Sizespectrum Based Differential Response of Phytoplankton To

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Phycological Research 2014

Size-spectrum based differential response of phytoplankton to nutrient and iron-organic matter combinations in microcosm experiments in a Chilean Patagonian Fjord

Jose L. Iriarte,1,2* Murat V. Ardelan,3 Luis Antonio Cuevas,4 Humberto E. González,5,2 Nicolas Sanchez3 and Sverne M. Myklestad6 1Instituto de Acuicultura and Centro de Investigación en Ecosistemas de la Patagonia-CIEP, Universidad Austral de Chile, Puerto Montt, 2COPAS Sur-Austral, 4Centro de Ciencias Ambientales EULA-Chile, Universidad de Concepción, Concepción, 5Instituto de Ciencias Marinas y Limnológicas and Centro de Investigación en Ecosistemas de la Patagonia-CIEP, Universidad Austral de Chile, Valdivia, Chile, Departments of 3Chemistry and 6Biotechnology, NTNU, Norwegian University of Science and Technology, Trondheim, Norway

teria through the interaction with organic ligands SUMMARY released by bacteria that eventually could increase solubility of the Fe dissolved fraction thus having The Patagonian fjords have been recognized as a major a positive effect on the small-sized phytoplankton region of relatively high primary productivity systems community. during spring–summer bloom periods, where iron- organic matter forms may be essential complexes Key words: Iron-dissolved organic matter, micro- involved in key growth processes connected to the phytoplankton, Patagonia fjord, polysaccharide, carbon and nitrogen cycles. We used two dissolved siderophore, Synechococcus. organic matter (DOM) types, marine polysaccharide and siderophore, as a model to understand how they affect the bioavailability of Fe to phytoplankton and bacteria and to assess their ecological role in fjord systems. A 10-day microcosm study was performed in INTRODUCTION the Comau Fjord during summer conditions (March Iron as a micronutrient is an essential enzymatic 2012). Pico-, nano-, and microphytoplankton abun- co-factor for photosynthesis, respiration, and macronu- dance, total chlorophyll-a and bacteria abundance, trient assimilation (Morel & Price 2003), nevertheless and bacterial secondary production estimates were micronutrient requirements can differ among phyto- analyzed in five treatments: (i) control (no additions), plankton taxa (Yang & Jiao 2002; Tsuda et al. 2005) (ii) only nutrients (NUT: PO4,NO3, Si), (iii) nutri- as did their impact as a limiting factor. In temperate + ents Fe(II), (iv) polysaccharide (natural diatoms coastal regions, in addition to the typical spring– extracted: 1–3 beta Glucan), and (v) Hexandentate summer conditions such as high solar radiation and Desferroxiamine B (DFB, siderophore). Our results macronutrients availability, we should also consider showed that while DFB reduced Fe bioavailability for the release of Fe from strong riverine sources as almost all phytoplankton assemblages in the fjord, another element that may influence the seasonal polysaccharide did not have effects on the iron cycle and magnitude of primary productivity (PP) and + bioavailability. At Nutrients Fe and Polysaccharide phytoplankton growth. Due to terrestrial (riverine), treatments, chlorophyll-a concentration abruptly aeolian dust, and water column-sediment sources of −3 increased from 0.9 to 20 mg m during the first 4–6 micronutrients and dissolved organic matter (DOM), days of the experimental period. Remarkably, at the the distribution, speciation, and transformation of dif- + Nutrients Fe treatment, the development of the ferent forms of micronutrients are more dynamic and bloom was accompanied by markedly high abundances complex in near-shore water than in oceanic water. In of Synechococcus, picoeukaryotes, and autotrophic nanoflagellates within the first 4 days of the experiment. Our study indicated that small plankton (phyto- < μ plankton 20 m and bacteria) were the first to *To whom correspondence should be addressed. + respond to dissolved Nutrients Fe compared to large Email: [email protected] sized micro-phytoplankton cells (>20 μm). This could Communicating editor: J. H. Kim. be at least partially attributed to biological utilization Received 6 April 2013; accepted 1 December 2013. of Fe (2 to 3 nM) by <20 μm phytoplankton and bac- doi: 10.1111/pre.12050

© 2014 Japanese Society of Phycology 2 J. L. Iriarte et al. spite of that, the importance of DOM as a significant Preliminary observations indicated that concentra- micronutrient ‘carrier’ for PP in coastal areas with tions of Fe in Comau Fjord are relatively low (2–3 nM, freshwater influence has not been fully elucidated. Ardelan et al. 2009b), compared to concentrations in Various DOM may have important effects on iron spe- those in other coastal areas (Bruland et al. 2001; ciation and hence its bioavailability to phytoplankton Öztürk & Bizsel 2002), suggesting a critical role in assemblages. Some DOM may convert available Fe the dynamics of phytoplankton in Patagonian fjords. form into not fully bioavailable Fe form (Öztürk & However, micronutrient availability, and in turn Bizsel 2002; Öztürk et al. 2002), thus it could be the primary productivity rate, can be affected by critical in marine areas where dissolved Fe (DFe) con- complexation with organic matter and colloids forma- centrations are already low. Furthermore, the additions tions (Öztürk et al. 2002). Terrestrial-derived organic of macronutrients to the system relative to Fe may also matter (e.g. humic acids), as well as biologically contribute to controlling the role of Fe on phytoplank- released agents (e.g. bacterial siderophores), are impor- ton structure and function. From the chemical point of tant DOM sources in aquatic systems. It has been view, both dissolved and particulate organic matter suggested that the complexation of micronutrients with (DOM and POM), as well as the lithogenic and DOM may reduce the availability of micronutrient biogenic fractions of the POM, constituted very elements, thus limiting the PP in aquatic systems complex pools with a high heterogeneity degree in sub- (Wells & Trick 2004). In iron-replete coastal waters it stance classes responsible for metal binding (Boyd has been observed that increasing concentrations of et al. 2010; Strmecki et al. 2010), usually loaded siderophores decreased the biological availability of with a highly variable amount of microelements such iron added to natural seawater (Wells & Trick 2004). as Fe. Thus, the growth response of phytoplankton Furthermore, the presence of hydroxycarboxylic acid exposed to different concentrations of natural DOM (e.g. glucaric acid) positively influenced iron availability would be difficult to analyse. A simple design to tackle to phytoplankton assemblages in the Southern Ocean this relevant problem is to assay single DOM com- (Öztürk et al. 2004). Marine polysaccharide and pounds and their capacity to form chelate complexes siderophore complexing capacities with Fe following with limiting micronutrients (i.e., DOM ligands +Fe) the growth of natural phytoplankton assemblages is still required for phytoplankton and bacterial growth. an open question (Hassler et al. 2011). We postulated Although current studies favor Fe availability as the that different phytoplankton species and/or functional main factor limiting PP in high nutrient low chlorophyll groups have different biological capacities to utilize (HNLC) oceanic regions (Bruland et al. 2001), biologi- these Fe-DOM complexes. Fe-DOM formation exerted cal availability of Fe–DOM interactions affecting cellular by heterotrophic bacteria in natural waters was also uptake processes of Fe on phytoplankton growth in addressed in this study, and the heterotrophic bacteria coastal areas require more attention. Specifically, the abundance and bacterial secondary production was growth and species composition of phytoplankton monitored to follow the bacterial evolution in our treat- assemblages in the ocean HNLC regions are often regu- ments in relationship with phytoplankton growth. The lated by iron (Tsuda et al. 2005), which in turn influ- main objective of this study was to determine the ences oceanic PP.From modeling results, the chemically effects of two different DOM on Fe bioavailability for important role of DOM in controlling free micronutrients pico-, nano-, and microphytoplankton abundance, in seawater (Hirose 2007), and their uptake by phyto- total autotrophic biomass, and species composition of plankton, is known. Due to seasonal climatologic- microphytopankton in Comau Fjord, Patagonia. For this oceanographic features in coastal temperate areas, purpose, an in situ experimental microcosm setting was temporal growth limitation by Fe can also be expected performed by using two different types of DOM to (Bruland et al. 2001; Hutchins et al. 2002; Öztürk et al. manipulate Fe bioavailability at the Comau Fjord during 2002; Torres & Ampuero 2009). Since Patagonian fjords austral summer. Knowledge is still scarce on phyto- have been suggested as major ‘CO2 sink’ areas, under- plankton capacity for biological uptake and macronu- standing the processes/factors that modulate the effi- trient metabolism (such as nitrate and ammonia), ciency of the biological pump in this ecosystem will be linked to the bioavailability of Fe, forming complexes in relevant. Early results strongly suggest that nitrogen a nutrient modified scenario in the Patagonia fjords source (mainly inorganic such as nitrate and ammonia) system where aquaculture may act as an important inputs could play a major role in sustaining relatively source of new nitrogen and DOM. Specifically, increas- large PP (mean = 3gCm−2 day−1), thus acting as a lim- ing aquaculture activities in Patagonian fjord ecosys- iting nutrient for phytoplankton growth in Patagonian tems may change water chemistry in the near future, by fjords (Iriarte et al. 2013). It is well known that NO3 introducing additional nitrogen as ammonia as well as and/or NO2 uptake by phytoplankton indeed depends on DOM. Here we hypothesized that due to this new envi- the amount of Fe available (via nitrate-nitrite reductase ronmental scenario, the amount and forms of DOM may enzymes) (Morel & Price 2003). have effects on Fe bioavailability. This in turn, may have

© 2014 Japanese Society of Phycology Phytoplankton and iron-organic matter 3 an effect on phytoplankton-bacterial structure and system is observed permanently at Comau Fjord consti- function in the fjord ecosystem. tuting the overall pattern of hydrographic conditions one surface layer (0 to 5 m: low salinity and nutrients) MATERIALS AND METHODS strongly influenced by river discharges from the Huinay and Vodudahue rivers), and a second more oceanic Study region: The nearly pristine Comau Fjord is (high salinity and nutrients) layer below 10 m with less located on the eastern border of the Pacific Ocean, in seasonal variability. In the Comau Fjord, during austral the southern region of Chile (42°S). The Comau Fjord is spring–summer months (October to February), the more than 30 km long, running mostly south to north, brackish layer (<5 m, 5–25 psu) is remarkably high in − but turning westward at its connection with the Gulf of silicic acid (>20 μM) but poor in NO3 and PO4, with Ancud through the Comau Channel (Fig. 1). Annual values lower than 2 μM and 1 μM, respectively, while − solar radiation and sea temperature showed a strong below the picnocline (10 m), nutrient-rich (NO3 : seasonality with low values during the winter months 5–20 μM; PO4: 1–2 μM; Si(OH)4: 10–20 μM) subant- and increasing values in late spring and summer (Iriarte arctic waters (SAAW) is observed. et al. 2013). Freshwater input into rivers in this region Microcosm Bioassay: We carried out one experiment is modulated by both pluvial and nival seasonal during summer conditions (March 2012) to assess the regimes. Oceanographically, the region can be consid- effects of DOM on Fe bioavailability on short-term shifts ered a transitional marine system, influenced by deep in the abundance of main phytoplankton size classes oceanic waters with high salinity and nutrients and by and bacterioplankton as well as the structure of the surface freshwater with low salinity and nutrients micro-phytoplankton assemblage. Before the experi- (except for silicic acid) (Iriarte et al. 2013). A two-layer ment, seawater was collected from a depth of 10 m

Fig. 1. Map of the sampling location for phytoplankton experiments in the Comau Fjord, Huinay Station (42.39°S, 72.44°W) performed in the austral summer (March 2012).

Table 1. Dissolved inorganic nutrient concentration in the subsurface water of Comau Fjord (oceanic water type at Day 0) used for seawater microcosm incubations throughout March 2012. Dissolved inorganic nutrient concentrations in experimental treated seawater after 6 and 10 days of ‘in situ’ incubation in the Comau Fjord (n = 3, for each treatment)

Nitrate (uM) Phosphate (uM) Silicic acid (uM)

Water type In situ Comau Fjord 14.01 1.01 15.81 Treatments All Treatments† 20.00 1–2 20.00 Experiment Control 7.95 0.60 15.70 6th Day Nutrients 3.36 0.25 2.0 Nutrients + Fe 2.43 0.50 38.6 Polysaccharide 0.60 0.55 5.93 Siderophore 28.79 3.61 29.92 Experiment Control 3.94 0.56 0.89 10th Day Nutrients 5.03 0.66 1.66 Nutrients + Fe 4.32 0.69 6.53 Polysaccharide 1.97 0.30 1.31 Siderophore 37.0 2.8 33.18

†Dissolved inorganic nutrient concentrations added every 2 day over the 10-day period.

(60% photosynthetically active radiation (PAR) irradi- complex (pFe) stability, (Boukhalfa & Crumbliss 2002). ance), an oceanic-influenced water depth in Comau Although there are several types of siderephores and Fjord (Iriarte et al. 2013), in an acid washed 1000-L most of them not autochthonous (especially DFB), we polyethylene tank using a peristaltic pump (Watson- used the most common siderephore model as a type of Marlow, Wilmington, MA, USA). We selected water from DOM. Here, we assumed that due to increased DOM from a depth of 10 m given the generally low nutrient aquaculture, bacterial activity may increase and produce concentrations in the austral summer (Table 1). The different types of siderephores (some of them might be experiment was conducted with native phytoplankton like DFB) released to the water column. (ii) Natural assemblages collected from one selected station polysaccharides produced by diatoms (Chaetoceros (Huinay Scientific Station) at the Comau Fjord in the sp. + Thalassiosira sp. diatoms extracted: 1–3 beta austral summer (March 2012). Phytoplankton was col- Glucan, extracted and supplied by Sverre M. Myklestad). lected using a Teflon diaphragm pump and collected at Five treatments were prepared for the bioassay (Table 1): a depth of 10 m (PAR: 90–180 μmol m−2 s−1), and CONTROL, consisting of untreated seawater without any added to each acid washed 30-L high density polyeth- nutrients addition; NUT, which included the addition of − 3− ylene (PE) bottle. All incubation carboys for the experi- NO3 ,PO4 , and Si(OH)4; Nutrients + Fe(II) (+4 nM), − 3− ment were acid washed. Although we note that the which included the addition of NO3 and PO4 ,Siand microcosms approach (30-L bottle) has its limitations, Fe; Polysaccharide, which consisted of the addition of we used it as a model system for asking our questions on 60 μM 1–3 Beta Glucan (natural diatoms extracted); phytoplankton populations and community properties Siderophore, which included the additions of DFB. The during a short time scale. In addition, the microcosms DOM additions were performed daily, restricting the experiment was useful for high replication throughout possible biochemical metamorphosis processes over time (days) and gave us statistical power over the time. Nitrate, orthophosphate, and silicic acid concen- observed results. Finally, microcosms gave us the control trations were maintained constant to natural concentra- to manipulate features of the system (i.e. nutrients, tions fluctuating between 20 μM, 2 μM, and 20 μM, Fe-DOM complexes) that are difficult to control and respectively (Table 1) in order to compare the effects of manipulate in highly heterogeneous natural environ- Fe-DOM on phytoplankton taxa, abundances and ments such as fjord ecosystems. The experiment was biomass. Three replicates of each treatment were incu- conducted by using natural phytoplankton assemblages bated in 30-L high-density PE carboys and randomly from Comau Fjord, using natural P:N:Si:Fe levels and placed in situ at 5 m depth under natural temperature enriched with P:N:Si and Fe (Sigma, St. Louis, MO, and light conditions near the pier. Carboys were incu- USA) to avoid nutrient limitation. As DOMs we selected bated for a 10-day period and samples were taken every the following compounds: (i) Hexadentate sidero- second day. During the experiment, irradiance ranged phore Desferroxiamine, (Desferroxiamine B, DFB, between 200 μmol m−2 s−1 and the environmental Sigma-Aldrich) iron carrier organic molecules which temperatures ranged from 14 to 16°C. In our experi- have higher redox potential and higher Fe(III)-Organic mental approach, total autotrophic biomass (Chl-a) and

© 2014 Japanese Society of Phycology Phytoplankton and iron-organic matter 5 size-class abundances (cell counts) were defined as All on-site sample processing was carried out in a main phytoplankton assemblage descriptors. These vari- closed room under clean air in a Class-100 laminar flow ables were selected given the phytoplankton response to hood (Air Clean Systems 400 Workstation) to avoid exogenous environmental factors such as inorganic contamination. Laboratory processing was done under nutrients, Fe-DOM supply expressed in chlorophyll-a constant laminar air flow in a class 100 clean laboratory biomass, and the relative contribution (cell numbers) to at the Department of Chemistry at Norwegian Science the total plankton assemblage made by the component and Technology University and later sent for analysis taxa in a sample (Iriarte et al. 2013). Although we are using a High Resolution Inductive Coupled Plasma uncertain of the Fe flux to elaborate on whether the Fe Mass Spectrometry (HR-ICP-MS) Element 2 (Thermo- supply rate is sufficient for uptake rate during phyto- Finnigan, Madison, CT, USA). A total of six replicates plankton bloom time, those Fe concentrations could be was run for methodological blanks analysis for both critical for the coastal waters of southern Chile. In Chelex and DGT samples (detection limit used here is addition, we would like to point out that this study was three times the standard deviation estimated from the not designed to work on ‘Fe limitation’ but on the type of measured method blank values). For details on meth- Fe-DOM formations, and therefore Fe bioavailability to odologies see Öztürk et al. (2002), Ardelan et al. phytoplankton assemblage. (2009a), and Ardelan et al. (2010). Seawater samples were collected for total Chelex Seawater Fe-FIA analyses: water samples for dissolved labile (TFeCh), dissolved Chelex labile (DFeCh), and dif- Fe (DFe) determination were collected from a glass-fiber fusive gradient in thin-films labile (FeDGT) iron during boat, either by a peristaltic pump (Masterflex, Oldham, January–February 2011. Subsurface (10 m) seawater Manchester, UK) or Teflon covered GO-FLO bottles samples were collected in front of the marine station in deployed with a polypropylene line during March 2012. All the middle of the fjord (∼2 km offshore) using an acid- equipment in contact with seawater was pre-cleaned, cleaned Teflon coated GO-FLO bottle and a polypropyl- soaked in high-purity 20% hydrochloric acid, sextuple- ene rope. rinsed with deionized water (Milli-Q-purified), and stored in Chelex samples: a volume of water (150 mL) was clean, sealed plastic bags (handling with clean disposable collected for total Chelex labile (TFeCh) and dissolved plastic gloves). DFe concentrations were measured by (filtered through a 0.2 μm acid washed filter Chemiluminescence Flow Injection Analysis (CL-FIA: (0.45 + 0.2 μm Sartorious Sartobran 300) Chelex labile Waterville Analytical, Waterville, ME, USA) (Bowie et al.

(DFeCh) iron samples, to which 0.8 mL of Chelex-100 1998). Water samples for DFe analysis were prefiltered solution (Ammonium Acetate buffer (C2H4O2.NH3)) was through Sartobran cartridges (0.2 μm with 0.4 μm prefil- added. Samples were placed in a shaker (65–80 rpm) ter, Sartorius, Goettingen, Germany) and collected in for 48–72 h and later transferred to acid-washed plastic acid-cleaned 250 mL low density polyethylene bottles polyethylene columns (Bio-Rad Laboratories, Hercules, (Nalgene, Rochester, NY, USA) and immediately acidified CA, USA), where the water was washed out and the (pH < 2) with ultrapure HCL (final concentration, Merck Chelex-100 (binding trace metals) was retained by a ultrapure, Whitehouse Station, NJ, USA). Immediate Fe resin at the bottom of the column. In the laboratory, analysis and speciation were carried out at the field station extraction of the trace metals was done in a two-step by using FIA-CL under a clean air laminar chamber (Air acidifying process, where 1 mL 2 M UP HNO3 was Clean System – Class 100 laminar flow). added, left for 5 min, and then gently shaken to Phytoplankton biomass and micro-phytoplankton re-suspend the Chelex-100. After 15 min, contents were abundance: Triplicate seawater samples (50 mL) for poured into new acid-washed PE tubes and 4 mL of chlorophyll-a (Chl-a) were filtered through a 0.7 μm

0.25 M UP HNO3 were added to the initial tubes left for micro filtration system glass-fiber filter and analyzed 10 min, and poured into the new polyethylene tubes, using a digital Turner P700 fluorometer (Parsons et al. obtaining a final 5 mL sample. 1984). For phytoplankton cell counts, 50-mL

DGT samples: Samples for DGT labile iron (FeDGT) subsamples were stored in clear plastic bottles, and were collected by placing three DGT samplers within a then preserved in a 1% Lugol iodine solution. volume (1500–2000 mL) of water, placed in a shaker Subsamples were placed in a sedimentation chamber (60–80 rpm) for 48–72 h and then frozen for posterior and left to settle for 24 h and the bottom of the analysis. In the laboratory, all DGT samples were set chamber was observed using an inverted microscope apart and the third layer (holding the metal binding (Utermöhl 1958). Micro-phytoplankton was identiﬁed resin) was transferred and 1 mL 3 M UP HNO3 was to genus or species level, when possible, and divided added. Polyethylene tubes containing the resin were into diatoms (Bacillariophyceae: Centric and Pennates) put on a shaker (60–80 rpm) for 12 h, after which the and thecate dinoﬂagellates (Dinophyceae).

HNO3 in the PE tubes was transferred to new acid- Pico- Nanoplankton abundances: The abundance of washed tubes and finally diluted (Milli-Q water) to a bacteria, cyanobacteria (Synechococcus), picophyto- final 5 mL sample volume. eukaryotes, and autotrophic nanoflagellates was

© 2014 Japanese Society of Phycology 6 J. L. Iriarte et al. estimated by flow cytometry. Water samples (4 mL) for water were relatively high (Table 1): 14.1 μM nitrate, analyses of pico- and nanoplankton abundance were 1.01 μM orthophosphate, and 15.81 μM silicic acid. collected during discrete days (days 1, 4, 8, 10). Major dissolved nutrients decreased at day 6 in NUT, Samples were preserved in glutaraldehyde (6.0% W/V Polysaccharide, and Nutrients + Fe (with the exception in 0.2 μm pre-filtered seawater). Subsamples of of silicic acid) treatments. On day 10, all dissolved 150 μL were processed on a FACSCalibur flow nutrients reached relatively low levels, but they were − cytometer equipped with a 488 nm, 15 mW ion-argon never depleted at those treatments (NO3 : 3.9–5.0 μM, 3- laser (Becton Dickinson, Oxford, UK). Identification of PO4 : 0.3–0.7 μM, Si(OH)4: 1.3–6.5 μM). At the end coccoid cyanobacteria (Synechococcus) and photosyn- of the experiment, the lowest dissolved nutrients were thetic eukaryotes was based on differences in side light found in the polysaccharide treatment (Table 1) sug- scatter and fluorescence in the orange (cyanobacteria) gesting that the addition of natural polysaccharides and red (eukaryotes) wavelengths. Abundance of enhance the uptake of nutrients by phytoplankton by heterotrophic bacteria was estimated from samples pre- making Fe readily available (Figs 2,3). viously stained with Sybr green I (Molecular Probes, Carlsbad, CA, USA). − 3− Inorganic Nutrients: Analyses of NO3 ,PO4 , and

Si(OH)4 were carried out in the experiment by collecting 50-mL water samples during the time course incubation. All samples were stored frozen until analysis (Parsons et al. 1984). All nutrients (nitrate, phosphate, and silicic acid) were added as a solution in Milli-Q water (18 Ω), purified for trace metals by passing through a previously prepared Chelex-100 column (Öztürk et al. 2002). Bacterial Productivity: Triplicate samples of 1.5 mL and a formalin-killed control were incubated with tritium- labeled leucine (Amersham, 1.15 × 1010 Bq mmol−1)ata final concentration of 50 nM (Simon & Azam 1989) in the dark at in situ temperature for 1 h. After incubation, samples were extracted with 100% trichloroacetic acid (TCA) and rinsed with cold 5% TCA and centrifuged at 14 000 g twice for 10 min before supernatant removal (Smith & Azam 1992). Counting cocktail (Ecoscint A, National Diagnostics, Hessle, UK) was added, and the uptake of radioactive leucine was measured on a Beckmann scintillation counter. Cell carbon produced per mol of leucine incorporated was based on the theoretical conversion factor proposed by Simon and Azam (1989) using the average value of 1.55 kgC mol−1 assuming no isotope dilution. Leucine incorporation was converted into biomass production using the carbon fraction of proteins of 0.86 (Simon & Azam 1989). Statistical analyses: Significant differences among treatments were evaluated using a parametric balanced one-way ANOVA (Zar 1984) with Chl-a as a dependent Fig. 2. Short-time course of pH values (a) and in vivo fluores- variable for each seasonal experiment. An a posteriori cence (b) in experiment performed in the summer season (March Tukey test was used to make multiple comparisons of 2012) in the Comau Fjord. Seawater ‘microcosms’ (20 L bottles), means of significant factors (P < 0.05). The variance including all microbial community (bacteria, pico-and nano- homogeneity for Chl-a was verified with a Bartlett test. autotrophs, micro-phytoplankton), were incubated under five different treatments: seawater without nutrients addition (Control), RESULTS seawater enriched with nitrate, orthophosphate, silicic acid (Nutrients), seawater enriched with nitrate, orthophosphate,

Relatively low concentrations of DFeCh and FeDGT were silicic acid + Fe (Nutrients + Fe(II)), seawater enriched with estimated at 10 m in the fjord with mean values of natural polysaccharide, and seawater enriched with siderophore. 2.0 nmol L−1 and 3.8 nmol L−1, respectively. At the Values are the means ± 1 standard deviations for samples from experiment, initial major nutrients in the sampled sea- duplicate bottles maintained until day 10.

Fig. 3. Short-time course of Chlorophyll-a concentration (mg m−3, a), Synechococcus (cells mL−1, b), pico-eukaryote (cells mL−1, c), autotrophic nanoﬂagellates (cells mL−1, d) and micro-phytoplankton abundance (cells L−1, e) in experiment performed in summer (March 2012) season in the Comau Fjord. Seawater ‘microcosms’ (20 L bottles), including all microbial community (bacteria, pico-and nanoautotrophs, micro-phytoplankton), were incubated under ﬁve different treatments: seawater without nutrients addition (Control), seawater enriched with nitrate, orthophosphate, silicic acid (Nutri- ents), seawater enriched with nitrate, orthophosphate, silicic acid + Fe (Nutrients + Fe(II)), seawater enriched with natural polysaccharide, and seawater enriched with siderophore. Values are the means ± 1 standard deviation for samples from duplicate bottles maintained until day 10. ◀

stant (8.5–8.7). In vivo fluorescence, pH values (Fig. 2), and total chlorophyll-a (Fig. 3a) were clearly related to dissolved nutrients, natural polysaccharide, and Fe (Table 1). Chlorophyll-a showed that at day 6 the response of the total phytoplankton community for nutrients + Fe, polysaccharide, and NUT additions have a significant effect on phytoplankton growth, reaching values around 20 mg Chl. a m−3, compared to control (without nutrients addition) and siderophore treatments (Bartlett Test, χ2 = 2.2, P > 0.05; ANOVA F-test, F = 4.0, P = 0.005; multiple comparisons Tukey Test, P < 0.05). Iron, nutrients and polysaccharide sources increased chlorophyll-a, with concomitant nitrate, orthophosphate, and silicic acid drawdown (Table 1), suggesting that phytoplankton biomass is modulated strongly by inorganic nutrients, Fe, and autochthonous organic substance availability. The growth of the two main size class phytoplankton groups of pico- and nanophytoplankton were simultaneously enhanced after the Fe + nutrients enrichment during the first days of the experiment. Synechococcus tended to peak on day 2 and remained significantly high and constant until day 8 (Fig. 3b) followed by picoeukaryote and nanoflagellates on day 4 in Fe + nutrients treatment (Fig. 3c,d). On average, the abundance magnitude of main phytoplankton groups revealed the dominance of Syne- chococcus (50 × 103 cells mL−1) and picophytoeukaryotes (82 × 103 cells mL−1) as well as of photoautotroph nanoflagellates (80 × 103 cells mL−1) between days 2 and 4 at nutrients + Fe treatment (Fig. 3d). We suggest that picoplankton populations were sensitive to iron enrichment and responded rapidly to relatively low concentrations (2–6 nM) within the first 2 and 4 days of the experiment. However, Synechococcus growth extended and dominated Values of pH, an indicator of autotroph photosynthetic during the entire experiment period (10 days) with response, initially increased in NUT, polysaccharide, and higher abundances (30–50 × 103 cells mL−1), where pico- nutrients + Fe treatments from 8.1 to 8.7 at day 4 eukaryote and nanoflagellates groups decreased in corre- (Fig. 2a). In the two highest treatments (NUT and spondence to initial abundances at day 6 (Fig. 3c,d). polysaccharide) pH increased slightly (8.8–9.0) from day 4 Microphytoplankton cell abundances (>20 μm) indicated to day 8, whereas Fe + nutrients remained relatively con- major changes of threefold increase in cell counts, with the

© 2014 Japanese Society of Phycology 8 J. L. Iriarte et al. highest values at days 4 and 6 for NUT and polysaccharide treatments (13–15 × 106 cells L−1). Diatom abundances for Fe + nutrients increased slightly at day 4 (7 × 106 cells L−1), then decreasing abruptly until day 10 to levels of siderophore treatment. Dinoflagellates showed low values during all experiments (5–51 × 103 cells L−1), and almost disappeared in all treatments. Although there were low cell abundances observed in siderophore treatment, there was no major change in the micro- phytoplankton community structure compared with polysaccharide and nutrients + Fe treatments. The initial phytoplankton assemblage consisted of several micro-sized phytoplankton taxa 95% dominated by centric and pennate diatoms such as Guinardia delicatula (Cleve) Hasle and Leptocylindrus minimus Cleve, Leptocylindrus danicus Cleve, Pseudo-nitzschia cf. delicatissima Peragallo and Pseudo-nitzschia cf. pungens Peragallo, Rhizosolenia setigera Brightwell, Proboscia alata (Brightwell) Sundström, Nitzschia longissima (Brébisson) Ralfs, Dactyliosolen fragilissimus (Bergon) Hasle, and dinoflagellates such as Cerarium fusus (Ehrenberg) Dujardin and Protoperidinium pellucidum Bergh. Although cell abundances and chlorophyll a increased abruptly during the 4–6 days period at NUT, polysaccharide, and nutrients + Fe treatments (Fig. 3e), there was no major change in community structure. At days 4 and 6 (high abundances days) the diatom community was dominated (95% of the total micro- phytoplankton cell counts) by a pennate diatom Pseudo- nitzschia cf. delicatissima, and a centric diatom Guinardia delicatula, showing higher growth rates (2.0– 2.2 day−1) than few other diatoms after all nutrient and polysaccharide enrichments. Total bacterial cell counts increased from initial abundances of 2 × 106 to near 8 × 106 cells mL−1 in nutrients + Fe and NUT treatments by day 6, whereas abundances remained low and constant at the control and polysaccharide treatments (Fig. 4a). Leucine incorporation rates showed similar dynamics in NUT, nutrients + Fe and polysaccharide treatments. Over days 0 to 6, the initial addition of nutrients was accompanied by a corresponding increase in bacterial Fig. 4. Dynamics in microbial growth during the experiments in μ −1 −1 production by a factor of about 6 (3.0 gCL h ). the summer season (March 2012). (a) Bacterial abundance (cells Bacterial production increased by a factor of 2 mL−1). (b) Bacterial Secondary Production, BSP (μgCL−1 h−1). (c) − − + μ 1 1 − from day 6 to 8 in nutrients Fe (3.6 gCL h ), Bacterial Growth Rate (BGR, d 1). Values are the means ± 1 μ −1 −1 polysaccharide (3.0 gCL h ), and siderophore standard deviations for samples from duplicate bottles maintained μ −1 −1 (1.6 gCL h ) treatments (Fig. 4b). Leucine incor- until day 10. poration rates fell slightly (down to 1 μgCL−1 h−1)by day 10, and this activity was still elevated and nearly three times the production observed in the control treat- to increase slightly in nutrients + Fe and NUT treatment (>0.5 μgCL−1 h−1). Bacterial production rate ments and after day 6, compared to control (Fig. 4c). levels are characteristics of the late summer periods at μ −1 −1 Comau Fjord (1.4 to 4.6 gCL h at 15 m depth; DISCUSSION Cuevas L.A., unpubl. data, 2006). In addition, bacterial growth (BGR) was only affected by polysaccharide addi- Dissolved organic matter types (e.g. hydroxycarboxylic tion during the first 6 days, whereas responses tended acid, siderophore, humic substances), could play

© 2014 Japanese Society of Phycology Phytoplankton and iron-organic matter 9 several roles as ‘micronutrient carriers’ (acting as elec- species decreased after day 4. Here, we suggest that for tron donors) for biological uptake and then for phyto- small cyanobacterial and picoeukaryotes the production plankton growth. In equivalent northern Hemisphere of siderophores increases their ability to utilize iron ecosystems (Norwegian fjords) phytoplankton growth from a Fe-deficient environment as has been observed has been shown to be sporadically limited by iron com- for cyanobacterial strain (Jiao et al. 2002; Eldridge plexes (Öztürk & Bizsel 2002). However, some major et al. 2004; Vraspir & Butler 2009; Liu et al. 2012). points are still in discussion regarding the role of trace After the addition of Fe, Synechococcus was a domi- metals-iron and DOM complexation in coastal areas: (i) nant component of the system, while pico-, nano-, and some DOM may contribute to coagulation of iron, thus microeukaryote organisms decreased after day 4, sug- removing it from the system; (ii) some DOM made gesting the possibility that Fe additions and its avail- complex with iron and retain it in soluble form; some of ability alter the competitive interactions between those them can be bioavailable but some others may not; (iii) groups in the fjord system. Large-scale iron fertilization availability of micronutrients needed to take into experiments have confirmed that phytoplankton blooms account its speciation, rate of supply and the species- induced by iron enrichments were dominated by large specific role of different forms of iron and phytoplank- diatoms (Boyd et al. 2007). Our result, on the other ton; (iv) some DOMs may contribute to transform iron hand, indicated that, small phytoplankton rather than from less to more bioavailable forms (or vice versa). The diatoms have been induced mostly by iron enrich- northern section of the Patagonia fjord (41–43°S) dis- ment in the coastal Patagonian ecosystem. Especially plays seasonal (spring-summer) relatively high primary cyanobacteria, Synechococcus growth are significantly productivity estimates (3–5gCm−2 day−1; Iriarte et al. higher in the Nut+Fe treatments than other treatments. 2013). In our study, relatively low total dissolved Fe Furthermore, nitrate drawdown, but not silicic acid was concentrations at subsurface waters were detected significant in nutrients + Fe treatment (Table 1), sug- during summer conditions (2–3 nM) and suggest the gesting that the increase in cell abundance of small possibility to observe ‘iron limitation’ depending on the cells was a direct consequence of the Fe availability existing forms of iron as well as its transformation in compared to the larger microphytoplankton species. fjords. For coastal waters these concentrations could be Experiments with coastal and oceanic phytoplankton relatively low due to coastal phytoplankton species may clones representing different algal groups and cell sizes have high Fe quota (Brand 1991), and large portion of suggest (due to evolutionary pressures) that iron uptake the Fe may not be bioavailable for phytoplankton in all species tend toward the maximum limits imposed growth. Low total dissolved Fe concentrations (1–3 nM) by diffusion and ligand exchange kinetics. Because in the Comau Fjord has previously been observed during of these physical/chemical limits on uptake, oceanic spring (December 2007) and summer (January 2011) species have been forced to decrease their cell size seasons (Ardelan M., unpubl. data, 2009), and were and/or to reduce their growth requirements for cellular consistent with the low Fe concentrations detected iron by up to eightfold (Sunda & Huntsman 1995). The in other coastal areas where Fe-limitation has been excretion of siderophores and the reduction of organic reported during spring bloom conditions (Bruland et al. iron-complexes at the cell surface are common reactions 2001; Öztürk et al. 2002; Hare et al. 2005). Specifi- of terrestrial plants, fungi and bacteria in response to low cally in the Comau Fjord, river concentrations ranged availability of iron. However, there is much less evidence 122.8 to 42.2 nmol L−1, 36.7 to 16.5 nmol L−1 and 5.0 for the use of these strategies by marine phytoplankton to 1.0 nmol L−1, for TFeCh, DFeCh and FeDGT frac- (Völker & Wolf-Gladrow 1999) and the binding mol- tions, respectively, (Sanchez N., unpubl. data, 2011). ecules and enzymes that mediate the biochemical role of In this study we tested the response of autotrophic trace metals in the marine environment. pico-, nano- and microphytoplankton abundances and Among micro-phytoplankton species, dominant micro-phytoplankton species composition to Fe-DOM species shifted from a diatoms/dinoflagellates assem- enrichment, which have been indicated as having an blage dominated by a centric diatom, Leptocylindrus effect on the phytoplankton community. Here we minimus, to an assemblage dominated by pennate addressed the main question: to what extent Fe, diatoms. In this experiment, the pennate diatoms polysaccharide, and siderophore types enhanced Nitzschia longissima and Pseudo-nitzschia cf. growth of particular phytoplankton size-classes, as well delicatissima numerically dominated the micro- as examining possible selection for certain micro- phytoplankton community at the Fe + Nutrients treat- phytoplankton species to those complexes. Our summer ment. It is noteworthy that pennate diatoms have been experiment indicated that increasing inputs of dis- the key group that bloom during iron enrichment experi- solved iron resulted in a rapid response of prokaryotic ments, such as Pseudo-nitzschia sp. (Trick et al. 2010; small cells and then a gradual shift in phytoplankton Marchetti et al. 2012), Thalassiothrix sp. (Marchetti community structure towards small eukaryotic and et al. 2012), Navicula sp. (Hare et al. 2005). In the nanoflagellates cells while abundances of large diatoms polysaccharide enrichment, the dominant diatoms were

© 2014 Japanese Society of Phycology 10 J. L. Iriarte et al. also Pseudo-nitzschia cf. delicatissima in addition to a also thank the Centro de Investigación de Ecosistemas centric diatom Guinardia delicatula. Major dissolved de la Patagonia (CIEP) and the Programa de Finan- nutrients, nitrate and silicic acid, were drawn down to ciamiento Basal COPAS–Sur Austral. Results from low levels in the polysaccharide treatment, suggesting January–February 2011 were part of the MSc. Thesis of that diatoms species take advantage until the end of Mrs. Nicolas Sanchez at Norwegian University of the experiment. Our results indicated that DFB addition Science and Technology, Norway. This is publication did not change the main species composition of 102 of the Huinay Scientific Field Station. diatoms and dinoflagellates relative to polysaccharide and nutrients + Fe treatments, suggesting no effects at species-specific level of DFB bioavailability. 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