Distribution patterns and phylogeny of marine stramenopiles in the north pacific ocean

Abstract

Marine stramenopiles (MASTs) are a diverse suite of eukaryotic microbes found in marine environments. Several MAST lineages are thought to contain heterotrophic nanoflagellates. However, MASTs remain uncultured and data on distributions and trophic modes are limited. We investigated MASTs in provinces on the west and east sides of the North Pacific Subtropical Gyre, specifically the East China Sea (ECS) and the California Current system (CALC). For each province, DNA was sampled from three zones: coastal, mesotrophic transitional, and more oligotrophic euphotic waters. Along with diatoms, chrysophytes, and other stramenopiles, sequences were recovered from nine MAST lineages in the six ECS and four CALC 18S rRNA gene clone libraries. All but one of these libraries were from surface samples. MAST clusters 1, 3, 7, 8, and 11 were identified in both provinces, with MAST cluster 3 (MAST-3) being found the most frequently. Additionally, MAST-2 was detected in the ECS and MAST-4, -9, and -12 were detected in the CALC. Phylogenetic analysis indicated that some subclades within these lineages differ along latitudinal gradients. MAST-1A, -1B, and -1C and MAST-4 size and abundance estimates obtained using fluorescence in situ hybridization on 79 spring and summer ECS samples showed a negative correlation between size of MAST-1B and MAST-4 cells and temperature. MAST-1A was rarely detected, but MAST-1B and -1C and MAST-4 were abundant in summer and MAST-1C and MAST-4 were more so at the coast, with maximum abundances of 543 and 1,896 cells ml(-1), respectively. MAST-4 and Synechococcus abundances were correlated, and experimental work showed that MAST-4 ingests Synechococcus. Together with previous studies, this study helps refine hypotheses on distribution and trophic modes of MAST lineages.

Fig 1

Study regions and sampling sites in the Central California and eastern Pacific Ocean provinces. (A) Location of the study regions within the North Pacific Ocean; (B) CALC sites sampled in October 2007; main stations are represented by white dots. (C and D) ECS sites sampled from April to May 2009 (C) and June to July 2009 (D). The color background represents sea surface salinity according to the gradient bars provided. Note the different scales between the top and bottom panels.

Fig 2

Abundance during spring and summer of MAST-1B (A and B, respectively), MAST-1C (C and D, respectively), and MAST-4 (E and F, respectively) in the ECS, as determined by FISH (units are cells ml⁻¹). Note the differences in the color scales.

Fig 3

Relationship between water temperature and cell diameter of each of the MAST groups enumerated as well as HNFs as a whole. Significant relationships were determined for HNFs (r = −0.70, P < 0.001, n = 79), MAST-1B (r = −0.39, P < 0.05, n = 41), and MAST-4 (r = −0.25, P < 0.05, n = 79) but not MAST-1C (r = −0.21, P = 0.91, n = 67). For MAST-1A, the relationship was also (negatively) significant, but the analysis involved very few cells. MAST and HNF cell sizes were estimated using different methods, the former by FISH (effectively, the cytoplasm) and the latter by DAPI staining (effectively, the nucleus).

Fig 4

Stramenopile phylogeny by ML methods of ECS (open squares) and CALC (solid squares) sequences. Nearly full-length 18S rRNA gene sequences from cultured or described organisms (gray) or environmental sequences (black) were used, resulting in 1,399 analyzed positions. ML and NJ bootstrap values are shown at nodes retaining >70% support (100 replicates total). Solid circles, significant support by both methods; gray circles, supported only by ML; open circles, only NJ support. Actual bootstrap values (ML/NJ) are shown for MAST clusters. The TrN+I+G substitution model (I = 0.206 and α = 0.438) was used, and four dinoflagellate sequences served as an outgroup. ECS clone names contain the cruise (897, spring; 905, summer) and station information, while in CALC clone names, the first number after the station identifier, i.e., 1, 8, and 3, corresponds to the sequenced size fractions 0.1 to <0.8 μm, 0.8 to <3 μm, and 3 to <20 μm, respectively; S and D indicate surface and DCM samples, respectively. Numbers in parentheses beside sequence names from our study indicate the number of clones represented by that sequence (all having >98% identity).

Fig 4

Stramenopile phylogeny by ML methods of ECS (open squares) and CALC (solid squares) sequences. Nearly full-length 18S rRNA gene sequences from cultured or described organisms (gray) or environmental sequences (black) were used, resulting in 1,399 analyzed positions. ML and NJ bootstrap values are shown at nodes retaining >70% support (100 replicates total). Solid circles, significant support by both methods; gray circles, supported only by ML; open circles, only NJ support. Actual bootstrap values (ML/NJ) are shown for MAST clusters. The TrN+I+G substitution model (I = 0.206 and α = 0.438) was used, and four dinoflagellate sequences served as an outgroup. ECS clone names contain the cruise (897, spring; 905, summer) and station information, while in CALC clone names, the first number after the station identifier, i.e., 1, 8, and 3, corresponds to the sequenced size fractions 0.1 to <0.8 μm, 0.8 to <3 μm, and 3 to <20 μm, respectively; S and D indicate surface and DCM samples, respectively. Numbers in parentheses beside sequence names from our study indicate the number of clones represented by that sequence (all having >98% identity).

Fig 5

Phylogenetic analysis of MAST-1 using NJ distance methods with partial-length sequences and containing representative ECS (open squares) and CALC (solid squares) sequences. Bootstrap values estimated from ML and NJ methods (100 replicates) are shown for nodes with >70% support, as described in the legend to Fig. 4. A total of 499 positions were analyzed, after masking and gap removal. Latitude and depth at the site of sequence origin are provided in color bars. Samples from latitudes of <20° are from the Cariaco Basin, Caribbean (A95, AA, AB, BC, and CA), Indian Ocean (IND58, IND60, IND70, and IND72), and Pacific Ocean (OLI); those from latitudes from 20° to 45° are from the Atlantic Ocean (AMT15_33, ENI, N5, N10, SSRP, and numerical sequence identifiers), Indian Ocean (IND1, IND2, IND31, and IND33), Mediterranean (BL and ME), and Pacific Ocean (TH and this study); those from latitudes from between 45° and 60° are from the Antarctic (ANT and DH), Atlantic Ocean (NA, AMT15_1, and OR), English Channel (RA), Framvaren Fjord (FV and SIF), and Helgoland (HA); and those from latitudes of >60° are from the Arctic Ocean (MD and NW), adjacent seas of the Arctic Ocean (NOR), Franklin Bay (CS), and Norwegian Sea (AD and CD). All clones were derived from the water column, except those from sediments (BAQ, TAGIRI, and DSGM).

Fig 6

Phylogenetic reconstruction of MAST-3 by NJ distance methods with partial-length sequences, including 7 representative sequences from ECS (open squares) and 27 representative CALC sequences (solid squares). A total of 471 positions were analyzed, after masking and gap removal; bootstrap values were estimated using ML and NJ methods with 100 replicates and are shown for nodes with >70% support. Clonal libraries represented, latitudinal ranges, and depths are as described in the legend to Fig. 5.

Fig 7

(A) Phylogenetic reconstruction of MAST-4 by NJ distance methods using partial-length 18S rRNA gene sequences. Nine representative CALC sequences (solid squares) were included, and 489 nucleotide positions were analyzed, after masking and gap removal. Bootstrap values were estimated using ML and NJ methods with 100 replicates and are shown for nodes with >70% support. Vertical red lines indicate probe mismatches with environmental sequences. (B) MAST-4 cells and ingested Synechococcus. The images are the result of an overlay of images from blue and green light excitation; red represents the MAST-4 cell, and yellow represents Synechococcus cells. Bar, 10 μm.

Fig 8

Phylogenetic reconstruction of MAST-7 on the basis of NJ distance methods and partial-length sequences. Our North Pacific sequences included 1 representative sequence from the ECS (open square) and 6 from the CALC (solid squares). A total of 515 nucleotide positions were analyzed, after masking and gap removal. Bootstrap values were estimated using ML and NJ methods with 100 replicates and are shown for nodes with >70% support.