# Calcification morphotypes of the coccolithophorid Emiliania huxleyi in the Southern Ocean: changes in 2001 to 2006 compared to historical data

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

Tilbrook, Alain Poisson, G. M. Hallegraeff

► To cite this version:

J. C. Cubillos, S. W. Wright, G. Nash, M. F. de Salas, B. Griffiths, et al., Calcification morphotypes of the coccolithophorid Emiliania huxleyi in the Southern Ocean: changes in 2001 to 2006 compared to historical data. Marine Ecology Progress Series, 2007, 348, pp.47-54. 10.3354/meps07058. hal-01245622

HAL Id: hal-01245622

https://hal.science/hal-01245622

Submitted on 19 Dec 2021

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Calcification morphotypes of the coccolithophorid *Emiliania huxleyi* in the Southern Ocean: changes in 2001 to 2006 compared to historical data

J. C. Cubillos<fnref n="1" />, S. W. Wright<fnref n="2" />,<fnref n="3" />, G. Nash<fnref n="2" />, M. F. de Salas<fnref n="1" />, B. Griffiths<fnref n="3" />,<fnref n="4" />, B. Tilbrook<fnref n="3" />,<fnref n="4" />, A. Poisson<fnref n="5" />, G. M. Hallegraeff<fnref n="1" />,*

ABSTRACT: We conducted a scanning electron microscopic survey of morphological variations in the calcareous nanoplankton species *Emiliania huxleyi* in Southern Ocean surface water samples collected along a transect from 43 to 64°S and 141 to 145°E during November 2001, October to February 2002/2003, 2003/2004, 2004/2005 and 2005/2006. The results were compared with historical data from a similar transect occupied in December to January 1983/1984 and January to February 1994 and 1995. While *E. huxleyi* was absent or extremely sparse (0.1 to 1 cells ml⁻<fnref n="1" />) south of 60°S in 1983/1984 and 1994/1995, this species was consistently present at about 100 cells ml⁻<fnref n="1" /> between 60 and 65°S during 2002 to 2006. The extended geographic range and/or increased southward abundance of this keystone species suggests a significant shift in Southern Ocean ecology over the past 2 decades, analogous to an observed recent range extension into the Bering Sea. Morphotype A 'overcalcified' mainly occurred north of the Subantarctic Front and was replaced by the weakly calcified Morphotype B/C between the Subantarctic Front and Southern Antarctic Circumpolar Current Front. This north-south shift in *E. huxleyi* calcification morphotypes closely tracked the north-south decline in the calcite saturation state as calculated from carbonate system measurements. Based on current evidence, no significant changes are apparent in the calcification status of *E. huxleyi* in the Southern Ocean during the past 12 yr. All cultured isolates from north of the Polar Front belonged to Morphotype A, while all strains isolated south of the Polar Front belonged to Morphotype B/C and their morphologies appeared conservative in culture. The north-south trend of decreased calcification of *E. huxleyi* in the Southern Ocean thus reflects the shift in dominance of one ecotype to another, rather than the environmental effect of decreased carbonatre ion concentrations and calcite saturation state on a single, 'apparently cosmopolitan', population.

KEY WORDS: Southern Ocean · *Emiliania huxleyi* · Calcite saturation state · Ocean acidification

Resale or republication not permitted without written consent of the publisher

## INTRODUCTION

Approximately half of the anthropogenic CO₂ released to the atmosphere since the beginning of the industrial revolution has dissolved in the ocean, leading to a reduction of surface pH by 0.1 U (Riebesell et

al. 2000, Orr et al. 2005). Under the IS92a scenario of continuing global CO₂ emissions from human activities, the average pH of the upper ocean is predicted to fall by 0.4 U to pH 7.8 by the year 2100. This pH is lower than has been experienced for millennia, and, critically, the predicted rate of change is 100 times

<footnote n="1">¹School of Plant Science, University of Tasmania, Private Bag 55, Hobart, Tasmania 7001, Australia</footnote>

<footnote n="2">²Australian Antarctic Division, Channel Highway, Kingston, Tasmania 7050, Australia</footnote>

<footnote n="3">³Antarctic Climate and Ecosystems Cooperative Research Centre, University of Tasmania, Private Bag 80, Hobart, Tasmania 7001, Australia</footnote>

<footnote n="4">⁴Wealth from Oceans Flagship, CSIRO, GPO Box 1538, Hobart, Tasmania 7001, Australia</footnote>

<footnote n="5">⁵Laboratoire d'Océanographie et du Climat: Expérimentations et Approches Numériques, Université Pierre et Marie Curie, 75252 Paris, France</footnote>
faster than ever experienced in the known history of our planet (The Royal Society 2005). The effects of ocean acidification are additional to, and are likely to exacerbate, the effects of climate change, yet research into the biological effects of high concentrations of CO₂ in the oceans is still in its infancy. It has been claimed that calcifying organisms will find it difficult to produce and maintain their calcareous shells. Of particular importance is the ubiquitous coccolithophorid nanoplankton *Emiliania huxleyi* that produces a shell (coccosphere) comprised of numerous calcite plates (coccoliths). Riebesell et al. (2000) experimentally simulated the effect of ocean acidification on a single North Atlantic strain (PML B92/11A; Morphotype A). Diminished calcification of *E. huxleyi* was accompanied by an increased proportion of malformed coccoliths and incomplete coccospheres. Extrapolation of these observations to the world's oceans raises the concern that ocean acidification will increase dissolution of calcium carbonate, causing a shift in the composition of the plankton community towards non-calcifying species. The Southern Ocean is particularly vulnerable due to a combination of low surface temperatures, which increase the solubility of CO₂ and calcium carbonate, and the incidence of large amounts of upwelled deep water (Orr et al. 2005). Thus, predicted changes in Southern Ocean chemistry during this century are expected to have far-reaching effects in altering the trophic structure and biodiversity of these globally critical ecosystems.

*Emiliania huxleyi* first appeared in the geological record 270 000 yr ago and is now the dominant coccolithophorid in the world's oceans. This species is widely considered to be a ubiquitous cosmopolitan marine microalga, occurring from tropical to polar waters. Most of our knowledge on calcification in *E. huxleyi*, however, has derived from experimental research on predominantly North Atlantic culture strains (Paasche 2001). Some morphological variations have appeared stable in culture for many years under widely varying growth conditions and have been interpreted to be genotypic in nature (Young & Westbroek 1991). The confirmation of immunological differences among Type A and B culture strains (Young & Westbroek 1991) has corroborated these ideas. Genotypic variation has proved to be the strongest single influence on coccolith morphology. Currently, some 6 morphotypes are recognized (Young et al. 2003), based on the fine structure of their coccoliths, but their precise delimitation can be difficult. Other variations in the structure of the coccoliths have been interpreted to be due to incomplete or abnormal (teratological) growth, coccosphere cell size variation, and degree of calcification (the latter often correlated with temperature). In the present study, widespread concern about

the potential effect of ocean acidification triggered a careful scanning electron microscopic analysis of *E. huxleyi* morphotypes in Southern Ocean samples collected from 43 to 64°S, 141 to 145°E, during November 2001 and October to February 2002/2003, 2003/2004, 2004/2005 and 2005/2006 and comparison with historic data from the same transect occupied from December to January 1983/1984 (Nishida 1986) and 1994/1995 (Findlay & Giraudeau 2000).

## MATERIALS AND METHODS

**Field.** The transect from Hobart (44°57′S, 147°20′E) to the French base Dumont d'Urville in Terre Adélie, Antarctica (65°50′S, 140°00′E), was sampled by the French MV 'l'Astrolabe' in 3 or 4 regular rotations per year from October to March in 2002/2003, 2003/2004, 2004/2005 and 2005/2006 (Fig. 1). Data from an earlier depth transect sampled by the Australian RV 'Aurora Australis' in November 2001 (Voyage 3) are also included. The positions of various frontal systems in the

<figure><img src="image_2.png" /><figcaption>Fig. 1. Transect occupied by regular MV 'l'Astrolabe' voyages between Tasmania and Antarctica, showing key frontal systems: Subtropical Front (STF), Subantarctic Front, Northern branch (SAF-N), Northern Polar Front (NPF), Southern Polar Front (SPF), Southern Antarctic Circumpolar Current Front, Northern branch (SACCF-N) and Southern Antarctic Circumpolar Current Front, Southern branch (SACCF-S) (adapted from Sokolov & Rintoul 2002). Southern Ocean stations south of 60°S previously occupied by Nishida (1986) in 1983/1984 and Findlay & Giraudeau (2000) in 1994/1995 are indicated</figcaption></figure>
Southern Ocean were interpreted from thermosalino-
graph records using the water mass definitions of
Sokolov & Rintoul (2002). Carbonate ion and calcite
saturation state were calculated using measurements
of total dissolved inorganic carbon and titration alka-
linity from Tilbrook et al. (2001). On board the MV
'l'Astrolabe', water samples were collected every 8 to
12 h from the clean water line, which constantly pumps
subsurface water (3 m depth) into the vessel. On
the RV 'Aurora Australis', samples from different
depths were collected with 10 l Niskin bottles mounted
on a rosette sampler. Subsamples of 250 ml were fil-
tered through a 25 mm diameter Nuclepore membrane
filter (1 µm pore size). Salt crystals were removed from
the filter by rinsing twice with 1 ml of distilled water,
and the filters were gently air-dried and stored over
silica gel.

**Laboratory.** Filters were prepared for scanning electron microscopy (SEM) by mounting onto a 1.2 cm diameter metal stub and sputter coating with 15 nm gold. Samples were examined using a JEOL 840 scanning electron microscope. A fixed magnification (1500×) was used for all quantitative observations, with higher magnification for morphotype identification. Only cells of *Emiliania huxleyi* were counted, but the presence of other species of coccolithophorids was recorded, as well as the abundance of other phytoplankton groups. For each sample, 200 cells were counted and classified into morphotypes, unless cell concentrations were too low, in which case 2000 fields of view in transects across the filter were observed. Quantitative *E. huxleyi* cell density was calculated, taking into account the number of cells viewed, number of SEM fields of view screened, section of filter area scanned, and volume of seawater filtered.

**Culturing of Southern Ocean strains.** On board the MV 'l'Astrolabe' in March 2006, 50 ml surface water samples were screened immediately after collection,

through a 20 µm plankton net to remove large or-
ganisms, and were maintained at 4°C in a 50 ml
polycarbonate flask topped up with a small amount of
Medium K (Keller et al. 1987). One month later, clonal
cultures (Culture Codes EHSO-1 to -14) were estab-
lished from single-cell isolates at the University of
Tasmania, using Medium K at 4 to 15°C (depending on
the ambient temperature of the sampling site), 35 psu
salinity and 100 µmol photons m⁻² s⁻¹ of photosynthet-
ically active radiation (PAR).

RESULTS

Water temperatures along the 43 to 64°S north-south transect ranged from 16 to 0°C, and salinities from 35.30 to 33.70 psu. Carbonate ion concentrations along the cruise track declined from 175 µmol l⁻¹ in warm subtropical waters to 95 µmol l⁻¹ at the southern end of the transect. Calcite saturation levels calculated from carbon measurements showed a decline in the saturation state from >4 in the north to values <2.5 south of 55°S. The steepest gradients in saturation state occurred in the transition between the Subantarctic Zone and the North Polar Front (Fig. 2). The range of calcification morphotypes commonly encountered in the Southern Ocean is illustrated in Fig. 3. Morphotypes shown in Fig. 3a,b,i,j correspond with Type A (var. *huxleyi*) of Young & Westbroek (1991) (referred to here as typical Type A) in that the coccoliths possess robust distal shield elements and curved elements in the central area, but the latter are obscured by overcalcification in Fig. 3c,d (hence referred to as Type A 'overcalcified', sensu Young et al. 2003). Morphotypes in Fig. 3e-g,k correspond with Type B/C of Young et al. (2003), in that they possess more delicate distal shield elements and have the central area open or covered by a thin membrane. This same morphotype

from the same locality has been previously
referred to as Type C by Findlay & Gi-
raudeau (2000) and Type K by Hiramatsu &
De Deckker (1996). Distal shield elements
of Morphotype A often tended to be
straight or concave compared to those of
Morphotype B/C, which were convex (com-
pare arrowed coccoliths in Fig. 3b,g). The
size of coccoliths can vary significantly on
the same coccosphere (notably obvious in
clonal cultures Fig. 3i-k) and hence was of
limited value in discriminating morpho-
types. Dissolution morphotypes (in which
the coccosphere appears to be partially
dissolved; Burns 1977) were rarely ob-
served (<1% of total cells). Fig. 3h depicts
a dissolution form where the distal shield

<figure><img src="image_3.png" /><figcaption>Fig. 2. Calcite saturation status calculated from TCO₂ and alkalinity measurements, along a typical 44 to 62°S north-south transect occupied by the RV 'Aurora Australis' in November 2001 (Tilbrook et al. 2001). Highly saturated surface water with calcite saturation >4 occurs in the north, while surface waters south of 55°S typically have values <2.5. The steepest gradients in saturation state occur in the transition between the Subantarctic Zone and the North Polar Front</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Fig. 3. *Emiliania huxleyi*. Diversity of morphotypes encountered in Southern Ocean and Australian waters. (a) Typical Type A from Sydney coastal waters. (b) Type A from 45°28′S, 146°93′E. (c) Type A overcalcified; field sample from 47°04′S, 146°70′E; 20 October 2004. (d) Type A overcalcified; field sample from 43°72′S, 147°37′E; 19 February 2003. (e) Type B/C field sample from 51°79′S, 142°84′E; 4 April 2006. (f) Type B/C field sample from 55°48′S, 140°73′E. (g) Type B/C; field sample from 43°74′S, 147°36′E; 2 March 2006; distal shield elements of Morphotype A often tended to be straight or concave compared to those of Morphotype B/C, which were convex (compare arrowed coccoliths in Panels b and g). (h) Dissolution morphotype; field sample from 59°12′S, 143°04′E; 24 January 2003. (i–k) Conservation of coccolith morphology in clonal cultures: (i) isolate of Type A from Tasmanian coastal waters; (j) isolate of Type A from 46°17′S, 146°04′E; 1 March 2006; (k) isolate of Type B/C from 53°21′S, 144°53′E; 28 February 2006. Scale bars = 1 µm (a–h); 10 µm (i–k)</figcaption></figure>

elements are almost unrecognizable and only the proximal shield is visible. While the 3 morphotypes (A, A 'overcalcified' and B/C, as originally defined by Young & Westbroek 1991) were mainly based on the size of the distal shield and the morphology of central area elements, in the Southern Ocean, these morphologies essentially represent a gradient from more to less calcified (Table 1), hence, our terminology of referring to them in the Southern Ocean as being equivalent to 'calcification morphotypes'.

Quantitatively, *Emiliania huxleyi* is a key species in the Southern Ocean phytoplankton, occurring at cell densities of up to 400 cells ml⁻¹. The dominant morphotype is B/C (Fig. 4), which has never previously been

studied in culture and which is distinct from the well-studied North Atlantic Type A. Morphotype A 'overcalcified' was dominant (60 to 80% of total *E. huxleyi*) north of 47 to 48°S (roughly the position of the Subantarctic Front), being replaced by the much more weakly calcified dominant Southern Ocean Morphotype B/C from 48 to 65°S (ranging down to the Southern Antarctic Circumpolar Current Front). Typical Morphotype A exhibited a partially overlapping distribution from 44 to 55°S (roughly from the Subtropical Front to the North Polar Front), but stretched south to 59°S in 2004/2005 and contracted again to 50°S in 2005/2006 (Figs. 4 & 5). In all years, Morphotype B/C was the only form of *E. huxleyi* present south of 57°S.
<table><caption>Table 1. *Emiliania huxleyi*. Morphological variants (sensu Young et al. 2003) identified in the present study. CaCO₃ (%): percentage of distal shield area covered</caption><thead><tr><th>Morphotype</th><th>Distal shield elements</th><th>Central area</th><th>CaCO<sub>3</sub> (%)</th><th>Geographic distribution</th></tr></thead><tbody><tr><td>Type A</td><td>Robust, straight, or concave</td><td>Elements curved</td><td>50–80</td><td>Dominant in Australian coastal waters</td></tr><tr><td>Type A overcalcified</td><td>Robust, straight, or concave</td><td>Elements curved; rarely visible</td><td>80–95</td><td>Dominant in Southern Ocean, 43–47° S</td></tr><tr><td>Type B/C</td><td>Delicate, convex</td><td>Irregular laths; open or covered by thin plain plate</td><td>30–45</td><td>Dominant in Southern Ocean, south of 50° S</td></tr></tbody></table>

All cultured isolates from north of the Polar Front belonged to Morphotype A, while all strains isolated south of the Polar Front belonged to Morphotype B/C (Table 2). These differing geographic distributions of *E. huxleyi* morphotypes in surface samples were also reflected in water column depth distributions down to 100–150 m depth (RV 'Aurora Australis', Voyage 3, November 2001; Fig. 6).

DISCUSSION

While *Emiliania huxleyi* was absent or extremely sparse south of 60°S in 1983/1984 (Nishida 1986) and 1994/1995 (Findlay & Giraudeau 2000), this species was consistently present at about 100 cells ml⁻¹ between 60 and 65°S during our surveys in the Australian sector of the Southern Ocean from 2002 to 2006. Hasle (1960) first reported the complete absence of *E. huxleyi* in the Antarctic Pacific based on a grid of stations at 53 to 70°S, 90 to 174°W covered by the 1947 to 1948 Norwegian Brategg Expedition. Thirty-five years later, Nishida (1986) occupied 5 stations, 9 depths each, south of 60°S in the same area covered by us, with *E. huxleyi* being absent or extremely sparse at 0.1 to 1 cells ml⁻¹ (Fig. 1). Another 10 yr later, Findlay & Giraudeau (2000) occupied 3 stations, 4 depths each, south of 60°S in the Australian sector of the Southern Ocean (Fig. 1) and found them to be devoid of *E. huxleyi*. These observations have led to the dogma of a north-south transition of calcareous towards siliceous nanoplankton (Nishida 1986). Winter et al. (1999) reported *E. huxleyi* from the Weddell Sea (69 to 71°S) during austral autumn 1992, but suggested that it may have been introduced by the Agulhas Current or a warm eddy system crossing the Brazil-Malvinas confluence into the Antarctic Circumpolar Current. The extended geographic range and increased southward abundance of this keystone species in the Australian sector of the Southern Ocean in the period 2001 to 2006 compared to historic data suggests a significant shift in Southern Ocean ecology over the past 2 decades. This is unlikely to be due to the approxi-

mate 0.1°C warming of the bulk ocean (Gille 2002), but may reflect enhanced surface warming or changes in zooplankton grazing populations (Atkinson et al. 2004, Hosie 2004) as a result of substantial

<figure><img src="image_5.png" /><figcaption>Fig. 4. *Emiliania huxleyi*. Surface cell density (log cells ml⁻¹) of different morphotypes (Types A, A overcalcified and B/C) along a regularly occupied transect in the Southern Ocean from 43 to 66°S. Cell counts have been averaged on a latitudinal basis for 4 different transects in each October to March period from the years 2002/2003 to 2005/2006. This species was consistently present south of 60°S, an area from which it was completely absent in previous surveys conducted in 1983/1984 and 1994/1995</figcaption></figure>
<figure><img src="image_6.png" /></figure>

Fig. 5. *Emiliania huxleyi*. Latitudinal and annual shifts in calcification morphotypes in the Southern Ocean, based on the presence or absence in surface samples collected by the MV 'I'Astrolabe' on a regular transect from 43 to 66°S between Tasmania and the Antarctic in 4 consecutive austral summers from 2002/2003 to 2005/2006. Different morphotypes exhibited differing geographic distributions with a distinct southward trend of decreased calcification evident in all years. Subtle year-to-year shifts in distribution patterns of calcification morphotypes are evident, but a full interpretation of their ecological significance awaits genetic and ecophysiological studies on newly established cultured Southern Ocean strains. The approximate positions of the South Tropical Front (STF), the North Polar Front (NPF) and the Southern Antarctic Circumpolar Current Front (SACCF, both branches) are indicated

retreat of the sea ice zone. We note that an appar-
ently analogous range extension of *E. huxleyi* has
also occurred in the Bering Sea since 1997 (Merico et
al. 2003), suggesting that this might be a bipolar
phenomenon.

It is likely that the morphotypic variations we ob-
served are genetically based, since these morphologies
appear to be conserved in clonal cultures (Fig. 3i–k).
*Emiliania huxleyi* has often been viewed as a single
cosmopolitan species, yet considerable evidence now
suggests otherwise. Brand (1982) first documented un-
expectedly high genetic differentiation in acclimatized
growth rates for multiple *E. huxleyi* clones established
from the same water sample. Barker et al. (1994) and
Medlin et al. (1996) used randomly amplified polymor-
phic DNA fingerprints (RAPDs) and amplification frag-
ment length polymorphisms (AFLPs) to reveal exten-

sive genetic diversity, both on a global scale and within
bloom populations, in both space and time. RAPDs
showed that all strains except for 1 pair were geneti-
cally distinct, and this appeared to be reflected in their
morphology and ecological distribution. Iglesias-Ro-
driguez et al. (2002, 2006) further corroborated a high
degree of polymorphism in 87 isolates of different geo-
graphical origin using AFLP and microsatellite loci.
Preliminary analysis of microsatellite loci in 3 southern
hemisphere strains (from Australia, New Zealand,
South Africa) has already indicated that they are
significantly different from northern hemisphere geno-
types (Iglesias-Rodriguez et al. 2006), and we expect
even greater genetic distances between Southern
Ocean and northern hemisphere strains in molecular
studies in progress on our cultured Southern Ocean
and Australian strains (Table 2). Studies on *E. huxleyi*
<table><caption>Table 2. *Emiliania huxleyi*. List of Southern Ocean and Australian culture strains established in the present study</caption><thead><tr><th>Strain code</th><th>No. of strains</th><th>Locality</th><th>Isolator</th><th>Morphotype</th></tr></thead><tbody><tr><td>EHSO2</td><td>1</td><td>Southern Ocean, 59°S, 143°E</td><td>de Salas & Cubillos</td><td>B/C</td></tr><tr><td>EHSO3</td><td>1</td><td>Southern Ocean, 58°S, 143°E</td><td>de Salas & Cubillos</td><td>B/C</td></tr><tr><td>EHSO4</td><td>1</td><td>Southern Ocean, 58°S, 143°E</td><td>de Salas & Cubillos</td><td>B/C</td></tr><tr><td>EHSO5</td><td>1</td><td>Southern Ocean, 57°S, 144°E</td><td>de Salas & Cubillos</td><td>B/C</td></tr><tr><td>EHSO6</td><td>1</td><td>Southern Ocean, 55°S, 144°E</td><td>de Salas & Cubillos</td><td>B/C</td></tr><tr><td>EHSO8</td><td>2</td><td>Southern Ocean, 53°S, 145°E</td><td>de Salas & Cubillos</td><td>B/C</td></tr><tr><td>EHSO10</td><td>2</td><td>Southern Ocean, 51°S, 146°E</td><td>de Salas & Cubillos</td><td>B/C</td></tr><tr><td>EHSO11</td><td>2</td><td>Southern Ocean, 49°S, 146°E</td><td>de Salas & Cubillos</td><td>A</td></tr><tr><td>EHSO12</td><td>1</td><td>Southern Ocean, 46°S, 146°E</td><td>de Salas & Cubillos</td><td>A</td></tr><tr><td>EHSO13</td><td>1</td><td>Southern Ocean, 46°S, 147°E</td><td>de Salas & Cubillos</td><td>A</td></tr><tr><td>EHSO14</td><td>1</td><td>Southern Ocean, 45°S, 147°E</td><td>de Salas & Cubillos</td><td>A</td></tr><tr><td>EHMP</td><td>36</td><td>Mercury Passage, Tasmania</td><td>de Salas & Cook</td><td>A</td></tr><tr><td>EHTB</td><td>81</td><td>Trumpeter Bay, Tasmania</td><td>de Salas</td><td>In progress</td></tr><tr><td>EHST</td><td>28</td><td>Stanley, Tasmania</td><td>Cook</td><td>In progress</td></tr><tr><td>EHWC</td><td>200</td><td>Western Tasmania</td><td>Cook</td><td>In progress</td></tr><tr><td>EHBI</td><td>15</td><td>Bicheno, Tasmania</td><td>Cook & de Salas</td><td>In progress</td></tr><tr><td>EHSB</td><td>15</td><td>Streaky Bay, South Australia</td><td>Cook & de Salas</td><td>In progress</td></tr><tr><td>EHCA</td><td>15</td><td>Cairns, Queensland, Australia</td><td>Cook & de Salas</td><td>In progress</td></tr><tr><td>EHGQ</td><td>9</td><td>Gladstone, Queensland, Australia</td><td>Cook</td><td>In progress</td></tr></tbody></table>

in the North Atlantic have long discriminated between the 2 morphotypes (A and B), which tend to show differing geographic and sometimes depth distributions (Van Bleijswijk et al. 1991, Young & Westbroek 1991). Schroeder et al. (2005) recently identified a genetic marker that confirmed the genetic basis for discrimination of these North Atlantic morphotypes.

The dominant morphotype in the Southern Ocean is B/C, which has never previously been studied in culture and which is distinct from the well-studied North Atlantic Type A. Morphotype A 'overcalcified' mainly occurred north of the Subantarctic Front, being replaced by the more weakly calcified Morphotype B/C between the Subantarctic Front and the Southern Antarctic Circumpolar Current Front. This north-south shift in *Emiliania huxleyi* calcification morphotypes closely tracked the north-south decline in calcite saturation state (Fig. 2). Based on current evidence, no significant changes have become apparent in the calcification status of *E. huxleyi* in the Southern Ocean during the past 12 yr. All cultured isolates from north of the Polar Front belonged to Morphotype A, while all strains isolated south of the Polar Front belonged to Morphotype B/C, and their morphologies appeared conservative in culture even when grown under a range of environmental conditions (authors' unpubl. data). The north-south trend towards decreasing calcification of *E. huxleyi* in the Southern Ocean thus reflects a shift in dominance of one ecotype to another, rather than the environmental effect of decreased carbonate ion concentrations and calcite saturation state on a single 'apparently cosmopolitan' population. A similar north-south trend of calcification morphotypes of *E. huxleyi*

has now also been confirmed for the Indian sector of the Southern Ocean (along a 45°E transect; Mohan et al. 2007), while in the northern hemisphere a reverse south-north trend exists off the coast of Japan when moving from the warm Kuroshio to the cold Oyashio Current (Hagino et al. 2005).

<figure><img src="image_7.png" /><figcaption>Fig. 6. *Emiliania huxleyi*. Depth distributions of the presence or absence of 3 different morphotypes along a transect occupied in the Southern Ocean by the Australian RV 'Aurora Australis' in November 2001 (Voyage 3 from 44 to 62°S). Surface distributions of the 3 different morphotypes reflect 150 m deep water column distributions. ○: *E. huxleyi* absent</figcaption></figure>
Different *Emiliania huxleyi* morphologies in the Southern Ocean and in other parts of the world’s oceans most likely reflect differing physiological and genetic properties and potentially deserve to be recognized as different varieties. Further studies using robust molecular tools are needed to discriminate between *E. huxleyi* genotypes in field populations and in culture to improve the confidence of our prediction of the future success of this key ocean plankton. Global extrapolations from research on limited culture strains may produce misleading results due to underestimation of the genetic diversity and ecophysiological plasticity of the plankton super-species (sensu Thierstein & Young 2004) *E. huxleyi*.

*Acknowledgements.* We thank the captain and crew of MV 'l'Astrolabe' for collaboration in the sampling program. This work was supported by IPSL, INSU and IPEV (France), the Australian Climate Change Reserach Programme, and the Australian Government's Cooperative Research Centres Programme through the Antarctic Climate and Ecosystems Cooperative Research Centre (ACE CRC).

## LITERATURE CITED

Atkinson A, Siegel V, Pakhomov E, Rothery P (2004) Long-term decline in krill stock and increase in salps within the Southern Ocean. Nature 432:100–103

Barker GLA, Green JC, Hayes PK, Medlin LK (1994) Preliminary results using the RAPD analysis to screen bloom populations of *Emiliania huxleyi* (Haptophyta). Sarsia 79:301–306

Brand L (1982) Genetic variability and spatial patterns of genetic differentiation in the reproductive rates of the marine coccolithophores *Emiliania huxleyi* and *Gephyrocapsa oceanica*. Limnol Oceanogr 27:236–245

Burns DA (1977) Phenotypes and dissolution morphotypes of the genus *Gephyrocapsa* Kamptner and *Emiliania huxleyi* (Lohmann). NZ J Geol Geophys 20:143–155

Findlay CS, Giraudeau J (2000) Extant calcareous nanoplankton in the Australian Sector of the Southern Ocean (austral summers 1994 and 1995). Mar Micropaleontol 40:417–439

Gille ST (2002) Warming of the Southern Ocean since the 1950s. Science 295:1275–1277

Hagino K, Okada H, Matsuoka H (2005) Coccolithophore assemblages and morphotypes of *Emiliania huxleyi* in the boundary zone between the cold Oyashio and warm Kuroshio currents off the coast of Japan. Mar Micropaleontol 55:19–47

Hasle GR (1960) Plankton coccolithophorids from the Subantarctic and Equatorial Pacific. Norw J Bot 8:77–88

Hiramatsu C, De Deckker P (1996) Distribution of calcareous nannoplankton near the subtropical convergence, south of Tasmania, Australia. Mar Freshw Res 47:707–713

Hosie G (2004) Southern Ocean CPR survey. Annual report. Sir Alister Hardy Foundation for Ocean Science, Plymouth, p 16–17

Iglesias-Rodriguez MD, Saez AG, Groben R, Edwards KJ, Batley J, Medlin LK, Hayes PK (2002) Polymorphic microsatellite loci in global populations of the marine coccolithophorid *Emiliania huxleyi*. Mol Ecol Notes 2: 495–497

Iglesias-Rodriguez MD, Schofield OM, Batley J, Medlin LK, Hayes PK (2006) Intraspecific genetic diversity in the marine coccolithophore *Emiliania huxleyi* (Prymnesio-

phyceae): the use of microsatellite analysis in marine phytoplankton population studies. J Phycol 42:526–536

Keller MD, Selvin R, Claus W, Guillard RRL (1987) Media for the culture of oceanic ultraphytoplankton. J Phycol 23: 633–638

Medlin LK, Barker GLA, Campbell L, Green JC, Hayes PK, Marie D, Wrieden S, Vaulot D (1996) Genetic characterisation of *Emiliania huxleyi* (Haptophyta). J Mar Syst 9:13–31

Merico A, Tyrrell T, Brown CW, Groom SB, Miller PI (2003) Analysis of satellite imagery for *Emiliania huxleyi* blooms in the Bering Sea before 1997. Geophys Res Lett 30:1337, doi: 10.1029/2002GL016648

Mohan R, Mergulhao LP, Gupta MVS, Rajakumar A, Thamban M, Anil Kumar N, Sudhakar M, Ravindra R (2007) Coccolithophores during the austral summer of 2004 along the 45°E transect in the Indian sector of Southern Ocean. Mar Micropaleontol (in press) doi: 110.1016/j.marmicro.2007.08.005

Nishida S (1986) Nanoplankton flora in the Southern Ocean, with special reference to siliceous varieties. Mem Nat Inst Polar Res Spec Issue 40:56–68

Orr JC, Fabry VJ, Aumont O, Bopp L and 24 others (2005) Anthropogenic ocean acidification over the twenty-first century and its impact on calcifying organisms. Nature 437:681–686

Paasche E (2001) A review of the coccolithophorid *Emiliania huxleyi* (Prymnesiophyceae), with particular reference to growth, coccolith formation and calcification—photosynthesis interactions. Phycologia 40:503–529

Riebesell U, Zondervan I, Rost B, Tortell PD, Zeebe RE, Morel FMM (2000) Reduced calcification of marine plankton in response to increased atmospheric CO₂. Nature 407: 364–367

Schroeder DC, Biggi GF, Hall M, Davy J, Martínez JM, Richardson AJM, Malin G, Wilson WH (2005) A genetic marker to separate *Emiliania huxleyi* (Prymnesiophyceae) morphotypes. J Phycol 41:874–879

Sokolov S, Rintoul ST (2002) Structure of Southern Ocean Fronts at 140°E. J Mar Syst 37:151–184

The Royal Society (2005) Ocean acidification due to increasing atmospheric carbon dioxide. Available at www.royalsoc.ac.uk (accessed on 1 October 2007)

Thierstein HR, Young J (eds) (2004) Coccolithophores: from molecular processes to global impact. Springer-Verlag, Berlin

Tilbrook B, Rintoul S, Sabine C (2001) Carbon dioxide, hydrographic, and chemical data obtained during the R/V *Aurora Australis* repeat hydrography cruise in the Southern Ocean: CLIVAR CO2 Repeat Section SR03–2001 (EXPOCODE AA0301), (29 October–22 November 2001). Carbon Dioxide Information Analysis Center, Oak Ridge National Laboratory, US Department of Energy, Oak Ridge, TN

Van Bleijswijk J, Van der Wal P, Kempers R, Veldhuis M, Young JR, Muyzer G, de Vrind-de Jong E, Westbroek P (1991) Distribution of two types of *Emiliania huxleyi* (Prymnesiophyceae) in the Northeast Atlantic region as determined by immunofluorescence and coccolith morphology. J Phycol 27:566–570

Winter A, Elbrachter M, Krause G (1999) Subtropical coccolithophores in the Weddell Sea. Deep-Sea Res 1 26:439–449

Young JR, Westbroek P (1991) Genotypic variation in the coccolithophorid *Emiliania huxleyi*. Mar Micropaleontol 18: 5–23

Young J, Geisen M, Cross L, Kleijne A, Sprengel C, Probert I, Østergaard J (2003) A guide to extant coccolithophore taxonomy. J Nanoplankton Res Spec Issue 1