Responses of diatoms to iron-enrichment (SEEDS) in the western subarctic Pacific, temporal and spatial comparisons

Progress in Oceanography Progress in Oceanography 64 (2005) 189–205 www.elsevier.com/locate/pocean

Responses of diatoms to iron-enrichment (SEEDS) in the western subarctic Pacific, temporal and spatial comparisons Atsushi Tsuda a,*, Hiroshi Kiyosawa b, Akira Kuwata c, Mamiko Mochizuki d, Naonobu Shiga d, Hiroaki Saito c, Sanae Chiba e, Keiri Imai a, Jun Nishioka f, Tsuneo Ono g a

Ocean Research Institute, University of Tokyo, 1-15-1 Minamidai, Nakano, Tokyo 164-8639, Japan Marine Biological Research Institute of Japan, 4-3-16 Yutaka, Shinagawa, Tokyo 142-0042, Japan c Tohoku National Fisheries Research Institute, Shinhama, Shiogama, Miyagi 985-0001, Japan Graduate School of Fisheries Sciences, Hokkaido University, 3-1-1 Minato-cho, Hakodate, Hokkaido 041-8611, Japan e Frontier Research System for Global Change, Showa, Kanazawa, Yokohama 236-0001, Japan f Central Research Institute of Electric Power Industry, Abiko, Chiba 270-1194, Japan g Hokkaido National Fisheries Research Institute, 116 Katsurakoi, Kushiro, Hokkaido 085-0802, Japan b

d

Available online 28 March 2005

Abstract Phytoplankton species composition was analyzed inside and outside of the iron-enriched patch during the SEEDS experiment. Before the iron-enrichment, the phytoplankton community consisted of similar proportions of pico-, nano- and micro-sized phytoplankton, and the micro-phytoplankton was dominated by the pennate diatom Pseudo-nitzschia turgidula. Although all the diatoms, except the nano-sized Fragilariopsis sp., increased during the two weeks of the observation period, the flora in the patch dramatically changed with the increase of phytoplankton biomass to a centric diatom-dominated community. Neritic diatoms, especially Chaetoceros debilis, showed higher growth rates than other diatoms, without any delay in the initiation of growth after the enrichment, and accounted for 90% of the micro-phytoplankton after day 9. In contrast, the oceanic diatoms showed distinct delays in the initiation of growth. We conclude that the responses of the diatoms to the manipulation of iron concentration were different by species, and the fast and intensive response of the phytoplankton to iron-enrichment resulted from the presence of a small amount of neritic diatoms at the study site. The important factors that determine the dominant species in the bloom are the potential growth rates under an iron-replete condition and the growth lag. Abundant species in the patch are widely distributed in the North Pacific and their relative contributions in the Oyashio area and at Stn KNOT are high from spring to summer. However, a characteristic difference of species composition between the SEEDS bloom and natural blooms was the lack of Thalassiosira and Coscinodiscus species in the patch,

*

Corresponding author. Tel.: +81 3 5351 6476; fax: +81 3 5351 6481. E-mail address: [email protected] (A. Tsuda).

0079-6611/$ - see front matter
2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.pocean.2005.02.008

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which usually account for a major part of the phytoplankton community under blooming conditions in the western North Pacific.
2005 Elsevier Ltd. All rights reserved. Keywords: Subarctic pacific; Phytoplankton; Diatom; Iron enrichment; Bloom; Species composition

1. Introduction In the subarctic Pacific, both the Alaskan Gyre (AG) and the Western Subarctic Gyre (WSG) are recognized as HNLC (high nitrate low chlorophyll) ocean waters (Banse & English, 1999) with a low concentration of dissolved iron (Martin & Fitzwater, 1988; Nishioka et al., 2003), while phytoplankton blooms occur at the some edges of the gyres and in neritic areas (Banse & English, 1999; Boyd & Harrison, 1999; Obayashi et al., 2001). The WSG shows a relatively high concentration of chlorophyll compared with the AG (Sugimoto & Tadokoro, 1997), and the relative abundance of diatoms is higher in WSG than AG throughout the year (Obayashi et al., 2001; Shiomoto & Asami, 1999). Moreover, WSG is characterized by a high export carbon flux having high contents of opal (Honda et al., 2002). Several mesoscale iron-enrichment experiments have been conducted in HNLC regions to test the iron-limitation hypothesis proposed by Martin (1990). Increases of phytoplankton biomass in the mesoscale experiments after manipulation of iron concentration have confirmed MartinÕs hypothesis (Boyd et al., 2000; Coale et al., 1996; Martin et al., 1994; Tsuda et al., 2003), as have the bottle incubation experiments (e.g., Boyd et al., 1996; de Baar & Boyd, 1999). The dominant phytoplankton taxon was diatoms in these experiments, and in the cases of the eastern equatorial Pacific and the Southern Ocean, pennate diatoms were dominant (Cavender-Bares, Mann, Chisholm, Ondrusek, & Bidigare, 1999; Gall, Boyd, Hall, Safi, & Chang, 2001). Diatoms are large-sized, autotrophic organisms with siliceous frustules and show relatively high sinking rates compared with those rates of nano- and picoplankton (e.g., Smetacek, 1985). Furthermore, many diatom species produce TEP (transparent exopolymers), which facilitate particle aggregation and accelerate sinking processes (Passow, 2002). The dominance of diatom cells is an important factor determining the fate of carbon assimilated by the phytoplankton communities by enhancing sedimentation. Actually, high export fluxes have been observed at the end of the diatom-dominated blooming in various marine environments (e.g., Odate & Maita, 1990; Saino et al., 1998; Welschmeyer & Lorenzen, 1985). In the present study, we investigated the development of a massive diatom-dominated bloom in a mesoscale in situ iron-enrichment experiment, SEEDS (Subarctic Pacific Iron Experiment for Ecosystem Dynamics Study) in the WSG, focusing on changes in the species composition and the specific growth responses of key diatom species. Furthermore, to examine the origin of seed stock for the massive bloom in the SEEDS experiment, seasonal data of the species composition of the Oyashio area and Stn KNOT (44N, 155E) in the western subarctic Pacific, where natural blooms occur (Imai, Nojiri, Tsurushima, & Saino, 2002; Mochizuki, Shiga, Saito, Imai, & Nojiri, 2002; Saito et al., 1998; Saito, Tsuda, & Kasai, 2002), were compared with the species composition observed in this study.

2. Materials and methods A meso-scale in situ iron-enrichment experiment (SEEDS) was conducted in the western subarctic gyre of the North Pacific (48.5N, 165E) from 18 July to 1 August 2001 aboard R.V. Kaiyo-Maru, Fisheries Agency of Japan (Fig. 1). The experiment consisted of a single addition of 350 kg iron as FeSO4 with 0.48 M of an inert tracer gas, sulphur hexafluoride (SF6), over an 8 · 10 km patch with a mixed layer depth of
10 m on 18 July 2001. Day 1 is defined as the 24-h period starting at 0:00 19 July 2001. The iron-patch

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65

Bering Sea

mc

Sea of Okhotsk

Ka

Latitude N

55

ha

tka

P.

60

50 il ur

SEEDS

K

45

KNOT

A4

40 35

nd

la

Is

140

150

160

170

Longitude E Fig. 1. Locations of mesoscale iron-enrichment experiment (SEEDS), and seasonal monitoring sites of diatom communities (KNOT and A4) in the western subarctic Pacific.

was followed from 19 July to 1 August 2001, by tracking the SF6 signals (Tsumune, Nishioka, Shimamoto, Takeda, & Tsuda, 2005). Hydrocast samplings were carried out 2, 4 7, 9, 11 and 13 days from the fertilization, both inside and outside the patch. Location of inside stations was determined by the SF6 monitoring, and outside samplings were carried out near a reference buoy that had been deployed about 37 km east from the center of the patch. However, the reference buoy moved east while the patch moved west. Therefore, we replaced the reference buoy to 30 km west of the center of the patch from day 9. Low concentration of SF6 (<3 fM) was also confirmed on each sampling day at the outside sampling stations. Phytoplankton samples were collected with a CTD multi-bottle sampler from depths of 5, 10, 20, 30 and 50 m. Other samples were collected during underway samplings using a clean pumping system connected by Teflon tubing to a towed fish suspended from the shipÕs side (2 m depth) (Tsumune et al., 2005). The locations of the sampling were determined by monitoring the SF6 or pCO2 concentrations, as the local maximum and minimum values, respectively. Cross-section samplings were also done on days 3, 8 and 12, down to 70-m depth with acid-clean Niskin bottles attached to a Kevlar wire. A part of the samples was transferred to a counting chamber and dominant phytoplankters larger than 10 lm were counted on board, with a microscope at magnifications between 100· and 400· with or without fixation. One liter of the samples was preserved with buffered formalin (2%, v/v). The samples were concentrated to 20–30 ml by settling, and an aliquot of the concentrated samples were used for identification and enumeration of the diatom cells with an inverted and a standard microscope (Hasle, 1978). In the dense samples (days 9 to 13 inside the patch), the settling process was omitted. Species identification and size measurement of diatom cells were done only for the samples from 5-m depth and the underway samples, because biological/chemical responses to the iron-enrichment were observed in the surface mixed layer (cf. upper 15-m depth, Tsuda et al., 2003). Counting and identification were done for at least 300 cells in each sample without replication. For the examination of girdle band structure of Pseudo-nitzschia spp., 10 ml of 60% HNO3 was added to 1 ml of concentrated subsample, followed by addition of a few grains of NaNO2. The sample was left to stand for 24 h, and then rinsed three times with distilled water to remove organic materials (Lundholm, Daugbjerg, & Moestrup, 2002). The cleaned samples were examined with SEM. For the nano-sized fraction of the phytoplankton, 300 ml of the samples were fixed immediately with glutaraldehyde (1.0%, v/v). Then subsamples of 1 to 100 ml stained with DAPI and FITC were gently filtered (<100 mmHg) onto black-stained Nuclepore filters (1 lm pore size), respectively, and nano-sized diatoms were counted and sized under an epifluorescent microscope at magnifications between 400· and 1000·.

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Cell volume of each diatom species was obtained from its approximate geometry. Volume estimates were converted to cellar carbon contents using the equation of Strathmann (1967). Size measurements of the cells were done between 2 and 6 months after the sampling. For size-fractionated chlorophyll-a concentration, 500 ml of seawater was sequentially filtered with Nuclepore filters (10, 2 and 0.2 lm pore size), and the pigments on the filters were extracted with 6 ml of N,N-dimethylformamide for 24 h (Suzuki & Ishimaru, 1990), then chlorophyll-a concentration was measured with a Turner Designs fluorometer (Welschmeyer, 1994). Vertical profiles of PAR (photosynthesis available radiation) were measured with a PRR 600 (Biospherical Instruments). For comparison of the SEEDS flora to diatom communities in the WSG, data from KNOT (44N, 155E) and A4 (4215 0 N, 14508 0 E) of the A-line monitoring (Saito et al., 1998) were used (Fig. 1). To compare the community structures in the surface mixed layer, we used the averages of values from 0 and 10 m depth at KNOT (Mochizuki et al., 2002), and the values at 10 m depth in the A4 data (Tsuda unpublished). A dissimilarity matrix based on differences in the species composition between the stations was obtained using the Bray–Curtis index (Bray & Curtis, 1957). An unweighted pair group clustering method using the arithmetic mean (UPGMA) was applied to the matrix to classify the stations into several groups with distinctive community structures. Non-metric multi-dimensional scaling (NMDS) (Kruskal & Wish, 1978) was used to visualize the similarity in diatom communities among the derived cluster groups. Goodness of fit of the NMDS plot was verified by the stress-value criteria constructed by Kruskal and Carmore (1971 in Domanski, 1984). The cluster analysis and NMDS were conducted with the BIOSTAT II version 3.5 statistical package (Pimental, 1994). These analyses were made on the samples taken on day 0 and outside of the patch, because the iron-induced bloom comprised only a small number of diatom species.

3. Results 3.1. Development of the bloom The water mass in which chlorophyll-a increased agreed with the iron-enriched water mass vertically and horizontally, as was evidenced by the cross-sections of chlorophyll-a and iron concentration on days 3, 8 and 12 (Fig. 2). Although the dissolved iron concentration in the patch was not discernible on day 12, the iron-enriched patch was clearly discernible by total iron concentration (dissolved and particulate). A large increase of phytoplankton in the surface mixed layer of the iron-enriched patch was observed between day 4 and day 7 (Fig. 3). The initial concentration of chlorophyll-a in the surface mixed layer was 0.8–0.9 mg m3, and the micro-phytoplankton (>10 lm) accounted for about 40% of the total concentration (>0.2 lm, Fig. 4). The chlorophyll-a in the patch increased to 17 mg m3 after day 9, and the maximum concentration of the underway surveys was 21.8 mg m3 on day 10. The increase of chlorophyll-a concentration was accompanied by the domination of micro-phytoplankton. Pico- and nano-phytoplankton also increased by 2 and 5 times, respectively, but the micro-phytoplankton increased by 45 times from day 0 to day 13 and accounted for over 90% of the total chlorophyll-a after day 9 (Fig. 4). Neither increase nor modification of the size-distribution of chlorophyll-a was observed below the surface mixed layer (Fig. 4). The bottom of the surface mixed layer in the patch was located around 10 m until day 4 and then deepened to 16–20 m, because of a relatively high wind stress during the later half of the observation period. The euphotic layer in the patch indicated by 1% of the surface photosynthetically available radiation rose from 46 to 12 m as a result of elevated chlorophyll-a concentration and was consequently shallower than the surface mixed layer after day 9 (Fig. 4). In the area outside of the patch, the chlorophyll-a concentration also increased from 0.8–0.9 to 1.8 mg m3 on day 9 and decreased to 1.1 mg m3 on day 13 (Fig. 3). The size distribution of chlorophyll-a did not change throughout the observation period (Fig. 4).

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Fig. 2. Sections of chlorophyll-a (mg m3), dissolved iron and total iron (nM) concentrations across the iron-enriched patch on day 3 (upper), 8 (middle) and 12 (bottom).

Fig. 3. Temporal variations of chlorophyll-a concentration inside (closed circles) and outside (open circles) the iron-enriched patch. The values on days 6, 8, 10 and 12 are average values in the patch during the underway survey (Tsuda et al., 2003), and others are average values in the surface mixed layer obtained by hydrocasts. Vertical bars denote 1 SD.

Before the iron-enrichment (day 0), nano-sized Fragilariopsis sp. (valve length 4–6 lm) numerically dominated the diatom assemblage (Fig. 5 and Table 1). In the micro-sized fraction, pennate diatoms (Pseudonitzschia turgidula and Neodenticula seminae) accounted for 75% of the micro-phytoplankton cell density,

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Fig. 4. Vertical profiles of total chlorophyll-a concentration (closed circles with solid lines, lower scale) and percentage of microphytoplankton chlorophyll-a (triangles with broken lines, upper scale) inside (upper panel) and outside (lower panel) the iron-enriched patch. Horizontal lines indicate euphotic layers (broken: 1% of surface irradiance) and bottom depth of the surface mixed layers (solid: 1 C of the surface temperature).

and centric diatoms (mainly Chaetoceros concavicornis, C. atlanticus) for 24% (Table 1 and Fig. 5). The species composition outside the patch was almost stable during the observation period (Table 1). Chaetoceros debilis was a minor component in the initial condition (0.9% of the micro-phytoplankton cell density). It grew exponentially until day 9, and it accounted for about 90% of micro-phytoplankton cell density between days 11 and 13 (Fig. 5). C. debilis showed a high net growth rate of 1.8 d1 between day 4 and 9, and then grew slowly (0.4 d1) until the end of the observation. Resting spores of C. debilis were not observed throughout the experiment. Other diatom species (Chaetoceros concavicornis, C. atlanticus, P. tugidula and N. seminae, Leptocyrindrus minimus, Eucampia groenlandica, Rhizosolenia spp.), except Fragilariopsis sp., also showed positive net growth after day 4, but the growth rates were lower than that of C. debilis (Table 2). We could not observe significant differences of net growth rates between the pennate diatoms and the centric diatoms other than C. debilis. We observed delays in the initiation of growth after iron-enrichment in C. concavicornis, C. atlanticus, P. turgidula and N. seminae (Table 2). In contrast, relatively high growth rates (>0.5 d1) was observed for C. debilis, E. groenlandica and L. minimus from the beginning of the observations. The diatom carbon was a minor component of the particulate organic carbon (POC) during the initial phase of the experiment comprising about 10% (Fig. 6). Chaetoceros concavicornis accounted for the largest part of the diatom carbon (21%), and the numerically dominant Fragilariopsis sp. only accounted for 6% at the beginning of the experiment. The diatom carbon increased exponentially until day 12, and the contribution of diatom carbon to the POC, especially that of C. debilis, increased with time, and finally diatom carbon almost agreed with the POC on day 13 (Fig. 6).

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Fig. 5. Temporal changes of cell density and composition of diatom species in the surface layer of the iron-enriched patch. The nanosized fraction is not included in the upper figure. Closed triangles; C. debilis, open triangles; N. seminae, open circles; Pseudo-nitzschia turgidula, closed circles; C. atlanticus, closed squares; C. concavicornis, open squares; Rhizosolenia spp., closed diamonds; E. groenlandica, open diamonds; L. minimus, hexagons; nano-pennates.

3.2. Comparison of the species composition with natural phytoplankton blooms Both spring and fall blooms were observed at Station KNOT (Fig. 7). The major components of the blooms were Thalassiosira spp. and Coscinodiscus spp. in spring and fall, respectively. Especially, Thalassiosira spp. was a major component of the diatom community throughout the year. C. debilis occurred almost throughout the year, although it made up only 3% of the total diatom cell density at most. Other diatom species which occurred abundantly in the SEEDS bloom (Pseudo-nitzschia spp., N. seminae, C. atlanticus, C. concavicornis, L. minimus, E. groenlandica, Rhizosolenia spp.) also occurred throughout the year and were relatively abundant from June to September (Fig. 7). In the Oyashio region (Station A4), diatom blooms were observed only in spring between April and May (Fig. 8). The cell densities during the blooming periods were higher than those at KNOT but lower than the SEEDS bloom. The spring blooms in this region are characterized by the abundant occurrence of pennate diatoms, Fragilariopsis spp. Thalassiosira spp. also occurred throughout the year, and species of that genus made up a major part of the diatom community during the bloom period (Fig. 8). Coscinodiscus spp. was a relatively important component after the spring blooms (June

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Table 1 Cell densities of the 20 dominant species of diatoms before iron-enrichment (day 0), and those on day 13 outside of the patch (cells ml1) Species

Day 0

Day 13-OUT

Fragilariopsis sp. 1 Pseudo-nitzschia turgidula Thalassiosira spp. Neodenticula seminae Chaetoceros concavicornis Chaetoceros sp. 2 Pseudo-nitzschia spp. Ch. atlanticus Chaetoceros sp. 3 Fragilariopsis sp. 2 Ch. convolutes Leptocylindrus minimus Ch. debilis Asteromphalus hyalinus C. distans Rhizosolenia (Proboscia) alata Eucampia groenlandica Thalassiothrix longissima

430 13.7 9.5 6.6 3.4 2.8 2.6 2.4 1.9 1.8 1.1 0.5 0.2 0.2 0.2 0.1 0.1 0.1

200 2.4 7.7 0.7 2.1 3.6 1.0 2.2 0.1 0.4 0.2 1.6 0.2 0.2 0.0 0.5 0.3 0.9

Underlined species are regarded as neritic species forming resting spores (McQuoid and Hobson, 1996).

Table 2 Net growth rates (d1) of the dominant diatom species in the iron enriched patch Day 0–4

Day 4–7

Day 7–13

Centric diatoms Chaetoceros concavicornis Ch. atlanticus Ch. debilis Eucampia groenlandica Leptocylindrus minimus

0.00 0.04 0.51 0.31 0.59

0.49 0.87 1.79 0.91 0.76

0.42 0.35 0.47 0.31 0.17

Pennate diatoms Pseudo-nitzschia turgidula Neodenticula seminae

0.10 0.13

0.75 0.51

0.29 0.39

Underlined species are regarded as meritic species forming resting spores (McQuoid and Hobson, 1996).

and July). C. debilis rarely occurred in the Oyashio region, and other SEEDS diatoms mainly occurred in spring. The initial diatom community in the SEEDS showed a high similarity to that outside the patch at the end of the observation (Figs. 9 and 10), which suggests that the background diatom community did not change throughout the observation period. This community made a subgroup 1 with those of Oyashio and KNOT in May (Figs. 9 and 10) at a 72% dissimilarity level. Subgroup 2 consisted of winter to early summer communities of Oyashio and KNOT, and both subgroups made a larger cluster at a 83% dissimilarity level. Numerically abundant species (indicated by >1% of the total cell number counted) were associated with both subgroups, although cell density of subgroup 1 was much higher than subgroup 2 (Table 3). Other communities were observed mainly during late summer to fall in Oyashio and KNOT.

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Fig. 6. Temporal changes of POC (diamonds), diatom carbon (closed circles) and carbon weight of C. debilis (open circles) in the surface layer (5 m) of the iron-enriched patch. POC data from Aono et al. (2005).

Fig. 7. Seasonal variations of diatom cell density (upper) and its composition (lower) in the surface water at Station KNOT (44N, 155E). The average of abundances at 0 and 10 m was used (from Mochizuki et al., 2002). SEEDS diatoms include N. seminae, Pseudonitzschia spp., C. atlanticus, C. concavicornis, L. minimus, E. groenlandica and Rhizosolenia spp.

4. Discussion 4.1. Pre-enrichment condition of the area and outside variation At the beginning of the iron-enrichment, the surface mixed layer of the studied area was regarded as in a HNLC condition (nitrate; 18.5 lM, silicate; 31.8 lM, chlorophyll-a; 0.85 mg m3) with a low concentration

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Fig. 8. Seasonal variations of diatom cell density (upper) and its composition (lower) in the surface water at Station A4 (4215 0 N, 14508 0 E). SEEDS diatoms include N. seminae, Pseudo-nitzschia spp., C. atlanticus, C. concavicornis, L. minimus, E. groenlandica and Rhizosolenia spp.

Fig. 9. Cluster dendrogram of the diatom communities (stations) based on the difference in diatom community structure. Communities were divided into a large Cluster A and five outliers at a 83% dissimilarity level. Cluster A was further divided into two subgroups (1 and 2) with two outliers at a 72% dissimilarity level. SED00IN is the initial community inside the iron patch and SED13OUT is the community outside the iron patch on the 13th day of the experiment. Names of the other communities consist of the location (OY: Oyashio, and KT: KNOT) and yy-mm, e.g., OY0005 stands for a Oyashio community in May, 2000.

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Fig. 10. Two-dimensional ordination plot of the diatom communities obtained by the NMDS (stress value: 17%). Each cluster group and subgroup (Fig. 9) is encircled. Note that the SEEDS communities (SED00IN and SED13OUT) overlap.

Table 3 Average cell density of diatom species for each cluster subgroup based on cluster analysis (Fig. 9) Rank

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Subgroup 1

Subgroup 2

Species

Cell density

(%)

Species

Cell density

(%)

Ch. concavicornis P. turgidula N. seminae E. groenlandica Ch. atlanticus T. nitzschioides Ch. debilis Co. criophilum L. minimus S. nipponica Ch. convolutes Cylindrotheca closterium T. longissima Ch. distans Rhizosolenia alata

5240 4030 3690 1450 1160 945 735 708 668 660 408 342 253 176 159

24.8 19.1 17.5 6.9 5.5 4.5 3.5 3.4 3.2 3.1 1.9 1.6 1.2 0.8 0.8

N. seminae Ch. concavicornis Thalassionema nitzschioides Lauderia annulata Corethron. criophilum Ch. convolutus Stephanopyxis nipponica Actinoptychus senarius Ch. debilis N. membranacea Navicula directa Thalassiothrix longissima Manguinea rigida Cy. closterium Manguinea fusiformis

658 179 68 25 17 12 10 10 7 6 3 3 3 2 2

65.1 17.7 6.8 2.5 1.7 1.2 1.0 0.9 0.6 0.6 0.3 0.3 0.3 0.3 0.3

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of dissolved iron (Kudo et al., 2005; Nishioka et al., 2003). These conditions were confirmed to cover at least 2600 km2 around the enrichment site (Tsuda et al., 2003). The bottom of the surface mixed layer in the area on 5 July (two weeks before the iron-enrichment) was located below 40 m depth, and pCO2 was around 400 ppm. These conditions suggest that there had been no bloom of phytoplankton before the iron-enrichment in the studied site. Moreover, the bottom of the surface mixing layer in the area rose to about 10 m just before the experiment. During the SEEDS experiment, chlorophyll-a concentration increased from 0.85 to 1.8 mg m3 outside the iron-patch (Fig. 3). These facts suggest that the period of this experiment corresponded with the local maximun of primary production. In the initial phytoplankton community, pico- and nano-phytoplankton were dominant and micro-phytoplankton (>10 lm) accounted for 40% of the total chlorophyll-a concentration at the beginning of the experiment (Fig. 4). The micro-phytoplankton community at the beginning of the iron-enrichment was dominated by Thalassiosira spp., Chaetoceros concavicornis, C. atlanticus, Pseudo-nitzschia spp. and N. seminae, which showed a high similarity to the community in the Oyashio and at KNOT in May, when local production maxima were observed (Figs. 7 and 8). They made a larger cluster with the samples from the Oyashio and at KNOT during winter to early summer (Figs. 9 and 10). Some dominant species of the cluster (C. concavicornis, C. atlanticus and N. seminae) were also commonly observed in the AG (Booth, Lewin, & Postel, 1993). In addition, relatively dominant Chaetoceros species (C. concavicornis, C. convolutus and C. atlanticus) belong to the subgenus Phaeoceros that is recognized as oceanic (Tomas, 1997), as is N. seminae (Hasle & Syvertsen, 1997). These observations suggest that the initial assemblage of diatoms was composed by widely distributed species and characterized by the dominance of the oceanic species. 4.2. Development of the massive diatom bloom After the iron-enrichment, chlorophyll-a concentration markedly increased from 0.85 to 17.8 ± 4.0 mg m3 inside the iron-patch and remained high until the end of the observation. The development of the massive bloom accompanied a floristic shift from a pico- and nano-phytoplankton dominated community to a micro-phytoplankton dominated community (Fig. 4). Among micro-phytoplankton species, dominance shifted from oceanic diatoms to neritic centric diatoms (Table 1). Furthermore, among centric diatom species, the population growth of C. debilis was particularly eminent, and it monopolized the phytoplankton community in the matured stage of the bloom, accounting for 90% of micro-phytoplankton cell density. To explore the dynamical mechanism of shifting of dominant diatoms in the SEEDS experiment, specific growth responses of key diatoms to the iron-enrichment were examined. The initially dominant oceanic diatoms, small Flagirariopsis sp., did not increase during the observation period. And the other oceanic diatoms, P. turgidula, N. seminae, C. concavicornis and C. atlanticus showed a delay about 4 days in the initiation of growth after the iron-enrichment, although these species increased after day 4 (Table 2). In contrast, neritic centric species, C. debilis as well as L. minimus and E. groenlandica, showed no delay in the growth response, and they sustained higher growth rates than the oceanic species throughout the observation period. Especially, the net growth rate of C. debilis was eminent, 1.8 d1 during days 4– 7, while those of other diatom species in the patch were 0.5–0.9 d1. The net growth rate of C. debilis was much higher than the maximum growth rate expected for diatoms at the in situ temperature of 9.5 C (
1.0 d1, Eppley, 1977; Suzuki & Takahashi, 1995). These observations indicate that responses to ironenrichment by diatoms differs strongly between oceanic species and neritic ones. The rapid response and fast growth of neritic centric diatoms led to their dominance in the SEEDS experiments, although neritic centric diatoms were only minor components of the phytoplankton community at the initial stage. Similar dominance of long-chain-forming Chaetoceros species was observed in an iron addition to coastally upwelled HNLC water (Hutchins & Bruland, 1998). The dominant species, C. debilis is known to be widespread in cold coastal waters (Hasle & Syvertsen, 1997), and blooms of this species are often observed in coastal environments (Rines & Hargraves, 1987, and

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references therein), especially at the initial stage of spring blooms (Guillard & Kilham, 1977). Actually, spring blooms by this species have been reported in the coastal regions of the subarctic and transition domain of the western Pacific (Nakata, 1982; Tsuda, Sugisaki, Takahashi, & Furuya, 1994). 4.3. Decay of the massive diatom bloom The net growth rates of the diatom species were slower after day 9 than those between days 4 and 7 (Table 2 and Fig. 5). The bottom of the euphotic layer in the patch rose from 46 to 12 m as a result of elevated chlorophyll-a concentration and was consequently shallower than the surface mixed layer after day 9 (Fig. 4). Macronutrients decreased greatly but were not depleted (nitrate >2.7 lM, silicic acid 5 lM) at the end of the observation (Nishioka et al., 2003; Tsuda et al., 2003). However, dissolved iron concentration was
0.15 nM after day 11 (Nishioka et al., 2003), which was a quarter of the half saturation constant of the iron-growth relationship for the ambient phytoplankton community (Noiri, Kudo, Kiyosawa, Nishioka, & Tsuda, 2005). These results indicate that the phytoplankton growth in the patch became lightand iron-stressed after day 9. Moreover, direct grazing on the dominant diatoms by heterotrophic dinoflagellates made a significant contribution to the mortality of the phytoplankton (Saito, Suzuki, Hinuma, Saino, & Tsuda, 2005), although mesozooplankton grazing was negligible (Tsuda, Saito, Nishioka, & Ono, 2005). 4.4. The seed source of the massive diatom bloom Survival and existence of neritic centric diatoms in the WSG was a key factor in the observed massive diatom bloom after the iron-enrichment. For large centric diatoms, especially neritic species, relatively large amounts of iron are necessary to grow compared to small phytoplankton (Brand, Sunda, & Guillard, 1983; Sunda & Huntsman, 1995). The western subarctic Pacific is a HNLC water receiving relatively high fluxes of aeolian dust from Asian desert and loess regions compared with the Southern Ocean or the equatorial Pacific (Duce & Tindale, 1991; Fung et al., 2000; Uematsu, Wang, & Uno, 2003). In addition, the subsurface dissolved iron concentration is higher in the WSG than in the AG (Nishioka et al., 2003). Suzuki et al. (2002) confirmed that the phytoplankton community in the WSG was less iron-stressed than that in the AG by measurements of photochemical quantum efficiencies of algal photosystem II using a fast repetition rate fluorometer. These observations suggest that fast-growing and neritic diatoms requiring high iron may survive in the WSG with an episodic supply of iron from dust and from the subsurface layer. Many neritic centric diatoms, including C. debilis and L. minimus in the SEEDS experiment, produce resting spores (McQuoid & Hobson, 1996). Resting spores and resting cells of diatoms exhibit adaptive dormancy (Kuwata, Hama, & Takahashi, 1993). Diatoms forming resting stages could survive weeks under nutrient depletion (Kuwata & Takahashi, 1999) and years under cold and dark conditions (McQuoid & Hobson, 1996). The occurrence year-round of neritic centric diatoms as well as their resting spores has been reported at Station KNOT in the WSG (Mochizuki et al., 2002), although resting spores were not observed in the surface layer during SEEDS. This suggests that neritic centric diatoms may survive and exist throughout the year in WSG waters as resting stages or vegetative cells. Concerning the seed source of neritic diatoms in the WSG, Mochizuki et al. (2002) suggested that they are supplied from the coast of the Kamchatka Peninsula and Kuril Islands to the oceanic area by eddies along the East Kamchatka Current (see also Sasaoka et al., 2002). The WSG attaches the coastal areas of the Kuril and Aleutian Islands, at the western and north-eastern ends, respectively (Fig. 1). Therefore, seed populations of potentially blooming, neritic diatoms may be supplied to the WSG from these coastal waters and survive as resting cells and/or resting spores, responding to episodic supply of iron from dust and the subsurface layer.

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4.5. Comparison with natural blooms and other Fe manipulation experiments A large difference between the iron-induced bloom and natural blooms in the western subarctic Pacific was the lack of Thalassiosira and Coscinodiscus species in the SEEDS bloom. Especially, Thalassiosira spp. was a relatively abundant centric diatom before the iron-enrichment but became a negligible component during the matured stage of the bloom. Thalassiosira spp. occurs throughout the year at KNOT and in the Oyashio region and composed a major part of the natural blooms (Figs. 7 and 8). Moreover, bottle incubation experiments with iron-enrichment in the WSG showed an increase of Thalassiosira spp. up to 5 mg m3 as chlorophyll-a (Harrison et al., 1999). The reason why Thalassiosira diatoms did not respond to the iron-enrichment is not presently known. However, bloom-forming Thalassiosira diatoms such as Thalassiosira nordenskioeldii were not observed in the samples collected before the iron-enrichment, suggesting that seed stocks of fast growing Thalassiosira diatoms were not present in the SEEDS experiment. Another difference was near monopolization by a single species in the iron-induced bloom. The ironinduced bloom in the Southern Ocean also comprised a single species of pennate diatom, Fragilariopsis kerguelensis (Gall et al., 2001). In natural phytoplankton blooms, several species of diatom share dominance (e.g., Figs. 7, 8), and there is a succession of dominant species accompanying the nutrient consumption processes (Margalef, 1978). In the case of SEEDS, only a limited number of diatom species with potentially high growth rate survived in the phytoplankton community during transport from the neritic area to the site of the iron manipulation, and near monopoly by a fast-growing diatom, C. debilis, occurred. Compared with the other mesoscale iron-enrichments experiments, the most notable characteristics of the SEEDS bloom was the fast and high accumulation of phytoplankton biomass in the patch. The maximum concentration of SEEDS bloom was several times higher than in previous mesoscale iron-enrichment experiments in the equatorial Pacific (IronEx II, Coale et al., 1996) and the Southern Ocean (SOIREE, Boyd et al., 2000). Both SEEDS and SOIREE showed chlorophyll peaks around day 10 (Fig. 2, Gall et al., 2001). However, it took 11 days to achieve a sixfold increase of chlorophyll-a concentration in SOIREE, while it took only 6 days in the SEEDS. The important factors producing the higher growth rates of phytoplankton in SEEDS were higher water temperature (7.4–9.5 C at the surface) and shallower surface mixed layer (10–20 m) compared with those of SOIREE (2.0 C and 65 m, respectively, Boyd & Law, 2001). In addition, the presence of a fast growing diatom, C. debilis, was important to achieving the massive accumulation of the phytoplankton, as mentioned above. 4.6. Conclusions In conclusion, the growth rates of almost all species of diatoms were enhanced by the iron-enrichment in the WSG, but responses to the manipulation of iron concentration differed greatly by species, even among centric diatoms. A drastic floristic shift occurred, because non-dominant neritic diatoms, especially C. debilis, grew faster than oceanic diatoms without delay in the growth response to the enrichment. The important factors determining the dominant phytoplankton species are the potential growth rates under an iron-replete condition and the growth lag. The scale of bloom after iron manipulation largely depends on the potential of the dominant species. However, the species becoming dominant must depend on the species that happen to be present at the moment of iron manipulation, and are therefore unpredictable. Diatoms have been the dominant phytoplankton in the matured stage of mesoscale iron-enrichment studies in three HNLC ocean areas (Boyd et al., 2000; Coale et al., 1996; Tsuda et al., 2003). Species composition of the dominant diatoms is intrinsically important to the fate of carbon assimilated by the phytoplankton community. The species composition determines the communityÕs growth rate, vulnerability to grazing, sinking rate and aggregation processes. The unpredictability of the bloom-forming species suggests that we have to be very prudent in applying large scale iron-enrichment as a tool for biogeoengineering.

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