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Submitted July 8th 2009 to Journal of Phycology
A new cell stage in the haploid-diploid life cycle of the colony-forming
Abstract
Few members of the well-studied marine phytoplankton taxa have such a complex and polymorphic life cycle as the genus Phaeocystis Lagerheim. However, despite the ecological and biogeochemical importance of Phaeocystis blooms, the life cycle of the major bloom-forming species of this genus remains illusive and poorly resolved. At least six different life stages and up to 15 different functional components of the life cycle have been proposed. Our culture and field observations indicate that there is a previously unrecognised stage in the life cycle of P. antarctica. This stage comprises non-motile cells that range in size from around 4.2 to 9.8 μm in diameter and form aggregates in which interstitial spaces between cells are small or absent. The aggregates (hereafter called attached aggregates, AAs) adhere to available surfaces. In field samples, small AAs, surrounded by mucilage, adopt an epiphytic life style and adher to setae or spines of diatoms. These, either directly or via other life stages, produce the colonial life stage. Culture studies indicate that bloom–forming, colonial stages release microzoospores that fuse and form AAs, which can proliferate on the bottom of culture vessels and which can eventually reform free-floating colonies. We propose that these AAs are a new stage in the life cycle of P. antarctica, which we believe to be the zygote, thus documenting sexual reproduction in this species for the first time.
Keywords: Phaeocystis, P. antarctica, life cycle stage, sexual reproduction, zygote
33 Introduction
Several Phaeocystis species have been identified world-wide, of which six have been formally described. There is at least one undescribed species and two of the described ones are likely to be complexes of cryptic species (Medlin and Zingone, 2007). Only three of the described species have been documented as forming colonies and dominating blooms, namely P.
pouchetii, P. globosa and P. antarctica (Medlin et al. 1994) with P. jahnii forming loose, small, occasionally palmelloid, colonies (Medlin and Zingone 2007).
Ecological relevance of Phaeocystis
P. antarctica can form virtually monospecific blooms in the Southern Ocean (SO), which can contribute >90% of total phytoplankton abundance and up to 65% of annual primary production. Blooms of P. antarctica have been shown to dominate both in deep (Arrigo et al.
1999) and shallow mixed layers in the SO (Bodungen et al. 1986) illustrating the ability of this species to adapt to varying light regimes. Blooms of P. globosa in temperate waters dominate in late spring in the aftermath of the diatom spring bloom following silica depletion and it has therefore been argued that diatoms out-compete Phaeocystis in the presence of high silica concentrations. However, the dominance of P. antarctica in the presence of high silica concentrations in field observations especially from the SO do not support this scenario (Smetacek et al. 2004). Iron availability and food web structure seem to be more critical factors in determining dominance of Phaeocystis in phytoplankton blooms. Relatively few species play such crucial roles in the trophic structure and biogeochemical cycles of the SO (Smetacek et al. 2004). Its peculiar physiology can profoundly influence tropho-dynamics, community composition and biogeochemical carbon and sulphur cycles in the Southern Ocean (Davidson & Marchant 1992). P. antarctica shows a much higher drawdown of carbon dioxide and nitrate per mole of phosphate and rate of new production than do diatoms (Arrigo et al. 1999). P. antarctica is known to be a major producer of dimethylsulphonioproprionate (DMSP), which is known not only to act possibly as cryoprotectant in algae (Kirst et al. 1991, Stefels 2000), to serve as an antioxidant system (Sunda et al. 2002) or possibly to maintain intracellular osmotic pressure (Vairavamurthy et al. 1985), but also to deter herbivores (Wolfe et al. 1997). In addition it is the precursor of volatile dimethylsulfide (DMS) that oxidises to sulphuric acid in the atmosphere and functions as a cloud condensation nuclei for cloud formation and thus impacts global albedo (Charlson et al. 1987).
The distribution of P. antarctica varies throughout the different regions in the SO. Whereas P.
antarctica blooms occur regularly in the Ross Sea gyre, in the Weddell Sea gyre they tend to be sporadic (Arrigo et al. 1999). One reason could be because of different grazer communities within these gyres (Smetacek et al. 2004). The optimal prey size can differ considerably between proto- and metazoan grazers and Long et al. (2007) have shown that chemical cues associated with certain grazers induced consumer-specific morphological changes in the bloom-forming species P. globosa. Chemical cues from grazing ciliates enhanced colony formation by >25%, whereas chemical cues from grazing copepods suppressed colony formation by 60-90%. So depending on the grazer present, P. globosa is able to alternate between colonial and single stage and illustrates the plasticity of this species to respond to changes in the structure of the zooplankton community and thereby reduce grazing-induced mortality. Their colony skin is also an effective defence against a wide range of grazers, such as protozoa. The colony surface is a tough outer coating composed of protein-carbohydrate complexes. It is highly permeable to ions and dissolved molecules of a size up to 2nm diameter, but impervious to viral attack (Hamm et al. 1999, Brussard et al. 1999, Smetacek et al. 2004). In addition early colonial stages and attached aggregates are often found adhered to setae or spines of diatoms that provide protection against potential grazers.
Morphological features of Phaeocystis
Colonial cells and two types of flagellates constitute the three morphotypes that have been described for P. antarctica (Rousseau et al. 2007). Colonial cells are assumed to be diploid and the cells are evenly distributed around the periphery of colonies (Vaulot et al. 1994, Rousseau et al. 2007). The flagellates, one bearing scales and producing filaments and stars, the other devoid of scales and not producing filaments or stars are assumed to have different ploidy levels. Whereas the scaly flagellate is assumed to be haploid, the naked one is assumed to be diploid. Despite P. antarctica´s pivotal role in pelagic ecosystems, little is known about its life cycle and sexual reproduction. The existence of a haploid-diploid lifecycle is well-supported for P. globosa and should possibly exist within the other colonial Phaeocystis species as for example in P. antarctica (Rousseau et al. 2007, Medlin and Zingone 2007), because haploid-diploid life cycles are common in most other haptophytes in the class Prymnesiophyceae (Edvardsen et al. 2000). Rousseau et al. (2007) speculated that the naked flagellate type of P. antarctica (without scales, filaments and stars and diploid) is formed and
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released by large colonies and subsequently attached to the spines of diatoms where it again forms new colonies in a vegetative cycle.
New cell stage in P. antarctica proposed
In this paper, we illustrate a fourth morphotype in the life cycle of Phaeocystis: the AAs (attached aggregates), which develops after the fusion of two scaled haploid flagellates forming a zygote. We assemble and synthesize data from various sources to hypothesize how this cell stage fits into the life cycle of P. antarctica as part of a sexual cycle.
Material and Methods Field material
The composition of the plankton community during the European Iron Fertilization Experiment (EIFEX), conducted in austral summer 2004 in the Polar Frontal Zone of the SO, was assessed by inverted light and epifluorescence microscopy (Axiovert 135 and 200, Zeiss, Oberkochen, Germany) both inside and outside the fertilized patch. Light micrographs were taken with a Zeiss AxioCam digital camera. Three cell types of P. antarctica were differentiated during routine counting of plankton samples: the solitary flagellate stage, large free-floating colonies and small compact epiphytic colonies believed to be derived from AAs.
Solitary flagellates and epiphytic colonies were counted in water samples of 200 ml obtained from Niskin bottles attached to a Conductivity Temperature Depth (CTD) rosette from 7 depths between 10 and 150 m. One set of samples was preserved with hexamine-buffered formaldehyde solution at a final concentration of 2% and one set with acid Lugol’s solution at a final concentration of 4%. Large free-floating colonies were concentrated by pouring the total content of two Niskin bottles (24 L) gently through 10-μm mesh net to a volume of 50 ml.
Culture material
Six rough cultures that contained free-floating colonies of Phaeocystis antarctica were collected from the sea ice or water column in Prydz Bay or near-shore waters off Davis Station, Antarctica between 1985 and 1990 and transferred into GP5 media (Loeblich &
Smith, 1968, Loeblich 1975). These were maintained under cool white fluorescent light on a 12h:12h light:dark cycle at around 2 ± 2°C and returned to the laboratory in Australia aboard the Research vessel Aurora Australis. Upon return to Australia, cultures were maintained in
laboratory culture at the Australian Antarctic Division (AAD) under the same conditions and individual colonies re-isolated into separate wells of a 36-well polystyrene culture plate (Linbro®) containing fresh GP5 medium.
In this study we investigated three cultures of these six (Table 2.1):
- DE12.2, showing colonies and colonial cells, both diploid, - T4.3. haploid flagellates, and
- KACTAS_A, supposed to contain haploid flagellates and diploid attached aggregates.
During EIFEX (February, 2004), Chains of the diatom Chaetoceros, to which Phaeocystis aggregates were adhered, were isolated into fresh F/2 medium (Guillard & Ryther 1962), cultured under cool white fluorescent light on a 16h:8h light:dark cycle at 4°C and new free-floating Phaeocystis colonies appeared.
Four strains (MB2-A6, MM3-B6, MM19-D3 and MM23-C4) were unintentionally isolated along with Chaetoceros chains whereas one of them was directly isolated (MM26-B1). All other P. antarctica cultures (MM-E1-B4, MM-E1-B5, MME1-B6, MM-E2-B5, MME2-C1, MM-E3-C3, MM-E4-B4, MM-E5-A4 and MM-E5-A5) were obtained by the serial dilution technique (Andersen P. & Throndsen J. 2003).
During the ANT XXII/4 cruise conducted in April 2005 cells of the diatom Corethron pennatum and Chaetoceros chains to which Phaeocystis aggregates were adhered were isolated into fresh GP5 media, cultured under cool white fluorescent light on a 12h:12h light:dark cycle at around 2°C and some new free-floating Phaeocystis colonial and some AA´s cultures appeared. Afterwards they were isolated several times to establish clonal cultures
Flow Cytometry
Three AAD strains (DE12.2, KACTAS_A A, and T4.3) were analyzed by flow cytometry (FC, FACSCalibur, Beckton Dickinson, USA) with respect to their ploidy level after specific double-stranded (ds)-DNA staining. Dye working solution of SybrGreen I (Invitrogen) was prepared freshly by diluting the stock solution from the supplier (10000x) 1:10 with DMSO (Sigma #D8418), followed by a 1:100 dilution with the sample (final dilution 10-3, final concentration 10x resp.). After 15 min of incubation in the dark the samples were analyzed by FC. The instrument was equipped with an air-cooled argon laser (15 mW, Ex. 488 nm). The
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samples where analyzed by visual inspection of 2D- dot plots (FL1 (green emission) vs FL3 (red emission) using the standard filter setup. Analyses were performed at the lowest flow rate (approx.12μl min-1, event range ~350 - 800 sec-1). Events were collected as logarithmic signals using 1024 channels. Manual gating was used to separate the respective Phaeocystis cells from the background noise and from bacteria followed by histogram analyses of the green ds-DNA fluorescence. Data were stored as list-mode files and subsequently analyzed with CellQuest (Version 3.3) and WinMDI (version 2.9; J. Trotter, The Scripps Institute, Flow Cytometry Core Facility, La Jolla, CA). Yellow-green fluorescent latex beads (0.94 μm diameter, Polyscience, USA) were used for intercalibration between different samples and served also as a reference for normalization of cellular optical properties. TruCount beads (Beckton Dickinson) were used for intercalibration and absolute volume calculation. Beads doublets and triplets were neglected during the analyses. For further details see del Giorgio et al. (1996) and Gasol and del Giorgio (2000).
Molecular methods - Genetic identification
PCRs were performed from single attached aggregates found in three cultured P. antarctica strains (KACTAS_A and T4.3 already used for FC, but for strain T4.3 an older culture was used where AAs were found) obtained by the AAD and three strains collected during EIFEX (see Table 2.1). AAs from each strain were added to 5μl 10x PCR buffer (15 μl) and served directly as template in the following PCR reaction. The ITS1 regions were amplified as described in Lange et al. (2002) with the following modifications: it was not necessary to add 10 μl of 50% acetamide (Sigma) to the PCR reaction. The amplifications of the ITS1 region were performed in a thermocycler (Eppendorf) with an initial denaturation step of 95 °C followed by 29 cycles of 72 °C for 4 min, 94 °C for 2 min, 45 °C for 2 min and a final extension step at 72 °C for 9 min. Sequences were aligned to the ITS alignment produced by Lange et al. (2002) and maintained in a database using the ARB program. Using all available ITS sequences from four Phaeocystis spp., we performed a DNA ML analysis within ARB using P. jahnii as an outgroup. This dataset was exported to PAUP (Swofford 2003) and a bootstrap analysis using 100 replicates in a weighted maximum parsimony analysis was performed. The data were reweighted in PAUP using a re-scaled consistency index.
Results Field material
AAs were observed during three Southern Ocean cruises, two of which were iron fertilization experiments: EisenEx in austral spring 2000 (data not shown) and EIFEX in austral summer 2004, and again during an austral autumn cruise in 2005 (ANT XXII/4, Isolation of P.
antarctica) in the Antarctic Peninsula region. The EisenEx/EIFEX experiments investigated the development of natural plankton assemblages following fertilization of the water column with iron. Both iron fertilization experiments induced phytoplankton blooms that were dominated by diatoms. Albeit being second in importance to the diatoms, both solitary flagellates and colonies of P. antarctica showed a marked response to the iron addition (Assmy et al. 2007, Assmy et al. in prep.). Three stages of P. antarctica were observed during both experiments: the solitary flagellate stage, large free-floating colonies and AAs. AAs of P.
antarctica bounded by an envelope were only found adhering to diatoms, especially to the setae of large Chaetoceros or spines of Corethron species respectively (Fig. 2.1). The abundance of free-floating colonies and AA cell stages appeared to increase simultaneously.
Fig. 2.2 shows the increase of AAs attached to diatoms in the iron fertilized area (in-patch), as well as its occurrence outside this area (out-patch) and the occurrence of flagellates and free-floating colonies over time (Fig. 2.2b, 2.2c). The temporal trend of these epiphytic AAs both inside and outside the patch closely followed those of Chaetoceros dichaeta and C. atlanticus (data not shown) to which they were mainly attached. The collapse of these two diatom species during the second half of the experiment is reflected in the concomitant decline of epiphytic AA colonies.
Culture material
Over the two weeks following isolation the isolated colonies of P. antarctica, obtained from the Australian Antarctic Division, adhered , to the surface of the culture well and fragmented to form small AAs that commonly contained 1, 2, 4, 8, or 16 cells (Fig. 2.3). Cells in small aggregates were frequently large (around 10 μm diameter) (Fig. 2.3i) containing from 2 to 5 plastids and a large nucleus. Interstitial spaces occurred between cells and the aggregates were surrounded by a tough but sticky skin (Figs. 2.3g-i). As AAs grew, the mean cell size declined to around 6.5 ± 1.5 μm diameter, whereas interstitial spaces between cells and the mucilaginous envelope disappeared (Figs. 2.3e-f). Following the initial formation of the AA stages, they spread over the surface of the culture flask. Further development of the AA stages
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occurred as the cell aggregates enlarged into discs that eventually intersected with one another until the entire surface of the culture vessel was covered with a brown mass of cells (Figs.2.3a-b). This could be considered a palmelloid stage,which is directly comparable in ploidy level to the free-floating colonial stage.
After free-floating colonies disappeared from the culture suspension, only AAs and microzoospores were observed, suggesting that these two cell stages were inter-changeable.
These microzoospores might be the naked flagellates mentioned by Rousseau et al. (2007).
They were not examined in electron microscopy. It cannot be ruled out that other life stages of low abundance in cultures may have been overlooked. However, simultaneous colonisation of the entire surface of the culture flask suggests that AAs were proliferated by a motile cell stage. At the time of proliferation, only motile or AAs cells 3.0 ± 0.3 μm in diameter were observed in the culture medium. The process of alternation between these life stages is hypothesized below (see Fig. 2.5 in section ploidy level).
AAs persisted in culture for > 3 years, which equals to 40 culture transfers into glass culture vessels in which 50 ml of fresh GP5 medium was inoculated with approximately 1 ml of parental culture. However, similar to field observations, free-floating colonies did eventually reappear in some cultures. The mechanism of this transition to free-floating, bloom-forming colonies was not directly observed but is presumed to be by mitosis because of the similarity in ploidy level of the two stages (see below).
Flow cytometric analyses of AAD strains
Three different strains were analyzed by FC (Fig. 2.4 and Table 2.2). The event frequency distribution of green ds-DNA fluorescence (Fig. 2.4) clearly revealed that the culture of DE12.2 contained only diploid cells (dashed line) as already microscopically observed whereas mainly haploid flagellates where found in T4.3 (dotted). Based on this particular event distribution patterns it turned out that KACTAS_A (solid line) showed a few haploid cells and a majority of diploid cells. It should be noted that the number of events per culture was normalized to the respective maximum counts for better comparison. The optical properties of KACTAS_A cells were also clearly different from T4.3 cells and more similar to DE12.2 cells in terms of size (Forward Scatter) and inner cellular complexity (Side Scatter).
Beside high green fluorescence both cultures, DE12.2 and KACTAS_A, contained relatively large cells which showed high inner granularity. In contrast, the haploid micro-zoospores of T4.3 were 2 to 3 times smaller and showed lower granularity and DNA fluorescence.
Ploidy level – microscopic observations
During microscopy we observed the formation of eight flagellates from an attached aggregate/zygote (Figs. 2.5a to 2.5d) in a sample of strain KACTAS_A from AAD. Four appeared sequentially and were photographed leaving an additional four cells within the AA that were not released.
Molecular methods - Genetic identification
Using partial DNA 18S data, strains obtained during EIFEX belong to the P. antarctica clade (data not shown). We performed a sequence analysis of ITS1 from single AA`s and from aliquots taken from cultures to resolve the relationship of these strains obtained by the AAD among other P. antarctica clades (see Fig. 2.6). Our ML analysis recovered two clades, which diverged after the outgroup (P. jahnii). One clade corresponded to the warm water species, Phaeocystis globosa, whereas the other corresponded to the cold water clade and contained both P. antarctica and P. pouchetii. In the Antarctica clade, sequence data from a single colony was not identical to an aliquot taken from the culture, suggesting that either the culture was not clonal or there were multiple copies of the ITS that could be preferentially amplified in a mixed assemblage. In the ML analysis, P. pouchetii was seen to arise from within P.
antarctica and there was not a clear separation of the two polar species as seen with the 18S and earlier ITS1 analyses (Lange et al., 2002). Here P. pouchetii is seen to be most closely related to the three AAD strains, and it is presumed that isolates from Prydz Bay gave rise to the Arctic species. In the weighted MP analysis, P. pouchetii was sister to all of the P.
antarctica strains as shown in Lange et al. (2002). Notice that within each clade the groupings of sister taxa were not all in the same in the two analyses.
Discussion
To our knowledge this is the first report of attached aggregates (AAs) from nature referring to it as a fourth morphotype within the life cycle of P. antarctica. Morphological similarities between attached aggregates in AAD cultures, previously believed to be culture artefacts, and those collected in the field samples (Fig. 2.1 & 2.3) further supported the significance of this new cell stage within the life cycle of P. antarctica. Our findings add a new morphotype to the three already described by Rousseau et al. (2007): the colonial cells and the two types of flagellates.
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A short schematic introduction of our proposed life cycle is shown in Fig. 2.7. We differentiate two phases: haploid and diploid, occurring in four different morphotypes: a) haploid flagellate bearing scales and filaments, b) diploid flagellate devoid of scales and filaments, c) diploid colonial cells and diploid attached aggregates (AAs).
The AA´s, as a proposed zygote, can follow two pathways. First, they can form new colonies through mitosis, or they can undergo meiotic division to form haploid flagellates with scales.
These flagellates are the haploid stage within the haplo-diploid life cycle and can reproduce mitotically or they can function as micro-zoospores (gametes) to reform the AA cell stage.
Thus, the AAs are a pivotal stage in the life cycle linking the diploid and haploid phases and represent the missing link in earlier work on life cycles in this genus.
We propose that the AAs represent the missing zygote stage in the life cycle of P. antarctica, which would support the existence of a haploid-diploid life cycle as observed in P. globosa and other haptophytes. Morphological differences between AAs and known life stages of Phaeocystis were likely overlooked in previous studies because field observations could misidentify or be unable to identify such AAs unless seen attached to diatoms as observed in this study. However, small groups of cells, presumably belonging to Phaeocystis, attached to diatoms in the field have been reported in the literature (Noethig 1988, Fryxell 1989, Garrison and Thomsen 1993, and Marchant and Thomsen 1994). The only previous report of a benthic, non-flagellate cell stage of Phaeocystis was by Kayser (1970). However, he reported that this life stage was solitary. Furthermore, Whipple et al. (2005) noted that this cell stage had not been observed since Kayser’s study and Rousseau et al. (1994) indicated that there was no evidence of a differentiated benthic stage in Phaeocystis.
AAs of P. antarctica were clearly derived from free-floating, bloom-forming colonies in cultured material, which were started from a single colony. During early development, AAs maintained considerable similarities to the colony stage. Individual non-flagellate cells were loosely distributed within a mucilaginous envelope. The only other life stage found to co-occur in culture with AAs was the characteristic Phaeocystis micro-zoospores (Kornmann 1955, Rousseau et al. 1994, Whipple et al. 2005). Valout et al. (1994) suggested that the presence of haploid micro-zoospores was strong evidence for sexual reproduction in the life cycle of Phaeocystis. Though we did not observe syngamy of micro-zoospores, the absence of other cell stages in the cultures containing only AAs and micro-zoospores suggest that AAs were proliferated by syngamy of haploid gametes. Therefore it is reasonable to suggest that the haploid micro-zoospores (gametes) fused and formed the new AA cell stage. Also
significant is that, in a culture only containing the two cell stages mentioned above, we observed the formation and release of eight flagellates from an AA cell stage, suggesting that meiosis had occurred at this point in time and formed four cells, which then reproduced mitotically to form another four cells (Fig. 2.5). The optical analyses of cells by FC showed that AA´s had almost the same amount of DNA as colonies and that haploid stages were also fairly similar to each other. This confirms our suggestions regarding the ploidy level of the three AAD strains and implies that FC might be a useful tool in studying DNA-related physiological processes of cells in culture and probably also in situ. We suggest that the flagellated micro-zoospores are part of the sexual cycle and represent the haploid gametes with scales, whereas the naked flagellated cells are part of diploid cycle and may reform the colonial stage or reproduce mitotically themselves. Garrison and Thomsen (1993) and Marchant and Thomsen (1994) reported that the naked zoospores, which are believed to be diploid, attached to the spines and processes of large diatoms like Corethron within 5-6 hours after release from the colony, where they subsequently formed new colonies (Fryxell 1989).
We suggest that upon attachment they form the AA from an asexual stage. Thus the AAs link the haploid micro-zoospore stage with the diploid colonial stage with its diploid zoospores.
Zygotes are known to form the over-wintering life stages as cysts in the Dinophyceae and akinetes in Pithophoraceae. Therefore, it is possible the proposed zygote of P. antarctica may perform a similar ecological function. The nature of an overwintering form and colony-forming cell of Phaeocystis, as well as the factors triggering the transition between free-living and colonial stages, is unknown (Rousseau et al. 2007). Furthermore, the occurrence of these AAs during the entire austral growth season (spring until autumn), together with the observed increase in abundance during an iron-induced diatom bloom (see Fig. 2.2), suggests that it may have other functions as well. It may also represent an intermediate stage between the solitary life stage and the free-floating colonies.
Small P. antarctica AAs occur as an epiphytic cell stage adhering to large, well armoured diatoms, such as Corethron and Chaetoceros, which provide protection against a range of smaller grazers, in particular ciliates (Fig. 2.1). Hart (1942) reported that Corethron is often associated with Phaeocystis close to the marginal ice zone, and that both species multiply rapidly when liberated from the ice in the summer. Thus, it is possible that P. antarctica blooms are enhanced by intimate association of attached aggregates with grazer-resistant diatoms. The frustules of large, heavily silicified diatoms provide mechanical protection against crustacean grazers (Hamm et al. 2003, Smetacek 2001), and their long setae and spines deter smaller protistan grazers. The AAs thus find protection on these large diatoms
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against a whole range of predators and thereby efficiently reduce grazer-induced mortality.
Similar to the free-floating colonial stage, formation of disc-like AAs may also provide protection against grazers by encasing cells in tough outer coatings (Hamm et al. 1999, Smetacek et al, 2004). Furthermore, adhesion of this cell stage to diatoms or to the under-surface of sea ice in autumn and release of colonies or micro-zoospores in austral spring would enable rapid colonisation of the water column. Such colonisation could explain why this species is commonly the first to bloom in Antarctic waters (Davidson & Marchant 1992), despite our observation that it was absent from the water column in early autumn time during ANT XXII/4, 2005.
To summarize, it appears that we have found a previously undescribed cell stage in the life cycle of P. antarctica. This cell stage may also occur in the life cycle of other Phaeocystis species because van Breemen (1905) observed colonies of P. globosa being produced by cells attached to Chaetoceros willei. Our evidence from cultures suggest that AAs are zygotes, thus documenting the sexual cycle in P. antarctica and with the two types of micro-zoospores present in this species, it is also likely that this species possesses the haploid-diploid life cycle typical of other haptophytes except that the haploid stage always occurs in the flagellate stage and the diploid stage can occur in both the flagellate and the colonial stage. The ecological function of the zygote either as a refuge from grazers, or as an overwintering stage, is unspecified at this time. Further studies are required to determine the significance of AAs on the ecology of P. antarctica.
Acknowledgements
This research was founded by the German Science Foundation (DFG) through a postgraduate research fellowship (ME 1480/2). Research at the AAD was supported by the Australian government’s Cooperative Research Centres Programme through the Antarctic Climate and Ecosystems Cooperative Research Centre (ACE CRC) and the Australian Antarctic Science program (project 40). PA was supported by the Bremen International Graduate School for Marine Sciences (GLOMAR) that is funded by the German Research Foundation (DFG) within the frame of the Excellence Initiative by the German federal and state governments to promote science and research at German universities.
We also like to thank Dr. B. Beszteri for his comments and input to the manuscript.
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