View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Electronic Publication Information Center J. Phycol. 35, 1322±1337 (1999) MORPHOLOGICAL AND GENETIC CHARACTERIZATION OF PHAEOCYSTIS CORDATA AND P. JAHNII (PRYMNESIOPHYCEAE), TWO NEW SPECIES FROM THE MEDITERRANEAN SEA1 Adriana Zingone 2 Stazione Zoologica A. Dohrn, Villa Comunale, I-80121 Naples, Italy Marie-JoseÁphe ChreÂtiennot-Dinet 3 Observatoire Oceanologique de Banyuls, Universite Pierre et Marie Curie I.N.S.U., C.N.R.S, Laboratoire Arago, F-66650 Banyuls-sur-Mer, France Martin Lange 4 and Linda Medlin4 Alfred Wegener Institute, Am Handelshafen, 12, D-27570 Bremerhaven, Germany Two new Phaeocystis species recently discovered in the available data on the evolution of the world's the Mediterranean Sea are described using light and oceans. electron microscopy, and their systematic position is discussed on the basis of an analysis of their nuclear- Key index words: Mediterranean Sea; Phaeocystis cor- encoded small-subunit ribosomal RNA gene (SSU data, sp. nov.; Phaeocystis jahnii, sp. nov.; phylogeny; rRNA) sequences. Phaeocystis cordata Zingone et Prymnesiophyceae; SSU rRNA gene analysis; tax- ChreÂtiennot-Dinet was observed only as ¯agellated onomy; ultrastructure unicells. Cells are heart shaped, with two ¯agella of Abbreviations: DMS, dimethyl sulphide; LM, light slightly unequal length and a short haptonema. The microscopy; MPN, most probable number; SDC, se- cell body is covered with two layers of thin scales. rial dilution culture; SSU rRNA, small-subunit ribo- The outermost layer scales are oval, with a faint ra- somal RNA gene diating pattern, a raised rim, and a modest central knob. The inner-layer scales are smaller and have a faint radiate pattern and an in¯exed rim. Cells swim Phaeocystis Lagerheim is a cosmopolitan genus with their ¯agella close together, obscuring the hap- that includes bloom-forming species that play a key tonema, pushing the cell, and causing it to rotate role in many nutrient-rich areas of the world's about its longitudinal axis while moving forward. oceans (Davidson and Marchant 1992, Baumann et Phaeocystis jahnii Zingone was isolated as a nonmo- al. 1994b). Phaeocystis blooms constitute a recurrent tile colony. It forms loose aggregates of cells em- stage in phytoplankton succession in Arctic, Antarc- bedded in a mucilaginous, presumably polysaccha- tic, and North Sea waters (Lancelot and Rousseau ride matrix without a de®nite shape or visible ex- 1994, Lancelot et al. 1998). Large-scale blooms may ternal envelope. The ¯agellated stage has the fea- also have a direct in¯uence on climate because tures typical of other Phaeocystis species. Cells are Phaeocystis cells contribute to the release of DMS rounded in shape and slightly larger than P. cordata. (Baumann et al. 1994a, Matrai et al. 1995), which The cell body is covered with extremely thin scales in¯uences cloud albedo and possibly the heat ¯ux of two different sizes with a very faint radiating pat- to the earth's surface (Charlson et al. 1987, Ayers et tern toward their margin. Swimming behavior is sim- al. 1997). Blooms directly interfere with human ac- ilar to that of P. cordata, with the ¯agella in a pos- tivities by producing net-clogging (Savage 1930, terior position as the cells swim. The SSU rRNA se- Chang 1983) or dense foams (Lancelot et al. 1987) quence analysis indicated that both species are dis- or by affecting wild-®sh taste (Levasseur et al. 1994). tinct from other cultivated Phaeocystis species In recent years, investigations have focused on sequenced to date. Regions previously identi®ed as Phaeocystis species with the aim of clarifying their speci®c for the genus Phaeocystis are not found in physiological characteristics, life-history traits, and P. jahnii, and new genus-speci®c regions have been potential role in the trophic chain, but many aspects identi®ed. P. cordata is more closely related to the are still to be unequivocally elucidated. colonial species P. globosa, P. antarctica, and P. One of the most striking characteristics of Phaeo- pouchetii and has branched prior to the divergence cystis species is their complex life cycle, with differ- of the warm-water P. globosa species complex from ent ¯agellate stages alternating with nonmotile sin- the cold-water species P. antarctica and P. pouchetii. gle-celled and colonial stages in a series that is not These results are discussed within a framework of yet fully understood (Kornmann 1955, Rousseau et al. 1994). This feature, coupled with the notable morphological similarity among Phaeocystis species, 1 Received 9 March 1999. Accepted 6 August 1999. has resulted in confused circumscriptions of the dif- 2 Author for reprint requests; e-mail [email protected]. ferent taxa within the genus. Nine Phaeocystis species 1322 PHAEOCYSTIS CORDATA AND P. JAHNII 1323 have been validly published, but information on pigments and genome size (Vaulot et al. 1994) was morphology and distinguishing features for most of isolated from the Gulf of Naples (Mediterranean them is rather super®cial (Sournia 1988). The most Sea). This strain did not form colonies or nonmotile widely accepted opinion for some years (Sournia cells. It consisted only of ¯agellated cells having 1988, Davidson and Marchant 1992) has been that morphological characters and swimming behavior a single species, P. pouchetii (Hariot in Pouchet) Lag- different from other Phaeocystis species. We provide erheim, existed, having a broad geographical distri- a morphological description of this species, Phaeo- bution, and that the only other distinct species in cystis cordata Zingone et ChreÂtiennot Dinet. Another the genus was P. scrobiculata Moestrup, described Phaeocystis strain isolated from the same area was ini- from New Zealand waters (Moestrup 1979). Subse- tially interpreted as the colonial stage of P. cordata quently, four different species have been recognized but subsequently released swarmers that were differ- on the basis of morphological and ecophysiological ent from those of P. cordata. We describe it as Phaeo- traits: P. pouchetii, P. scrobiculata, P. globosa Scherffel, cystis jahnii Zingone and provide information on its and P. antarctica Karsten (Moestrup and Larsen distinctive morphological features. A sequence anal- 1992, Baumann et al. 1994b). These species are also ysis of the SSU rRNA gene was done to assess the distinct in terms of their geographic range. Of the position of the two new species within Phaeocystis. ®ve remaining species, P. fuscescens (Braun) De Toni and P. giraudyi (DerbeÁs et Solier) Hamel have de®- MATERIALS AND METHODS nitely been shown not to belong to this genus (Sour- Cultures. A unialgal culture of Phaeocystis cordata, Phaeonap1, nia 1988), whereas P. brucei Mangin, P. amoeboidea was established by serial dilution of a seawater sample collected with a Niskin bottle in the Gulf of Naples, at the station MC BuÈttner, and P. sphaeroidea BuÈttner have not been (408499 N; 148159 E), at 20 m on 15 March 1991. The culture was studied since they were described. isolated and grown in K±Si medium (Keller et al. 1987) and kept Recently, pigment complement, genome size at 188 to 258 C. Irradiance was at 100 mmol quanta´m22´s21 emit- (Vaulot et al. 1994), and genetic similarities (Medlin ted from Osram L36W/20 cool-white ¯uorescent tubes with a 12: et al. 1994) have been utilized as valuable characters 12 h LD (light:dark) cycle. Three more strains were examined for comparison of live material and body scales: Phaeonap2, iso- to substantiate and circumscribe Phaeocystis species. lated from the same site at0mon28March 1994, and MEDNS2 These studies have con®rmed that, besides the pre- and MEDNS3, isolated by Nathalie Simon from the northwestern viously mentioned four valid species, other strains Mediterranean Sea (408459 N; 6879 E, 20 July 1993, and 428519 N; can be separated on the basis of more than one 38479 E, 30 July 1993) and kindly provided by D. Vaulot. Addi- tional information and illustrations were obtained from other character, and these probably correspond to as yet mixed dilution cultures and natural samples collected at the sta- undescribed species or subspecies. tion MC (Mare Chiara) on different sampling dates. Unfortunately, progress made in understanding Phaeocystis jahnii strains were obtained from a serial dilution these new species' separation based on genetic and culture (SDC) of a natural sample collected in surface waters of biochemical characteristics has not been supported by the Gulf of Naples (St. MC) on 6 March 1996. This was a mixed culture in which nonmotile cells and mucilaginous colonies of adequate morphological discrimination. The shape similar cells were noticed along with Phaeocystis-like ¯agellates and and internal organization of Phaeocystis colonies has other species. Colonies of four to eight cells were isolated by mi- been described for all the species, with the exception cropipetting and placed into multiwell plates ®lled with K±Si me- of P. scrobiculata, for which only ¯agellate stages are dium. After a few days, colonies were transferred to culture tubes with the same medium. Single nonmotile cells were subsequently known. However, complete ultrastructural data are isolated from these cultures by micropipetting, and two cultures not yet available to compare both colonial and ¯ag- were established, A1 and B5, and grown as previously described. ellated stages of all species. Size and ornamentation Cultured material of both species was observed and photo- pattern of organic scales covering the ¯agellated cells graphed using a Zeiss Axiophot microscope equipped with phase constitute a reliable character in prymnesiophyte tax- contrast and differential interferential contrast. Data on the distribution of Phaeocystis spp. were obtained from onomy but are described only for P. globosa (as P. SDCs (Throndsen 1995) established from seawater samples from pouchetii in Parke et al. 1971) and P. scrobiculata (Moes- the Gulf of Naples and from the Sicily Channel. Cell concentra- trup 1979).