Seasonal Variability in Phytoplankton Carbon Biomass and Primary Production, and Their Contribution to Particulate Carbon in the Neritic Area of Sagami Bay, Japan

Plankton Benthos Res 14(4): 224–250, 2019 Plankton & Benthos Research © The Plankton Society of Japan

Seasonal variability in phytoplankton carbon biomass and primary production, and their contribution to particulate carbon in the neritic area of Sagami Bay, Japan

1,2, 2 2 2,3 Koichi Ara *, Satoshi Fukuyama , Takeshi Okutsu , Sadao Nagasaka & 4 Akihiro Shiomoto

1 Department of Marine Science and Resources, College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa 252–0880, Japan 2 Research Division in Biological Environment Studies, Graduate School of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa 252–0880, Japan 3 Department of Bioenvironmental and Agricultural Engineering, College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa 252–0880, Japan 4 Department of Ocean and Fisheries Sciences, Faculty of Bioindustry, Tokyo University of Agriculture, Abashiri, Hokkaido 099–2493, Japan Received 27 August 2018; Accepted 6 June 2019 Responsible Editor: Akira Ishikawa doi: 10.3800/pbr.14.224

Abstract: Seasonal variations in environmental variables, chlorophyll a (Chl-a), particulate carbon and nitrogen (PC and PN, respectively), phytoplankton carbon biomass (Ph-C) and primary production were investigated at a neritic station in Sagami Bay, Kanagawa, from January 2008 to December 2013. Size-fractionated Ph-C was converted from cell volume by microscopic observation, adding valuable data for this area. During spring blooms, the micro-size fraction (>20 µm) comprised the majority of the total Chl-a and total Ph-C, whereas during other periods the pico- and nano- size fraction (<20 µm) comprised a larger proportion, indicating that phytoplankton standing crops were affected by sunlight conditions and physicochemical properties of the water. In February–March, phytoplankton biomass increased and formed the ﬁrst peak of spring blooms under increasing sunlight intensities (>15.7 MJ m−2 d−1), high nutrient concentrations and balanced molar ratios. From the regression equations of size-fractionated Ph-C-Chl-a relationships, the mean Ph-C/Chl-a ratio was 5.3–7.7, 29.2–32.6 and 22.1–25.1 for the <20 µm, >20 µm and total fraction, respectively. The Ph-C/Chl-a ratio (1.8–128.8) was regulated by irradiance and nutrients. Growth rate (ca. 0–3.7 d−1) was positively correlated with irradiance and assimilation number, and negatively with the Ph-C/Chl-a ratio. The depth-integrated primary production (DIPP) was 0.15–5.43 g C m−2 d−1. On the basis of the 0–50 m depth-integrated values, the total Ph-C and DIPP accounted for 1.3–34.4% and 1.3–30.9% d−1 of PC, respectively, indicating that PC variations depended on the total Ph-C and DIPP.

Key words: growth rate, particulate carbon, phytoplankton carbon biomass, primary production, Ph-C/Chl-a ratio

oceanic areas (e.g. Cloern et al. 2014, Costello & Chaud- Introduction hary 2017). The quantity and size composition of phyto- In general, coastal areas are highly productive habitats plankton assemblages are strongly related to food web for aquatic organisms, attributable principally to the great- structure, which influences energy flow and material (car- er phytoplankton standing crops (i.e. Chl-a, Ph-C) and pri- bon) cycles in marine ecosystems, including the amount mary production rates correlated to intermittently higher of higher trophic level productivity (Sommer et al. 2002, concentrations of land-derived nutrients in addition to Friedland et al. 2012). Hence, quantifying phytoplankton those derived from the open ocean, than found in offshore standing crop, size composition and primary production is essential to understand the food web structure and biologi- * Corresponding author: Koichi Ara; E-mail, [email protected] cal productivity of the marine ecosystem, and is significant Phytoplankton carbon biomass and primary production in Sagami Bay 225 in evaluating the role of phytoplankton in marine carbon Studies on the phytoplankton assemblages in the coastal cycles (Llewellyn et al. 2005). waters of Sagami Bay have dealt with aspects of the sea- Seasonal variations in size-fractionated (i.e. pico-, nano- sonal variations in total and size-fractionated Chl-a con- and micro-) phytoplankton standing crop (i.e. Chl-a and/ centrations (Tatara & Kikuchi 2003, Baek et al. 2007, Ara or Ph-C) have been extensively studied in subarctic and & Hiromi 2007, 2008, 2009, Baki et al. 2009, Ara et al. temperate estuarine and coastal waters in Japan, e.g. the 2011a, b, 2013, Okutsu et al. 2012, Fukuyama 2013), size- Oyashio region off Akkeshi (Shinada et al. 2001), Funka fractionated Ph-C (Ara & Hiromi 2009) and primary pro- Bay and adjacent waters (Maita & Odate 1988, Odate et duction (Ara & Hiromi 2007, 2009, Ara et al. 2011b, 2013). al. 1993, Shinada et al. 2005, 2008), Oginohara Bay (Ishi- There has been no attempt, however, to assess the sea- nomaki Bay) (Kamiyama et al. 2005), Tokyo Bay (Han sonal variations in phytoplankton standing crop in terms & Furuya 2000), Sagami Bay (Ara & Hiromi 2009), Ago of Chl-a and Ph-C and in primary production rate in rela- Bay (Tanimura et al. 2008), Hiroshima Bay (Lee et al. tion to environmental parameters (i.e. weather conditions, 1996, Kamiyama et al. 2005), Tosa Bay (Ichikawa & Hiro- physicochemical properties), and their contribution to PC ta 2004) and Uchiumi Bay (Tomaru et al. 2002), as well as in these waters. In the present study, we aimed to elucidate primary production (references in Ara et al. 2011b). These (1) the seasonal variations in size-fractionated phytoplank- studies found drastic seasonal variations with consider- ton standing crop in terms of Chl-a and Ph-C, PC and PN able peaks of Chl-a, Ph-C and primary production rate in concentration and primary production, (2) the size-frac- spring–autumn, depending on the trophic state; in oligo- tionated and total Ph-C/Chl-a ratio (i.e. conversion factor trophic open coastal waters, their peaks occurred only in to estimate carbon biomass from Chl-a), (3) growth rate of spring, whereas in eutrophic estuarine waters the peaks the phytoplankton assemblage, and (4) the contribution of occurred intermittently in summer–autumn in addition to Ph-C and primary production to PC, over 6 years (2008– in spring. However, almost all of these studies were based 2013) in the neritic area of Sagami Bay. on the determination of size-fractionated Chl-a, with studies on size-fractionated Ph-C being limited, except in Fun- Materials and Methods ka Bay and adjacent waters (Maita & Odate 1988, Odate et al. 1993, Shinada et al. 2005, 2008), the Oyashio region off Field investigation Akkeshi (Shinada et al. 2001) and Sagami Bay (Ara & Hi- romi 2009). In addition, many of those studies were based A series of field investigations were conducted mostly on field investigations conducted only once in a month or every two weeks (i.e. twice a month) on 144 occasions in a season, except Ara & Hiromi (2007, 2009) and Ara from January 2008 to December 2013, at a station (Lat. et al. (2011b, 2013), which investigated size-fractionated 35°16.3′N, Long. 139°29.6′E; local depth: ca. 56 m) located Ph-C and/or primary production twice a month for mul- in the neritic area off Enoshima Island (ca. 4 km off the tiple years in the innermost open area, near Enoshima Is- coastline), Fujisawa, Kanagawa, of Sagami Bay (Fig. 1). land, of Sagami Bay. On each date, sampling was always done during daytime Sagami Bay is a semi-circular embayment, located on (07:30–12:00 a.m.). the southern coast of central Honshu, the main island of The procedure for measuring physicochemical proper- Japan, and facing the western North Pacific Ocean (Fig. ties (i.e. water temperature, salinity and dissolved inor- 1). It is located in a transition zone where hyper-eutrophic ganic nutrients), Chl-a concentration and primary produc- Tokyo Bay Water flows out to the Pacific Ocean. The water tion at this station have been previously published by e.g. quality environment in the wide-mouthed Sagami Bay has Ara & Hiromi (2008, 2009) and Ara et al. (2011a, b), an maintained much better conditions than in the neighboring outline of which is briefly given as follows. Prior to sample semi-closed Tokyo Bay (Ara & Hiromi 2008, Ara et al. collection, water temperature and salinity were measured, 2011a, b), which is attributed to less freshwater discharge using a Memory STD (AST-1000/P-64K, Alec Electronics, with smaller nutrient and pollutant load from rivers, and Japan). Transparency was measured using a Secchi disk. their shorter residence time in estuarine areas due to much Water samples for assessments of cyanobacteria (size: 0.2– more frequent exchange between estuarine and offshore 2 µm) and autotrophic nanoplankton (ANP, size: 2–20 µm) ocean waters, compared to Tokyo Bay (Kawabe & Yoneno were taken at depths of 0, 10, 20, 30, 40 and 50 m, using 1987, Iwata & Matsuyama 1989, Yamada & Matsushita a Kitahara bottle. Water samples for microphytoplankton 2006). In the innermost open waters of Sagami Bay, es- (size: >20 µm), Chl-a, particulate matter (PM) and dis- pecially in the upper layer around Enoshima Island, Chl- solved inorganic nutrients were taken at depths of 0, 5, 10, a concentrations and primary production rates have been 20, 30, 40 and 50 m, using duplicate Van Dorn bottles. higher than in any other open area of this bay, and these The samples for cyanobacteria (40 ml) and ANP (100 ml) have been attributed to higher nutrient concentrations due were transferred into γ-ray-sterilized polypropylene bottles to nutrient-rich freshwater discharge from the Sakai and and HCl-sterilized glass bottles, respectively. For micro- Hikiji Rivers (Yamada & Matsushita 2005, 2006, Ara & phytoplankton, 10 or 20 L of water samples were concen- Hiromi 2007, 2008, 2009, Ara et al. 2011a, b). trated using a hand-net (20 µm mesh opening size), trans- 226 K. Ara et al.

Fig. 1. Map showing the sampling station in Sagami Bay with isobaths in meters. ferred into 100 ml bottles. These samples were immediately using an Autoanalyzer (AACSIII, Bran+Luebbe, Germa- preserved in 1% glutaraldehyde (final concentration). ny) with detection limits of 0.05, 0.02 and 0.03 µM, respec- For Chl-a, the seawater was size-fractionated into tively (Parsons et al. 1984a, Hansen & Koroleff 1999). For <20 µm and >20 µm by sieving through a 20 µm-mesh PM, 1 L of seawater aliquots were filtered through pre- screen, and 0.5 L of unsieved and size-fractionated sea- combusted (at 400°C for 4 hrs) Whatman GF/F glass fiber water aliquots were filtered through Whatman GF/F glass filters (47 mm). The filters were dried completely at 60°C fiber filters (47 mm). Chl-a concentration was determined for 2 hrs and stored in a vacuum desiccator. PC and PN on with a fluorometer (TD-700, Turner Designs, USA) after the glass fiber filters were determined using a CN Corder grinding in 90% acetone (Holm-Hansen et al. 1965, Par- (MT-700II, Yanaco, Japan). sons et al. 1984a). The fluorometric determination was cal- Primary productivity (PP) was determined by the in situ ibrated with a Standard Chl-a reagent derived from Chlo- 13C tracer method (Hama et al. 1983). Photosynthetically rella (Wako Pure Chemical Industries, Japan). Dissolved active radiation (PAR) was measured using an underwa- inorganic nutrient concentrations, i.e. dissolved inorganic ter quantum sensor and a light meter (LI-192SA/LI-250, + − − nitrogen (DIN: NH4 +NO3 +NO2 -N), phosphate (DIP: LI-COR, USA). Water samples were taken from depths 3− PO4 -P) and silicate [DSi: Si(OH)4-Si], were determined corresponding to 100, 50, 25, 10, 5 and 1% photon fluxes Phytoplankton carbon biomass and primary production in Sagami Bay 227

Table 1. Conversion factors or formulae to estimate carbon biomass (C) for phytoplanktonic organisms. CV: cell volume (µm−3). Plankton Conversion factors or formulae Reference Cyanobacteria 153 fg C cell−1* Verity et al. (1992), Hamasaki et al. (1999)

Autotrophic nanoplankton (ANP) log10pg C=0.863 log10CV−0.363 Verity et al. (1992) Diatoms (<3,000 µm3) pg C=0.288 CV 0.811 Menden-Deuer & Lessard (2000) Diatoms (>3,000 µm3) pg C=0.117 CV 0.881 Menden-Deuer & Lessard (2000) Dinoflagellates pg C=0.760 CV 0.819 Menden-Deuer & Lessard (2000) * based on the averaged biovolume (BV) of 0.30 µm−3 (Hamasaki et al. 1999) and the equation from biovolume to carbon (C, pg C cell−1) by Verity et al. (1992): C=0.433 BV 0.863. of that just above the sea surface using a Niskin bottle low) in 6–954 fields (1 field: 0.01 mm2) were counted. For (volume: 12 L). After removing large zooplankton with a ANP, >20 cells (>5 cells when abundances were low) in 200 µm-mesh screen, seawater samples were immediately 6–744 fields (1 field: 0.01 mm2) were counted. On each sam- transferred into HCl-sterilized polycarbonate bottles (vol- pling date, after sample collection and preservation, cyano- ume: 0.5 L; one initial, two light and one dark bottles at bacteria and ANP were stained, filtrated and slide-prepared 13 each depth). After the addition of NaH CO3 (Cambridge within 5–7 hrs, then counted and measured within 6–11 hrs. Isotope Laboratories, USA, ca. 10% of the total inorganic Microphytoplankton from 0.2–2% subsamples were trans- carbon in ambient water), the bottles were placed at the ferred onto plankton counting plates (size: 40×78 mm; Ri- same depths at which the water samples were taken, and gosha, Japan) fitted with outer frames, identified to species were incubated in situ for 24 h. The in situ incubation was level, and counted under a binocular microscope (magnifi- usually started at 08:30–10:00 a.m. The water samples in cation: ×200; Eclipse E600, Nikon, Japan). Cell sizes were initial, incubated light and dark bottles were then filtered measured for all analyzed ANP cells and for 50 cells per through precombusted (at 400°C for 4 h) Whatman GF/F sample or all cells when samples contained <50 cells for filters (47 mm). Dissolved inorganic carbon (DIC) concen- each microphytoplankton species/genus, using an eyepiece tration in the initial bottles was determined using a Total micrometer to determine the cell volume according to their

Organic Carbon (TOC) Analyzer (TOC-VCPH, Shimadzu, spherical, prolate spheroidal, cylindrical and geometric con- Japan). The filters were dried at 60°C for 1–2 h, treated figurations (Kovala & Larrance 1966, Hillebrand et al. 1999, with HCl fumes for 3 h to remove inorganic carbon, com- Sun & Liu 2003, Olenina et al. 2006). The carbon biomass pletely dried at 60°C for 1–2 h and stored in a vacuum of each phytoplankton was calculated from cell number or desiccator. The isotopic concentration of 13C and 12C was cell volume, using conversion factors or formulae (Table 1). determined using a mass-spectrometer (ANCA-SL, Europe Other data sources Scientific, UK or INTEGRA-2, SerCon, UK). The dark uptake was always corrected for primary productivity. The Sunlight duration (SD) data were obtained from an depth-integrated primary production was calculated by the AMeDAS (Automated Meteorological Data Acquisition integral of primary productivities in the euphotic zone, i.e. System) recorded at the Tsujido Station, Fujisawa (Lat. from the sea surface to the depth corresponding to 1% of 35°19.2′N, Long. 139°27.0′E), of the Japan Meteorological sea surface sunlight intensity. Agency (2014). Global solar radiation (SR) data were obtained from the UV-Observation at Shonan, Fujisawa (Lat. Plankton sample treatments and calculations 35°19.3′N, Long. 139°27.3′E), of Shonan Institute of Tech- Cyanobacteria were stained with 4′6-diamidino-2-phenology (2014) through the Monitoring Network for Ultra- nylindole (DAPI, final concentration: 1 µg mL−1) for 10 violet Radiation established by the Center for Global Envi- minutes, and 4–20 ml aliquots of the samples were filtered ronmental Research, National Institute for Environmental (suction vacuum pressure: ≤10 mmHg) onto 0.2 µm What- Studies. The measuring points of SD and SR are located man Nuclepore Track-Etch Membranes (25 mm). ANP were ca. 6.6 and 6.5 km from our sampling station, respectively, stained with DAPI (final concentration: 0.1 µg mL−1) and and the distance between these two points is ca. 0.5 km. fluorescein-4-isothiocyanate (FITC-I, final concentration: Daily SD (hrs) were calculated from the cumula- 3 µg mL−1) for 10 minutes, and 20 ml aliquots of the sam- tive SD (min) measured every 10 minutes. Daily SR val- ples were filtered (suction vacuum pressure: ≤10 mmHg) ues (MJ m−2 d−1) were calculated from the cumulative SR onto 0.8 µm Nuclepore Track-Etch Membranes (25 mm). (kW m−2) measured every 10 minutes, using the conversion Cyanobacteria and ANP were inspected with blue excitation factor of (MJ h−1) kW−1 to be 3.6. For each sampling date, under an epifluorescence microscope (magnification: ×400; SD and SR were calculated as the integral during the in situ BX50, Olympus, Japan) to detect the orange-fluorescent cy- incubation time (i.e. 24 hrs) for primary productivity mea- anobacterial and red-fluorescent ANP chlorophyll. For cya- surement (SDPP and SRPP, respectively). Daily SR on dates nobacteria, >300 cells (>150 cells when abundances were when it was not measured (e.g. in June 2008–May 2009, 228 K. Ara et al.

March–April 2011 and some dates during the other periods) to-day and seasonal variations (Fig. 2). Daily SD and daily was calculated from daily SD using the obtained equations SR were low in December–January (monthly max.: 9.0–9.8 of linear daily SD-daily SR relationships in each month of hrs and 10.9–13.6 MJ m−2 d−1, respectively), and the high- 2008–2013 (r2=0.846–0.927, p<0.0001 for each, on aver- est values in each year were observed in May–August age 0.003–0.03% margins of error for each). The estimated (monthly max.: 11.7–13.7 hrs and 24.8–29.3 MJ m−2 d−1, re-

SR and SRPP are shown for the seasonal variations, whereas spectively). SDPP and SRPP varied from 0 to 13.5 hrs and these were excluded from the regression analysis. from 2.0 to 27.9 MJ m−2 d−1, respectively (Fig. 2). Transparency varied from 2.2 to 26.8 m (Fig. 2). Al- Data analysis though transparency varied drastically during the study For each sampling date, phytoplankton 13C-derived spe- period, it was greater in December–February (monthly ciﬁc growth rate (GR, d−1) was estimated by the following mean: 14.1–16.6 m) than in April–July (monthly mean: equation (Furuya 2015, Gasol et al. 2016): 7.0–9.7 m).

GR= ln[1 + (PP/Ph-C)], Temperature, salinity and dissolved inorganic nutrients where PP is net primary productivity (µg C L−1 d−1), and Water column temperature and salinity varied from Ph-C is phytoplankton carbon biomass (µg C L−1). 12.83 to 28.36°C and from 29.25 to 34.67, respectively, Canonical correspondence analysis (CCA), a direct gra- and they showed similar seasonal variations during all six dient analysis technique, was used to examine the rela- years (Fig. 3), as observed at the same site for an earlier pe- tionships between environmental variables [i.e. daily SD riod (Ara & Hiromi 2008, Ara et al. 2011a, b). In autumn– and SR (hrs and MJ m−2 d−1, respectively), mixed layer spring (late October/November–early April), the water depth (MLD, m), Brunt-Väisälä frequency (N, s−1), and column conditions were vertically well-mixed with low mean water temperature (°C), salinity, DIN, DIP and DSi temperatures-high salinities. The water column became concentration (µM) in the euphotic zone] and phytoplank- gradually stratiﬁed in April–May, and then a thermo- and ton standing crops [i.e. mean total Chl-a, Chl-a <20 µm halocline developed at 20–30 m depth in summer (June– and >20 µm (µg L−1), total Ph-C and carbon biomass September), with the surface waters at high temperatures- (µg C L−1) of cyanobacteria, ANP, microphytoplankton low salinities, rising to 26–28°C and declining to 29–32,

(Ph-CM) and dominant microphytoplankton genera in the respectively. In autumn (October), the depth of the upper euphotic zone]. Here, MLD was defined as the depth of mixed layer deepened to the bottom, and the thermo- and sea surface density σt+0.125 σt (Δ0.125σt is equivalent to halocline disappeared (Fig. 3). −1 3− Δ0.5°C). Brunt-Väisälä frequency (N, s ) was calculated as DIN, PO4 -P and Si(OH)4-Si concentrations ranged from 0.62 to 28.58 µM, from below the detection lim- g d it (0.02 µM) to 0.94 µM and from 0.12 to 29.94 µM, re- N =  dz spectively. In autumn–spring (October/November–early 3− March), DIN, PO4 -P and Si(OH)4-Si concentrations were where g is gravitational acceleration (9.8 m s−2), ρ is the high (mostly >3, >0.15 and >3 µM, respectively) through- mean density at the sea surface (1027 kg m−3), and dρ/dz out the water column (Fig. 3). In spring–summer, very low 3− is density σt gradient between sea surface and 50 m depth. DIN, PO4 -P and Si(OH)4-Si concentrations (<2, <0.1 Brunt-Väisälä frequencies and phytoplankton carbon bio- and <2 µM, respectively) were frequently observed in the −2 −1 masses were transformed [(×10 s ) and log10 (n+1), re- upper mixed layer (0–20 m depth), whereas these remained spectively] to reduce the effects of extreme values. The high below the stratified layer (30–50 m depth) (Fig. 3, Ara significance (p<0.05) of environmental variables to ex- & Hiromi 2008, Ara et al. 2011a, b). plain the variability of abiotic data in CCA was tested by Chl-a and PM forward selection of the permutation test (999 unrestrict- ed permutations) using the XLSTAT for Microsoft Excel Total Chl-a concentration varied from 0.04 to (Mindware, Japan). 14.25 µg L−1. Total Chl-a concentrations were intermittent- The period of the first spring phytoplankton bloom was ly very high throughout the water column or in the upper defined by 5% above annual median values of mean phyto- layer (0–20 m depth) during spring phytoplankton blooms plankton carbon biomass (PCB: Ph-CM and total Ph-C) and in February–May (Fig. 4, Ara & Hiromi 2008, Ara et al. Chl-a (total and >20 µm) in the euphotic zone in each year 2011a, b). Spring blooms in 2010 (max. 12.2 µg L−1) were of 2008–2013, respectively (Ferreira et al. 2014). greater than those in other years (max. 4.1–7.5 µg L−1). To- tal Chl-a concentrations remained high in the upper layer (0–20 m depth) during summer blooms (June–September), Results especially in June 2008 and August 2011, at which the maximum Chl-a concentration in each year was record- SD, SR and transparency ed in the upper layer (0–5 m depth). Autumn blooms in −1 Daily SD, daily SR, SDPP and SRPP showed drastic day- 2010 (max. 4.3 µg L ) were greater than those in other Phytoplankton carbon biomass and primary production in Sagami Bay 229

Fig. 2. Seasonal variations in sunshine duration (SD), global solar radiation (SR) and transparency from January 2008 to December 2013. Thin line: daily data; open circle: the integral SD and SR during the in situ incubation period (24 hrs) for primary productivity measurements, respectively. years (max. 0.9–2.0 µg L−1). In December–January, the >20 µm fraction were sporadically observed in June and total Chl-a concentrations were low (mostly <1 µg L−1) August 2008, July 2010, January, June and November 2011, throughout the water column. Relatively high Chl-a con- July–August 2012 and October–December 2013 (Fig. 4). centrations (>2 µg L−1, “winter blooms”) were observed in PC and PN ranged from 0.08 to 1.77 mg L−1 and from December 2010–January 2011 and December 2013 (Fig. 4). 0.03 to 0.27 mg L−1, respectively. PC and PN concentra- Chl-a <20 µm and >20 µm varied from below the de- tions were low throughout the water column in Novem- tection limit (0.02 µg L−1) to 5.24 µg L−1 and from below ber–early February. In March–October, high concentra- the detection limit to 9.04 µg L−1, respectively. Of the to- tions were frequently observed in the upper layer (0–20 m tal Chl-a, the >20 µm fraction comprised the majority in depth), whereas these remained low in the lower layer (30– February–May, accounting for an average at 0–50 m depth 50 m depth) (Fig. 4, Ara et al. 2011b). of 59.5–78.5% (monthly mean), especially during spring PC and PN concentrations were directly proportional to blooms (>80%) (Fig. 4, Ara & Hiromi 2008, Ara et al. the total Chl-a concentration for all data in 2008–2013, 2011a, b). In June–August, the <20 and >20 µm fractions respectively (Fig. 5, Ara et al. 2011b). From the slopes of accounted for an average at 0–50 m depth of approximately these regression equations obtained for PC-Chl-a and PN- half (monthly mean: 48.5–51.8% for the >20 µm fraction), Chl-a relationships, the mean ratio of C:Chl-a and N:Chl-a respectively. During the other periods, i.e. in September– was 104.0 and 15.6, respectively. There was a signiﬁcantly January, the <20 µm fraction was greater, accounting linear correlation between the molar C and N of PM for all for an average at 0–50 m depth of 52.5–61.5% (monthly data in 2008–2013. From the slope of the regression equa- mean). In June–January, large proportions (>60%) of the tion obtained for the molar C-N relationship, the mean mo- 230 K. Ara et al.

+ − − Fig. 3. Seasonal variations in vertical proﬁles of water temperature, salinity, dissolved inorganic nitrogen (DIN: NH4 +NO3 +NO2 -N), phos- 3− phate (PO4 -P) and silicate [Si(OH)4-Si] concentrations in Sagami Bay, from January 2008 to December 2013. Arrows denote sampling dates. Phytoplankton carbon biomass and primary production in Sagami Bay 231

Fig. 4. Seasonal variations in vertical proﬁles of total chlorophyll a (Chl-a) concentration, mean Chl-a size fraction (Chl-a <20 µm and >20 µm) at 0–50 m depth, particulate carbon and nitrogen (PC and PN, respectively) concentrations in Sagami Bay, from January 2008 to December 2013. Arrows denote sampling dates. lar C:N ratio of PM was estimated to be 6.63 (Fig. 5). high (>5 µg C L−1) in June–October/November (Fig. 6). Higher biomasses (>10 µg C L−1) were sporadically ob- Phytoplankton carbon biomass served in the upper layer (0–10 m depth) in June and Sep- The cyanobacterial biomass varied from 0.01 to tember 2008, July–September 2009, and July and Septem- 22.12 µg C L−1, overall mean±SE of 1.76±0.08 µg C L−1. ber 2010. It showed essentially a consistent seasonal pattern during The ANP biomass varied from 0.07 to 31.37 µg C L−1, the study period: the biomasses were low (<1 µg C L−1) overall mean±SE of 3.71±0.11 µg C L−1. ANP biomasses throughout the water column in January–May, and were were low (mostly <5 µg C L−1) throughout the water col- 232 K. Ara et al.

(mostly <1 µg L−1) throughout the water column, while high biomasses (>10 µg C L−1) were observed in January 2010, January 2011 and December 2013.

Regarding Ph-CM, diatoms were predominant during the study period, accounting for an average at 0–50 m depth of 81.1% (overall mean), whereas large proportions (an average at 0–50 m depth of >50%) of dinoﬂagellates were observed sporadically in spring–autumn, especially in May–September 2008 (60.4–98.9%), May–September 2009 (56.5–97.6%), July–August 2011 (69.9–76.5%), May– July 2012 (63.7–95.0%), and June–July 2013 (59.3–80.3%) (Fig. 6). In December 2012, silicoﬂagellates comprised a large proportion (an average at 0–50 m depth of 59.3%) of

Ph-CM, whereas during other periods these accounted for an average at 0–50 m depth of only 0–5.6%. The genus Chaetoceros (e.g. C. afﬁnis Lauder, C. cur- visetus Cleve, C. devilis Cleve) was predominant during the study period, accounting for an average at 0–50 m

depth of 31.3% (overall mean) of Ph-CM (Fig. 6). The greatest contributor to Ph-CM was Chaetoceros in Janu- ary, March and August–December (monthly mean: 29.1–66.3%), whereas it was Ceratium [e.g. C. furca (Eh- renberg) Clapalède et Lachmann, C. fusus (Ehrenberg) Dujardin, C. kofoidii Jørgensen] in May–July (monthly mean: 25.4–54.3%), Ditylum brightwellii (West) Grunow ex Van Heurck in February (monthly mean: 33.2%), and Cerataulina [e.g. C. dentata Hasle, C. pelagica (Cleve) Hendey] in April (monthly mean: 27.7%). In January, Au- gust and November, Coscinodiscus (e.g. C. curvatulus Crunow, C. gigas Ehrenberg, C. wailesii Gran et Angst) comprised large proportions (monthly mean: 19.4–25.0%)

of Ph-CM. Eucampia zodiacus Ehrenberg and Thalas- siosira (e.g. T. mara Takano, T. paciﬁca Gran et Angst) comprised large portions in March–April and December (monthly mean: 16.1–19.8%) and in April and December (monthly mean: 18.3–19.0%), respectively. Rhizosolenia [e.g., R. alata forma gracillima (Cleve) Gran, R. bergonii Péragallo, R. stolterfothii Péragallo] comprised large portions in May and October (monthly mean: 21.2–26.2%) of

Ph-CM (Fig. 6). The total Ph-C (i.e. cyanobacterial, ANP plus micro- Fig. 5. Particulate carbon and nitrogen (PC and PN, respec- phytoplankton) varied from 0.87 to 348.89 µg C L−1, over- tively) plotted against total chlorophyll a (Chl-a), and the molar C all mean±SE of 23.37±1.25 µg C L−1. The variation pat- of particulate matter (PM) plotted against the molar N of PM for all sampling dates in 2008–2013. tern of the total Ph-C was quite similar to that of Ph-CM (Fig. 6). Of the total Ph-C, Ph-CM was the most predominant during the study period, accounting for an average at umn in October/November–February/March, depending 0–50 m of 3.4–98.4% (overall mean: 76.9%), while during on the year (Fig. 6). Higher values (>10 µg C L−1) were spring blooms in February–May it comprised the major- sporadically observed in the upper layer (0–20 m depth) in ity, accounting for an average at 0–50 m depth of ca. 90% April–September of each year. or more. During the other periods, i.e. in summer–win-

The microphytoplankton biomass (Ph-CM) var- ter (June–December), the proportions of cyanobacteria ied from 0.01 to 316.60 µg C L−1, overall mean±SE of plus ANP were frequently greater: cyanobacteria were the −1 17.50±1.20 µg C L . Ph-CMs were intermittently high greater contributors during the ﬁrst half of these periods, (>10 µg C L−1) throughout the water column in February– whereas during the latter half of these periods they were May or in the upper layer (0–20 m depth) in June–Sep- substituted by ANP (Fig. 6). tember (Fig. 6). In December–January, Ph-CMs were low The Ph-C/Chl-a ratio varied from 1.8 to 128.8. The ra- Phytoplankton carbon biomass and primary production in Sagami Bay 233 234 K. Ara et al.

Fig. 6. Seasonal variations in vertical distribution of cyanobacterial, autotrophic nanoplankton (ANP), microphytoplankton and total phytoplankton biomass, mean composition of microphytoplankton and total phytoplankton at 0–50 m depth, and the ratio of total phytoplankton biomass to total chlorophyll a (Phyto-C/Chl-a) in Sagami Bay, from January 2008 to December 2013. Arrows denote sampling dates. tios were low (<10) throughout the water column in No- riod of active vertical mixing of seawater and were ex- vember/December–April/May (Fig. 6). Higher ratios (>50) cluded from the regression analysis (Fig. 8). The relation- were sporadically observed in the upper layer (0–10 m ship between three day-mean solar radiation just before depth) in September 2008, August 2009, March–April and each sampling date (SR3dmSD) and the Ph-C/Chl-a ratio was August 2012, and June 2013. positive and well described by a logarithmic regression.

The cyanobacterial plus ANP biomass (Ph-CPN), Ph- The Ph-C/Chl-a ratio was directly proportional to tempera- CM and total Ph-C showed significantly linear correlations ture, showing a linear correlation. The Ph-C/Chl-a ratio with Chl-a <20 µm, Chl-a >20 µm and total Chl-a for all decreased with increase in nutrient concentrations, show- sampling dates and for each season of 2008–2013, respec- ing significantly exponential and logarithmic correlations 3− tively (Fig. 7). From the slopes of the regressions, the mean with DIN and with PO4 -P and Si(OH)4-Si, respectively C:Chl-a ratio was 5.3–7.7 (overall mean: 6.2) for Ph-CPN, (Fig. 8). 29.2–32.6 (overall mean: 30.1) for Ph-C , and 22.1–25.1 M Relationships between environmental variables and bi- (overall mean: 22.6) for the total Ph-C. otic data In the euphotic zone, the variations in the Ph-C/Chl-a ratio showed significant correlations with irradiance (i.e. The horizontal axis (CCA AX1) is a linear combination SR), temperature and nutrient concentration, where a few of environmental variables that best explains the variation inconsistently high Ph-C/Chl-a ratios observed at interme- in a matrix of phytoplankton standing crop in terms in diate SR3dmSDs, low-to-mid temperatures and low-to-high Chl-a and Ph-C, while the vertical axis (CCA AX2) ex- nutrient concentrations were observed only during the pe- plains the remaining variance in these biotic data (Fig. 9). Phytoplankton carbon biomass and primary production in Sagami Bay 235

Fig. 7. Biomass of pico- plus nanophytoplankton (Ph-CPN), microphytoplankton (Ph-CM) and total phytoplankton (Ph-C) plotted against chlorophyll a <20 µm (Chl-a<20 µm), >20 µm (Chl-a>20 µm) and total chlorophyll a (Chl-a), respectively, for all sampling dates in 2008–2013 and in each season of 2008–2013.

The eigenvalues of CCA AX1 and CCA AX2 were 0.072 respectively. Application of forward selection by the per- and 0.037, respectively, which accounted for 78.6% of the mutation test confirmed that the first two axes were highly cumulative variance in the biotic data. The explained vari- significant (p<0.0001). ance of CCA AX1 and CCA AX2 was 51.6% and 27.0%, A decreasing demand of mixed layer depth, salinity, dai- 236 K. Ara et al.

Fig. 8. The ratio of total phytoplankton biomass to total chlorophyll a (Ph-C/Chl-a) plotted against three day-mean solar radiation just 3− before each sampling date (SR3dmSD), water temperature, DIN, DIP (PO4 -P) and DSi [Si(OH)4-Si] concentration for all sampling dates in 2008–2013. Regression analysis was done using the data during the period of active vertical mixing of seawater (dark gray circle) and during the summer stratified (light gray circle) together, where the inconsistently high Ph-C/Chl-a ratios (light open circle) observed only during the period of active vertical mixing of seawater were excluded from regression analysis. Data on Phyto-C/Chl-a, water temperature, DIN, DIP and DSi are expressed as mean±SE in the euphotic zone. For each relationship, logarithmic, linear or power approximation was applied to obtain higher correlation coefficients in the regression analyses. ly SD, DIP and DIN and an increasing demand of water Chl-a, Chl-a >20 µm, Chaetoceros and Rhizosolenia were temperature and Brunt-Väisälä frequency were observed located in the second quadrant in spring, and they were right along CCA AX1 (Fig. 9). In addition, a decreasing correlated with daily SR. Total dinoflagellates, Ceratium demand of DSi and an increasing demand of daily SR were and Chl-a <20 µm and cyanobacteria were found in the found upwards CCA AX2. Total Ph-C, Ph-CM, ANP and first and fourth quadrant in summer and summer–autumn, Coscinodiscus were found around the border of the third respectively, and these dinoflagellates were correlated to and fourth quadrants of the bioplot, mainly in winter, and Brunt-Väisälä frequency, whereas Chl-a <20 µm was cor- were correlated to DSi, DIN and DIP values (Fig. 9). To- related to water temperature (Fig. 9). tal diatoms, Eucampia, Thalassiosira, Cerataulina and Ditylum were located in the third quadrant in spring, and Primary production total diatoms were correlated strongly to daily SD. Total The depth of the euphotic zone (DEZ) varied from 8.8 m Phytoplankton carbon biomass and primary production in Sagami Bay 237

Fig. 9. CCA ordination diagram of the correlations between environmental variables (arrows) and biotic data (points) and of sampling dates in Sagami Bay, from January 2008 to December 2013. SD: daily SD; SR: daily SR; MLD: mixed layer depth; BV: Brunt-Väisälä frequency; T: water temperature; S: salinity; N: DIN; P: DIP; Si: DSi; 1: total Chl-a; 2: Chl-a <20 µm; 3: Chl-a >20 µm; 4: total Ph-C; 5 cyanobacteria; 6: ANP; 7: Ph-CM; 8: total diatoms; 9: total dinoflagellates; 10: Chaetoceros; 11: Ceratium; 12: Coscinodiscus; 13: Eucampia; 14: Thalassiosira; 15: Cerataulina; 16: Ditylum; 17: Rhizosolenia. to the bottom, with an overall mean±SE of 28.9±0.8 m. In the euphotic zone, GR showed significant correlations −1 −1 PP varied from ca. 0 to 1324.5 µg C L d . PPs were with SRPP, assimilation number and Ph-C/Chl-a ratio, re- low (<50 µg C L−1 d−1) throughout the water column spectively (Fig. 11). GR increased exponentially with in- in October/November–February/March, and were high crease in SRPP and assimilation number, while it decreased (>300 µg C L−1 d−1) in the upper layer (0–20 m depth) in exponentially with increase in the Ph-C/Chl-a ratio. March–October (Fig. 10), as observed at the same site for DIPP showed significantly positive correlations with an earlier period (Ara & Hiromi 2007, 2009, Ara et al. SDPP and SRPP, total chlorophyll a concentration at the 2011b, 2013). surface (Chl-a0 m), depth-integrated total chlorophyll a in Chl-a-specific productivity (i.e. assimilation number) the euphotic zone and at 0–50 m depth (Chl-aEZ and Chl- −1 −1 varied from ca. 0 to 10.74 µg C µg Chl-a h . Low assim- a0–50 m, respectively), whereas it showed significantly neg- ilation numbers (<1 µg C µg Chl-a−1 h−1) were frequently ative correlations with transparency and DEZ (Fig. 12). observed throughout the water column in December–Feb- Among these abiotic/biotic variables, SRPP and Chl-a0 m, ruary and below 10–20 m depth in March–November (Fig. showing higher correlation coefficients, were used for mul- 10). Higher numbers (>5 µg C µg Chl-a−1 h−1) were spo- tiple regression analysis, while there was no statistically radically observed in the upper layer (0–10 m depth) in significant correlation between SRPP and Chl-a0 m (p>0.05 March–September, especially in August 2010 and Septem- for all sampling dates in 2008–2013 and in each season of ber 2013 (>10 µg C µg Chl-a−1 h−1). 2008–2013). With the present multiple regression model, GR varied from ca. 0 to 3.77 d−1. GRs were low 77.1% and 56.7–77.7% of variance in DIPP for all sam- (<0.5 d−1) throughout the water column in September 2008 pling dates in 2008–2013 and in each season of 2008–2013 and below 3.0 to 45.6 m depth, which varied drastically was explained by combining SRPP and Chl-a0 m (Table 2). −1 during the study period (Fig. 10). Higher GRs (>1.5 d ) SRPP showed greater contribution to DIPP variations for were frequently observed in the upper layer (0–10 m depth) all sampling dates and in winter, spring and summer of in February/March–October/November. On each sampling 2008–2013, whereas Chl-a0 m showed greater contribution date, the maximum GR was 0.28–3.77 d−1, while the depth- in autumn of 2008–2013. integrated GRs in the euphotic zone were 0.20–2.95 d−1. The depth-integrated primary production in the eu- Discussion photic zone (DIPP) varied from 0.15 to 5.43 g C m−2 d−1, overall mean±SE of 1.27±0.09 g C m−2 d−1. Low DIPPs Seasonal variations in nutrients, Chl-a and phytoplank- (<0.5 g C m−2 d−1) were frequently observed in Oc- ton biomass tober–February of each year (Fig. 10). DIPPs were high (>3 g C m−2 d−1) in April–May and June 2008, April–May In the neritic area of Sagami Bay, Chl-a concentration, 2009, March and May–August 2011. Ph-C and their size composition revealed seasonal varia- 238 K. Ara et al.

Fig. 10. Seasonal variations in vertical profiles of primary productivity, chlorophyll a (Chl-a)-specific productivity (assimilation number) and growth rate and in the depth-integrated primary production (DIPP) in Sagami Bay, from January 2008 to December 2013. Arrows denote sampling dates. tions, and these variations were essentially similar to each concentrations and Ph-Cs, the pico- and/or nano-size frac- other during all six years (Figs. 4, 6), as has been formerly tion (<20 µm) comprised larger portions (Figs. 4, 6). The observed in other temperate coastal waters (Llewellyn et proportion of the micro-size fraction (>20 µm) in terms of al. 2005, Shinada et al. 2005, 2008, Gasol et al. 2016). Dur- Chl-a and Ph-C exhibited direct relationships to the total ing spring blooms with much higher Chl-a concentrations Chl-a and total Ph-C, respectively, and there were signifi- and Ph-Cs, the micro-size fraction (>20 µm) comprised cantly positive correlations among them (logarithmic ap- the majority of the total Chl-a and total Ph-C, whereas proximation, average at 0–50 m depth: r2=0.430 and 0.682 during other periods of relatively high and/or low Chl-a for Chl-a and Ph-C, p<0.0001 for each, respectively), as Phytoplankton carbon biomass and primary production in Sagami Bay 239

(i.e. Redfield ratio: Si : N : P=16 : 16 : 1) in February– March of each year (Fig. 13, Redfield et al. 1963, Brzez- inski 1985, Justić et al. 1995, Kamatani et al. 2000, Ara et al. 2011a). In addition, the timing of the first phytoplankton peak followed the dates when SR exceeded ca. 15.7 MJ m−2 d−1 for the first time in early February–mid March in 2008–2011. However, in late March in 2012 and 2013 it was found a few weeks later than those dates: in late February–early March in 2012 and 2013 Chl-a and PCBs maintained low levels despite SR having already exceeded ca. SR >15.7 MJ m−2 d−1 (Fig. 13). This can be plausibly explained by the interval of sampling dates and phytoplankton growth rate. In fact, on and/or just after the dates of SR>15.7 MJ m−2 d−1 for the first time in late February–early March in 2012 and 2013, relatively high Chl-a concentrations (i.e. the possible first peak) were recorded by continuous mooring observation using a Chl-a/ temperature meter (Compact-CLW, Alec Electronics, Ja- pan) suspended at 10 m depth, at a mooring station (Lat. 35°16′22.6”N, Long. 139°29′21.2″E; local depth: ca. 56 m) near the sampling station (Ara, unpublished data). Actu- ally, during this period nutrient concentrations declined considerably (Fig. 13), which would be due to active nutrient uptake by phytoplankton, and implies that during this period the first peak of spring blooms probably occurred. Such relatively high Chl-a values (i.e. the possible first peak) were not recorded by the continuous mooring observation from late January to the first phytoplankton peak in late February–early March in 2008–2011 or before the possible first peak in late March 2012 and 2013 (Ara, unpublished data), suggesting that there was no other possible first peak. The time needed for the phytoplankton assemblage to increase can be estimated, under the as- sumption that logarithmic growth is maintained, by the following equation (Parsons et al. 1984b): N =N×exp Fig. 11. Growth rate (GR) plotted against solar radiation dur- 2 1 [µ(t –t )], where µ is growth rate (GR, d−1), and N and N ing the in situ incubation period (24 hrs) for primary productivity 2 1 1 2 are phytoplankton biomass (total Chl-a or total Ph-C) at measurements (SRPP), the ratio of total phytoplankton biomass to total chlorophyll a (Ph-C/Chl-a), and assimilation number (ASN) times t1 and t2 in days, respectively. From early January to for all sampling dates in 2008–2013. Data on GR, Ph-C/Chl-a and the first phytoplankton peak in each year, except in 2008, −1 ASN are expressed as mean±SE in the euphotic zone. For each the highest GR in the euphotic zone was 1.35–2.02 d relationship, power or exponential approximation was applied to (Fig. 10), which is comparable to or slightly lower than obtain higher correlation coefficients in the regression analyses. that (2.03–2.28 d−1) estimated by the following equation

(Eppley 1972): log10 µ=0.0275T–0.070, where T is water similarly observed for Chl-a in various waters (Fu et al. temperature (°C). In 2008–2013, the lowest total Chl-a and

2009, Strom et al. 2016). total Ph-C from early January to their first peaks (N1) were We examined the timing of the first peak of spring 0.29–0.70 µg L−1 and 1.67–8.17 µg C L−1, while their high- −1 blooms since it differed depending on the year, although est values during spring blooms (N2) were 1.95–5.42 µg L the variations in weather conditions (i.e. SD, SR) and and 40.6–260.4 µg C L−1, respectively. As a result, the time physicochemical properties (i.e. water temperature, salin- (t2–t1) taken from N1 to N2 was calculated to be 0.8–1.4 and ity, nutrient concentration) were similar to each other in 1.1–2.6 days in terms of Chl-a and Ph-C, respectively. This each year (Figs. 2, 3, 12). The timing of the first peak of indicates that at our study site the phytoplankton assem- Chl-a (total and >20 µm) and PCB (total Ph-C and Ph- blage could start to increase and attain the maximum lev-

CM) followed the time of lowest Brunt-Väisälä frequen- els of Chl-a and Ph-C within 1–3 days when the upper lay- cies, higher nutrient concentrations than threshold concen- er (i.e. the euphotic zone) reached higher levels of sunlight trations for phytoplankton (diatom) growth (DIN=1 µM, intensity (SR>15.7 MJ m−2 d−1) for the ﬁrst time in each DIP=0.1 µM and DSi=3 µM) and balanced molar ratios year under high nutrient concentrations and balanced mo- 240 K. Ara et al.

Fig. 12. The depth-integrated primary production (DIPP) plotted against sunshine duration and solar radiation during the in situ incubation period (24 hrs) for primary productivity measurements (SDPP and SRPP, respectively), transparency (Tra), depth of the euphotic zone (DEZ), total chlorophyll a concentration at the surface (Chl-a0 m), depth-integrated total chlorophyll a in the euphotic zone and at 0–50 m depth (Chl- aEZ and Chl-a0–50 m, respectively) for all sampling dates in 2008–2013. For each relationship, exponential or power approximation was applied to obtain higher correlation coefﬁcients in the regression analyses. Phytoplankton carbon biomass and primary production in Sagami Bay 241

Table 2. Multiple regression statistics for the depth-integrated production in the euphotic zone (DIPP: Y, g C m−2 d−1) with respect to the −2 −1 integral SR during in situ incubation time for primary productivity measurements (SRPP: X1, MJ m d ) and total chlorophyll a concentra- −1 tion at the surface (Chl-a0 m: X2, µg L ). a and b are partial regression coefﬁcients, and c is constant.

ln Y=a ln X1+b ln X2+c Period r2 n p a b c All sampling dates 0.800 0.551 −2.260 0.771 125 <0.0001 Winter (December–February) 0.651 0.510 −2.112 0.535 32 <0.0001 Spring (March–May) 0.770 0.482 −2.000 0.751 32 <0.0001 Summer (June–September) 0.769 0.397 −2.052 0.648 41 <0.0001 Autumn (October–November) 0.295 0.762 −1.197 0.777 20 <0.0001 lar ratios (Fig. 13), which would be suitable conditions for increases in rainfall and nutrient-rich freshwater discharge microphytoplankton (diatom) growth. Thus, in the present from rivers and also to the expansion of nutrient-rich estu- study, the first peak of spring blooms must have occurred arine water of neighboring Tokyo Bay Water into Sagami in mid February–early March in 2012 and 2013, although it Bay, when the seasonal stratification interfered with nutri- could not be observed by our shipboard sampling at inter- ent supplies to the upper layer from deeper ones (Figs. 3, vals of mostly two weeks. 4, 6, Ara & Hiromi 2008, Ara et al. 2011a, b). Although in In February–May, Chl-a and Ph-C showed dras- general phytoplankton specific growth rate increases with tic variations within a few weeks: during these periods, a decrease in cell size (volume) (Finkel et al. 2010), growth phytoplankton peaks (i.e. high Chl-a concentrations and rate and predominance in the mixed phytoplankton assem- Ph-Cs) and high nutrient concentrations appeared alter- blage can vary depending on nutrient concentration: un- nately over time in each year (Fig. 13), presumably af- der high nutrient conditions, micro-size diatoms enhance fected principally by nutrient concentrations and molar growth and/or assimilation rates and dominate the phyto- ratios (Ara et al. 2011a). Under higher levels of sunlight plankton assemblage, whereas as nutrient concentrations intensity (SR>15.7 MJ m−2 d−1), microphytoplankton (dia- decline to deficient conditions, pico- and/or nano-phyto- toms) had enhanced growth rates and standing crops (i.e. plankton have higher growth and/or assimilation rates and

Chl-a>20 µm, Ph-CM), while they took up and removed outcompete micro-size diatoms (Ishizaka et al. 1983, Fu- substantial amounts of nutrients from seawater (Fig. 13). ruya et al. 1986, Örnólfsdóttir et al. 2004, Cermeño et al. As a result, the first peak of Chl-a and Ph-C declined to 2005). In addition, pico- and/or nanophytoplankton often the lowest levels due to P-, Si- and/or N-deficiencies and dominate the phytoplankton assemblage in various high- unbalanced molar ratios. Then, nutrients were compen- temperature and low-nutrient environments, in which cy- satively supplied to the upper layer (i.e. euphotic zone) anobacterial abundance, biomass and/or growth rate are from deeper ones by active vertical mixing of seawater, dependent principally on temperature, and less on nutrient while microphytoplankton (diatoms) were scarce in the concentration (Mitbavkar et al. 2009, Chen & Liu 2010, water column. The second and third phytoplankton peaks Chen et al. 2014). Actually, at our study site, cyanobacte- would appear and then decline, similarly to the first one, ria and/or ANP exhibited much higher biomasses in high- although the former and latter were apparently unclear in temperature and low-nutrient environments (i.e. in the 2012 and in all six years, respectively (Fig. 13). These sug- upper mixed water in summer–autumn), while they were gest that under high nutrient conditions (i.e. DIN>1 µM, frequently greater contributors to the total Ph-C (Figs. 3, DIP>0.1 µM and DSi>3 µM) sunlight intensity was a 6, 9, Ara & Hiromi 2009). Thus, in the neritic area of Sa- trigger factor to start and attain the first phase of spring gami Bay, nutrient declines (i.e. P, Si and/or N deficiency) blooms, and that under sufficient sunlight conditions (i.e. and temperature rises in the upper layer (0–20 m depth) in SR>15.7 MJ m−2 d−1) nutrients are the principal external spring–summer induced the variations in phytoplankton factors affecting Chl-a and Ph-C variations during the size composition in terms of Chl-a and Ph-C, from spring mid–final phase of spring blooms. blooms skewed toward the micro-size fraction (>20 µm) to During the summer stratified period, Chl-a concentra- summer blooms gradually shifting towards the pico-fractions and Ph-Cs maintained relatively high values (“sum- tion (<2 µm) through the nano-fraction (2–20 µm) (Figs. mer blooms”) in the upper layer (0–20 m depth) (Figs. 4, 3, 4, 6). 6), as has been observed in various temperate estuarine Relatively high Chl-a concentrations and Ph-Cs and coastal waters. During this period, the pico- and nano- (>2 µg L−1 and >100 µg C L−1, respectively) were irregu- size fraction (<20 µm) frequently comprised large portions larly observed in December 2010–January 2011 and De- of the total Chl-a and total Ph-C, while the micro-size cember 2013, and these could be termed “winter blooms”. fraction (>20 µm) intermittently comprised the majority. During all other winter periods in December–January Chl- This was dependent on nutrient supplies due to temporary a concentrations and Ph-Cs stayed at low levels (Figs. 4, 242 K. Ara et al.

Fig. 13. Temporal variations in microphytoplankton and total phytoplankton carbon biomass (PCB: Ph-CM and total Ph-C, respectively), chlorophyll a (Chl-a: total and >20 µm), daily solar radiation (SR), Brunt-Väisälä frequency (N), DIN, DIP, DSi and the molar ratio of N:P, Si:P and Si:N in the euphotic zone in Sagami Bay, from January to May in 2008–2013. Data are means±SE. Open circle (○) in PCB denotes

Ph-CM calculated from Chl-a>20 µm using the mean C: Chl-a ratios from the slope of the Chl-a>20 µm-Ph-CM regression equations obtained for each season in the present study. Dark shading denotes the period of ﬁrst spring phytoplankton bloom in terms of PCB and Chl-a in each year of 2008–2013, respectively. Arrows denote the dates of SR>15.7 MJ m−2 d−1 for the ﬁrst time in mid February–early March in 2012 and 2013. Light shading denotes the period of low Chl-a and PCBs despite after SR exceeded SR >15.7 MJ m−2 d−1. Phytoplankton carbon biomass and primary production in Sagami Bay 243

6). At our study site, winter blooms were rarely observed Ara & Hiromi 2009, Kamiyama et al. 2009), and is lower during an earlier period (Ara & Hiromi 2008, Ara et al. than in and near Funka Bay (pico-phytoplankton: 0.61– 2011a). Just before and/or during these winter blooms in 80.3 µg C L−1, Odate et al. 1993, Shinada et al. 2008) (Ta- 2010–2011 and 2013, northwest-ward currents were inter- ble 3). The ANP biomass obtained in the present study mittently recorded by continuous observations at the sur- is comparable to that found off Cape Esan near Funka face off Jogashima Island, Miura (Shiro Toida, Kanagawa Bay (<5–>40 µg C L−1, Shinada et al. 2008), which is Prefectural Fisheries Technology Center, personal com- lower than at the same site in Sagami Bay in 2003–2005 munication). Thus, the apparent winter blooms could be (1.14–191.77 µg C L−1, Ara & Hiromi 2009), and is higher induced by the expansion of neighboring Tokyo Bay Water than in Funka Bay (2–10 µm: 0.57–7.88 µg C L−1, Odate

(Furushima & Sugimoto 1995, Ara & Hiromi 2008, Ara et et al. 1993). Ph-CM obtained in the present study is com- al. 2011b), which would contain a much higher phytoplank- parable to that found in the Oyashio region off Akkeshi ton standing crop (Chl-a) all year round (Han & Furuya (2.5–410.7 µg C L−1, Shinada et al. 2001) and off Cape 2000, Kubo et al. 2017). Esan near Funka Bay (<10–>300 µg C L−1, Shinada Field studies have shown that diatoms dominate micro- et al. 2008), and is higher than in Funka Bay (>10 µm: phytoplankton assemblages in high turbulence, low tem- 0.90–193.01 µg C L−1, Odate et al. 1993). The total Ph-C perature and high nutrient environments, such as the pe- obtained in the present study is comparable to that found riod of active vertical mixing of seawater in winter–early in the Oyashio region off Akkeshi (8–418 µg C L−1, spring, whereas dinoflagellates outcompete diatoms in low Shinada et al. 2001), which is lower than in Tokyo Bay turbulence, high temperature and low nutrient environ- (mean±SD: 392.7±300.5 µg C L−1, Furuya & Maru- ments in late spring–summer (Margalef 1978, Irwin et al. mo 1983), and is higher than that in and near Funka Bay 2012, Xie et al. 2015). Actually, at our study site diatoms (3.3–245.2 µg C L−1, Odate et al. 1993, Shinada et al. 2005, dominated Ph-CM during the study period, especially dur- 2008) (Table 3). ing spring blooms (February–May) induced by nutrient The Ph-C/Chl-a ratio (i.e. conversion factor to estimate supplies due to active vertical mixing of seawater, as al- carbon biomass from Chl-a) varied widely depending on ready mentioned above, whereas large relative proportions the depth and season (Fig. 6), and these ratios (1.8 to 128.8) and predominance of dinoflagellates were found during the obtained in the present study are comparable to the range summer stratified period (Figs. 3, 6, 9), as has been previ- (ca. 0 to 150) obtained by microscopic observations for ously observed for microphytoplankton abundance in the estimating phytoplankton (i.e. pico-, nano-plus microphy- coastal waters of Sagami Bay (Tatara & Kikuchi 2003, toplankton) carbon biomass in other waters (Furuya et al. Baek et al. 2007, Ara & Hiromi 2009, Ara et al. 2011a, 1986, Furuya 1990, Chang et al. 2003, Llewellyn et al.

Fukuyama 2013). Regarding Ph-CM, larger diatoms (e.g. 2005), which are within the wide ranges (ca. 0 to >400) Eucampia, Coscinodiscus, Ditylum) dominated during the obtained by similar methods in field studies (Falkowski et initial–mid phase of spring blooms, and were substitut- al. 1985, Cloern et al. 1995, Pérez et al. 2006). ed by medium-sized (e.g. Cerataulina, Rhizosolenia) and The Ph-C/Chl-a ratio has been based on cell volume smaller ones (e.g. Chaetoceros, Thalassiosira) during the conversion by microscopic observations or on the regres- mid–final phase of spring blooms, and then smaller ones sion equations of particulate organic carbon (POC)-Chl-a maintained dominance in Ph-CM from summer through relationships. In the present study, linear regression equa- winter (Fig. 6). tions of Ph-C-Chl-a relationships were obtained for all Although the variation pattern of phytoplankton size sampling dates in 2008–2013 and in each season of 2008– composition in terms of Ph-C was mostly similar to that in 2013 (Fig. 7), as has been similarly found for the relatively terms of Chl-a, the proportions of the micro-size fraction narrow ranges of Chl-a, POC and Ph-C in other waters (>20 µm) in terms of Ph-C (overall mean: 73.6%) were (Furuya et al. 1986, Llewellyn et al. 2005, Lü et al. 2009, higher than those in terms of Chl-a (overall mean: 56.1%) Graff et al. 2015), whereas for their wider ranges non-lin- (Figs. 4, 6). On the basis of an X2 test, there was a statis- ear relationships could be fitted by a power function (Sath- tically significant difference between phytoplankton size yendranath et al. 2009, Jakobsen & Markager 2016). In the composition in terms of Chl-a and Ph-C (p<0.05). Thus, present study, however, there were statistically significant in the neritic area of Sagami Bay, phytoplankton size com- differences between the two regression equations of PC- position in terms of Ph-C does not strictly equal that in Chl-a and Ph-C-Chl-a relationships (p<0.0001, Figs. 5, terms of Chl-a. 7). The values of slope and y-intercept of the PC-Chl-a relationship were significantly higher than those of Ph-C- Phytoplankton biomass and the Ph-C/Chl-a ratio Chl-a relationship (p<0.0001 for each). Thus, in Sagami The cyanobacterial biomass obtained in the present Bay estimating phytoplankton carbon biomass from PC study is comparable to that found in other bays and coastal concentration using the regression equation of PC-Chl-a waters in Japan, e.g. at the same site in 2003–2005, off relationship can lead to a significant overestimate. Estimat- Manazuru in Sagami Bay and in Oginohara Bay (Ishi- ing phytoplankton carbon biomass from cell volume by nomaki Bay) (0.03–34.21 µg C L−1, Hamasaki et al. 1999, microscopic observations would be closer to actual values 244 K. Ara et al.

Table 3. The size-fractionated and total phytoplankton carbon biomass obtained on the basis of cell volume conversion by microscopic observations in embayment, coastal and neritic waters in Japan. Number in parenthesis denotes mean carbon biomass during the period in each study.

Region Period and depths Size Biomass (µg C L−1) Reference

the Oyashio region off Akkeshi Jul. 1997–May 1998, 0–50 m Pico+nano (0.2–20 µm) (7.3–58.1) Shinada et al. (2001) Micro (>20 µm) (2.5–410.7) Total 8–418 Funka Bay Feb.–Aug. 1989, 0–90 m Pico (0.2–2 µm) 0.61–80.3 Odate et al. (1993) ANP (2–10 µm) 0.57–7.88 Micro (>10 µm) 0.90–193.01 Total 3.3–202.1 off Usujiri near Funka Bay May 1997–Jun. 1999, 0–50 m Total 6.3–245.2 Shinada et al. (2005) off Cape Esan near Funka Bay May 1997–Jun. 1999, 0–200 m Pico (0.2–2 µm) <5–>40 Shinada et al. (2008) ANP (2–20 µm) <5–>40 Micro (>20 µm) <10–>300 Total (4.4–59.1) Oginohara Bay (Ishinomaki Bay) Jul. 2002–Jul. 2004, 0–10 m Cyanobacteria (0.2–2 µm) 0.1–18.4 Kamiyama et al. (2009) Tokyo Bay Jul. 1979, 0–5 m Total (392.7±300.5) Furuya & Marumo (1983) off Manazuru, Sagami Bay Aug.–Dec. 1996, 0–75 m Cyanobacteria (0.2–2 µm) 0.06–12.55 Hamasaki et al. (1999) off Enoshima Island, Sagami Bay Jan. 2003–Dec. 2005, 0–10 m Cyanobacteria (0.2–2 µm) 0.03–34.21 Ara & Hiromi (2009) ANP (2–20 µm) 1.14–191.77 off Enoshima Island, Sagami Bay Jan. 2008–Dec. 2013, 0–50 m Cyanobacteria (0.2–2 µm) 0.009–22.12 (1.80±0.08) This study ANP (2–20 µm) 0.07–31.37 (3.62±0.11) Micro (>20 µm) 0.012–316.60 (17.50±1.02) Total 0.87–348.89 (20.41±1.06)

Pico: cyanobacteria plus picoeukaryotes. and demand a greater amount of time and effort, rather 2002, Lü et al. 2009), the Ph-C/Chl-a ratio decreased ex- than chemical analysis of Chl-a concentrations. In Sagami ponentially and logarithmically with increase in DIN and 3− Bay, the total and size-fractionated (<20 µm and >20 µm) in PO4 -P and Si(OH)4-Si, respectively (Fig. 8). This indi- phytoplankton biomass (i.e. total Ph-C, Ph-CPN and Ph-CM, cates that at our study site the Ph-C/Chl-a ratio (>30) was 3− respectively) can be easily calculated from total and size- controlled by nutrient concentrations, when DIN, PO4 -P fractionated Chl-a using the mean Ph-C/Chl-a ratios from and Si(OH)4-Si concentrations were lower than ca. 4, 0.3 the slopes of the regression equations obtained in the pres- and 5 μM, respectively. The Ph-C/Chl-a ratio increased ent study. The mean Ph-C/Chl-a ratios obtained for micro- with increase in temperature (Fig. 8), as observed in other phytoplankton (>20 µm) in the present study were close to waters: the ratio was higher in the warmer upper water or those (25–30) for growing phytoplankton (predominantly in warmer months associated with low nutrient concentra- diatoms) with excess nutrients in seawater (Parsons et al. tions, and was lower in the colder deeper water or in colder 1984b). months associated with high nutrient concentrations (Fu- The Ph-C/Chl-a ratio can be highly regulated in re- ruya 1990, Odate et al. 1993, Taylor et al. 1997, Pérez et al. sponse to variations in environmental variables such as 2006). Here, the Ph-C/Chl-a ratio would be affected princi- irradiance, temperature and nutrients (Geider 1987, An- pally by nutrient concentrations as mentioned above, rather dersson & Rudehäll 1993, Lü et al. 2009). Actually, during than temperature, since the Ph-C/Chl-a ratio decreases the study period, the Ph-C/Chl-a ratio showed significant sharply with an increase in temperature in nutrient-suffi- correlations with irradiance (i.e. SR3dmSD), temperature and cient culture experiments (Geider 1987, Strzepek & Price nutrient concentrations (Fig. 8). The ratio was directly pro- 2000, Behrenfeld et al. 2005). Actually, at our study site in portional to SR3dmSD, as similarly observed in other waters the euphotic zone temperature exhibited negative correla- 3− (Geider 1987, 1993, Behrenfeld et al. 2005, Llewellyn et tions with DIN, PO4 -P and Si(OH)4-Si concentrations, al. 2005). The higher Ph-C/Chl-a ratios (>50) were spo- respectively (linear approximation, r2=0.269, 0.403 and radically found in the upper mixed water (0–10 m depth) in 0.134, respectively, p<0.0001 for each). summer, suggesting that the higher ratios followed nutri- Primary production and growth rate ent-depleted conditions (Figs. 3, 6), as similarly observed in other waters and culture experiments (Riemann et al. The values of Chl-a-specific productivity (i.e. assimi- 1989, Furuya 1990, Taylor et al. 1997, Chang et al. 2003). lation number) obtained in the present study are compa- In addition, as similarly observed in other waters (Verity rable to those obtained at the same site in 2002–2008 (ca. Phytoplankton carbon biomass and primary production in Sagami Bay 245

0–19.56 µg C µg Chl-a−1 h−1, Ara & Hiromi 2009, Ara regression equations obtained in the present study (Fig. et al. 2011b) and in other coastal waters in Japan (ca. 0 to 12), while these estimates include on average 25.5, 14.7, 10–20 µg C Chl-a−1 h−1, Yamaguchi & Imai 1996, Tada et 21.1, 23.5, 14.7, 16.1 and 15.2% margins of error for all al. 2001, Han & Furuya 2000). sampling dates, respectively. Moreover, multiple regression Comparison of GRs in the present study with those analysis was done, since it is difficult to calculate close in other studies is difficult because of the difference in to actual DIPPs from the single abiotic/biotic variables, the method of GR estimates (e.g. PP determination, Ph-C especially SDPP, transparency and DEZ, due to their low estimates, equation employed for estimating GR, phyto- correlation coefficients and large margins of error. Conse- plankton size composition). Nonetheless, the highest GR quently, DIPP showed significant correlations with com- −1 (3.77 d ) obtained for the phytoplankton assemblage in the bined SRPP and Chl-a0 m, where these correlation coeffi- present study is lower than that found in some other wa- cients were higher than those for the single abiotic/biotic ters, e.g. the Sargasso Sea (5.8 d−1, by short incubation, not variables (Table 2, Fig. 12). For estimates closer to actual convertible to 24 h rate, Sheldon & Sutcliffe 1978), Pacific values in Sagami Bay, DIPP can be empirically calculated −1 Ocean (4.6 d , Koblenz-Mischke et al. 1976), but is higher by combining SRPP and Chl-a0 m, using the multiple regres- than many other waters (≤2.9 d−1, Furnas 1990, Duhamel sion equation obtained in the present study (Table 2), and et al. 2007, Finkel et al. 2010, Laws 2013). these estimates include on average 7.4%, 6.5%, 5.0%, 7.8%

GR was principally regulated by irradiance (i.e. SRPP) and 5.7% margins of error for all sampling dates, winter, (Fig. 11), as has been frequently observed in former stud- spring, summer and autumn, respectively. ies (Falkowski et al. 1985, Geider 1987, 1993, Cloern et al. 1995, Arin et al. 2002), but not by water temperature Contribution of total Ph-C and DIPP to PC (linear approximation, r2=0.001, p>0.05). This fact was Natural seawater contains a complex variety of living unexpected, since GR has been observed to be proportion- and non-living organic matter as well as inorganic par- al to water temperature in previous studies (Eppley 1972, ticles (Volkman & Tanoue 2002, De La Rocha & Passow Ishizaka et al. 1983, Montagnes & Franklin 2001). 2007). In the present study, the variations in Chl-a and The Ph-C/Chl-a ratio can be closely related to pho- PM (i.e. PC, PN) showed a similar pattern and signifi- tosynthetic efficiency (activity) and to phytoplankton cant correlations with each other (Fig. 5). The mean mo- growth rate: these lower ratios followed higher growing lar C:N ratio (6.63) of PM obtained in the present study (photosynthetic) activities of phytoplankton. Actually, in was close to the phytoplanktonic C:N ratio (Redfield et al. the present study, GR variations showed the opposite pat- 1963, Montagnes et al. 1994, Menden-Deuer & Lessard tern of Ph-C/Chl-a variations, and GR decreased with in- 2000, Sarthou et al. 2005). At our study site, particulate crease in the Ph-C/Chl-a ratio (Figs. 6, 10, 11), as simi- organic carbon and nitrogen (POC and PON, respectively) larly observed under nutrient-limiting conditions in other comprised the majority of PC and PN (mean: >90% for waters (Cullen 1982, Geider 1993, Cloern et al. 1995, Le each) all year round, respectively (Ara, unpublished data). Bouteiller et al. 2003). In addition, assimilation number In addition, at our study site phytoplankton were the great- variations showed a similar pattern to GR variations, and est contributors, accounting for on average ca. 50%, to GR increased with increasing assimilation number (Figs. the 0–50 m depth-integrated carbon biomass of the total 10, 11), as similarly observed in other waters (Glover 1980, plankton (pico-, nano-, micro- and mesoplankton) all year Laws et al. 1987, Erga 1989, Berg et al. 2017). round (Ara & Hiromi 2009). This indicates that at our The mean DIPPs in 2008–2013 (1.27 g C m−2 d−1) deter- study site, PC variations could depend on the total Ph- mined in the present study are similar to those obtained C, although the contribution of the total Ph-C to PC var- at our study site in 2002–2004 (1.08 g C m−2 d−1, Ara and ied vertically and temporally, accounting for 0.3–42.5% Hiromi 2007), in 2003–2005 (1.00 g C m−2 d−1, Ara and (overall mean±SE: 7.2±0.2%). Similar to PC variations, Hiromi 2009) and 2002–2008 (0.99 g C m−2 d−1, Ara et al. PN variations corresponded to phytoplanktonic-N, which 2011b). The highest DIPPs obtained in the neritic area of accounted for 0.3–44.4% (overall mean±SE: 5.4±0.2%), Sagami Bay (4.64–5.90 g C m−2 d−1, Fig. 10, Ara & Hiromi using the conversion factor of phytoplanktonic-C/N (w/w) 2007, 2009, Ara et al. 2011b) are lower than in hyper-eu- ratio of 5.68, obtained in the present study. trophic waters e.g. Tokyo Bay, Mikawa Bay and Osaka On the basis of 0–50 m depth-integrated values, the total Bay (13.6–16.42 g C m−2 d−1), but are higher than any other Ph-C accounted for 1.3–34.4% of PC, which was higher bays and coastal waters in Japan, including other coastal in March–June (monthly mean: 7.2–12.3%), and lower in and offshore waters of Sagami Bay (see Table 3 in Ara et October–January (monthly mean: 3.8–5.6%) (Fig. 14). The al. 2011b). contribution of the total Ph-C to PC obtained in the pres- DIPP showed significant correlations with abiotic/biot- ent study was comparable to the range in other productive ic variables (i.e. SDPP, SRPP, transparency, DEZ, Chl-a0 m, regions (∼10–30%), e.g. high latitudes, and estuarine and Chl-aEZ, Chl-a0–50 m), respectively (Fig. 12). For rough es- coastal waters, but lower than in less productive regions, timates, DIPP can be calculated from SDPP, SRPP, trans- e.g. subtropical latitudes and the open ocean (∼30–70%) parency, DEZ, Chl-a0 m, Chl-aEZ or Chl-a0–50 m using the (Andersson & Rudehäll 1993, Graff et al. 2015, Arteaga 246 K. Ara et al.

Fig. 14. Seasonal variations in the contribution of phytoplanktonic-C (total Ph-C) and of the depth-integrated primary production (DIPP) to PC in the whole water column (0–50 m depth).

−0.27 et al. 2016). Daily phytoplanktonic new carbon produc- and IHDF=1.413 BV , respectively, where BV is biovol- tion (i.e. DIPP) was estimated to be 1.3–30.9% d−1 (overall ume (µm3). For HNF and protozoan microzooplankton in- −1 mean±SE: 8.3±0.5% d ) of the 0–50 m depth-integrat- gestion rates, a Q10=2.8 was adopted for temperature cor- ed PC, which was higher in March–September (month- rection (Sherr et al. 1988, Hansen et al. 1997). This was ly mean: 8.1–12.1% d−1), and lower in October–February then converted to daily rates of food requirement by multi- (monthly mean: 3.4–5.6% d−1) (Fig. 14). DIPPs were equiv- plying by 24 (hrs). Food requirement by mesozooplankton alent to 13.8–756.5% d−1 (overall mean±SE: 121.5±14.5% [i.e. copepods (N–C6) and non-copepods (e.g. appendicu- d−1) of the 0–50 m depth-integrated total Ph-C. larians, cladocerans, crustacean nauplii, malacostracans, During the study period, daily vertical PC flux, which ostracods, thaliaceans, molluscan larvae, polychaete lar- was measured by a sediment trap moored at 40 m depth vae)] was estimated using the equation (Omori & Ikeda for 24 h in parallel with primary productivity measure- 1984): FR=Res/(As–Gr), where Res is respiration rate (µL −1 −1 ments on all sampling dates during the study period at our O2 ind. h ), As (assimilation efficiency) and Gr (gross study site, was 0.22±0.01 g C m−2 d−1 (overall mean±SE), growth efficiency) were assumed to be 0.7 and 0.3, respec- which was equivalent to 1.7±0.1% d−1 (overall mean±SE) tively (Ikeda & Motoda 1978, Ikeda 1985). Res was esti- of the 0–40 m depth-integrated PC (Ara unpublished data). mated by the multiple-regression model proposed by Ikeda In addition, the carbon budget at our study site is assessed (1985): ln Res=0.5254 + 0.8354 ln WC + 0.0601T, where by an estimation of daily food requirement (i.e. potential T is ambient water temperature (°C), and WC is individual carbon removal rates) by phytoplankton feeders [i.e. het- weight (mg C). Oxygen respired was converted to carbon erotrophic nanoflagellates (HNF), microzooplankton, her- using a respiratory quotient of 0.97 (Gnaiger 1983), and to bivorous and omnivorous mesozooplankton] to DIPP plus daily rates by multiplying by 24 (hrs). The depth-integrat- total Ph-C. Food requirement (FR: µg C L−1 d−1) by HNF ed food requirement at depths of 0–50 m by HNF and mi- and protozoan microzooplankton [i.e. naked ciliates, tin- crozooplankton was calculated as the integral of the food tinnids and heterotrophic dinoflagellates (HDF)] was es- requirement at depths of 0, 10, 20, 30, 40 and 50 m, where- timated using the following equation: FR=ΣN×Wc×I, as for mesozooplankton it was calculated by multiplica- where N is abundance (cells or ind. L−1), Wc is individual tion of the food requirement and depth (50 m) (Ara & Hi- weight (µg C), and I is maximum specific ingestion rate romi 2007, Okutsu et al. 2012). Consequently, on the basis (h−1) (Ara & Hiromi 2009). Here, I was estimated for HNF of 0–50 m depth-integrated values, the amount of excess

(IHNF), ciliated protozooplankton (ICPROTOZOO) and HDF carbon calculated by subtracting daily food requirement (IHDF) by the regression equations obtained by Hansen et by phytoplankton feeders from DIPP plus total Ph-C was −0.16 −0.20 −2 −1 al. (1997): IHNF=0.912 BV , ICPROTOZOO=1.259 BV calculated to be 0.26±0.19 g C m d (overall mean±SE), Phytoplankton carbon biomass and primary production in Sagami Bay 247 which was equivalent to 1.7±1.4% d−1 (overall mean±SE) surface ocean. Global Biogeochem Cycles 30: 1791–1810. of PC (Okutsu et al. 2012, Ara, unpublished data). There- Baki MA, Motegi C, Shibata A, Fukuda H, Shimode S, Kikuchi fore, for at least six years from 2008 through 2013, PC, T (2009) Temporal changes in chlorophyll a concentrations Ph-C and DIPP revealed neither increasing nor decreasing and bacterial, viral, and heterotrophic nanoflagellates abun- trends (Figs. 4, 6, 10), which could be explained by the dances in the coastal zone of Sagami Bay, Japan: implication well-balanced carbon budget between the excess carbon in of top-down and bottom-up effects. Coast Mar Sci 33: 29–38. the water column and vertical flux, although the horizontal Baek SH, Shimode S, Kikuchi T (2007) Reproductive ecology of PC export was not evaluated in the present study. dominant dinoflagellate, Ceratium fusus, in the coastal area of Sagami Bay, Japan. J Oceanogr 63: 35–45. Behrenfeld MJ, Boss E, Siegel DA, Shea DM (2005) Carbon- Acknowledgements based ocean productivity and phytoplankton physiology from space. Global Biogeochem Cycles 19: GB1006; 1–14. We thank Mr. Kazuharu Yuasa, captain/owner of the Berg GM, Driscoll S, Hayashi K, Ross M, Kudela R (2017) Vari- fishery boat “Genshun-maru”, for his continuous help- ation in growth rate, carbon assimilation, and photosynthetic ful assistance in fieldwork. We thank Mr. Shiro Toida, efficiency in response to nitrogen source and concentration in Kanagawa Prefectural Fisheries Technology Center, phytoplankton isolated from upper San Francisco Bay. J Phy- for providing current data off Jogashima Island, Miura. col 53: 664–679. Thanks are also due to all members who participated in Brzezinski MA (1985) The Si : C : N ratio of marine diatoms: “Project SHONAM” for helping with fieldwork and ana- interspecific variability and the effect of some environmental lyzing samples. This research was financially supported variables. 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