Life Cyle and Sexuality of the Freshwater Raphidophyte Gonyostomum Semen (Raphidophyceae)1
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J. Phycol. 42, 859–871 (2006) r 2006 by the Phycological Society of America DOI: 10.1111/j.1529-8817.2006.00240.x LIFE CYLE AND SEXUALITY OF THE FRESHWATER RAPHIDOPHYTE GONYOSTOMUM SEMEN (RAPHIDOPHYCEAE)1 Rosa Isabel Figueroa Instituto Oceanogra´fico Vigo, Cabo Estai-Canido, 36200 Vigo, Spain and Karin Rengefors2 Limnology, Department of Ecology, Lund University, Ecology Building, 22362 Lund, Sweden Previously unknown aspects in the life cycle of Key index words: AFLP; cysts; encystment; Go- the freshwater flagellate Gonyostomum semen (Eh- nyostomum semen life cycle; raphydophyceans; sex- renb.) (Raphidophyceae) are described here. This uality; SSU. species forms intense blooms in many northern temperate lakes, and has increased in abundance Abbreviation: AFLP, amplified fragment length po- and frequency in northern Europe during the past lymorphism decades. The proposed life cycle is based on obser- vations of life cycle stages and transitions in cul- tures. Viable stages of the life cycle were Gonyostomum semen is the most common freshwater individually isolated and monitored by time-lapse member of the raphidophyceans, and like several of its photography. The most common processes under- marine counterparts it is considered a nuisance alga. G. taken by the isolated cells were: division, fusion semen often forms intensive blooms (Pithart et al. 1997, followed by division, asexual cyst formation, and Wille´n 2003) and may dominate the phytoplankton sexual cyst formation. Motile cells divided by two community by as much as 98% for extended periods different processes. One lasted between 6 and 24 h (Le Cohu et al. 1989). This alga adversely affects lakes and formed two cells with vegetative cell size and used for recreation, as it discharges mucilaginous with or without the same shape. The second division strands upon contact, thereby covering bathers with a process lasted between 10 and 20 min and formed slimy layer causing itching and other allergic reactions two identical cells, half the size of the mother cell. (Cronberg et al. 1988). The distribution of G. semen is Planozygotes formed by the fusion of hologametes widespread, and has been described in various loca- subsequently underwent division into two cells. tions in Europe, Asia, Africa, North America, and South Asexual cyst-like stages were spherical, devoid America (Eloranta and Ra¨ike 1995). In the last decades, of a thick wall and red spot, and germinated in there has been an increase in the abundance and oc- 24–48 h. Heterogamete pairs were isogamous, and currence of this flagellate in Nordic countries including formed an angle of 0–901 between each other. Plano- Sweden, Finland, and Norway (Hongve et al. 1987, zygote and sexual cyst formation were identified Cronberg et al. 1988, Lepisto¨ et al. 1994), while before within strains established from one vegetative cell. the 1980s, the occurrence of these blooms was rare. The identity of these strains, which was studied by G. semen is most common in brown-water lakes, i.e. lakes with moderate to high humic content (50–60 mg an amplified fragment length polymorphism analy- À 1 À 1 sis, was correlated with the viability of the planozy- Pt Á L or approximately 10 mg DOC Á L ) and slight gote. Resting cyst germination was described using acidity, which are typically found in forested areas cysts collected in the field. The size and morphol- (Cronberg et al. 1988). However, principal component ogy of these cysts were comparable with those analyses show no direct connection with pH, but rather formed sexually in culture. The excystment rate with humic content and nutrients, especially phospho- was higher at 241 C than at 19 or 161 C, although the rus (Cronberg et al. 1988, Eloranta and Ra¨ike 1995). cell liberated during germination (germling) was Although peat processing and general eutrophication only viable at 161 C. The placement of G. semen of forest lakes have been suggested as a probable cause within the Raphidophyceae family was confirmed (Eloranta and Ra¨ike 1995), the recent spreading of this by sequence analysis of a segment of the 18S rib- species remains largely unexplained. osomal DNA. G. semen can form extremely dense blooms, reach- ing a biomass of over 1800 mg chl a Á L À 1 (Pithart et al. 1997). However, an explanation behind the bloom formation and competitive advantage of G. semen 1Received 30 August 2005. Accepted 26 April 2006. over other algae is lacking. Vertical migration has 2Author for correspondence: e-mail [email protected]. been suggested as one important adaptation allowing 859 860 ROSA I. FIGUEROA AND KARIN RENGEFORS G. semen to maximize both nutrient uptake and cells from a water sample of Lake Dagstorpsjo¨n in August photosynthesis (Eloranta and Ra¨ike 1995). Another 2002. The cultures were grown in modified WC culture me- important characteristic of G. semen is that it has an dium (MWC) (Guillard and Lorenzen 1972) and maintained 1 À 2 À 1 overwintering benthic stage or resting cyst (Drouet and at 15 Cat30mmol photons Á m Á s on a 12:12 light:dark (L:D) photo-cycle. Two strains from single-cell isolations, Cohen 1935). The resting cyst is considered as an ad- named G2 and G3, were established by the isolation of indi- aptation to survive during unfavorable environmental vidual cells from this culture. conditions (Fryxell 1983), including predation (Hans- Life cycle study. Observations and size determination were son 1996, Hansson 2000). A life cycle with an alterna- carried out on vegetative cells, cysts formed in laboratory, tion between a benthic and a planktonic stage is a trait and natural cysts. For monitoring of individual cells, different that G. semen has in common with a number of other life cycle stages of G. semen (Table 1) were isolated from six different treatments: (i) MWC-replete medium, (ii) medium freshwater and marine harmful algal bloom formers, without nitrate (N) added, (iii) medium without phosphate including other raphidophytes, dinoflagellates, and (P) added, (iv) medium without N nor P added, (v) medium cyanobacteria. In these species, the cyst stage has without N and placed 72h in darkness, and (vi) medium been shown to play an important role in bloom initi- without P and placed 72 h in darkness. Both cultures were ation (Anderson and Wall 1978) and seasonal succes- exposed to each of the treatments. Duplicate sterile polysty- sion (Anderson and Rengefors 2006). Moreover, in the rene petri dishes (Iwaki, Tokyo, Japan, 35 mm diameter) were inoculated with exponentially growing cells (4000– dinoflagellates, resting cysts are known to form follow- 6000 cells Á mL À 1) to a final concentration of 700 cells Á mL À 1 ing sexual reproduction, making the cyst stage impor- in a total volume of 10 mL of each treatment medium. Fifty tant in connection to gene recombination and genetic cells or pairs of cells with different morphology were indi- diversity of populations (Pfiester and Anderson 1987). vidually isolated, photographed, and separately transferred Despite the importance of the life cycle in the ecol- to wells of tissue culture plates (Iwaki, 6.4 mm diameter) ogy of phytoplankton, it has to date been poorly in- filled with MWC medium. Culture plates were incubated un- vestigated in G. semen. Previous works have reported der the same conditions as those previously described for culture maintenance. The isolated cells were checked at least aspects of asexual reproduction (Drouet and Cohen once daily and were photographed with a CANON EOS dig- 1935) and of isolated steps of the sexual cycle (Cronb- ital camera. They were also measured at  630 magnification erg 2005). Nevertheless, a detailed description has never been given of sexual reproduction, in which sin- gle cells are followed through the process, nor has TABLE 1. Morphologies most commonly observed in clonal there been a study of mating type in cultures, two as- cultures of Gonyostomum semen after 5 days of incubation in pects of great importance in phytoplankton life cycles. MWC medium. Marine raphidiophytes, a small taxon whose members are almost all classified as ‘‘harmful’’ species, have in con- Morphology and numbers of cells isolated from each trast been studied in more detail. Despite previous re- clone (G2/G3) Processa ports of sexual cyst formation (Nakamura et al. 1990) in these marine relatives, the cyst stage was later claimed to Division type 1 be haploid (Yamaguchi and Imai 1994, Itakura et al. 1996). Chattonella antiqua (Hada) and Chattonella marina 10/10 (Subrahmanyan) Hara et Chihara have diplontic life his- tories, in which vegetative cells are diploid and cysts are Division type 2 haploid (Yamaguchi and Imai 1994). A similar life cycle is also suggested for Heterosigma akasiwo (Hada) Hada (Imai 7/6 et al. 1993a), which, like G. semen (Salonen and Rosenb- erg 2000), exhibits diel vertical migrations (Watanabe et al. 1983, Yamochi and Abe 1984, Nagasaki et al. 1996). Fusing pairs of hologametes Here, we investigated the life cycle of G. semen by following the development and fate of the morpholog- 10/10 ical types that were commonly observed in culture. The individual monitoring of these cells allowed us to report undescribed aspects in the life cycle of raphidiophytes, Fusing pairs of heterogametes such as two kinds of vegetative division, fusion of holo- and heterogametes, and both asexual and sexual cyst 7/3 formation. Part of the small subunit (SSU) of the rib- osomal DNA was sequenced in order to provide a mo- Temporary cysts lecular identification of G. semen, and to determine its 5/5 phylogenetic relationship with other raphidophytes. Cytoplasmic reduction MATERIAL AND METHODS 4/5 Cultures. In these experiments, cultures of G. semen were aClassification based on results from this study. established through the isolation of several individual motile MWC, modified WC culture medium.