Review Paper

A taxonomic review of the genus Phaeocystis

Linda Medlin • Adriana Zingone

Received: 20 October 2005 / Accepted: 22 August 2006 / Published online: 15 March 2007 © Springer Science+Business Media B.V. 2007

Abstract Phaeocystis is recognized both as a nuisance and as an ecologically important phyto-plankton species. Its polymorphic life cycle with both colonial and flagellated cells causes many taxonomic problems. Sequence variation among 22 isolates representing a global distribution of the genus has been compared using three molecular markers. The ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) spacer is too conserved to resolve species. The most conserved 18S ribosomal deoxyribonucleic acid (rDNA) analysis suggests that an undescribed unicellular Phaeocystis sp. (isolate PLY559) is a sister taxon to the Mediterranean unicellular Phaeocystis jahnii; this clade branched prior to the divergence of all other Phaeocystis species, including the colonial ones. The internal transcribed spacer (ITS) region shows sufficient variation that some spatial population structure can be recovered, at least in P. antarctica. P. globosa and P. pouchetii have

Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, Bremerhaven, 27570, Germany e-mail: [email protected] A. Zingone

Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Naples, Italy multiple different ITS copies, suggestive of cryptic species that are still able to hybridize. A molecular clock has been constructed that estimates the divergence of the cold water colonial forms from the warm-water colonial forms to be about 30 Ma and the divergence of P. antarctica and P. pouchetii to be about 15 Ma. A short description of the colonial stage and the flagellated stage for each formally recognized species is provided. Morphological information is also provided on a number of undescribed species. These include the strain Ply 559, consisting of non-colonial cells with peculiar tubular extrusomes, a second non-colonial species from the north western Mediterranean Sea producing a lot of mucus, and a colonial species with scale-less flagellates found in Italian waters. In addition, three flagellated morphotypes with scales different from those of P. antarctica were reported in the literature from Antarctic waters. The picture emerging from both molecular and morphological data is that the number of species in the genus is still underestimated and that cryptic or pseudocryptic diversity requires a sound assessment in future research of this genus. Based on all published observations, an emended description of the genus is provided.

Keywords Molecular clock • Phaeocystis antarctica • P. cordata • P. globosa • P. jahnii • P. pouchetii • P. scrobiculata • rDNA analysis

Introduction

Phaeocystis Lagerheim is a cosmopolitan bloom-forming alga that is often recognized both as a nuisance alga and an ecologically important member of the phytoplankton (Davidson 1985; Lancelot etal. 1987; Smith et al. 1991; Davidson and Marchant 1992; Baumann et al. 1994; Schoemann etal. 2005; Veldhuis and Wassmann 2005). Its various life forms can make large-scale blooms that are often avoided by fish (Chang 1983) and appear detrimental to the growth and reproduction of shellfish and macrozooplankton (Davidson and Marchant 1992) or are ichthyotoxic (Shen et al. 2004). Massive areas of pollution are created when dissolved organic compounds released by Phaeocystis during declining bloom conditions accumulate, foam and then wash onshore (Lancelot et al. 1987). Phaeocystis is a major contributor to the global sulphur budget by releasing substantial quantities of dimethylsulfide propionate (DMSP) (Keller et al. 1989; Baumann et al. 1993), which is metabolized to dimethylsulfide (DMS) as the cells are grazed or infected and lysed by viruses. It may play yet another important ecological role with its production of ultraviolet B (UV-B)-absorbing compounds (Marchant etal. 1991; Davidson and Marchant 1992).

Phaeocystis has a polymorphic life cycle with both colonial and flagellated cells (Kornmann 1955; Whipple etal. 2005). The colonial stage, with cells very loosely interconnected and enclosed in a thin skin (Hamm et al. 1999), is most easily recognized, although some species may form mucilaginous colonies or do not seem to have a colonial stage. Thousands of cells can occur in a colony that may reach 2 cm in diameter (Jahnke and Baumann 1987; Verity etal. 1988; Rousseau et al. 1994; Davidson and Marchant 1992). Colony sizes of 3 cm or more have been reported in blooms from China (Shen et al. 2004). The difficulty in assigning a specific name to the colonial stage has caused much taxonomic confusion. Flagellated cells have two parietal chlorop-lasts and two flagella, which may be equal or unequal in length and heterodynamic. A short haptonema is present between the two flagella, which may or may not have a swollen end. The flagellated cells may be naked or have two layers of different shaped organic scales. Some flagellated cells also produce groups of filaments, which are extruded from the cell and assume a characteristic pattern.

The genus was erected by Lagerheim in 1893 to accommodate the colonial stage of an alga described originally as Tetraspora poucheti by Hariot in Pouchet (1892). Phaeocystis pouchetii (its correct orthography) occurs in cold waters and in its globular, lobed colonies, cells are arranged in packets of four (see Jahnke and Baumann 1987 for illustrations). Phaeocystis globosa was described by Scherffel (1900) from temperate waters and forms spherical colonies with cells arranged homogeneously within the colony (Jahnke and Baumann 1987), whereas older stages can assume distorted pear shapes (Bätje and Michaelis 1986). Early workers separated P. pouchetii and P. globosa based on different distributions and colonial morphologies until Kornmann (1955) doubted the differentiation between the two species. From his life-cycle studies, he considered that P. globosa cell types appeared to be juvenile forms of P. pouchetii. Since that report, colony morphology has been judged an unreliable specific character.

Sournia (1988) reviewed the diagnostic features of Phaeocystis, and discussed the reliability of the nine valid species published since the last century. He discarded two species from the genus, P. fuscescens (Braun) De Toni and P. giraudyi (Derbès and Solier) Hamel, because they did not fit the genus and probably not even the class characteristics. The descriptions of four species, two from cold waters, P. antarctica Karsten and P. brucei Mangin, and two from temperate waters, P. amoeboidea Büttner and P. sphaeroidea Büttner, were all judged as very superficial. The poor illustrations and unlikely features, including one chloroplast per cell and no haptonema (Büttner 1911), were probably the reasons why the two temperate species have never been mentioned again in the literature. For similar reasons the two Antarctic species were reported rarely and not studied again. As for the two most frequently recorded species, P. pouchetii (Hariot in Pouchet) Lagerheim and P. globosa Scherffel, they had been studied in more detail yet no element was available to keep them separate. Therefore Sournia suggested that the name P. pouchetii or, better, P. cf. pouchetii should have been used for the colonial Phaeocystis species pending new information. The only other reliable species was Phaeocystis scrobiculata Moestrup, described with modern methods but known only from the flagellated state (Moestrup 1979). Without convincing information on the differentiation between P. pouchetii and P. globosa (see paragraph below), most marine ecologists until the mid 1990s followed Sournia's advice and reported Phaeocystis colonies as P. pouchetii (the older name) or as Phaeocystis sp. to avoid confusion.

Baumann and Jahnke (1986), Jahnke and Baumann (1986, 1987) and Jahnke (1989) regarded this as over-simplification. Their observations on colony shape maintenance in long term culture showed that both juvenile and older stages of P. globosa and P. pouchetii were distinct and these data supported the recognition of the two entities as separate species. Also, detailed studies of the temperature and light tolerances suggested separation at the species level. P. globosa was a temperate species and P. pouchetii was a cold-water form. A third, unnamed colonial species from Antarctic waters was recognized by Baumann et al. (1993), which had a combination of features of P. globosa and pouchetii, as suggested earlier by Moestrup and Larsen (1992). The colonies resembled those of P. globosa (Larsen and Moestrup 1989), whereas temperature tolerance was similar to that of P. pouchetii. Notably, strains from the Antarctic had different pigment spectra (Buma et al. 1991; Vaulot et al. 1994) and DNA content (Vaulot et al. 1994). First indications that further undescribed species could exist were provided by Pienaar (1991, 1996) who illustrated a Phaeocystis flagellate with cup-shaped scales from South African waters suggesting it was a new species but not publishing a formal description.

To resolve the species issue in Phaeocystis, a molecular analysis of various clones was begun (Lange 1997). Medlin et al. (1994) were the first to propose that P. globosa and P. pouchetii were separate species based on genetic evidence. They also showed that colonial Phaeocystis from the Antarctic was genetically distinct from the other two species. They resurrected the species P. antarctica described by Karsten (1905) for the colonial isolates previously termed P. globosa or pouchetii from these waters. Zingone et al. (1999) added two more species to the genus, P. cordata and P. jahnii, but these were basically unicellular species, although the latter species could make simple clusters of cells that could be termed colonial. In this review we summarize the molecular and morphological information available to date for Phae-ocystis species and provide evidence that a high diversity is still hidden in the genus.

Molecular analysis

A global distribution of the prymnesiophyte genus Phaeocystis was compared using nuclear-encoded 18S rDNA genes and two non-coding regions, the ribosomal DNA internal transcribed spacer 1 (ITS1) separating the 18S rDNA and 5.8S rDNA genes and the plastid ribulose-1,5-bis-phosphate carboxylase/oxygenase (RUBISCO) spacer flanked by short stretches of the adjacent large and small subunits (rbcL and rbcS) (Lange et al., 2002). The RUBISCO spacer regions were highly conserved and generally uninformative among all Phaeocystis strains (Lange et al. 2002). The 18S rDNA analysis suggests that an unde-scribed unicellular Phaeocystis sp. (isolate PLY559) is a sister taxon to the occasionally colonial Mediterranean P. jahnii with which it forms a clade that diverges at the same time from a clade with all other Phaeocystis species, including those forming typical colonies wrapped in a skin (Fig. 1). In this latter clade, the unicellular P. cordata diverges before the colonial ones, which can be divided into a cold-water complex (P. pouch-etii from the Arctic and P. antarctica from the Antarctic) and a warm-water complex consisting of P. globosa. Thus, all of the variation seen earlier using morphological and physiological criteria had a strong genetic basis and separation at the species level was warranted for all colonial species originating from major climatic regions, i.e., the Arctic, the Antarctic and temperate/tropical regions.

In contrast, ITS1 exhibited substantial inter-and intra-specific sequence divergence and showed more resolution among the strains (Fig. 2). Markedly different copies of the ITS1

Fig. 1 Maximum-likelihood phylogeny (fastD-NAml) of 17 Phaeocystis species/strains and other prymnesiophytes inferred from 18S rDNA. The class Pavlovophyceae was used as outgroup. Bootstrap values are placed on the nodes that are identical from ML/NJ/MP analyses. The scale bar corresponds to two base changes per 100 nucleotides. Redrawn from Lange et al. (2002)

Pavlova cf. salina

Pavlova ccmp1416

100/100

Pavlova cf. salina

Pavlova ccmp1416

Pavlova ccmp 1394 Pavlova gyrans

Chrysochromulina throndsenii Chrysochromulina scutellum 100/1 oo i— Chrysochromulina strobilus

Chrysochromulina campanulifera Chrysochromulina leadbeaterii Imantonia rotunda Chrysochromulina chiton

Prymnesium parvum Chrysochromulina brevifilum Chrysochromulina ericina Coccolithus pelagicus

Reticulosphaera japonensis Pleurochrysis carterae Isochrysis galbana Emiliania huxleyi

L Phaeocystis sp. PLY 559

i uu/1 uu Phaeocystis jahnii -

— Phaeocystis cordata -

_ Phaeocystis antarctica i|" Phaeocystis antarctica ACC Phaeocystis antarctica Phaeocystis antarctica Phaeocystis antarctica 97/ioo1h Phaeocystis pouchetii P360 99/100 i Phaeocystis pouchetii SK34

r Phaeocystis globosa North Sea I Phaeocystis globosa South Africa I—II Phaeocystis globosa Surinam 99/100 I Phaeocystis globosa Gulf of Mexico P Phaeocystis globosa Thailand \{ Phaeocystis globosa Eastern Atlantic Phaeocystis globosa Galapagos

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