Size fraction and class composition of …Size fraction and class composition of phytoplankton in the Antarctic marginal ice zone along the 140 E meridian during FebruaryeMarch 2003

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Polar Science 2 (2008) 109e120http://ees.elsevier.com/polar/

Size fraction and class composition of phytoplankton in theAntarctic marginal ice zone along the 140�E meridian during

FebruaryeMarch 2003

Fuminori Hashihama a,*, Toru Hirawake b, Sakae Kudoh b, Jota Kanda a, Ken Furuya c,Yukuya Yamaguchi a, Takashi Ishimaru a

a Tokyo University of Fisheries, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japanb National Institute of Polar Research, Kaga 1-chome, Itabashi-ku, Tokyo 173-8515, Japan

c Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received 11 January 2008; revised 21 April 2008; accepted 9 May 2008

Available online 24 May 2008

Abstract

We investigated the size fraction and pigment-derived class compositions of phytoplankton within the euphotic zone of theAntarctic marginal ice zone between 63.3�S and 66.5�S along the 140�E meridian on two consecutive cruises in the late australsummer and early austral autumn of 2003. We observed significant temporal and spatial variations in phytoplankton size and tax-onomic composition, although chlorophyll a concentrations were generally below 1 mg l�1 during both periods. Microphytoplank-ton (>20 mm), mainly diatoms, were prominent in the euphotic zone in the southernmost area around 66.5�S during late summer. Inthe rest of the study area during both cruises, the phytoplankton community was dominated by pico- and nano-sized populations(<20 mm) throughout the euphotic zone. The small-size populations mostly consisted of diatoms and haptophytes, although chlor-ophytes were dominant in extremely cold water (�1.5�C) below the overlying warm water around 65.5�S during late summer. Fromlate summer to early autumn, chlorophytes declined in abundance, probably due to increasing temperature within the euphotic zone(�1 to 0�C). These pico- and nano-phytoplankton-dominated populations were often accompanied by relatively high concentra-tions of ammonium, suggesting the active regeneration of nutrients within the small-size plankton community.� 2008 Elsevier B.V. and NIPR. All rights reserved.

Keywords: Phytoplankton; Size fraction; Class composition; Marginal ice zone; Ammonium

1. Introduction

Several studies have documented the biomass, sizefraction, and taxonomic composition of phytoplanktonin the marginal ice zone of the Southern Ocean (Chiba

* Corresponding author. Department of Ocean Sciences, Tokyo

University of Marine Science and Technology, 4-5-7 Konan,

Minato-ku, Tokyo 108-8477, Japan. Tel.: þ81 3 5463 0452.

E-mail address: [email protected] (F. Hashihama).

1873-9652/$ - see front matter � 2008 Elsevier B.V. and NIPR. All rights

doi:10.1016/j.polar.2008.05.001

et al., 2000; Saggiomo et al., 1998; Savidge et al.,1995; Wright and van den Enden, 2000). Areas ofcontrasting phytoplankton density have been recog-nized in this zone from austral spring to austral autumn,including areas with low phytoplankton biomass andthose in which phytoplankton blooms occur. Thesedistinct biomass regimes tend to be accompanied byvariability in phytoplankton composition and sizestructure.

reserved.

110 F. Hashihama et al. / Polar Science 2 (2008) 109e120

In the marginal ice zone of the eastern Australiansector of the Southern Ocean, low phytoplankton bio-mass was observed off Adelie Land between 135�Eand 145�E from January 1996 to February 1996 (Chibaet al., 2000) and between 128�E and 150�E duringMarch 1996 (Wright and van den Enden, 2000); inthese waters, the surface concentration of chlorophylla (Chl a) barely exceeded 1 mg l�1. The factors respon-sible for low biomass are relatively complex: the hori-zontal intrusion of Antarctic Circumpolar Currentswith low Chl a content, the weakened irradiance andstratification that accompanies seasonal transition,and selective grazing by herbivores have all been sug-gested as underlying factors (Chiba et al., 2000; Wrightand van den Enden, 2000). Microscopic analysisrevealed that the low biomass off Adelie Land duringJanuaryeFebruary 1996 was dominated by nano-sizedeukaryotic cells (Chiba et al., 2000). Furthermore, pig-ment chemotaxonomic analysis indicated that the phy-toplankton community in the eastern Australian sectorduring March 1996 was dominated by diatoms andhaptophytes, and that significant increases in prasino-phytes occurred within a temperature-minimum layerrelative to other layers (Wright and van den Enden,2000).

In contrast to the low-biomass regime, elevatedChl a concentrations, which exceeded 1 mg l�1, werelocally observed in the Bellingshausen Sea fromNovember 1992 to December 1992 (Savidge et al.,1995), in the Ross Sea from November 1994 toDecember 1994 (Saggiomo et al., 1998), and in thewestern part of the Australian sector between 93.5�Eand 104.5�E during February 1996 (Wright and vanden Enden, 2000). These blooms were commonlyobserved to follow vertical stabilization of the watercolumn with ice melting (Saggiomo et al., 1998;Wright and van den Enden, 2000). Size-fractionatedChl a analysis in conjunction with microscopic exam-ination revealed that the bloom in the BellingshausenSea during NovembereDecember 1992 was dominatedby micro-sized diatom species and occasionally thehaptophyte Phaeocystis (Savidge et al., 1995). In addi-tion, size-fractionated Chl a analysis indicated that the-bloom in the Ross Sea during NovembereDecember1994 was dominated by microphytoplankton (Saggiomoet al., 1998).

Compared with the number of studies undertakenin areas of phytoplankton blooms, few studies haveinvestigated phytoplankton composition in conjunc-tion with the size fraction in the low-biomass waterswithin the marginal ice zone of the eastern Australiansector. Although a microscopic examination has been

conducted in this area (Chiba et al., 2000), little isknown of the composition of small flagellates due totheir small size and poor preservation in conventionalfixatives (Furuya and Marumo, 1983; Gieskes andKraay, 1983). A previous pigment chemotaxonomicanalysis has provided information on the compositionof the overall phytoplankton population, includingflagellate forms, but information is lacking regardingthe size structure of phytoplankton within the marginalice zone of the eastern Australian sector (Wright andvan den Enden, 2000). The present study reports onconcurrent analyses of phytoplankton size fractionand pigment-derived class composition for the mar-ginal ice zone along the 140�E meridian off AdelieLand, conducted from February to March 2003. Wealso describe the relationships of these parameters tohydrographic variability.

2. Materials and methods

2.1. Field investigations

The Antarctic marginal ice zone along the 140�Emeridian was investigated during 3e7 February 2003(late summer) as part of a cruise undertaken by RT/VUmitaka-maru of Tokyo University of Fisheries, Japan,and from 26 February 2003 to 5 March 2003 (earlyautumn) during a cruise undertaken by the R/V Tan-garoa as part of the 44th Japanese Antarctic ResearchExpedition (JARE-44). Measurements were taken from63.5�S to near the coast at 66.5�S during late summer(Fig. 1a) and from 63.3�S to the thick sea-ice at 65.6�Sin early autumn (Fig. 1b). Vertical profiles of tempera-ture, salinity, and density in the upper 200 m of thewater column were determined using a conductivityetemperatureedepth (CTD) sensor. The BrunteVaisalafrequency (N), which represents the vertical densitygradient index, was obtained from the density profilesusing the equation described by Millard et al. (1990).The mixed layer depth (Zm) was defined as the depthof the maximum change in density over a 1 m depthinterval within 10e200 m depth in the water column,corresponding to the depth at which the maximumvalue of N was observed at each station (Fig. 2).

Seawater samples were collected in Niskin bottlesusing a rosette system attached to the CTD. Samplesintended for analyses of nutrients and fluorometricChl a were collected at 6e11 depths in the upper200 m during both cruises. During the late summercruise, light depths relative to the surface were deter-mined using a PAR sensor (Biospherical, USA). Sam-ples for size-fractionated Chl a analysis were collected

Fig. 1. Locations of measurement stations in the marginal ice zone along the 140�E meridian off Adelie Land of the Australian sector during the

late summer (a) and early autumn cruises (b).

111F. Hashihama et al. / Polar Science 2 (2008) 109e120

at 100%, 12.5%, and 1% light depths, and samples forplant pigment analysis using high-performance liquidchromatography (HPLC) were collected at 100%,50%, 25%, 12.5%, 6%, 3%, and 1% light depths. Dur-ing the early autumn cruise, samples for size-fraction-ated Chl a analysis were collected at 0, 25, and 80 mdepth, and those for plant pigment analysis were col-lected at 0, 10, 30, 50, 60, and 80 m. These samplingdepths were shallower than the 1% light depths as de-termined using a PAR sensor (Biospherical, USA). Theproperties investigated at each station are summarizedin Table 1.

Nutrient concentrations were determined using anauto-analyzer (Bran&Luebbe, Germany) based onstandard colorimetric methods for nitrate, ammonium,silicate, and phosphate (data for phosphate were notrecorded during the R/V Tangaroa cruise). Samplesfor fluorometric Chl a analysis were filtered ontoa Whatman GF/F filter, and pigments were extractedwith N,N-dimethylformamide (Suzuki and Ishimaru,1990). The extracts were assayed using a TurnerDesign fluorometer (USA) (Strickland and Parsons,

1972). Samples for size-fractionated Chl a analysiswere filtered onto a 20 mm nylon mesh, 10 and 2 mmNuclepore filters, and a Whatman GF/F filter to obtainfractions nominally >20 mm, 10e20 mm, 2e10 mm,and 0.7e2 mm, respectively. Extraction and assays ofthe fractionated samples followed the same proceduresas those described above for fluorometric Chl aanalysis.

2.2. HPLC pigment analysis

Samples for plant pigment analysis by HPLC werefiltered onto a Whatman GF/F filter. The filter waskept frozen in liquid nitrogen while aboard the vesseland then stored in a freezer (�80�C) onshore untilanalysis. Pigment analysis followed the method of Za-pata et al. (2000). The frozen filter was soaked in 95%methanol in an ultrasonic bath for 5 min with the addi-tion of canthaxanthin solution (Danish Hydraulic Insti-tute [DHI] Water and Environment, Denmark) as aninternal standard. The extract was filtered througha 0.2 mm pore Whatman polytetrafluoroethylene filter

66 65.5 64.5 63.565 64

0

50

C00 PP9 C01 C03 C05 C07 C09 C10C08C02 C04 C06

100

150

2000

50

100

150

2000

50

100

150

200

Dep

th

(m

)

0

50

100

150

2000

50

100

150

200

Latitude (°S)

65.5 64.5 63.565 64Latitude (°S)

C11 C12 C13 C14 C15

0

50

K 7 6.1

7.1K

6.2 6.17

100

150

2000

50

100

150

2000

50

100

150

200

Dep

th

(m

)

0

50

100

150

2000

50

100

150

200

5 4a b

Fig. 2. Vertical distributions of temperature, salinity, BrunteVaisala frequency (N), ammonium concentration, and chlorophyll a concentration in

the upper 200 m of the water column during late summer (a) and early autumn (b). Black squares in those panels showing temperature and salinity

denote the depth of the mixed layer (Zm). The locations of measurement stations are shown along the top edge of the uppermost figures.

112 F. Hashihama et al. / Polar Science 2 (2008) 109e120

and mixed with water (extract/water [5:1 v/v]). Themixture (500 ml) was injected into a HPLC system(Shimadzu, Japan) fitted with a 3.5 mm Symmetry C8

column (4.6 � 150 mm, Waters, USA).Separated pigments were identified based on reten-

tion times and the absorption spectra of standard pig-ments (peridinin [Perid], 190-butanoyloxyfucoxanthin[But-fuco], fucoxanthin [Fuco], 190-hexanoyloxyfu-coxanthin [Hex-fuco], prasinoxanthin [Pras], violaxan-thin [Viola], alloxanthin [Allo], chlorophyll b [Chl b],and Chl a), and quantified in terms of weight from thepeak area calibrated against that of standard pigments.Perid and Fuco as standard pigments were preparedbased on the method of Repeta and Bjørnland (1997)from cultures of Ceratium fusus and Ditylum sol,

respectively. But-fuco, Pras, Viola, Hex-fuco, andAllo were purchased from DHI Water and Environ-ment (Denmark). Chl b and Chl a were purchasedfrom Sigma Chemicals (USA). All samples, fromthe time of on-board filtration to injection into theHPLC, were handled under dim light. All solventsused for extraction and elution were either of HPLCgrade, super special grade, or JIS special grade(Wako, Japan).

2.3. CHEMTAX analysis

The relative contributions to Chl a of diatoms, hap-tophytes, dinoflagellates, cryptophytes, prasinophytes,and chlorophytes were calculated using the CHEMTAX

Table 1

List of station locations, observation dates, and properties observed at each station during the two cruises

Cruise Station Latitude (�S) Date Profile Hydrocast

Umitaka-maru C00 66.47 3-Feb-03 T, S Nut, Chl, FChl, HPLC

PP9 66.18 3-Feb-03 T, S Nut, Chl, FChl, HPLC

C01 66.00 4-Feb-03 T, S Nut, Chl

C02 65.90 4-Feb-03 T, S

C03 65.83 4-Feb-03 T, S Nut, Chl

C04 65.77 4-Feb-03 T, S

C05 65.67 4-Feb-03 T, S Nut, Chl

C06 65.58 4-Feb-03 T, S

C07 65.50 4-Feb-03 T, S Nut, Chl

C08 65.42 4-Feb-03 T, S Nut, Chl, FChl, HPLC

C09 65.32 5-Feb-03 T, S Nut, Chl

C10 65.17 5-Feb-03 T, S Nut, Chl

C11 65.00 5-Feb-03 T, S Nut, Chl, FChl, HPLC

C12 64.67 6-Feb-03 T, S Nut, Chl

C13 64.33 6-Feb-03 T, S Nut, Chl

C14 64.00 7-Feb-03 T, S Nut, Chl, FChl, HPLC

C15 63.50 7-Feb-03 T, S Nut, Chl, FChl, HPLC

Tangaroa (JARE-44) K 65.59 26-Feb-03 T, S Nut, Chl, FChl, HPLC

7 65.43 26-Feb-03 T, S Nut

7.1 65.53 27-Feb-03 T, S Nut

6.2 65.38 28-Feb-03 T, S Nut

6.1 65.08 28-Feb-03 T, S Nut

5 63.99 4-Mar-03 T, S Nut, Chl, FChl, HPLC

4 63.25 5-Mar-03 T, S Nut, Chl

The available data from profilers indicate temperature (T) and salinity (S). The hydrocast data indicate nutrients (Nut), chlorophyll a (Chl), size-

fractionated chlorophyll a (FChl), and plant pigments analyzed using high performance liquid chromatography (HPLC).

113F. Hashihama et al. / Polar Science 2 (2008) 109e120

method (Mackey et al., 1996, 1997). An initial ratiomatrix in the calculation was determined based onthe work of Jeffrey and Wright (1997) for diatoms, di-noflagellates, cryptophytes, and prasinophytes; Zapataet al. (2004) for haptophytes; and Burczyk et al.(1981) for chlorophytes (Table 2a). In a previous che-motaxonomic study conducted in the marginal ice zone

Table 2

Pigment:Chl a ratios used in the CHEMTAX calculations, showing initial (

Perid But-fuco Fuco Hex-fuc

(a) Initial

Diatoms 0.75

Haptophytes 0.07 0.01 0.79

Dinoflagellates 1.06

Cryptophytes

Prasinophytes

Chlorophytes

(b) Final

Diatoms 0.73

Haptophytes 0.22 0.02 0.65

Dinoflagellates 1.06

Cryptophytes

Prasinophytes

Chlorophytes

of the Australian sector (Wright and van den Enden,2000), haptophytes were divided into two categoriescharacterized by coccolithophorids (Type 3) andPhaeocystis antarctica (Type 4); however, coccolitho-phorids are generally sparse or absent between 63�Sand 65�S around the 140�E meridian (Cubillos et al.,2007; Findlay and Giraudeau, 2000; Nishida, 1986).

a) and final ratio matrices (b)

o Pras Viola Allo Chl b Chl a

1

1

1

0.23 1

0.22 0.11 0.02 1

0.01 0.55 1

1

1

1

0.23 1

022 0.11 0.02 1

0.01 0.61 1

114 F. Hashihama et al. / Polar Science 2 (2008) 109e120

Furthermore, Type 4 is characterized by a ratio of But-fuco to (Fuco þ Hex-fuco) in excess of 0.02, whileType 3 is defined by the presence of Fuco and Hex-fuco and the absence of But-fuco (Mackey et al.,1996, 1997). The combined pigment data obtainedin the present study yield a mean ratio of But-fuco to(Fuco þ Hex-fuco) of 0.09 � 0.07 (n ¼ 53), muchhigher than 0.02 despite the inclusion of diatom-derived Fuco. This finding implies that Type 4 hapto-phytes, rather than Type 3, dominate the present studyarea. Thus, in the present study haptophytes werecharacterized solely on the pigment composition ofPhaeocystis antarctica (Zapata et al., 2004), withoutthat of coccolithophorids. Wright and van den Enden(2000) also included cyanobacteria as characterized byzeaxanthin; however, we did not include cyanobacteriabecause zeaxanthin was undetected in all samples.

The final pigment ratio matrix obtained by CHEM-TAX analysis (Table 2b) was used to calculate the con-tributions of algal classes to Chl a. Slight variationsfrom the initial ratios to the final ratios were foundfor several classes in the CHEMTAX calculations,but these were within the acceptable range proposedby Mackey et al. (1996, 1997); therefore, our initialassumptions were considered to be reasonable.

The algal classes in the present study were determinedbased on pigment composition and pigment ratios normal-ized to Chl a of the phytoplankton species. It must benoted that pigment groups do not provide an exact matchwith taxonomic phytoplankton classes because, forexample, the pigment composition of the prasinoxan-thin-lacking prasinophyte Pyramimonas (Latasa et al.,2004) is similar to that of chlorophytes, and thehaptophyte Pavlova has the same pigment compositionas diatoms (Zapata et al., 2004). Thus, in some cases apigment group may comprise several taxonomic classes.

3. Results

3.1. Hydrographic structure and Chl a distribution

Vertical distributions of temperature, salinity, and Nin the upper 200 m of the water column showed signif-icant temporal and spatial variations (Fig. 2). Exceptfor the two southernmost stations during late summer,warm (>0�C) and less saline (<34.0) water wasobserved within the surface mixed layer above Zm,and high N values greater than 0.02 s�1 were observedaround Zm. The hydrographic structure clearly indicatedstratification with ice melt water; the overlying upperwater was vertically separated from the cold water(<0�C) that existed below Zm. In the three

northernmost stations (north of 64.5�S), the cold wa-ter was observed as a thin layer between 50 and100 m depth, characterized as a temperature-mini-mum layer, as described in Bindoff et al. (2000) andWright and van den Enden (2000). In the southernmostarea around 66.5�S, the temperature (�1 to 0�C) and sa-linity (34.2e34.4) were vertically uniform, with rela-tively low N values of <0.01 s�1.

During early autumn at the two northernmoststations (north of 64�S), warm (>0�C) and less saline(<34.0) water was observed within the surface mixedlayer above Zm, and the temperature-minimum layeroccurred between 50 and 100 m depth (Fig. 2). In thesouthernmost area around 65.5�S during early autumn,Zm was deeper (near 100 m) than during late summer(near 50 m). Furthermore, the N values around 65.5�Sduring early autumn had decreased to <0.02 s�1

(<0.04 s�1 during late summer). Thus, the stratificationweakened at around 65.5�S during early autumn.

High concentrations of nutrients were observed inthe upper 200 m of the water column: nitrate >27 mMand silicate >25 mM during both late summer and earlyautumn, and phosphate >1.6 mM during late summer(data not shown). These nutrients were never depletedover the study period. The ammonium concentrationranged from 0 to 0.6 mM in the upper 200 m of the wa-ter column during both periods (Fig. 2). A concentra-tion of >0.2 mM was generally observed in the upper80 m during both periods; this was also observed inthe water column below Zm around 66.5�S during latesummer and around 65.5�S during early autumn.

Chl a concentrations in the upper 200 m of the wa-ter column during both periods ranged from 0.01 to2.04 mg l�1 (Fig. 2). During late summer, a small in-crease in Chl a concentrations (>1.5 mg l�1) was ob-served in the upper 100 m in the southernmost area.Northward from this area, the concentration in the up-per 200 m gradually declined, and a subsurface Chl a-maximum layer developed in the cold water just belowZm. In contrast to late summer, the overall Chl a con-centration during early autumn (0.01e0.65 mg l�1)was relatively low, with a northesouth gradient. Thevertical depth of the zone of Chl a concentrationsexceeding 0.2 mg l�1 was broader at the southernmoststation (K; 0e150 m) than at a nearby station duringlate summer (C08; 0e100 m).

3.2. Relative contributions of size-fractionatedChl a to total Chl a

The relative contributions of size-fractionated Chla to total Chl a at all stations in both periods showed

115F. Hashihama et al. / Polar Science 2 (2008) 109e120

vertically uniform variations (Fig. 3). During late sum-mer, micro-sized cells of >20 mm were prominent inthe southernmost area (C00: 66e75%; PP9: 41e50%). In other areas, pico- and nano-sized cells of10e20 mm (5e33%), 2e10 mm (15e46%), and 0.7e2 mm (20e36%) were generally more abundant thanmicro-sized cells (11e51%). During early autumn (sta-tions K and 5), micro-sized cells of >20 mm made onlysmall contributions (7e25%), while pico- and nano-sized cells of 10e20 mm (10e25%), 2e10 mm (25e41%), and 0.7e2 mm (25e47%) were collectivelydominant.

0PP9; 66.1°C00; 66.47°S [3-Feb-03]

C11; 65.00°S [5-Feb-03] C14; 64.00°

20

40

60

80

80

>20 m10–20 m2–10 m0.7–2 m

0

20

40

60

Dep

th

(m

)

0

20

5; 63.99°SK; 65.59°S [26-Feb-03]

40

60

0 20 40 4060 80 100Relative ab

800 20

Fig. 3. Relative contributions of size-fractionated chlorophyll a to total c

summer and early autumn.

3.3. Relative contributions of algal classes to Chl a

Using data for chemotaxonomic algal pigments(Table 3), the relative contributions of algal classes toChl a at each station during both periods were derivedvia CHEMTAX analysis (Fig. 4). During late summer,diatoms contributed most of the Chl a in the southern-most area (C00: 92e93%; PP9: 62e90%), where thecontributions of diatoms was vertically uniform. In thearea between 65�S and 65.5�S (C08 and C11), diatoms(45e59%) and haptophytes (30e40%) collectivelydominated within the surface mixed layer above Zm,

S [3-Feb-03]

S [7-Feb-03] C15; 63.50°S [7-Feb-03]

[4-Mar-03]

undance (%)

C08; 65.42°S [4-Feb-03]

0 20 40 60 80 100

60 80 100

hlorophyll a within the euphotic zone at eight stations during late

Table 3

Concentrations of chemotaxonomic algal pigments (ng l�1) measured during the two cruises

Cruise Station Depth (m) Perid But-fuco Fuco Hex-fuco Pras Viola Allo Chl b

Umitaka-maru C00 0 26 11 599 27 0 0 0 0

4 17 11 507 26 0 0 0 0

10 19 11 522 26 0 0 0 0

16 23 11 528 25 0 0 0 0

22 21 9 532 23 0 0 0 0

27 22 8 496 18 0 0 0 0

36 20 8 481 18 0 0 0 0

PP9 0 11 21 168 16 0 4 6 20

9 12 17 163 16 0 5 6 23

17 13 16 197 17 0 4 7 20

24 17 15 317 20 0 5 7 21

31 22 21 443 27 0 5 6 21

42 0 0 12 2 0 0 0 0

C08 0 10 17 54 22 0 0 0 0

4 8 14 47 20 0 0 3 0

12 9 15 51 22 0 0 4 0

22 8 16 76 23 0 0 3 0

33 0 24 99 29 0 0 4 13

43 11 26 109 26 4 5 4 33

60 6 15 101 37 6 12 4 97

C11 0 8 15 57 32 0 0 0 0

4 8 15 57 34 0 0 2 0

11 9 16 63 34 0 0 3 0

21 8 15 57 30 0 0 2 0

32 7 14 55 31 0 0 2 0

43 7 18 65 37 0 0 2 0

60 14 29 137 59 7 5 4 46

C14 0 0 0 35 17 0 0 0 0

5 0 0 36 17 0 0 0 0

10 0 0 32 15 0 0 0 0

30 0 0 36 18 0 0 0 0

47 0 4 35 23 0 0 0 0

62 6 11 50 30 0 0 0 0

76 22 24 107 43 0 0 2 7

C15 0 0 3 32 16 0 0 0 0

3 0 3 33 18 0 0 0 0

11 0 3 34 16 0 0 0 0

22 0 3 38 20 0 0 0 0

40 0 4 44 23 0 0 0 0

57 7 8 37 29 0 0 0 0

76 7 23 132 31 0 0 0 7

Tangaroa (JARE-44) K 0 0 5 23 13 0 0 0 0

10 0 7 42 24 0 0 0 0

30 0 6 34 24 0 0 0 10

50 0 7 39 29 0 0 0 11

60 0 11 46 32 0 0 0 13

80 0 8 38 28 0 0 0 12

5 0 0 0 29 8 0 0 0 0

10 0 0 55 20 0 0 0 0

30 0 0 74 20 0 0 0 0

50 15 9 46 18 0 0 0 0

60 15 23 120 66 0 0 0 0

80 27 24 161 44 0 0 0 23

116 F. Hashihama et al. / Polar Science 2 (2008) 109e120

117F. Hashihama et al. / Polar Science 2 (2008) 109e120

whereas the contributions of chlorophytes increased to20e45% in the extremely cold water (�1.5�C; Fig. 2)below the surface mixed layer, compared with 0e9%within the surface mixed layer itself. Diatoms weredominant (44e76%) to the north of 64.5�S (C14and C15), followed by haptophytes (24e45%); theircontributions were vertically uniform compared withthose in the area between 65�S and 65.5�S.

At the southernmost station (K) during early au-tumn, diatoms (46e72%) and haptophytes (26e38%)

0PP9; 66.18°C00; 66.47°S [3-Feb-03]

C11; 65.00°S [5-Feb-03] C14; 64.00°

20

40 Chlorophytes

60

80

80

PrasinophytesCryptophytesDinoflagellatesHaptophytesDiatoms

0

20

40

60

Dep

th

(m

)

0

20

5; 63.99°SK; 65.59°S [26-Feb-03]

40

60

0 20 40 20 4060 80 100Relative ab

800

Fig. 4. Relative contributions of algal classes to chlorophyll a within the e

Gray dashed lines indicate the depth of the mixed layer (Zm).

collectively dominated the upper 80 m of the water col-umn: chlorophytes were relatively rare (0e17%; Fig. 4).The contributions of these populations throughout thewater column were more uniform than those at a nearbystation during late summer (C08). Diatoms dominated(46e84%) at 64�S (station 5), followed by haptophytes(15e42%); their vertical variations were as uniform asthose to the north of 64.5�S during late summer (C14and C15). The contributions of dinoflagellates, crypto-phytes, and prasinophytes were minor throughout the

S [3-Feb-03]

S [7-Feb-03] C15; 63.50°S [7-Feb-03]

[4-Mar-03]

undance (%)

C08; 65.42°S [4-Feb-03]

Zm

0 20 40 60 80 100

60 80 100

uphotic zone at eight stations during late summer and early autumn.

120

80

40

Ch

lo

ro

ph

ytes-C

hl a

(n

g l–1)

0

Temperature (°C)

-2 -1 0 1 2

Fig. 5. Relationship between temperature and CHEMTAX-derived

chlorophyll a concentration of chlorophytes at all sampling depths

during late summer and early autumn.

118 F. Hashihama et al. / Polar Science 2 (2008) 109e120

observation periods, although slight increases in thesealgal groups were observed in the cold waters immedi-ately below Zm (Fig. 2).

4. Discussion

Despite an abundance of macronutrients (nitrate,silicate, and phosphate) in the study area throughoutthe observation periods, the Chl a concentration barelyexceeded 1 mg l�1 (Fig. 2), as observed in previousstudies (Chiba et al., 2000; Wright and van den Enden,2000). Nevertheless, temporal and spatial changes inthe size fraction and composition of phytoplanktonwere significant (Figs. 3 and 4); this variability wasassociated with stability of the water column and thedistribution of ammonium (Fig. 2).

Diatoms (Fig. 4) with micro-sized cells (Fig. 3) dom-inated only in the southernmost area during late summer(C00 and PP9); however, no marked elevations in theChl a concentrations were observed in this area(<2.04 mg l�1; Fig. 2). The water column stabilizationthat occurs with ice melting induces blooms of largephytoplankton (Saggiomo et al., 1998). In the marginalice zone of the western part of the Australian sector,Wright and van den Enden (2000) reported that diatomblooms occurred in conjunction with thermal stratifica-tion, and that the Chl a concentration reached a maxi-mum of 3.4 mg l�1, indicating the dominance of largecells. Thus, the weak stratification in the southernmostarea of the present study during late summer (Fig. 2) po-tentially prevented the optimal growth of large diatoms.

The phytoplankton community throughout the restof the study area for both periods was dominated bypico- and nano-sized diatoms and haptophytes (Figs.3 and 4). Their large contributions to biomass did notdecrease even with the weakening of stratificationaround 65.5�S during early autumn (Fig. 2). Many re-ports have proposed that the success of the haptophytePhaeocystis antarctica in deep-mixing waters is due tosuperior adaptation to low-light conditions (e.g. Arrigoet al., 1999; Moisan et al., 1998; Schoemann et al.,2005). Furthermore, small diatoms have an advantageover large diatoms in terms of light harvesting, as largecells have a larger package effect than small cells(Finkel, 2001). The dominance of small diatoms andhaptophytes during early autumn could be ascribed totheir adaptation to low-light conditions.

A significant increase in chlorophytes was observedin the extremely cold water around 65.5�S during latesummer (Fig. 4). The chlorophyte Chlamydomonas haspreviously been found in Antarctic sea-ice (Palmisanoand Garrison, 1993), Greenland sea-ice (Ikavalko and

Gradinger, 1997), and Arctic sea-ice (Weissenberger,1998). According to Weissenberger (1998), Chlamydo-monas ranges in diameter from 5 to 20 mm. Moreover,Barlow et al. (1998) reported that Chl b was detected atice melt stations in the marginal ice zone within theBellingshausen Sea from November 1992 to December1992 and in ice cores collected from this area; the au-thors suggested that the chlorophytes originated frommelting ice. The occurrence of chlorophytes in thepresent study could also be related to melting ice.

A mesocosm experiment undertaken in the Arcticregion over the period from natural ice freezing inDecember 1994 to melting in April 1995 revealeda transition from a Chlamydomonas-dominated popula-tion to a diatom-dominated population (Weissenberger,1998). This result suggests that chlorophytes declinewith increasing temperature. In support of this pro-posal, all of our data show that the abundance of chlor-ophytes decreased with an increase in temperaturefrom �1.5�C to 2�C (Fig. 5). Thus, the decline ofchlorophytes around 65.5�S during early autumn couldbe ascribed to the temperature increase in the upper100 m of the water column that occurred in associationwith weakened stratification (�1 to 0�C; Fig. 2).

Koike et al. (1986) reported that in coastal waters ofthe southern Scotia Sea from February 1981 to March1981, approximately 80% of phytoplankton nitrogenwas assimilated in the form of ammonium, with mostof the uptake being associated with pico- and nano-phytoplankton. The authors also demonstrated thatactive ammonium production by microzooplankton oc-curred simultaneously with uptake by pico- and nano-phytoplankton. In the present study, the contributions

119F. Hashihama et al. / Polar Science 2 (2008) 109e120

of pico- and nano-phytoplankton to total Chl a weregenerally above 70% in waters with ammoniumconcentrations of >0.25 mM (Fig. 6). Wheeler andKokkinakis (1990) reported the inhibition of nitrate up-take by ammonium in the oceanic subarctic Pacific, evenat 0.1e0.3 mM ammonium concentration, just <1% ofthe total inorganic nitrogen. Moreover, in the marginalice zones in the Scotia and Weddell Seas, a predomi-nance of ammonium uptake (f-ratio: <0.5) generallyoccurs when ammonium stocks exceed 1.7% of the totalinorganic nitrogen (Goeyens et al., 1995). The meanconcentrations of ammonium and nitrate in the upper80 m at all observation stations in the present studywere 0.31 � 0.14 mM and 29.0 � 2.0 mM, respectively,and the ammonium stocks as a proportion of the totalinorganic nitrogen were 1.1 � 0.5%, similar to the valuesdescribed in Wheeler and Kokkinakis (1990) andGoeyens et al. (1995). Thus, the growth of pico- andnano-phytoplankton was probably supported by ammo-nium, resulting in significant regenerated production.

The present study area was under high-nitrate, low-chlorophyll conditions, within which, the dominanceof small, ammonium-dependent phytoplankton is likelyascribed to low iron availability, as iron is essential inthe reduction of nitrate and nitrite (Raven, 1988). Insitu iron enrichment at 61�S, 140�E significantlystimulated a bloom of micro-sized diatoms during theSouthern Ocean Iron RElease Experiment (SOIREE)(Gall et al., 2001). Furthermore, Sohrin et al. (2000)reported a low labile iron concentration of0.14 � 0.12 nM (n ¼ 97) throughout the upper 100 m

100

80

40

60

20

00 0.1 0.2 0.3 0.4 0.5 0.6R

elative ab

un

dan

ce o

f <

20 m

size fractio

n

to

to

tal C

hl a

(%

)

Ammonium ( M)

Fig. 6. Relationship between ammonium concentration and relative

abundance of the <20 mm size fraction to total chlorophyll a concen-

tration at all sampling depths during late summer and early autumn.

Open circles denote the contributions of <20 mm size fraction above

70% in waters with ammonium concentrations of >0.25 mM.

of the water column in the area between 50�S and65�S along the 140�E meridian from December 1994to January 1995, suggesting that iron was a limitingfactor of primary production. Although in the presentstudy we cannot evaluate the nature of iron availabilitybecause of a lack of rigorous iron data collected duringthe two cruises, the phytoplankton community, partic-ularly microphytoplankton, may suffer iron stress inthe marginal ice zone along the 140�E meridian.

5. Conclusions

The present study demonstrates spatial changes inthe size fraction and class composition of phytoplank-ton within the Antarctic marginal ice zone along the140�E meridian during late summer and early autumn.The ecosystem over the study area was dominated bypico- and nano-phytoplankton. Large diatoms only oc-curred in the southernmost area during late summer;however, this event was not accompanied by a signifi-cant elevation in Chl a concentrations. In contrast, therest of the study area was characterized by temporaland spatial variations in the compositions of pico-and nano-phytoplankton. In particular, the decline ofchlorophytes was closely related to increasing tem-perature. The elevated ammonium concentrationsassociated with the dominance of pico- and nano-phy-toplankton suggest that active regenerated productionis driven within the small-size plankton community.

Acknowledgments

We are grateful to Drs. S. Takeda, Y. Narumi, andM. Miki for their useful comments regarding thisstudy. We thank the captains and crew of the RT/VUmitaka-maru and R/V Tangaroa for their supportduring the cruises, along with students of Tokyo Uni-versity of Fisheries and the scientists of JARE-44.We also thank Dr. T. Omura and Ms. K. Sato for theirassistance in maintaining algal culture.

References

Arrigo, K.R., Robinson, D.H., Worthen, D.L., Dunbar, R.B.,

DiTullio, G.R., VanWoert, M., Lizotte, M.P., 1999. Phytoplank-

ton community structure and the drawdown of nutrients and

CO2 in the Southern Ocean. Science 283, 365e367.

Barlow, R.G., Mantoura, R.F.C., Cummings, D.G., 1998. Phytoplank-

ton pigment distributions and associated fluxes in the Bellingshau-

sen Sea during the austral spring 1992. J. Mar. Sys 17, 97e113.

Bindoff, N.L., Rosenberg, M.A., Warner, M.J., 2000. On the circula-

tion and water masses over the Antarctic continental slope and

rise between 80 and 150�E. Deep-Sea Res. II (47), 2299e2326.

120 F. Hashihama et al. / Polar Science 2 (2008) 109e120

Burczyk, J., Szkawran, H., Zontek, I., Czygan, F.-C., 1981. Caroten-

oids in the outer cell-wall layer of Scenedesmus (chlorophyceae).

Planta 151, 247e250.

Chiba, S., Hirawake, T., Ushio, S., Horimoto, N., Satoh, R.,

Nakajima, Y., Ishimaru, T., Yamaguchi, Y., 2000. An overview

of biological/oceanographic survey by the RTV Umitaka-Maru

III off Adelie Land, Antarctica in JanuaryeFebruary 1996.

Deep-Sea Res. II (47), 2589e2613.

Cubillos, J.C., Wright, S.W., Nash, G., de Salas, M.F., Griffiths, B.,

Tilbrook, B., Poisson, A., Hallegraeff, G.M., 2007. Calcification

morphotypes of the coccolithophorid Emiliania huxleyi in the

Southern Ocean: changes in 2001 to 2006 compared to historical

data. Mar. Ecol. Prog. Ser. 348, 47e54.

Findlay, C.S., Giraudeau, J., 2000. Extant calcareous nanoplankton in

the Australian Sector of the Southern Ocean (austral summers

1994 and 1995). Mar. Micropaleontol. 40, 417e439.

Finkel, Z.V., 2001. Light absorption and size scaling of light-limited

metabolism in marine diatoms. Limnol. Oceanogr. 46, 86e94.

Furuya, K., Marumo, R., 1983. The structure of phytoplankton com-

munity in the subsurface chlorophyll maxima in the western

North Pacific Ocean. J. Plankton Res. 5, 393e406.

Gall, M.P., Boyd, P.W., Hall, J., Safi, K.A., Chang, H., 2001. Phyto-

plankton processes. Part 1: Community structure during the

Southern Ocean Iron RElease Experiment (SOIREE). Deep-Sea

Res. II (48), 2551e2570.

Gieskes, W.W.C., Kraay, G.W., 1983. Dominance of cryptophyceae

during the phytoplankton spring bloom in the central North Sea

detected by HPLC analysis of pigments. Mar. Biol. 75, 179e185.

Goeyens, L., Treguer, P., Baumann, M.E.M., Baeyens, W., Dehairs, F.,

1995. The leading role of ammonium in the nitrogen uptake regime

of Southern Ocean marginal ice zones. J. Mar. Sys. 6, 345e361.

Ikavalko, J., Gradinger, R., 1997. Flagellates and heliozoans in the

Greenland Sea ice studied alive using light microscopy. Polar

Biol. 17, 473e481.

Jeffrey, S.W., Wright, S.W., 1997. Qualitative and quantitative HPLC

analysis of SCOR reference algal cultures. In: Jeffrey, S.W.,

Mantoura, R.F.C., Wright, S.W. (Eds.), Phytoplankton Pigments

in Oceanography: Guidelines to Modern Methods. UNESCO,

Paris, pp. 343e360.

Koike, I., Holm-Hansen, O., Biggs, D.C., 1986. Inorganic nitrogen

metabolism by Antarctic phytoplankton with special reference

to ammonium cycling. Mar. Ecol. Prog. Ser. 30, 105e116.

Latasa, M., Scharek, R., Le Gall, F., Guillou, L., 2004. Pigment suites

and taxonomic groups in Prasinophyceae. J. Phycol. 40, 1149e1155.

Mackey, M.D., Mackey, D.J., Higgins, H.W., Wright, S.W., 1996.

CHEMTAXda program for estimating class abundances from

chemical makers: application to HPLC measurements of phyto-

plankton. Mar. Ecol. Prog. Ser. 144, 265e283.

Mackey, M.D., Higgins, H.W., Mackey, D.J., Wright, S.W., 1997.

CHEMTAX User’s Manual: A Program for Estimating Class

Abundances from Chemical MarkersdApplication to HPLC

Measurements of Phytoplankton Pigments. CSIRO Marine Labo-

ratories Report 229, Hobart.

Millard, R.C., Owens, W.B., Fofonoff, N.P., 1990. On the calculation

of the Brunt-Vaisala frequency. Deep-Sea Res. 37, 167e181.

Moisan, T.A., Olaizola, M., Mitchell, B.G., 1998. Xanthophyll

cycling in Phaeocystis antarctica: changes in cellular fluores-

cence. Mar. Ecol. Prog. Ser. 169, 113e121.

Nishida, S., 1986. Nanoplankton flora in the Southern Ocean, with

special reference to siliceous varieties. Mem. Nat. Inst. Polar

Res. Spec. Issue 40, 56e68.

Palmisano, A.C., Garrison, D.L., 1993. Microorganisms in Antarctic

sea ice. In: Friedmann, E.I. (Ed.), Antarctic Microbiology. Wiley,

New York, pp. 167e218.

Raven, J.A., 1988. The iron and molybdenum use efficiencies of

plant growth with different energy, carbon and nitrogen sources.

New Phytol. 109, 279e287.

Repeta, D.J., Bjørnland, T., 1997. Preparation of carotenoid stan-

dards. In: Jeffrey, S.W., Mantoura, R.F.C., Wright, S.W. (Eds.),

Phytoplankton Pigments in Oceanography: Guidelines to Modern

Methods. UNESCO, Paris, pp. 239e260.

Saggiomo, V., Carrada, G.C., Mangoni, O., Ribera d’Alcala, M.,

Russo, A., 1998. Spatial and temporal variability of size-fractionated

biomass and primary production in the Ross Sea (Antarctica) during

austral spring and summer. J. Mar. Sys. 17, 115e127.

Savidge, G., Harbour, D., Gilpin, L.C., Boyd, P.W., 1995. Phyto-

plankton distributions and production in the Bellingshausen

Sea, Austral spring 1992. Deep-Sea Res. II (42), 1201e1224.

Schoemann, V., Becquevort, S., Stefels, J., Rousseau, V.,

Lancelot, C., 2005. Phaeocystis blooms in the global ocean and

their controlling mechanisms: a review. J. Sea Res. 53, 43e66.

Sohrin, Y., Iwamoto, S., Matsui, M., Obata, H., Nakayama, E.,

Suzuki, K., Handa, N., Ishii, M., 2000. The distribution of Fe in the

Australian sector of the Southern Ocean. Deep-Sea Res. I 47, 55e84.

Strickland, J.D.H., Parsons, T.R., 1972. A Practical Handbook of

Seawater Analysis. Fish. Res. Board. Can. Bull 167.

Suzuki, R., Ishimaru, T., 1990. An improved method for the determi-

nation of phytoplankton chlorophyll using N, N-dimethylforma-

mide. J. Oceanogr. Soc. Japan 46, 190e194.

Weissenberger, J., 1998. Arctic Sea ice biota: design and evaluation

of a mesocosm experiment. Polar Biol. 19, 151e159.

Wheeler, P.A., Kokkinakis, S.A., 1990. Ammonium recycling limits

nitrate use in the oceanic subarctic Pacific. Limnol. Oceanogr. 35,

1267e1278.

Wright, S.W., van den Enden, R.L., 2000. Phytoplankton community

structure and stocks in the East Antarctic marginal ice zone (BROKE

survey, January-March 1996) determined by CHEMTAX analysis

of HPLC pigment signatures. Deep-Sea Res. II (47), 2363e2400.

Zapata, M., Rodrıguez, F., Garrido, J.L., 2000. Separation of chloro-

phylls and carotenoids from marine phytoplankton: a new HPLC

method using a reversed phase C8 column and pyridine-contain-

ing mobile phases. Mar. Ecol. Prog. Ser. 195, 29e45.

Zapata, M., Jeffrey, S.W., Wright, S.W., Rodrıguez, F., Garrido, J.L.,

Clementson, L., 2004. Photosynthetic pigments in 37 species (65

strains) of Haptophyta: implications for oceanography and

chemotaxonomy. Mar. Ecol. Prog. Ser. 270, 83e102.