0.038 0.024 0.022 0.402 0.976 0.061 216-S52C1 −0.945a −0.909b −0.864b −0.398 0.084 ND (n=6) 0.005 0.012 0.027 0.434 0.874 223-S52C1 −0.418 0.050 0.816b 0.810 0.668 ND (n=6) 0.410 0.925 0.048 0.051 0.147

Significant values are marked in italics. The two rows at each cell represents “r” and “p” value, respectively a Correlation is significant at the 0.01 (two-tailed) b Correlation is significant at the 0.05 (two-tailed) 828 Q. Li et al.

a Aplanochytrium EU851170 Aplanochytrium stocchinoi AJ519935 Aplanochytrium Colonization on “marine snow” 50 Aplanochytrium sp. FJ810216 52 Uncult marine eukaryote DQ103777 84 Aplanochytrium kerguelense AB022103 52-5-1(4) 1-2-5 (3) 88 52-2-8 (1) 50 6-3-9 (6) 82 1-1-7 (6) Oblongichytrium Uncult eukaryote EF100267 L

56 abyrinthulea 88 Uncult eukaryote EF100415 78 Uncult marine eukaryote GQ344784 73 Uncul eukaryote GU825121 1-2-4 (1) 56 Oblongichytrium sp FJ799794 bc87 Oblongichytrium sp. FJ799798 Oblongichytrium sp. AB290575 99 Ulkenia

66 Oblongichytrium sp. FJ821480 Thraustochytriidae Free living cell Thraustochytriidae sp. FJ010826 98 Ulkenia profunda isolate DQ023615 99 Ulkenia profunda L34054 Zoospore 86 1-5-1 (1) 86 89 Uncult labyrinthulid clone FJ800622 98 Uncult marine eukaryote EF539096 52-1-2 (1) 59 Uncult eukaryote AY256317 Uncult eukaryote HM369554 70 Uncult eukaryote GU825677 Unk 6-2-11(1) 99 6-1-13 (1) non-Labyrinthulea Stramenopile Fig. 3 Microscopic photogram of thraustochytrid cells from Hawaiian waters. Scale bar 5 μm 0.01 Fig. 5 Maximum likelihood tree based on thraustochytrids-specific 18S has been eliciting much attention of mycologists is the ecolog- rRNA gene sequences derived from Hawaiian waters. Amplicons re- ical niches and living habitats of thraustochytrids. As Fig. 3 trieved by us are marked in bold, followed by the numbers belong to the same OUT labeled in brackets. Three digital abbreviations of the showed, a patchy distribution of thraustochytrids being at- specie names stand for site number (1 =S1C1,6 =S2C6,52 = S52C1), tached to marine snow particles were commonly found in our depth (1 =5m,2 =45m,3 =75m,4 =100m,5 =125m,and6 =200m), water samples. This is the very first microscopic evidence to and clone number, respectively show their ecological niches in Hawaiian waters, and future isolation work needs to be carried out since these particles doubled in July 2010 at station S2C6, neither thraustochytrids provide good resource of thraustochytrids. In addition, these nor bacterial numbers increased instantaneously (Fig. 2). Thus, findings suggested their possible dependence on particulate it is safe to speculate that other factors such as POC, in partic- organic matters and potential ecological function in hydrolyz- ular dead particulate organic materials [10, 36, 37], are the more ing them. Spatially, thraustochytrids biomass exceeded that of likely reason controlling their population, rather than the total bacterioplankton at several stations and depths during HOT biomass of living primary production. What was the most likely 216 (Fig. 4). This was particularly the case at the base of explanation was that the accumulation of photoautotrophs dur- euphotic zone, where bacteria decreased dramatically ing the summer months had led to a large detrital aggregates suggesting that they make a significant contribution towards increase during the fall, namely the maximum in fall and the the organic carbon pool. Temporally, the maximum value of minimum in winter of particulate matter standing stocks [38]. their abundance occurred in late fall (one order of magnitude Thus, the subsequent bloom of thraustochytrids would occur. higher) at station ALOHA, with no obvious change observed in Our results also supported the early findings in the Arabian Sea, either photosynthetic biomass (chlorophyll) or environmental where high numbers of thraustochytrids were observed in parameters during HOT-216 (Suppl. Fig. 1). Even for cruise association with mucus particles in September, the HOT-223, when the total amount of chlorophyll almost end of the highly productive summer monsoon [36].

Fig. 4 Relative percentage of biomass contribution of thraustochytrids and bacterioplankton in the water column (six bars from top to bottom at each station represent depth of 5, 45, 75, 100, 125, and 200 m, respectively) during all three cruises Ecology of Planktonic Thraustochytrids 829

Additionally, the likely seasonal species composition shift of mechanisms driving the seasonal variation of their population POC produced by phytoplankton at ALOHA [39, 40] can be remain a challenging yet intriguing topic area to be further another possible reason influencing the thraustochytrids com- investigated. Information provided by the present study adds munities, (known as) the substrate-specific relationship be- an additional dimension of our own general perspective on tween thraustochytrids and POC [5, 8, 41]. Our results support marine microbial ecosystems. the hypothesis that a seasonal late fall bloom and winter–spring demise of thraustochytrids was what most likely occurred at Acknowledgments This work was partially funded by National Natural station ALOHA. Further investigation should guarantee these Science Foundation of China grant 31170109 (GYW) and NOAA grants NA04OAR4600196(GYW) and NA09AOR4170060 (GYW). 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