The life cycle of Phaeocystis state of knowledge and presumptive role in ecology

Marie-Josèphe Chrétiennot-Dinet •

Anita Jacobsen • Peter Verity • Stuart Whipple

Received: 9 March 2006 / Accepted: 19 May 2006 / Published online: 13 April 2007 © Springer Science+Business Media, B.V. 2007

Abstract Despite numerous investigations, the number and role of morphotypes involved in the life cycle of Phaeocystis species remain under debate. This is partly due to the application of different methodologies such as light, transmission, scanning electron microscopy and flow cytometry on specific samples. This heterogeneity of approaches results in the incomplete morpho-metric description of the different cell types existing within one species according to relevant criteria and the indetermination of the ploidy level of each observed stage. We review here the different morphotypes observed within each of the six Phaeocystis species recognized up to now. Four different cell types have been observed. In common to all six species is the occurrence of a

Ecologie des Systèmes Aquatiques, ULB, CP 221, Boulevard du Triomphe, 1050 Brussels, Belgium e-mail: [email protected]

M.-J. Chrétiennot-Dinet

Observatoire Biologique de Banyuls, UMR 7621, Lab. Arago, BP 44, 66651 Banyuls-sur-Mer, France

A. Jacobsen

Department of Biology, University of Bergen, PO Box 7800, 5020 Bergen, Norway

Skidaway Institute of Oceanography,

10 Ocean Science Circle, Savannah, GA, 31411, USA

scaly flagellate producing star-forming filaments (all species except P. jahnii) or not (P. globosa and P. jahnii). In three colony-forming species, P. globosa, P. pouchetii and P. antarctica, three morphotypes are observed: a flagellate with scales and filaments, a colonial cell, and a flagellate devoid of scales and filaments. In the non-colony-forming species, P. scrobiculata and P. cordata, only flagellates with scales and filaments have been observed. While suspected in P. pouchetii and P. antarctica, a haploid-diploid life cycle has only been evidenced for P. globosa. The two main prominent features of this cycle are that sexuality is prevalent in colony bloom formation and termination and that two types of vegetative reproduction exist. The ecological relevance of alternating haploid and diploid stages is not clearly apparent on the basis of existing ecological studies.

Keywords Ecological niche • Haploid-diploid • Life cycle stages • Morphotype • Phaeocystis species • Sexual processes

Introduction

The genus Phaeocystis is a worldwide colony-blooming species that has a significant role in bio-geochemical cycles (Schoemann et al. 2005) including the global sulphur cycle (Liss et al. 1994). Despite numerous investigations devoted to its ecophysiology and the role and impact of colonies in ecosystem processes, knowledge of some major biological features of the genus is still limited. Such is the case for the Phaeocystis life cycle and its controlling mechanisms that, 50 years after Kornmann's (1955) classic paper, remain under debate. Problems of taxonomic confusion, lack of fine morphometric description of the different cell types within one species, and inadequacy of cell nomenclature have precluded a complete understanding of the Phaeocystis life cycle.

Since early description of the genus Phaeocys-tis by Lagerheim in 1893, the number of inclusive species has long been a matter of discussion (e.g. Kornmann 1955; Kashkin 1963; Parke et al. 1971; Sournia 1988; Medlin et al. 1994). This is mainly because the criteria used to distinguish Phaeocys-tis species were based on phenotypic characters such as the morphometry of the colonial stage, and/or physiological and biochemical properties (Jahnke and Baumann 1987; Baumann et al. 1994; Vaulot et al. 1994). Six species are now recognized based on small subunit (SSU) rDNA sequence analysis and morphological characterization. These are: P. antarctica Karsten, P. globosa Scherffel, P. pouchetii (Hariot) Lagerheim, P. jahnii Zingone, P. scrobiculata Moestrup and P. cordata Zingone et Chrétiennot-Dinet (Moestrup 1979; Medlin et al. 1994; Zingone et al. 1999; Edvardsen et al. 2000; Lange et al. 2002). Colonial forms have been reported for the first four species. It is now considered that probably more than six Phaeocystis species exist (Lange et al. 2002; Medlin and Zingone this issue).

Comparative descriptions of cell types existing within one species using morphometric criteria, i.e., presence/absence of body scales, flagella, haptonema and star-forming filaments, and ploidy levels have been made (e.g. Zingone et al. 1999; Peperzak etal. 2000a). However, a complete study of all morphotypes occurring within one species is still missing (Lancelot and Rousseau 2002). Our current knowledge of Phaeocystis cell types relies on composite independent investigations combining light (LM), transmission (TEM) and scanning electron microscopy (SEM) as well as flow cytometry. Each of these methodologies provides part of the information needed for a complete identification of the morphotype. LM is useful for observations of cell shape, size, number, presence of flagella, and swimming activity. SEM and TEM with higher resolution and magnification are needed for morphological and ultrastructural details of the cell covering, appendages and organelles. Flow cytometry is required for determining the ploidy levels of each cell type. In addition, it is essential that sample preservation and fixation procedures be fully described because such procedures may lead to methodological biases. Use of fixatives can indeed cause cell shrinkage, loss of appendages (Peperzak et al. 2000a; Wassmann et al. 2005) or colony disintegration, releasing colonial cells into the medium, and therefore lead to possible misinterpretation (Wassmann et al. 2005). This mixed approach results in a confuse nomenclature of the various cell types, i.e. solitary flagellates and nonflagel-lates, free-living single cells, colonial flagellates, motile free-living cells, swarmers, zoids, microfla-gellates and microzoospores. These terms are often used loosely, and this can lead to misinterpretation of life cycle events.

The number and role of cell types involved in the life cycle of the six Phaeocystis species, and whether these are the same within each species, are still among the main questions not yet resolved (Lancelot and Rousseau 2002). Of particular interest is the identification of the stage persisting between two colony bloom events, as well as the nature of colony-forming cells. The persistence of Phaeocystis as a flagellate between two colony blooms has been suggested (Kornmann 1955; Parke et al. 1971; Veldhuis et al. 1986; Verity et al. 1988b), but the type of flagellate was never described from field observations due to its low cell density and possible confusion with other nanoplanktonic species. On the other hand, senescent colonies or aggregates have also been proposed as over-wintering forms of P. globosa (Cadee 1991). Still unknown are factors responsible for the transition between life stages. The ecological significance of the different life cycle stages, flagellates and colonies, has recently been discussed by Verity and Medlin (2003). Further investigation is however needed to discriminate between the different flagellates that have been identified within some species.

In this paper, we review and synthesize the available information gained from field and culture observations on the different morphotypes occurring within each of the six Phaeocystis species, focusing on cell morphology and ploidy level. We also present unpublished data on the morphology of P. globosa and P. antarctica cells. The role of the different morphotypes within the life cycle will be addressed based on field and culture observations on their occurrence. Finally, the ecological relevance of the free-living and colonial stages will be discussed based on knowledge of physiology and trophic significance of the various morphotypes. Because it is the best-known species, P. globosa will be used as a model.

Morphotypes among Phaeocystis species

This section reviews the different cell types reported for the six Phaeocystis species which are considered here according to their revised taxo-nomic status as recommended by Baumann et al. (1994), Medlin et al. (1994) and Vaulot etal. (1994). This is particularly relevant for the species globosa, which has long been referred to as pouchetii in the previous literature (e.g., Parke etal. 1971; Kayser 1970; Admiraal and Venekamp 1986; Veldhuis etal. 1991; Davidson and Marchant 1992a). The seasonal distribution of the different cell types in the natural environment will also be considered in order to assess their role in the life cycle.

Morphotypes of P. globosa

A careful analysis of the literature published since the first description of P. globosa cells by Scherffel (1899, 1900) suggests that four morphotypes exist: diploid colonial cells, diploid flagellates, and two types of haploid flagellates.

Colonial cells

Colonial cells have 2-4 parietal chloroplasts, are deprived of body scales, haptonema, and flagella and are embedded in a mucilaginous matrix (Scherffel 1899; Kornmann 1955). They possess on their flagellar pole two short appendages, the role and nature of which being presently unknown (Fig. 1; Rousseau et al. submitted). Reported size ranges for live colonial cells are 4.5-8.0 |m (Kornmann 1955) and 5.8-10.4 |m (Rousseau et al. submitted) while they are 5.68.3 |m (Peperzak et al. 2000a) and 4.6-7.8 |m (Rousseau et al. submitted) for Lugol and Lugol-glutaraldehyde fixed cells. These cells are diploid (Cariou et al. 1994; Vaulot et al. 1994). They are evenly distributed in the colony 15-20 |m beneath a thin skin, and are weakly interconnected with dilute gel (Kornmann 1955; van Rijs-sel et al. 1997; Hamm et al. 1999). The colony skin is strong and semipermeable with pore size 1.04.4 nm in diameter, and presents plastic and elastic properties (Hamm et al. 1999). Diameter of colonies typically ranges from 10 |m to 8-9 mm (Kornmann 1955; Jahnke and Baumann 1987; Rousseau et al. 1990), but may occasionally reach 20-30 mm (Kayser 1970; Gieskes and Kraay 1975; Chen et al. 2002). Colonies are originally spherical but deviate into nonspherical shapes when growing larger or when subjected to hydrodynam-ical stress (Kornmann 1955; Bätje and Michaelis 1986; Rousseau et al. 1994).

Non-motile free-living cells from colonial origin have also been reported in P. globosa cultures. These cells are morphologically similar to colonial cells: same size range, lack of flagella, haptonema (Kornmann 1955; Rousseau et al. 1990; 1994; Peperzak 1993; Peperzak et al. 2000a; Dutz and Koski 2006), body scales (Peperzak et al. 2000a), thread-like filaments and stars (Peperzak et al. 2000a; Dutz and Koski 2006) and were assumed to have the same ploidy level, i.e., diploidy (Peperzak et al. 2000a). On this basis, non-motile free-living and colonial cells should not be considered anymore as distinct morphotypes (Peperzak et al. 2000a; Dutz and Koski 2006).

Haplo id flagellates

The fine structure of the flagellate stage of P. globosa was first described as P. pouchetii from TEM by Parke et al. (1971). These cells have been reported as swarmers (Scherffel 1900), micro-zoospores (Kornmann 1955), small and large zoids (Parke et al. 1971), microflagellates

Fig. 1 SEM photographs of P. globosa cells observed in the Belgian coastal waters. Small-sized Xagellates with stars and Wlaments but no scales observed (A) in February during the pre-bloom period; bar = 3 ^m and (B) in May still embedded within the colony mucus at the end of the colonial stage; bar = 2 ^m; colonial cells with the typical short appendages observed (C) in the early stage of the bloom within Chaetoceros setae; bar = 4 ^m and (D) at the end of the bloom just before formation of haploid Xagellates; bar = 2.4 ^m. H: hapto-nema; Fl: Xagella; S: star; F: Wlament; M: mucus; A: short appendage; Ch: Chaetoceros cell"

(Veldhuis et al. 1986) and micro- and mesoflagel-lates (Peperzak et al. 2000a). They have a rounded shape, and are smaller than colonial cells, with a diameter of 3-5 ^m (Kornmann 1955); 3-6 ^m (Parke et al. 1971) and 3.6-5.8 ^m (Rousseau et al. submitted) for live cells observed under LM. Their small size has long been used for distinguishing them from other P. globosa cell types (e.g. Veldhuis et al. 1986) before Vaulot et al. (1994), using flow cytometry, demonstrated they were the haploid stage of P. globosa.

These flagellates are capable of rapid vegetative reproduction (Kornmann 1955; Parke et al. 1971; Rousseau et al. 1994; Vaulot et al. 1994) and swim very actively (Kornmann 1955; Parke et al. 1971). They possess two equal heterody-namic flagella, 10-15 ^m in length, and a short haptonema (3-4 ^m) characterized by a distal swelling. They present an anterior depression and two golden-brown plastids (Parke et al. 1971). The cell body is covered by two types of organic scales showing a pattern of radiating ridges, visible on both sides (Parke et al. 1971;

Peperzak etal. 2000a). Two types of haploid flagellates were distinguished by Parke et al. (1971). One type, 3-6 ^m in size, possesses two superficial, bright vesicles located on the body surface (Parke et al. 1971). These vesicles release filaments, 20 ^m in length, 0.05 ^m in diameter, and made of alpha-chitin (Chretiennot-Dinet et al. 1997), which form a highly characteristic pentagonal star (Parke et al. 1971). The function of these filaments is unknown. It has been hypothesized that they could act as anchors for attachment to solid structures before colony initiation (Chretiennot-Dinet 1999), or alternatively have a role in defense against grazers (Peperzak et al. 2000a; Dutz and Koski 2006). The other haploid flagellate, 3-5 ^m in size, lacks the vesicles and filaments (Parke et al. 1971; Peperzak et al. 2000a). Such a difference in size and ability to produce filaments has also been observed in field samples for cells described as zoids (Manton and Leadbeater 1974 cited in Peperzak et al. 2000a) or meso- and micro flagellates (Peperzak et al. 2000a).

These flagellates were observed in senescent cultures, swimming inside spherical colonies of various sizes, and were associated with colony disappearance (Kornmann 1955). In the natural environment and in mesocosms, large numbers of these flagellates were repeatedly reported, at the decline of P. globosa colony blooms, either inside spherical colonies (Scherffel 1899, 1900; Cadee 1991; Peperzak etal. 1998, 2000a), or released into the medium leaving ghost colonies or as free-living flagellates (Jones and Haq 1963; Admiraal and Venekamp 1986; Veldhuis et al. 1986; Verity et al. 1988b; Escaravage et al. 1995). These small-sized flagellates were observed invariably associated with the presence of chitinous filaments and stars, a feature specific to the haploid stage of P. globosa Scherffel (Vaulot et al. 1994; Zingone et al. 1999). Released from colonies at the end of the bloom, these flagellates were recorded at different periods of the year in the water column of the Channel and the Southern Bight of the North Sea, at cell densities varying from 80 x 103 cells L-1 to 220 x 103 cells L-1 (Fig. 1; Rousseau and Chretiennot-Dinet, unpublished data). They apparently represent the life stage persisting between two blooms of colonial cells, as suggested by Kornmann (1955) and Parke et al. (1971).

Diploid flagellates

The third morphotype of P. globosa is a flagellate of the same size range as colonial cells when observed live, i.e., 4.5-8.0 |m (Kornmann 1955), 6-7 |m (Peperzak et al. 2000a) and 6.1-9.3 |m (Rousseau et al. submitted) containing two flagella and one haptonema but lacking scales, filaments and stars (Rousseau et al. submitted). This flagellate has been reported as an asexual swarmer by Kornmann (1955), as a large flagellate by Cariou et al. (1994) and Rousseau et al. (1994), and as a macroflagellate by Peperzak et al. (2000a). It typically appears in cultures within 24 h when colonial cells are released mechanically from the colony (Kornmann 1955; Rousseau et al. 1990; Cariou et al. 1994). This flagellate is diploid as no ploidy change was found during the transformation of colonial cells into flagellates (Cariou et al. 1994; Rousseau et al. 1994).

These flagellates are able to rapidly form new colonies within a day after adhesion to a surface (Kornmann 1955; Kayser 1970; Cariou et al. 1994; Rousseau et al. 1994). Inanimate particles (Rousseau et al. 1994), walls of culture vessels (Kayser 1970; Cariou etal. 1994), and diatoms (weisse etal. 1986; Boalch 1987; Rousseau etal. 1994) have been observed as adhesion sites. This property of attachment to surfaces, specific to this life stage, led to the assumption that a benthic stage, acting as an overwintering form, exists in the natural environment (Kayser 1970). It is not clear if this flagellate is able to mitotically divide. Its rapid transformation into a colony suggests it is short-lived (Kornmann 1955; Rousseau et al. 1994), but vegetative multiplication was also reported by Kayser (1970). The short lifespan of this morphotype could well explain why it is observed only occasionally in the field (Kornmann 1955; Peperzak et al. 2000a; Rousseau et al. submitted).

Such flagellates were occasionally observed inside colonies in cultures by Kornmann (1955) who considered them as a distinct cell types, the macrozoospores, and in the natural environment (Peperzak et al. 2000a).

Morphotypes of P. pouchetii

At the present time, two P. pouchetii cell types have been confirmed based on EM observations and cytometric analysis: a diploid colonial cell and a diploid flagellate (Jacobsen 2002). However, other reports suggest that a larger flagellate could exist within the P. pouchetii life cycle (Sukhanova and Flint 2001; Wassmann et al. 2005).

Colonial cells

Diploid colonial cells are in the size range of 57 |m, have an anterior longitudinal groove and are deprived of filamentous appendages and scale coverings (Jacobsen 2002). Actively growing colonial cells are typically distributed in groups on lobes of cloud-like colonies (Jahnke and Baumann 1987; Gunkel 1988; Baumann et al. 1994; Rousseau et al. 1994; Jacobsen 2002). Phaeocystis pouchetii colonies, which are spherical up to 0.1 mm (Rousseau et al. 1994), have a maximum size of 1.5-2 mm (Jahnke and Baumann 1987). They are characterized by a delicate mucus which disrupts easily compared to the solid mucilage of P. globosa (van Rijssel et al. 1997; Jacobsen 2002; Wassmann et al. 2005). Non-motile free-living cells morphologically similar to colonial cells, i.e., deprived of flagella, haptonema, filamentous appendages, scales, can be found together with the colony stage due to colony disruption (Eilert-sen 1989; Jacobsen 2002; Wassmann et al. 2005).

Flagellates

A filament-bearing flagellate has been described in detail by Jacobsen et al. (1996) and Jacobsen (2002) on the basis of LM and TEM observations. This flagellate, which originates from a colony when brought into culture, is round and has an average diameter of 5 im when live. It has two golden-brown parietal chloroplasts, two hetero-dynamic equally long (11 |m) flagella, and a short non-coiling haptonema (1.5 |m). The cell body is covered by two types of scales, both with radiating ridges visible on both surfaces. The filaments are coiled inside one or two superficial vesicles, and form five-ray stars when unwound. Contrary to the filament-producing cell of P. globosa, this flagellate was assumed to be diploid (Jacobsen 2002). This flagellate was observed during winter, increasing in abundance prior to colonial development, and has been thought to be the precursor of the colonial stage (Jacobsen 2002; Jacobsen and Veldhuis 2005). Such flagellates were observed inside colonies at the end of spring blooms, being subsequently released in the water column (Jacobsen 2002).

From LM observation of field samples, two types of flagellates were reported within the P. pouchetii life cycle based on size criteria, i.e., large (6 |m; Sukhanova and Flint 2001; Wassmann et al. 2005) and small heart-shaped (34 |m; Wassmann et al. 2005) cells. The large flagellates co-occurred with colonies and were abundant, sometimes being dominant over colonial cells in terms of cell density (Sukhanova and Flint 2001; Wassmann et al. 2005). The small flagellates were found free-living (Wassmann et al. 2005) or within decaying colonies before their disappearance (Lagerheim 1896; Wassmann etal. 2005). They might well correspond to the flagellate described by Jacobsen (2002), with the size difference explained by cell shrinkage due to the use of Lugol-glutaraldehyde as sample fixative. Interestingly, small flagellate formation and their further release into the water column have been shown to originate from colonies assuming a spherical shape (Gunkel 1988; Whipple et al. this issue).

Morphotypes of P. antarctica

Three morphotypes have been observed in P. antarctica: colonial cells and two types of flagellates. Two ploidy levels have been recorded.

Colonial cells

The colonial cell of P. antarctica has no flagella, no haptonema, no scales, and no vesicles or star-forming filaments (Davidson 1985). This cell has an anterior depression and two short appendages similar to those observed on P. globosa colonial cells (Fig. 2; Chrétiennot-Dinet and Rousseau, unpublished). The size range reported for colonial cells of P. antarctica is quite large, depending on preservation and fixation procedures. Size range includes: 10 im for live cells (Moestrup and Larsen 1992), and 3.2-7.9 |m (Mathot etal. 2000), 4.7-5.6 |m (Vaulot etal. 1994), 4-6 |m (this study) for Lugol-gluraldehyde fixed cells. Colonial cells of P. antarctica, assumed to be dip-loid (Vaulot et al. 1994), are evenly distributed along the periphery of colonies characterized by a solid mucus. Field P. antarctica colonies are typically spherical but can include numerous morphs with a maximum size of 9 mm (Baumann et al. 1994; Marchant and Thomsen 1994).

Flagellates

One P. antarctica flagellate bears scales and produces filaments and stars; the other is devoid of scales, filaments and stars. The scale-bearing flagellate was described in detail by Davidson (1985): it has an anterior depression, two chlorop-lasts with a large central pyrenoid, and bears two flagella and an haptonema. Cell size ranges between 3.5 |m and 7 |m when fixed with

Fig. 2 SEM photographs of P. antarctica (strain CCMP 1871): (A) Colonial cells with four plastids and the two short appendages typical of the colonial cell on the flagellar pole; bar = 2 pm: (B) Flagellates co-occurring with colonies (diploid?) deprived of scales and filaments; bar = 6 pm. H: haptonema; Fl: flagella; A: short appendage

Fig. 2 SEM photographs of P. antarctica (strain CCMP 1871): (A) Colonial cells with four plastids and the two short appendages typical of the colonial cell on the flagellar pole; bar = 2 pm: (B) Flagellates co-occurring with colonies (diploid?) deprived of scales and filaments; bar = 6 pm. H: haptonema; Fl: flagella; A: short appendage

glutaraldehyde and observed with TEM. The two flagella are equally long (6-10 pm). The haptonema presents a bulbous tip and is 1.5-2 pm long. The body cell is covered by two types of scales with a pattern of radiating ridges visible on both sides. Thread-like material is regularly arranged within circular posterio-lateral vesicles. When deployed, the threads have a length of 25 pm and form a pentagonal star.

In the natural environment, such scale-bearing flagellates increase in number at the beginning of the colonial bloom, and decline during the bloom; they are present in large numbers after the bloom (Davidson and Marchant 1992b). Similar flagellates, that have a diameter of 5.2 pm when fixed with glutaraldehyde, two flagella of 14.3 pm in length, and filamentous appendages of 40 pm in length forming a pentagonal star, were found at the ice edge of the Weddell Sea during the austral summer (Buck and Garrison 1983). Flagellates with thread-like material were observed in the Bransfield Strait region during post-bloom period (Kang and Lee 1995). On the other hand, an increase in the abundance of small-sized free-living flagellates (2.4-5.5 pm when fixed with glutaraldehyde) was observed in the Ross Sea in early austral spring just before colony formation occurred (Mathot et al. 2000; Smith et al. 2003). Although no fine morphological description was provided, these small flagellates could well correspond to the flagellates described by Buck and Garrison (1983) and Davidson (1985), the size difference resulting from cell shrinkage and preparation for microscopic observations. Their relative abundance decreased to a minimum, while colonial cells dominated in late spring before they were observed again inside colonies (Smith et al. 2003) or as free-living cells (Putt et al. 1994) at the end of the bloom.

The second P. antarctica flagellate type has the same size as the colonial cell, ranging from 6.5 pm (Garrison and Thomsen 1993) to 7.5 pm (Fryxell 1989). This flagellate bears two flagella and a haptonema but lacks scales, filaments and stars (Fig. 2, Garrison and Thomsen 1993; Marchant and Thomsen 1994; Chretiennot-Dinet and Rousseau, unpublished). This flagellate was formed inside and released from spherical or elongated colonies at the ice edge during austral spring (Fryxell 1989; Garrison and Thomsen 1993; Marchant and Thomsen 1994). Some 5-6 h after formation, it was found attached to the spines and processes of large diatoms (Garrison and Thom-sen 1993; Marchant and Thomsen 1994) where it subsequently formed new colonies (Fryxell 1989).

Morphotypes of P. jahnii

Two cell types have been described for P. jahnii: colonial cells and flagellates (Zingone et al. 1999).

Colonial cells

Phaeocystis jahnii colonial cells possess the two short appendages typical of P. globosa and P. antarctica colonial cells, have a size of 6-8.5 pm when live and are irregularly distributed in loose, irregular colonies. Non-motile free-living cells of the same size have also been reported (Zingone et al. 1999).

Table 1 Different morphotypes reported for the six Phaeocystis species recognized to date

Measured or assumed ploidy levels are indicated — not observed

Flagellates Flagellates Flagellates without scales, Colonial with scales, with scales threads and stars Same size cells threads and stars range as colonial cells

P. globosa P. pouchetii P. antarctica P. jahnii P. cordata P. scrobiculata

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