J. Phycol. 34, 945±951 (1998) THE PHYCOBILIN SIGNATURES OF CHLOROPLASTS FROM THREE DINOFLAGELLATE SPECIES: A MICROANALYTICAL STUDY OF DINOPHYSIS CAUDATA, D. FORTII, AND D. ACUMINATA (DINOPHYSIALES, DINOPHYCEAE)1 Christopher D. Hewes2 Polar Research Program, Scripps Institution of Oceanography, University of California-San Diego, La Jolla, California 92093-0202 B. Greg Mitchell, Tiffany A. Moisan, Maria Vernet Marine Research Division, Scripps Institution of Oceanography, University of California-San Diego, La Jolla, California 92093-0218 and Freda M. H. Reid Marine Life Research Group, Scripps Institution of Oceanography, University of California-San Diego, La Jolla, California 92093-0218 ABSTRACT tion of photosynthetic cellular organelles (Margulis The absorbance and ¯uorescence emission spectra for 1970) that resulted in ancestral trees having com- three species of Dinophysis, D. caudata Saville-Kent, D. mon and divergent links (Gibbs 1981, McFadden fortii Pavillard, and D. acuminata ClapareÁde et Lach- and Gilson 1995, Liaud et al. 1997). mann, were obtained through an in vivo microanalytical However, relatively recent insights into the dy- technique using a new type of transparent ®lter. The pig- namics and function of unicellular marine plankton, ment signatures of these Dinophysis species were compared assisted by tools developed and now used to observe to those of Synechococcus NaÈgeli, a cryptophyte, and two individuals, may require a new evaluation of these wild rhodophytes, as well as those of another dino¯agellate, classical paradigms. The use of epi¯uorescence mi- a diatom, and a chlorophyte. Phycobilins are not consid- croscopy expanded the perceptions of taxonomists, ered a native protein group for dino¯agellates, yet the ab- who began to identify organisms in relation to nat- sorption and ¯uorescence properties of the three Dino- ural pigmentation and histochemical properties to physis species were demonstrated to closely resemble phy- supplement classi®cations based on morphology. cobilins and chlorophylls of Rhodomonas Karsten (Cryp- Today, we now know that what previously had been tophyceae). Analyses of Dinophysis species using considered phytoplankton includes both photo- epi¯uorescence microscopy found no additional nucleus or trophs and heterotrophs, and a signi®cant number nuclear remnant as would be contributed by an endosym- of taxa compose a wide spectrum having mixotroph- biont. ic or symbiotic lifestyles. Dino¯agellates (considered dinokaryotic) are thought to represent an ancient Key index words: absorbance spectra; Cyclopore ®lter; Di- branching from the evolutionary development to- nophysis; endosymbiosis; ¯uorescence spectra; nucleus; ward the eukaryotes (Taylor 1980, Rizzo 1987). phycobilin; toxic algae Some investigators consider dino¯agellates to be heterotrophic protists that derived their chloroplasts Abbreviations: chl, chlorophyll, DAPI, diamidino-2- from multiple endosymbiotic events and did not phenylindole-2-HCL; FTF, ®lter±transfer±freeze technique; evolve parallel with the evolution of their morphol- MeOH, methanol; PEB, phycoerythrobilin; PUB, phycou- ogy and general structure of their order (see Dodge robilin 1987). One of the primitive dino¯agellate genera, Dino- Traditionally, diatoms (containing fucoxanthin) physis, has gained recent recognition because it in- can be distinguished from dino¯agellates (contain- cludes toxic species that produce dinophysotoxin re- ing peridinin), cryptophytes (containing chloro- sponsible for diarrhetic shell®sh poisoning, which phyll [chl] c and C-phycoerythrobilin [PEB]), and has closed ®sheries in Europe and Asia (Hallegraeff red algae (lacking chl c but having R-PEB) based on 1995). Dinophysis is one of several dino¯agellate gen- pigment analysis. Spectral data of bulk water sam- era that contain both photosynthetic and heterotro- ples can be interpreted in terms of the types of phy- phic species. Although not all dinophysoid species toplankton that contribute based on these charac- have been examined with epi¯uorescence micros- teristics (Yentsch and Phinney 1982). The types of copy, approximately half those investigated have pigments contained in archetypical algal cells also been found to be heterotrophic, and many of the provide a convenient paradigm to explain evolution- remaining, under blue-light excitation, ¯uoresce ary development involving endosymbiotic acquisi- yellow to orange, (see cover of J. Phycol. 34(6); Les- sard and Swift 1986, Geider and Gunter 1988, Hal- 1 Received 31 December 1997. Accepted 7 August 1998. legraeff and Lucas 1988, Schnepf and ElbraÈchter 2 Author for reprint requests; e-mail [email protected]. 1988, Giacobbe 1995). This is in contrast to the red 945 946 CHRISTOPHER D. HEWES ET AL. ¯uorescence that represents chlorophyll±peridinin- malized at 680 nm then averaged and smoothed, as were absor- containing chloroplasts of ``traditional'' dino¯agel- bance spectra. Taxa examined were the dino¯agellates D. fortii, D. acuminata, lates. Such species were suggested to have phycobi- D. caudata, and Prorocentrum micans (Ehrenberg); a centric diatom lins, and ultrastructural studies indicate the thyla- Eucampia sp.; and both the individual cells composing thalli (Red koid structure of their chloroplasts is similar to that Alga 1) and an unidenti®ed unicellular type (Red Alga 2; 5±10 found in cryptophytes (Hallegraeff and Lucas 1988, mm diameter) from the class Rhodophyceae. All natural samples were collected mid-September through mid-October 1997. Nu- Schnepf and ElbraÈchter 1988, Lucas and Vesk merous attempts to quantify Dinophysis species abundance using 1990). Further physiological study on these species ®ltration (up to 1 L seawater) and settling (150 mL) techniques has been hampered by the lack of success in obtain- (Reid 1983) were made without success. ing them in culture. Therefore, study of dinophy- Synechococcus cultures of WH-8103, WH-7803, and WH-7805, soid species has been limited to wild forms and re- grown using standard culture plating techniques (Toledo and Pal- enik 1997), were also examined for pigmentation. Colonies were stricted by methods where concentration or isola- scooped from agar and squeezed between a coverslip and micro- tion of individuals from natural populations permit- scope slide. Our microanalytical system cannot resolve spectra of ted their investigation. individual particles ,3 mm in diameter accurately; therefore, ab- Here, we report in vivo absorbance and ¯uores- sorbance and ¯uorescence emission spectra were determined for aggregates of Synechococcus cells. Native wild Synechococcus sp. pres- cence emission spectra for three species of yellow- ent in our net samples were sometimes found in small aggregates ¯uorescing Dinophysis: D. fortii, D. acuminata, and D. devoid of nonphycoerythrin photosynthetic cells. These aggre- caudata. They represented a minor component of gates did not provide enough material to obtain good absorbance the Southern California Bight plankton communi- spectra, but ¯uorescence emission spectra were obtained. ties (,1 cell/L) at the time of sampling. Absorbance The nuclear contents of dino¯agellates were examined by epi- ¯uorescence microscopy using 49,69-diamidino-2-phenylindole-2- and ¯uorescence emission spectra of individual cells HCL (DAPI)-stained samples prepared by a modi®ed ®lter±trans- were determined in vivo by spectral microphoto- fer±freeze (FTF) technique (Hewes and Holm-Hansen 1983). Net metry to examine and compare pigment composi- phytoplankton samples were ®ltered onto polycarbonate ®lters tions. Epi¯uorescence microscopical technique was until a thin ®lm of seawater remained. Methanol (MeOH) was added to extract chlorophyll in a gradient of up to 100% by con- employed to further determine whether a crypto- tinuously ®ltering and slowly adding increased concentrations phycean endosymbiont's nucleus or nuclear rem- (starting at 25%) of 48 C MeOH. Cells were left in contact with nants could be found. Methodology for the spectral 100% MeOH for at least 10 min. Rehydration of the cells was analysis of pigments on very rare individuals within made by continuously ®ltering and slowly adding chilled deion- a population is described, presenting a potentially ized water. Cells were stained with DAPI, then washed again with distilled water and prepared for combined epi¯uorescence and important in vivo technique for assessing other spe- transmitted light microscopy using 10% glycerine for the mount- cies-speci®c characteristics of algae. The in vivo spec- ing medium. tral signatures of these Dinophysis species are com- Cultures of the green alga Dunaliella sp. and a cryptophyte, pared to those of other algae of both cultured and Rhodomonas sp., were processed for microanalysis using three methods. Cells were ®ltered onto 3.0-mm-pore Cyclopore ®lters, wild types. frozen to about 2608 C with aerosol freezing spray, allowed to thaw, and subsequently examined for absorbance spectra. These METHODS cells also were prepared using FTF with and without 0.5% glutar- Natural phytoplankton surface samples were collected by dip- aldehyde ®xation (30 s ®xation, followed by FSW wash for 3 min) ping a 20 mm mesh plankton net into surface waters off the and were mounted in 10% glycerine. Absorbance spectra of ®ve Scripps Institution of Oceanography pier in La Jolla, California individual cells for each species and preparation were zeroed at (328509 N, 1178109 W). Samples were ®ltered through either a 750 nm then averaged to provide representative spectra. Addi-