2011 - Phytoplankton Composition and Biomass Across the Southern Indian Ocean

8/11/2019 2011 - Phytoplankton Composition and Biomass Across the Southern Indian Ocean

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Phytoplankton composition and biomass across the southern Indian Ocean

Louise Schluter a,n, Peter Henriksenb, Torkel Gissel Nielsen b,c, Hans H. Jakobsen c,b

a DHI Water Environment & Health, Agern Alle 5, DK-2970 Hrsholm, Denmarkb National Environmental Research Institute, Aarhus University, Department of Marine Ecology, Frederiksborgvej 399, P.O. Box 358, DK-4000 Roskilde, Denmarkc National Institute of Aquatic Resources, DTU Aqua, Section for Oceanecology and Climate, Technical University of Denmark, Jgersborg Alle1, DK-2920 Charlottenlund, Denmark

a r t i c l e i n f o

Article hi story:

Received 7 September 2010

Received in revised form9 February 2011

Accepted 14 February 2011Available online 2 March 2011

Keywords:

Phytoplankton

Pigments

Indian Ocean

HPLC

CHEMTAX

Galathea3

a b s t r a c t

Phytoplankton composition and biomass was investigated across the southern Indian Ocean. Phyto-

plankton composition was determined from pigment analysis with subsequent calculations of group

contributions to total chlorophyll a (Chl a) using CHEMTAX and, in addition, by examination in themicroscope. The different plankton communities detected reflected the different water masses along a

transect from Cape Town, South Africa, to Broome, Australia. The first station was influenced by the

Agulhas Current with a very deep mixed surface layer. Based on pigment analysis this station was

dominated by haptophytes, pelagophytes, cyanobacteria, and prasinophytes. Sub-Antarctic waters of

the Southern Ocean were encountered at the next station, where new nutrients were intruded to the

surface layer and the total Chl a concentration reached high concentrations of 1.7 mg ChlaL1 with

increased proportions of diatoms and dinoflagellates. The third station was also influenced by Southern

Ocean waters, but located in a transition area on the boundary to subtropical water. Prochlorophytes

appeared in the samples and Chl a was low, i.e., 0.3mg L1 in the surface with prevalence of

haptophytes, pelagophytes, and cyanobacteria. The next two stations were located in the subtropical

gyre with little mixing and general oligotrophic conditions where prochlorophytes, haptophytes and

pelagophytes dominated. The last two stations were located in tropical waters influenced by down-

welling of the Leeuwin Current and particularly prochlorophytes dominated at these two stations, but

also pelagophytes, haptophytes and cyanobacteria were abundant. Haptophytes Type 6 (sensu Zapata

et al., 2004), most likely Emiliania huxleyi, and pelagophytes were the dominating eucaryotes in thesouthern Indian Ocean. Prochlorophytes dominated in the subtrophic and oligotrophic eastern Indian

Ocean where Chlawas low, i.e., 0.0430.086 mg total ChlaL1 in the surface, and up to 0.4 mg ChlaL1

at deep Chla maximum. From the pigment analyses it was found that the dinoflagellates of unknown

trophy enumerated in the microscope at the oligotrophic stations were possibly heterotrophic or

mixotrophic. Presence of zeaxanthin containing heterotrophic bacteria may have increased the

abundance of cyanobacteria determined by CHEMTAX.

& 2011 Elsevier Ltd. All rights reserved.

1. Introduction

The Indian Ocean is one of the largest yet the least studied

oceans in the world. In October 2006, the Danish expedition,Galathea 3, crossed the southern Indian Ocean from Cape Town in

South Africa to Broome in north-western Australia. The cruise

passed the nutrient-rich Agulhas Current, which runs along the

east coast of South Africa, encountered sub-Antarctic waters of

the Southern Ocean, passed the oligotrophic subtropical mid part

of the Indian Ocean, and approached the western coast of

Australia where down-welling occurs due to influence of the

Leeuwin Current. This gave a unique opportunity to compare the

various phytoplankton communities influenced by the oceano-

graphy in the different areas of this large ocean.

Nano- and picoplankton are important components of phyto-

plankton in the oceans, and in oligotrophic oceans 80% of thephytoplankton communities are composed by cells smaller than

3 mm (Goericke, 1998). Classical microscopy is insufficient for

identification and quantification of these prominent phytoplank-

ton components in the large oceanic regions. Methods have been

developed especially suited for detecting cells that were pre-

viously overlooked, e.g., epifluorescence microscopy, electron

microscopy, and cell-flow cytometry (reviewed by Jeffrey et al.,

1999), which have greatly improved the knowledge of especially

picoplankton and documented their key role in the oligotrophic

ecosystems (Zapata, 2005). More recently molecular techniques

have been applied (e.g., Moon-van der Staay et al., 2001; Dez

et al., 2001). However, these methods cannot yet be used on a

Contents lists available atScienceDirect

journal homepage: www.elsevier.com/locate/dsri

Deep-Sea Research I

0967-0637/$- see front matter& 2011 Elsevier Ltd. All rights reserved.

doi:10.1016/j.dsr.2011.02.007

n Corresponding author. Tel.: 45 4516 9557; fax:45 4516 9292.

E-mail address: [email protected] (L. Schluter).

Deep-Sea Research I 58 (2011) 546556
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routine basis for measuring the whole size range of phytoplank-

ton. Instead the fast and automated chemotaxonomic method,

pigment analysis by High Performance Liquid Chromatography

(HPLC), is increasingly used as a method for measuring the

composition and chlorophyll a (Chl a) biomass of phytoplankton

groups (e.g., Wright and Jeffrey, 2006). Following the HPLC

analysis of pigments the Chl a biomass of the individual phyto-

plankton groups can be calculated by using the CHEMTAX

program developed byMackey et al. (1996). CHEMTAX calculatesthe contribution from different phytoplankton groups to Chl a

based on ratios between accessory pigments and Chla, which are

loaded into the program together with the field measurements of

pigment concentrations. Knowledge on the phytoplankton com-

munities in the area sampled is important to achieve trustworthy

results of the CHEMTAX calculated biomasses (Wright et al.,

1996; Ansotegui et al., 2003; Irigoien et al., 2004). General

agreement between results of microscopy and pigment analyses

including the CHEMTAX calculation of the biomass of the phyto-

plankton groups has been found in samples from many different

environments, i.e., freshwater, estuaries and coastal waters, and

oceanic regions (Andersen et al., 1996; Schluter et al., 2000,

2006;Henriksen et al., 2002;Havskum et al., 2004).

Monitoring of phytoplankton by the pigment method to

describe the general structure and composition of phytoplankton

groups has been conducted in oligotrophic oceans (e.g.,Gibb et al.,

2000; Marty et al., 2002; Sakamoto et al., 2004; Veldhuis and

Kraay, 2004). Knowledge on phytoplankton assemblages in ocea-

nic regions of the southern hemisphere is, however, still limited.

In the Indian Ocean only a few investigations on phytoplankton

communities encompassing the whole size range of phytoplank-

ton have been published. Barlow et al. (2007) analyzed phyto-

plankton by HPLC in surface samples only on transects in the

different oceans of the southern hemisphere, and found relatively

low biomass and prokaryote dominance in the Indian Ocean. Not

et al. (2008) used pigment analysis and subsequent CHEMTAX

analysis to measure phytoplankton in two regions of the Indian

Ocean with special emphasis on the picoeucaryotes.

The water column structure has impact on the nutrient supplyto the euphotic zone. In general well-mixed nutrient-rich oceans

will support classic food chains with large phytoplankton and

large copepods, whereas oligotrophic stratified waters are domi-

nated by small phytoplankton where the productivity is based on

nutrients regenerated from microbial driven food webs. Knowl-

edge about the phytoplankton communities is essential to

improve our understanding of the plankton community structure

and productivity of the southern Indian Ocean. In order to get a

better understanding of the structure and function of the phyto-

plankton in the different regions in the large Indian Ocean, which

are influenced by different water masses each with different

physical characteristics (Visser et al., submitted), more investiga-

tions on the vertical and horizontal distribution, abundance and

composition of the whole size range of the phytoplankton com-

munities are needed. The aim of the present paper is to analyze

and characterize the phytoplankton communities across the

Indian Ocean in relation to the different oceanographic regimesby HPLC supplemented by classic microscopy to gain new

information on the primary producers in this poorly studied area.

2. Material and methods

2.1. Sampling strategy

Water samples were collected in 30-L Niskin bottles mounted

on a rosette with a Seabird 9/11 CTD on a transect with

seven stations from Cape Town in South Africa to Broome in

north-western Australia (Fig. 1) during late spring, October

18November 5, 2006. A detailed description of the sampling

and the oceanography of the transect can be found inVisser et al.

(submitted). Briefly, water samples from 10, 30, 60, 100, and

200 m depth were taken at all stations. Furthermore, in between

these casts discrete surface samples were taken along the transect

by a seawater intake system positioned approximately 5 m below

the ocean surface, which enabled water sampling of surface water

while cruising.

2.2. HPLC analyses

A Shimadzu LC-10A HPLC, composed of one pump (LC-

10ADVP), photodiode array detector (SPD-M10A VP), SCL-10ADVP

System controller with Class-VP software, temperature-controlled

auto sampler (set at 4 1C), column oven (CTO-10ASVP) and a

degasser, was installed in one of the six laboratory containers

mounted on the quarterdeck of the vessel. In order to hamper thenoise on the power supply generated by the vessel, the electricity

in all the laboratory containers was EMC secured (electromag-

netic compatibility) according to military standards and supplied

from a UPS (universal power supply) grounded to a single node in

each container. The baseline noise of the HPLC was only slightly

increased compared to land based HPLC analyses.

The samples for HPLC analysis were filtered in dim light onto

25 mm Whatman GF/F filters and immediately frozen in liquid

Fig. 1. Cruise track with station locations superimposed on map of surface temperature.

L. Schluter et al. / Deep-Sea Research I 58 (2011) 546556 547


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nitrogen. The samples were analyzed onboard within 3 days after

sampling. The filters were placed in a syringe mounted with a

0.2 mm Teflon syringe filter and 2 mL 95% acetone containing

0.025 mg mL1 vitamin E acetate as internal standard was added.

The samples were sonicated in the syringes for 10 s on ice with a

Sonics Vibra Cell Ultrasonic processor, and filtered directly into

HPLC vials. The vials were placed in the cooling rack (4 1C) of the

HPLC together with a parallel set of vials with injection buffer

(90:10, 28 mM aqueous tetrabutyl ammonium acetate (TBA), pH6.5; methanol). The samples were mixed with buffer using the

auto injector by programming it to make a mix in the loop of

buffer and sample in the ratio 5:2. A total volume of 500 mL was

injected. The method used for HPLC analysis was the Van

Heukelem and Thomas (2001)method, with an Eclipse XDB C8,

4.6 mm150 mm column (Agilent Technologies). Solvent A:

(70:30) methanol: 28 mM aqueous TBA (hydroxide titrant, JT

Baker HPLC reagent V365-07), pH 6.4, solvent B: 100% methanol.

Solvents were mixed using linear gradients along the following

time program: 0 min: 95% A, 5% B, 22 min: 5% A, 95% B, 30 min:

95% A, 5% B, 31 min: 100% A, 0% B, 34 min: 100% A, 0% B, 35 min

5% A, 95% B, 41 min: Stop. The flow rate was 1.1 mL min 1 and

the temperature of the column oven was set at 60 1C. The HPLC

was calibrated with pigment standards from DHI Lab Products,

Denmark. The internal standard was detected at 222 nm, while

the rest of the pigments were detected at 450 nm. Peak identities

were routinely confirmed by on-line PDA analysis.

A QA threshold procedure, application of limit of quantitation

(LOQ) and limit of detection (LOD), was applied to the pigment

data as described by Hooker et al. (2005) to reduce the uncer-

tainty of pigments found either in low concentrations or not

detected at all, causing false positives or false negatives, which

frequently occur when pigments are quantified near the

detection limit.

To investigate if all phytoplankton cells were collected on the

filters used in this study to collect the whole phytoplankton

community (Whatman GF/F, nominal pore size 0.7 mm), subsam-

ples of filtrates (the volumes were not recorded) of two surface

samples from station 6 were collected and filtered onto GEOsmonics polycarbonate 0.2 mm filters, extracted and analyzed

by HPLC as described above.

2.3. Enumeration, identification, and biomass estimates of

autotrophic protists

Samples from 10 and 60 m were collected from the rosette

immediately after it landed on deck, fixed in acidic Lugols (final

conc. 5%) and stored in 300 mL brown bottles at 5 1C. Samples

were subsequently counted within 3 months from the sampling

date. Sample aliquots were allowed to settle in 100 mL Utermohl

settling chambers for 24 h and analyzed in an inverted micro-

scope. Cellso20 mm were observed using an objective of 40X,while larger cells were analyzed by a 10X objective. Depending on

the abundance of the species in consideration, a fraction of or the

entire sample was counted. Each counted cell was assigned to a

morphological group and size class. The size classes were made of

10 mm equivalent spherical diameter (ESD) intervals and a

volume was assigned using the appropriate morphology volume

relationship equations. Except for a few rare species most of the

thecate dinoflagellates could be identified to genera. Hence,

nutritional mode could be deduced from the literature. The naked

dinoflagellates presented a challenging taxonomical problem, and

onlyGymnodinium spiraleand members of the genusCochlodinium

spp. were identified as heterotrophic (data on heterotrophic

protozoans will be presented in a later publication, Jonasdottir

et al., in preparation). The cellular carbon content of protists was

estimated from the taxon-specific ESD:carbon relationships

(Menden-Deuer and Lessard, 2000).

2.4. CHEMTAX analyses

The pigment concentrations were loaded into the CHEMTAX

program to calculate the Chl a biomass of the individual phyto-

plankton groups (Mackey et al., 1996). The pigment data set was

divided in two oceanographic regions: the samples taken duringthe first part of the cruise, where the Agulhas Bank encounters

sub-Antarctic waters of the Southern Ocean until 861E longitude

(south-western (SW) Indian Ocean), and samples taken from

911151E longitude (south-eastern (SE) Indian Ocean) (Fig. 1),

where total Chla was below 0.1 mg L1. These two data sets were

further divided into two sets: surface samples and samples from

and below the vertical Chl a maximum (Chl amax).

The pigment ratios used as input values for the CHEMTAX

calculations were fromSchluter et al. (2000),Higgins and Mackey

(2000), Gibb et al. (2001), Rodrguez et al. (2002), and Higgins

et al. (in press). The CHEMTAX program version 1.95 was used to

construct 60 different ratio matrices from the initial ratios for

each of the four data sets. Monovinyl (MV) Chl a was used for

calculating the biomass of all other groups than prochlorophytesfor which divinyl (DV) Chl a was used. 10% (n6) of the ratios

creating the lowest residual root mean square were averaged and

run repeatedly until the ratios became stable.

3. Results

3.1. The oceanography of the southern Indian Ocean

The oceanography of the cruise track (Fig. 1) is described in

details in Visser et al. (submitted). Briefly, the first station was

characterized by the confluence of the circumpolar circulation

and the water masses of the Agulhas Bank, which is a region of

elevated biological production. Station 2 was located where the

waters masses of the Agulhas Bank meet the converging sub-tropical and sub-Antarctic fronts just north of the Crozet Plateau

in the Southern Ocean. Station 3 was situated on the subtropical

front where relatively cold and fresh Antarctic intermediate

waters are subducted under the salty-warm waters of the sub-

tropical gyre. Stations 4 and 5 were located in the subtropical gyre

with little mixing from winds or from meso-scale eddies and

oligotrophic conditions. Stations 6 and 7 were situated in tropical

waters influenced by the Indonesian through flow where station

7 was on the shelf break of the North-West Australian shelf and

influenced by coastal condition (Fig. 1).

3.2. Phytoplankton biomass: results of pigment analyses

The different oceanic regimes crossed were reflected in thephytoplankton biomass and diversity. The phytoplankton biomass

in the surface of the SW Indian Ocean was between 0.2 and

0.4 mg ChlaL1 with an increase to 1.4 mg ChlaL1 in the area

with influence of Southern Ocean water (Fig. 2). In the SE Indian

Ocean the Chla biomass dropped at the surface to concentrations

of 0.040.09 mg ChlaL1 (Fig. 2). At the first stations in the SW

Indian Ocean the depth profile showed a Chl amax at around

3040 m, while the Chl amaxwas located deeper, i.e., at around

100 m, in the middle part of the Indian Ocean (Fig. 3). Towards

the western coast of Australia, the Chl amax was again located

higher up in the water column at around 60 m ( Fig. 3).

The HPLC results revealed presence of DV Chl a, the

diagnostic pigment of prochlorophytes, in samples from station

3 and onwards. Zeaxanthin, another important pigment in

L. Schluter et al. / Deep-Sea Research I 58 (2011) 546556548


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prochlorophytes, was detected in the samples from the first part

of the cruise where DV Chl a was absent, indicating presence of,

e.g., cyanobacteria, prasinophytes, and/or chlorophytes. Crypto-

phytes were detected in practically all samples by the diagnostic

pigment alloxanthin. The presence of MV Chl c3 as well as

190-butanoyloxyfucoxanthin (190-but) and 190-hexanoyloxyfu-

coxanthin (190-hex) in almost all samples across the Indian Ocean

indicated presence of at least haptophytes Type 6; Emiliana

huxleyi and Gephyrocapsa oceanica according to Zapata et al.

(2004), and probably also pelagophytes (Andersen et al., 1993).

Peridinin, the diagnostic marker of dinoflagellates, prasinoxanthin

as well as other pigments present in prasinophytes and/or

chlorophytes (Chl b, lutein, violaxanthin, neoxanthin) were

detected in many samples. Calculation of the pigment ratios:

prasinoxanthin/Chl b and lutein/Chl b (Schluter and Mhlenberg,

2003) for SW Indian Ocean, where DV Chl b was absent andtherefore did not interfere these calculations due to coelution of

DV and MV Chl b, indicated that both prasinophytes with

prasinoxanthin (prasinophytes Type 3, Higgins et al., in press,

and prasinophytes without prasinoxanthin and including chlor-

ophytes (prasinophytes Type 1, Higgins et al., in press) were

present in the samples.

The output ratios from the CHEMTAX analyses are shown in

Table 1. The ratios of peridinin/Chl a in dinoflagellates and

prasinoxanthin/Chl a in prasinophytes were both horizontally

and vertically relatively constant, while fucoxanthin/Chl a in

diatom ratios were constant across different water masses, but

differed vertically with higher ratios in the deeper part of the

water column (Table 1). The fucoxanthin/Chlaratios inE. huxleyi-

Type 6 haptophytes were variable around 0.1 showing no cleardifference for the different data sets, but the ratio of the

diagnostic pigment 190-hex to Chl a was higher in the SE Indian

Ocean and increased in the deeper part of the water column

(Table 1). 190-but/Chl a in pelagophytes and alloxanthin/Chl a

ratios in cryptophytes were lower in the deeper parts of the water

column in the SW Indian Ocean, but tended to increase in the SE

Indian Ocean. Such different responses were also obvious in the

two subtypes of prasinophytes Types 1 and 3; Chl b/Chla ratios of

prasinophytes with prasinoxanthin were relatively stable across

the southern Indian Ocean, while those of prasinophytes Type 1

(incl. chlorophytes) were approx. twice as high in the SW Indian

Ocean (Table 1). While zeaxanthin/Chl a ratios in the upper part

of the water column were more than twice as high as in the

deeper waters for cyanobacteria, they were more than 5 times

higher for prochlorophytes (Table 1). Both groups had higher

ratios in the eastern part of the Indian Ocean.

The phytoplankton groups calculated by CHEMTAX revealed

that the increase in Chl a at station 2 were caused by a general

increase in the Chl a biomass of most phytoplankton groups

(Fig. 3), but particularly dinoflagellates and diatoms were abun-

dant (Table 2). The phytoplankton populations in the surface

waters after station 2 were dominated by haptophytes (Fig. 2). At

station 4 the phytoplankton biomass in the surface dropped to a

total Chl a biomass less than 0.05 mg ChlaL1, and became

dominated by prochlorophytes, but haptophytes, cyanobacteria,

and pelagophytes also constituted an important part of the

phytoplankton population (Fig. 2,Table 2).

The CHEMTAX calculations showed that diatoms as well as

dinoflagellates were only sporadically present when prochloro-

phytes became dominating in the SE Indian Ocean. Both verticallyand horizontally, haptophytes and pelagophytes constituted a

significant part of the phytoplankton biomass, and pelagophytes

even exceeded the biomass of prochlorophytes at Chl amax at

station 5 (Fig. 3, Table 2). Except at station 2 cyanobacteria also

appeared to constitute an important part of the phytoplankton

population in the Indian Ocean (Fig. 3).

The filtrates of the GF/F filtered samples collected onto 0.2 mm

filters showed that all phytoplankton cells were collected on

the GF/F filters in this study, since the Chl a concentrations in the

0.2mm samples were below the limit of detection. However, the

pigment zeaxanthin was detected on the 0.2 mm filters as the only

accessory pigment indicating that non-autotrophic cells with a size

less than the nominal size of GF/F filters of approx. 0.7 mm contain-

ing this photo-protective pigment were present in the samples.

3.3. Phytoplankton biomass: results of microscopy

The phototrophic protists 45 mm identified by inverted micro-

scopy showed a dominance of unidentified flagellates at most

stations except station 2, where the highest biomass of

280 mg C L1 was measured at 60 m depth and diatoms and

dinoflagellates dominated, and at station 7 off the Australian west

coast (Table 3). At this westernmost station pennate diatoms

o20 mm and dinoflagellates of unknown trophy dominated. The

autotrophic thecate dinoflagellates constituted an insignificant

biomass except at station 2, where species of the genera Gonyau-

lax, Heterocapsa, Prorocentrum, and Torodinium were observed.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

21.10St. 1

22.10 23.10St. 2

24.1025.1026.10 27.10St. 3

27.10 28.10 29.10

gC

hlaL-1

0

0.02

0.04

0.06

0.08

0.1

30.10St. 4

31.10St. 5

2.11St. 6

4.11St. 7

Prochlorophytes

Diatoms

Cyanobacteria

Prasinophytes type 1

PelagococcusHaptophytes type 6

Cryptophytes

Dinoflagellates


Fig. 2. Biomass of the phytoplankton population calculated by CHEMTAX in the surface in the southern Indian Ocean. The x -axis shows the sampling dates and the

stations. The dates sampled in between stations are sampled with the seawater intake system described in Material and methods.



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Naked dinoflagellates of unknown trophy were present in most

samples (Table 3). Assigning nutrition strategy to this group is

challenging. Regression between the biomass of dinoflagellate

estimated in microscope averaged over 10 and 60 m against the

biomass of dinoflagellates determined by HPLC from all stations

except station 2 revealed that that the slope of the regression line

was not different from 0 (N6; P0.31, when including dino-

flagellates of unknown trophy and P0.79 when excluding dino-

flagellates of unknown trophy). Station 2 was not included in this

analysis as the food web at this station appeared very differentfrom the remaining stations. As the dinoflagellates of unknown

trophy did not add any significance to the relationship between

the biomass estimated by HPLC and microscopy, respectively, the

dinoflagellates of unknown trophy were most likely either mixo-

trophic with a low pigment content or strictly heterotrophic.

4. Discussion

4.1. Phytoplankton in relation to the oceanography of the southern

Indian Ocean

The different phytoplankton communities measured were reflect-

ing the different water masses of the southern Indian Ocean. The first

station sampled, station 1, was influenced by the Agulhas Current

with a very deep mixed surface layer, down to 300 m (Visser et al.,

submitted), where total Chl a was 0.4 mg L1 at the surface. Flagel-

lates and athecate dinoflagellates were detected by microscopy

(Table 3). HPLC measurements suggested that phytoplankton was

in fact dominated by haptophytes, pelagophytes, and cyanobacteria.

Prasinophytes Types 1 and 3 were also important at this station. This

is comparable to the results of Not et al. (2008), who also found

cyanobacteria, i.e., Synechococcus, and picoeucaryotes (pelagophytes,

haptophytes, and chlorophytes/prasinophytes) to dominate at themore coastal, nutrient-rich stations in the Indian Ocean. At station

2 sub-Antarctic waters of the Southern Ocean were encountered,

reducing the surface temperatures and the salinity (Visser et al.,

submitted). The Southern Ocean is one of the high-nutrient low-

chlorophyll (HNLC) regions, but at this station the total Chl a

concentration reached relatively high values for oceanic regions of

1.4mg Chl a L1 in the surface (Fig. 2) with a Chlamaxof 1.7 mg L1 in

30 m depth. The high Chl a concentration was accompanied by

increased proportions of diatoms and dinoflagellates in the phyto-

plankton population detected by both methods (Tables 2 and 3). This

is commonly found in areas with a supply of new nutrients since

these opportunistic taxa are particularly well suited to take advantage

of excess nutrients (Fogg, 1991; Claustre, 1994). High nutrient

concentrations were indeed measured from the surface throughout

Table 1

Output ratios of pigment/chlorophyll a from the CHEMTAX calculations for the four different data sets analyzed (see text for details).

Chlc3 Chlc2 Chlc1 MV Chlc3 Peri 190-but Fuco Neox Pras Viol 190-hex Allo Zeax Lut Chlb

South-western Indian Ocean, surface

Prasinophytes Type 3 0.111 0.377 0.050 0.020 0.784

Dinoflagellates 0.456 0.698

Cryptophytes 0.065 0.307

Haptophytes Type 6 0.195 0.084 0.036 0.006 0.032 0.781

Pelagophytes 0.131 0.512 1.108 0.222Prasinophytes Type 1 0.091 0.111 0.038 0.054 0.793

Cyanobacteria 1.378

Diatoms 0.032 0.026 0.307

Prochlorophytes 0.466 0.147

South-western Indian Ocean, from and below chlorophyll a maximum





Pelagophytes 0.365 0.176 0.471 0.085


Cyanobacteria 0.650

Diatoms 0.140 0.012 0.809


South-eastern Indian Ocean, surface

Prasinophytes Type 3 0.123 0.488 0.074 0.016 0.905Dinoflagellates 0.271 0.735



Pelagophytes 0.188 0.334 0.821 0.095


Cyanobacteria 2.692

Diatoms 0.105 0.014 0.370


South-eastern Indian Ocean, from and below chlorophyll amaximum





Pelagophytes 0.769 0.344 1.059 0.095


Cyanobacteria 0.781

Diatoms 0.163 0.029 0.719Prochlorophytes 0.156 1.184

Abbreviations: Chl, chlorophyll; MV, monovinyl; peri, peridinin; 19 0-but, 190-butanoyloxyfucoxanthin; fuco, fucoxanthin; neox, neoxanthin; pras, prasinoxanthin; viol,

violaxanthin; 190-hex, 190-hexanoyloxyfucoxanthin; allo, alloxanthin; zeax, zeaxanthin; lut: lutein.



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the mixed layer, and preceding days of rough weather conditions had

intruded new nutrients, and possibly iron enrichment from the Crozet

Plateau (Pollard et al., 2007) to the area (Visser et al., submitted).Station 3 was also influenced by the Southern Ocean waters and at

this station prochlorophytes appeared for the first time in the samples

and Chl a was relatively low, i.e., 0.3 mg L1 in the surface waters,

with prevalence of haptophytes particularly in the surface. Further-

more, pelagophytes and cyanobacteria were abundant (Fig. 3), indi-

cating that this station was located in a transition area on the

boundary to subtropical water. Stations 4 and 5 were located within

the subtropical gyre with little mixing and general oligotrophic

conditions. This was reflected in the composition and biomass of

phytoplankton with low surface concentrations of Chl a and a

maximum of 0.2mg Chl a L1 at 100 m depth at both stations,

showing dominance of prochlorophytes, cyanobacteria, and small

flagellates (Fig. 3) typical for oligotrophic areas where regenerated

nutrients are the only nutrient source. The last two stations, 6 and 7,

were located in tropical waters influenced by down-welling of the

Leeuwin Current. However, the presence of pennate diatoms at

station 7 suggests a complex exchange of ocean water and coastalwater masses that inject Si to the water column. Particularly

prochlorophytes dominated at these two stations (Figs. 2 and 3),

but also pelagophytes, haptophytes, and cyanobacteria were abun-

dant. These cells were not detected by the microscopy method used,

by which only larger cells were identified. The Chl amaxat station

7 located on the shelf break influenced by coastal conditions was

situated higher up in the water column at 60 m and picoprocaryotes

(cyanobacteria and prochlorophytes) contributed 4567% to the total

Chla biomass in and above Chl amax(Table 2). This is comparable to

the range of 5565% found byHanson et al. (2007) for picoprocar-

yotes in deep Chl a maximum (DCM) in the Leeuwin Current in

coastal waters of western Australia in close vicinity of our station 7,

with haptophytes as the other primary contributor (2132%). How-

ever, contrary to the present study the phytoplankton population in

0

50

100

150

200

0

m

g Chl a L-1

St. 1

0

50

100

150

200

0

m

g Chl a L-1

St. 2

0

50

100

150

200

0

m

St. 3

0

50

100

150

200

0

m

St. 4

0

50

100

150

200

0

m

St. 5

0

50

100

150

200

0

m

St. 6

0

50

100

150

200

0

m


DinoflagellatesCryptophytes

Haptophytes type 6

Pelagophytes


Cyanobacteria

Diatoms

ProchlorophytesSt. 7

0.05 0.1 0.15 0.1 0.2 0.3 0.4 0.5 0.6

0.02 0.04 0.06 0.080.04 0.08 0.12 0.16

0.01 0.02 0.03 0.04 0.05 0.06 0.07

0.04 0.08 0.12 0.16

0.05 0.1 0.15 0.2

Fig. 3. Depth distribution of the biomass of the individual phytoplankton groups as Chla concentration calculated by CHEMTAX at the stations sampled.



7/11

the surface waters of the study ofHanson et al. (2007) was dominated

by cyanobacteria and haptophytes, while prochlorophytes were

virtually absent. Hanson et al. (2007) did, however, not determine

the diagnostic pigment of prochlorophytes, DV Chl a, by HPLC. Instead

this group was calculated by CHEMTAX using the pigments zeax-

anthin and Chl b. Nevertheless, Barlow et al. (2007) did indeed

measure DV Chl a, indicative of prochlorophytes, and the DV

Chl a/total Chl a ratio was 0.4 in the surface transect of the Indian

Ocean at 201S, the station closest to the Australian coast placed at

approx. 1131E, close to our station 7 (1151E, 201S). This is comparable

to the present study where this ratio was 0.47 at station 7. Barlow

et al. (2007) sampled only surface water and found dominance by

prokaryotes and low total Chl a biomass down to 0.02mg

total Chl a L1 at a transect more northerly (201S) than ours. In the

oligotrophic SE Indian Ocean we measured total Chl a concentrations

from 0.043 to 0.086 mg Chl a L1

in the surface (Fig. 2).Barlow et al.

Table 2

Contribution in percentage of the different phytoplankton groups to chlorophyll a biomass obtained by CHEMTAX.

Station Depth

(m)

Prasinophytes

Type 3

Dinoflagellates Cryptophytes Haptophytes

Type 6

Pelagophytes Prasinophytes

Type 1

Cyanobacteria Diatoms Prochlorophytes

1 10 9 0 5 38 12 11 9 15 0

30 8 0 5 18 28 11 24 6 0

60 9 0 7 17 29 11 21 5 0

100 9 0 8 16 30 10 21 6 0

2 10 1 16 3 27 9 7 2 36 0

30 3 13 5 15 29 11 3 23 0

60 5 3 3 13 32 6 1 35 0

100 5 13 6 18 22 12 3 18 0

3 10 0 1 1 62 10 0 9 5 13

30 0 0 1 26 22 1 25 13 11

60 0 0 0 13 26 0 32 0 29

100 4 0 1 12 56 4 8 0 15

4 10 3 2 8 12 10 9 10 7 39

30 3 2 6 17 16 9 7 6 36

60 3 2 6 18 19 9 4 1 39

100 1 1 2 15 18 5 19 0 40

5 10 3 3 10 29 16 12 6 0 20

30 3 2 8 23 21 11 8 0 25

60 3 1 5 28 26 9 2 0 26

100 2 1 2 21 31 6 11 5 22

6 10 3 2 7 15 11 9 13 3 38

30 3 3 7 19 9 9 13 2 36

60 3 3 8 21 14 8 11 3 29

100 1 1 2 10 16 2 20 1 47

7 10 2 3 3 9 4 7 17 8 47

30 2 3 2 9 5 6 13 6 54

60 7 3 4 11 16 5 11 8 34

80 4 3 3 11 25 4 10 11 29

100 2 2 5 16 33 4 3 15 20

Average 3 3 5 19 20 7 12 8 31

Table 3

Biomasses of autotrophic protists across the Indian Ocean and the sum of all groups at 10 and 60 m depths. Units mg C L1.

Diatoms Flagellates45 lm Autotrophic thecate

dinoflagellates

Autotrophic athecate

dinoflagellates

Athecate dinoflagellates of

unknown trophy

Sum of all

groups

Station 10 m 60 m 10 m 60 m 10 m 60 m 10 m 60 m 10 m 60 m 10 m 60 m

1 0.09 0.07 0.03 1.00 0.47 0.46 0.05 0.05 1.44 1.73 2.08 3.30

2 6.74 133.92 9.15 34.35 31.91 57.17 0.82 1.31 56.06 53.41 104.68 280.16

3 0.23 0.12 7.75 3.25 0.93 1.35 0.12 0.13 4.31 3.05 13.34 7.90

4 0.02 0.01 2.22 10.97 0.57 1.09 0.09 0.11 2.53 3.81 5.44 16.00

5 0.00 0.08 4.02 3.31 0.65 1.27 0.06 0.03 2.49 2.93 7.23 7.63

6 0.09 0.14 3.15 0.02 0.16 0.64 0.06 0.23 1.04 6.95 4.51 7.99

7 0.44 2.55 0.02 0.10 0.77 1.00 0.07 0.11 2.79 3.04 4.08 6.81



8/11

(2007)found total Chl a of 0.09 mg L1, which is comparable to this

study. Prochlorophytes were, however, even more concentrated in

the DCM (Fig. 3) where total Chl a reached 0.4 mg Chl a L1.

4.2. The use of CHEMTAX for determining phytoplankton

composition and biomass

Analyzing phytoplankton pigments by HPLC gives the advan-tage of getting information on the whole phytoplankton commu-

nity by one method, which cannot be achieved in one single step

by other methods. However, the subsequent application of pro-

grams like CHEMTAX requires subjective interpretations on

which phytoplankton groups and pigment/Chl a ratios to load

into CHEMTAX. In order to diminish the uncertainty on these

taxonomical interpretations,Higgins et al. (in press) have made a

guide for quantitative chemotaxonomic interpretation of pigment

data. Briefly, it is important to obtain as much information as

possible on which phytoplankton groups to expect in the samples,

i.e., also by using alternative methods to pigment analysis, since

the results of the CHEMTAX analyses rely on the input to the

program. Furthermore, the pigment ratios and phytoplankton

groups chosen to load into CHEMTAX should reflect the phyto-

plankton communities sampled. It is recommended to divide the

pigment data into subsamples of populations with equal environ-

mental conditions (light adaptations, water mass properties, etc.),

and carefully select the initial pigment/Chlaratios, using multiple

starting pigment ratios. Then to run the CHEMTAX calculations

repeatedly by optimizing the input ratios in order to minimize the

residual root mean square error (Higgins et al., in press). These

approaches were used in this study. The pigment data set was

divided to represent two oceanographic regions, SE and SW

Indian Ocean. Although the phytoplankton populations, detected

at the different stations sampled indicated, that even more sub-

grouping of this large ocean could be considered, the number of

samples was limited and we found it more important to divide

the dataset vertically.

The choice of which phytoplankton groups to load intoCHEMTAX was supported by microscopic enumerations made

by inverted microscope, showing presence of diatoms, dinofla-

gellates, and unidentifiable flagellates (Table 3). Peridinin is a

diagnostic marker of dinoflagellates, but the pigment method

only found peridinin containing dinoflagellates to be a significant

part of the phytoplankton population at station 2, and absent or

sporadically present in the rest of the samples (Fig. 3, Table 2).

Some dinoflagellates have acquired their chloroplast and pig-

ments from other taxa and contain, e.g., fucoxanthin and its

derivates (De Salas et al., 2003). If present, these organisms will

have been included in the haptophytes by the CHEMTAX analyses.

The dinoflagellate genera Ornithocercus, Histioneis, Parahistioneis

and Citharistes, Amphisolenia and Triposoleniaobserved by micro-

scopy had ectosymbiotic cyanobacteria, while the latter two alsohad endosymbionts of eukaryotic origin (Farnelid et al., 2010;

Tarangkoon et al., 2010). Although, ecto- and endosymbiotic

mixotrophy is found in a wide range of oceanic dinoflagellate

species, their abundance is o2 cells L1 (Tarangkoon et al., 2010)

and thus of minor importance. The discrepancy between the

HPLC pigment method and microscopic analysis may be

explained by heterotrophic nutrition in the dinoflagellates, which

may be dominant yet invisible to pigment analysis, except for

what they have consumed (Higgins et al., in press). Hence,

we suggest that most of the dinoflagellates enumerated in the

microscope as dinoflagellates of unknown trophy were most

likely heterotrophic.

Prochlorophytes, cryptophytes, and prasinophytes Type 3

were detected by their unique diagnostic pigments DV Chl a,

alloxanthin, and prasinoxanthin, respectively. Cyanobacteria are

usually detected by the non-specific pigment zeaxanthin, which

was present at all stations. Since Synechococcus has long been

documented by flow cytometry as an important constituent of the

prokaryotic algal community in many oceanic regions, including

the Indian Ocean (Not et al., 2008), this group was included in the

CHEMTAX calculations. The presence of prasinophytes

Type 1 (including chlorophytes) could be identified by pig-

ment ratios as mentioned in the results.The presence of 190-hex indicated presence of haptophytes.

Zapata et al. (2004) investigated pigments of haptophytes and

found 8 different types based on their pigment content, where

3 types contained 190-hex. One of the pigments, MV Chl c3which

was detected in this study, has been found to be strongly

associated with the globally important species Emiliania huxleyi

and one other species, Gephyrocapsa oceanica, grouped as hapto-

phytes Type 6 inZapata et al. (2004). MV Chl c3 was detected in

most samples in this study along with 190-hex and 190-but, and

haptophytes Type 6 was consequently included in CHEMTAX

(Table 1). This group was found to constitute an important

part of the phytoplankton population in the Indian Ocean, and

particularly in the SW Indian Ocean haptophytes Type 6 tended

to dominate in the surface waters, but were also abundant

in the deeper parts of the water column in the SE Indian Ocean

(Fig. 3).E. huxleyihas been found to dominate the coccolithophore

populations in various regions of the worlds oceans (e.g.,Boeckel

et al., 2006; Lipsen et al., 2007; Siegel et al., 2007; Gravalosa

et al., 2008), and this study shows that haptophytes Type 6,

i.e., E. huxleyi and G. oceanica, are important in the Indian Ocean

too. Flagellates were grouped as unknown flagellates by micro-

scopy. Since acidic Lugols iodine was used to fix the samples, the

coccoliths were most likely dissolved (Sournia, 1978), which

made the identification of coccolithophorids impossible.

Haptophytes types 15 do not contain the characteristic

pigments 190-hex and 190-but (Zapata et al., 2004) and if present,

they were included in the groups of diatoms by CHEMTAX. An

examination of the diatoms carbon/Chl a (C/Chla) ratios showed

that these were varying with an average of 132 and whenexcluding station 2, 60 m, the C/Chl a ratio was in average 27

(data not shown). The very high C/Chl a ratio at station 2, 60 m

together with a generally low photosynthetic activity (Visser

et al., submitted), indicates a decline of the bloom encountered

at this station. Although uncertainty exists on counting and

biomass estimations made by microscopy and particularly dia-

toms of varying C/Chl a ratios (Schluter and Mhlenberg, 2003),

the potential inclusion of haptophytes Types 15 in the group

diatoms by the CHEMTAX calculations did not lead to low C/Chl

a ratios of diatoms. Thus haptophytes types 15 seem to have

been of minor importance in the Indian Ocean. Furthermore,

except at station 2 diatoms determined by pigment analysis

usually only constituted a few percentages and 15% at maximum

(Table 2), which also indicates that any haptophytes types15 included in diatoms by CHEMTAX were of insignificant

importance.

The 190-hex/190-but-ratios of all data in the present data set

were 2.471.1 (average7standard deviation, n223). E. huxleyi

and G. oceanica contain no or very little 190-but and according

toZapata et al. (2004)the Type 6 haptophytes, which were found

to contain 190-but, had 190-hex/190-but-ratios of at least 50. This

indicates that other 190-but containing algae were present in the

samples of this study. In the study of Andersen et al. (1996)

pelagophytes were found to be an important phytoplankton

group both by pigments and electron microscopy in the Atlantic

and Pacific Oceans, and subsequently, the pelagophytes have been

identified by 190-but in various oligotrophic waters (e.g.,Bidigare

and Ondrusek, 1996; Steinberg et al., 2001; Suzuki et al., 2002;



9/11

Marty et al., 2008). Consequently, pelagophytes were included in

the CHEMTAX calculations, and were found to comprise an

important part, on average 20%, of the phytoplankton population

in the Indian Ocean, particularly in the deeper parts of the ocean

(Table 2). This agreed well with results ofNot et al. (2008)from a

more northerly transect in the Indian Ocean, where pelagophytes

were found by HPLC to constitute a similar part of the phyto-

plankton population. As found in other oceans (e.g., Andersen

et al., 1996;Steinberg et al., 2001) haptophytes and pelagophyteswere the most abundant eucaryotes in the Indian Ocean con-

tributing profoundly to the Chl amax (Fig. 3, Table 2). In the

present study pelagophytes tended to be placed even lower in the

water column than the haptophytes, a pattern also found in a few

other studies, e.g., in the Atlantic Ocean (Veldhuis and Kraay,

2004) and in the Mediterranean (Marty et al., 2008).

4.3. Effects of applying QA threshold

The QA threshold procedure applied to the data set in this

study, where the baseline noise was slightly increased due to the

noise from the power supply generated by the vessel, resulted in

an improved CHEMTAX solution. This is apparent from an

evaluation of the residuals (pigment content unexplained by theCHEMTAX solution as root mean square, data not shown). The

residuals from the CHEMTAX analyses, carried out after the QA

threshold procedure was applied, were up to 15 times lower

when compared to the residuals achieved before applying LOD

values to the results, thus proving a better data fit when LOD

values were applied. The reason for this is that the QA procedure

reduced the uncertainties contributed by false negatives and

false positives, which occurs when pigments are quantified near

the method detection limit (Hooker et al., 2005). In the present

data set these pigments were secondary pigments like neox-

anthin, violaxanthin, MV Chlc3, and lutein. Instead of zero values

in the spread sheet, when such pigments could not be detected

and quantified, the LOD values applied caused that CHEMTAX

included these pigments in the calculations. For example whenlow concentrations of prasinoxanthin were detected the acces-

sory pigments neoxanthin and lutein of prasinophytes Type

3 were often below the detection limit. Applying LOD values

instead of zeroes for these secondary pigments improved the

CHEMTAX calculations, thus causing a better data fit.

4.4. Presence of other zeaxanthin containing organisms in the

southern Indian Ocean

Analyses of organisms not retained by the GF/F filters used to

filter the samples revealed that all autotrophic pico-sized algae

were in fact collected by the filters, since no Chlawas detected on

0.2 mm filters after passage of the GF/F filters. The 0.2 mm filters

did, nevertheless, retain some zeaxanthin-containing organisms,probably a marine bacterium such asParacoccus zeaxanthinifaciens

(formerlyFlavobacterium;Berry et al., 2003). In a recent study in

Antarctic waters (Wright et al., 2009) high zeaxanthin concentra-

tions caused an unrealistic high contribution of cyanobacteria by

the CHEMTAX calculations, and bacteria rather than cyanobac-

teria were the most likely source to zeaxanthin. However, in the

Antarctic waters the bacteria were retained by GF/F filters

(Wright et al., 2009), indicating that the size of such pigmented

marine bacteria is variable or that the Antarctic bacteria were

attached to aggregates such as mucilage. If such larger sized

bacteria also were present in the Indian Ocean they would have

influenced the CHEMTAX calculations and increased particularly

the biomass of the cyanobacteria, which was calculated from the

zeaxanthin concentration (Table 1). The zeaxanthin/Chla ratios of

cyanobacteria obtained by the CHEMTAX calculations were 1.38

in the SW Indian Ocean, but 2.69 in the SE Indian Ocean (Table 1),

and the latter value is higher than ratios of high light treated cells

of Synechococcus sp. cultures (Schluter et al., 2000; Henriksen

et al., 2002). Although zeaxanthin is a photo-protective pigment

and zeaxanthin/Chl a ratios did show 2 and 5 times changes as

function of depth/light intensity for cyanobacteria and prochlor-

ophytes, respectively (Table 1), the high zeaxanthin/Chl a ratio of

cyanobacteria in the SE Indian Ocean indicated that other zeax-anthin containing cells may have been retained on the GF/F filters

too. CHEMTAX calculated a variable contribution of cyanobacteria

with an average of 12% (Table 2). In the oligotrophic parts of the

Pacific Ocean the biomass ofSynechococcus was found to always

constitute less than 10% by flow cytometry (Campbell et al., 1994,

1997; Blanchot et al., 2001). Of the few studies of picoplankton

conducted in the oligotrophic parts of the Indian Ocean Not et al.

(2008)used flow cytometry to enumerate cells ofProchlorococcus

andSynechococcus. Although no biomass estimations were made

the cell numbers appear comparable to the results of Blanchot

et al. (2001) from the Pacific Ocean. A seasonal succession

towards prokaryote dominance during high temperatures and

irradiance in summer has been demonstrated across the global

ocean basins in the subtropical southern hemisphere (Barlow

et al., 2007). While the study ofNot et al. (2008)was carried out

during late fall, this study was carried out during late spring and a

higher prokaryotic proportion of phytoplankton should be

expected from station 4 and onwards where oligotrophic condi-

tions prevailed. Unfortunately, no flow cytometry measurements

were conducted, and in some occasions the cyanobacteria con-

stituted up to 20% (Table 2) of the phytoplankton population,

which might indicate that zeaxanthin from non-photosynthetic

bacteria could have biased the biomass of cyanobacteria deter-

mined by CHEMTAX. The presence and size range of such

zeaxanthin containing bacteria that might interfere with deter-

mination of cyanobacteria by CHEMTAX definitely need special

attention in future investigations.

5. Conclusion

The difference of the pigment/Chl a ratios (Table 1) and the

large variety in the phytoplankton communities encountered

across the southern Indian Ocean reflects the complexity of this

under-sampled ocean, which is influenced by confluences of

water currents, upwelling and down-welling resulting in produc-

tive mixing areas to oligotrophic regions. The microscopy method

used in this study (inverted microscope) was providing only

limited, yet important, information on 45 mm algae. Valuable

information on the phytoplankton communities in the southern

Indian Ocean was obtained by combining the results from the two

phytoplankton identification methods. It could be deduced that

most of the dinoflagellate community of unknown trophy in theoligotrophic regions of the Indian Ocean were likely heterotrophic

species. For the different subtypes of haptophytes and pelago-

phytes the CHEMTAX setup could be justified by using the

information on diatom biomasses achieved by microscopy. In

order to obtain more information on the pico-sized phytoplank-

ton populations special microscopy methods are needed, which,

however, seldom are feasible when many samples need to be

analyzed. Since the pigment method certainly has limitations too

(e.g., Higgins et al., in press), and microscopic analyses also

introduce taxonomical misinterpretations even by competent

taxonomists (Culverhouse et al., 2003; Culverhouse, 2007), a

combination of several methods is warranted when examining

phytoplankton communities like those sampled in the southern

Indian Ocean.



10/11

Acknowledgments

The sampling was conducted during the third Danish Galathea

expedition. We thank the Captain of HMDS Vdderen, Carsten

Schmidt, and his crew for excellent assistance in connection with

our sampling. The project was supported by grants from Knud

Hjgaards Fond and the Danish Natural Sciences Research Coun-

cil. We are grateful to Simon Wright, Australian Antarctic Divi-

sion, for receiving CHEMTAX ver. 1.95, and to Jacob L. Hyer,Danish Meteorological Institute for preparingFig. 1. The present

work was carried out as part of the Galathea3 expedition under

the auspices of the Danish Expedition Foundation. This is

Galathea3 contribution no. P77.

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