Photosynthesis, excretion, and growth rates of Phaeocystis colonies and solitary cells PETER G . VERITY, T H E O D O R E J . SMAYDA and EGlL SAKSHAUG Verity, P. G., Smayda. T. J . & Sakshaug. E . 1991: Photosynthesis, excretion, and growth rates of Phaeocysfis colonies and solitary cells. Pp. 117-128 i n Sakshaug. E.. Hopkins, C. C. E. & Britsland. N. A. (eds.): Proceedings of the Pro Mare Symposium on Polar Marine Ecology. Trondhcim, 12-16 May 1990. Polar Research IO(1). Unialgal cultures of the prymnesiophyte. Phaeocystis cf. pouchefii, were isolated from Norwegian and United States coastal waters. Manipulation of the nutrient medium resulted in populations overwhelmingly dominated by either colonies or solitary cells of Phaeocystis. Both morphotypes were grown under a range of irradiances at 0". 2". 5 " . 10" and 20°C. Photosynthesis was measured as incorporation of H"C03-, and excretion as accumulation of DOI4C during 24-hour incubations; growth rates of solitary cells were determined concurrently from changes in abundance. Both morphotypes exhibited temperature-dependent asymptotic increases in pigment-specific photo- synthesis with irradiance. Saturation intensities increased with temperaturc. Cell division by Phaeoc.vstis solitary cells exhibited a functional response similar to photosynthcsis, although growth apparently saturated at lower irradiances. C:Chla ratios were positively correlated with irradiance and inversely related to temperature, while C:N ratios were insensitive to these environmental parameters. Colonies had higher C:Chla and C:N ratios than solitary cells. Pigment-specific excretion rates wcrc linear functions of irradiance, and exhibited temperature-dependent positive correlations with photosynthesis. Percent extra- cellular release (PER) by both morphotypes was inversely related to tempcraturc. At low temperatures (0-5°C). solitary cells had higher photosynthesis rates than colonies at all irradiances. Their excretion rates, however, were also higher, such that the PER of solitary cells cxceeded those of colonies at 0°C and low irradiances at 2°C. No differences were detectable at 5°C. At higher temperatures. photosynthesis by solitary cells still generally exceeded that by colonies, but the colonies excreted con- siderably more DOC. Thus, while solitary cells are more efficient at utilizing light for photosynthesis, they do not necessarily channel a larger proportion into biomass production. Colonies, howcvcr. appear to be particularly stressed by higher temperatures and irradiances. Peter G. Verify, Skidaway lnsfitute of Oceanography, P . 0. Box 13687. Saoannah, Georgia 31416. USA; Theodore J . Smayda. Graduate School of Oceanography, Uniuersify of Rhode Island. Kingston. Rhode Island 02881. USA; Egil Sakshaug, Trondhjem Biological Sfafion, Uniuersify of Trondheim. Bynesoeien 46, N-7018 Trondheim, Norway. Introduction Phaeocystis cf. pouchetii (Hariot) Lagerheim, a prymnesiophyte, is one of few marine phyto- plankton taxa which exhibits phase alternations between free-living solitary cells and a gelatinous colonial aggregation of non-motile cells. While both stages are capable of rapid vegetative growth (Kornmann 1955; Kayser 1970), the colonies occur most prominently in the plankton. Thou- sands of non-motile cells (3-10 pm) may be con- tained within colonies up to 10-20 mm in diameter (Gieskes & Kraay 1975; Verity et al. 1988a, b; Weisse & Scheffel-Moser 1990). The free-living solitary cells, of similar size, can be released from colonies, may persist indefinitely in the plankton, and may initiate formation of new colonies. In addition, macrospheres and microspores have been described (Kornmann 1955; Parke et al. 1971) which behave like asexual gametes. While they persist in some cultures, they have not been reported from natural populations, and their func- tion remains obscure. Blooms of colonial Phaeocystis have been reported for 100 years (Pouchet 1892; Gran 1902). They are prominent in both coastal and oceanic waters, near the ice edge in both polar regions (Braarud 1935; Biggs 1982), and exhibit cross- shelf distributional gradients influenced by frontal dynamics (Iverson et al. 1979a, b). Colony blooms are particularly well documented in Norwegian fjords (Sakshaug 1972; Heimdal 1974; Eilertsen et al. 1981), in the Norwegian Sea (Smayda 1958; Paasche 1960), and in the North Sea (Cadee & Hegeman 1986). In contrast, archived samples from the Continuous Plankton Recorder (CPR) imply a long-term decline in Phaeocystis in all open sea areas of the North Sea (Owens et al. 118 P . G . Verity. T. J . Smayda & E . Sakshaug 1989). This trend, however, may be applicable only to colonies since the CPR mesh size (ca. 250 pm) excludes small colonies and all solitary cells. This apparent conflict in historical trends of abundance emphasizes that the majority of the data are restricted to observations of Phaeocysris colonies, and that our knowledge of i n situ behavior is based almost exclusively on colonial stage dynamics. The occurrence of solitary cells as an alternate planktonic stage complicates quan- titation of Phaeocysris bloom inception and its environmental regulation. Although the role of life cycle phenomena has recently received atten- tion (Verity et al. 1988b). the abundance and temporal development of solitary cells has not generally been assessed, primarily due to dif- ficulties in recognition of living Phaeocysris in natural samples containing other phytoflagellates of similar size and morphology. This dearth of data biases our understanding of Phaeocysris bloom dynamics. For example, Phaeocysris is usu- ally considered to be bipolar, with a preference for boreal and colder waters and the winter period in temperate seas (Kashkin 1963). It occurs exten- sively in polar seas up to the ice edge, and it has been reported from sea ice in both the Arctic (Hsiao 1980) and Antarctic (Whitaker 1977). But this bipolar nature is now suspect, as fhaeocysris cf. poucherti also occurs in subtropical and tropical water (Guillard & Hellebust 1971 ; Leadbeater 1974; Estep et al. 1984: Verity unpubl. data). Yet it rarely (if at all) produces the prodigious blooms commonplace in cold waters. Why? The major structural difference between col- onial and solitary stages is the large gelatinous matrix of the former. Significant amounts of pho- tosynthate are directed towards its manufacture (Guillard & Hellebust 1971; Lancelot & Mathot 1985; Veldhuis & Admiraal 1985) which, pre- sumably, is energetically expensive. Surprisingly, though, little is known of the relative photo- synthetic and excretion potentials of the two mor- photypes. It was the purpose of this study to provide such data and to investigate the role of temperature and irradiance i n influencing these parameters. Methods Unialgal cultures of Phaeocysris cf. pouchefii (see Sournia 1988) were isolated from the Tromse sound near T r o m s ~ . Norway, and grown in batch culture using polycarbonate flasks. Manipulation of nutrient concentrations in sterilized "f" medium (Guillard 1975) resulted in cultures over- whelmingly dominated by colonies (f/50 - Silicate) or by solitary cells (f/2 - Silicate). Simi- lar responses to nutrient concentrations have been observed for other Phaeocystis clones (Guillard & Hellebust 1971: Verity & Smayda 1989; Turner et al. 1990). For both treatments, > 9 5 % of all cells belonged to a single morphotype, e.g. col- onies or solitary cells. All media were prepared from the same batch of seawater Colony cultures could not be cleaned entirely of their bacterial populations, probably due to their residual occurrence on the gelatinous sheaths of the colonies. Healthy colonies, however. have few bacteria attached to their sur- faces (Verity et al. 1988b). Cultures of solitary cells were deliberately not made axenic in order to better intercompare results with colonies. In both cases bacterial abundances (determined in DAPI-stained epifluorescence microscope counts) were < 2 x 10'' cells/ml-', and bacterial biomass was an insignificant proportion of algal biomass. Each morphotype was cultured at four to six irradiances and 14: 10 L : D photoperiods at O", 2", 5 " . lo", and 20" C. Cool-white fluorescent light was attenuated using neutral density screen, and irradiance was measured with a Biospherical Instruments QSL-100 quantum sensor. Cultures were adapted to experimental conditions for > 10 generations, after which identical aliquots were used to measure carbon, nitrogen, and chloro- phyll a (chl a ) contents; photosynthesis and excretion rates; and population growth rates (of solitary cells only). For proximate analyses, samples were collected on 0.45 pm Gelman A E filters using low vacuum pressures (< 50 mm Hg difference between atmospheric pressure and vacuum pressure in the flask). Chl a was extracted in the dark after grind- ing i n 90% acetone, and measured fluoro- metrically before and after acidification (Holm- Hansen et al. 1965). Filters for carbon (C) and nitrogen ( N ) determinations were pre-combusted at 400" C for 1 hour. Carbon and nitrogen were measured with a HP185B CHN analyzer (Sharp 1974). Growth and photosynthesis were measured as follows: two sets of 250-ml polycarbonate bottles, previously soaked in dilute HCI and washed in Photosynthesis, excretion. and growth rates 119 over time t (Eppley 1972). Note that I4C uptake rates described here approximate net rather than gross particulate carbon production, due to the relatively long incubations (24 hours). Statistical tests were conducted according to Sokal & Rohlf (1969), with significance levels of P > 0.05 unless otherwise noted. Correlations between photosynthesis and growth rates were analyzed by functional (geometric mean) re- gressions as both parameters were subject t o inde- pendent errors in measurement (Ricker 1973; Laws & Archie 1981). deionized water, were gently filled with aliquots of the acclimated stock cultures. One set received an inoculum of 1.85 x lo5 Becquerels (5 pCi) of NaH14C03 (New England Nuclear), prepared using trace metal clean techniques (Fitzwater et al. 1982). The other set was unaltered and was used to measure cell and colony size and abun- dance, as well as growth rates of solitary cells. Both sets were returned to their acclimation irradiances and incubated for 24 hours. In addition, an extra 14C bottle was wrapped in aluminum foil to provide a dark control. The I4C incubations were terminated by col- lecting phytoplankton on Gelman 0.45 pm AE glass fiber filters in triplicate subsamples. The filters were rinsed, placed in glass vials, acidified for 1 hour with 0.1 ml of 5 N HCI to drive off residual inorganic 14C, and suspended in 5 m l of Aquasol I1 scintillation cocktail. Release of dissolved organic carbon (DOC) was determined using methods modified after Verity (1981). Trip- licate 8-ml aliquots of the 14C filtrate from each bottle were pipetted into 10ml glass test tubes. The pH was lowered t o 3.0 by acidification with HCI, and samples were bubbled with air for 1 hour to purge inorganic carbon. Five ml of each replicate were counted in 16 ml of Aquasol 11. All samples were counted t o an accuracy of at least 5% using a Beckman LS150 liquid scin- tillation counter. Quenching was corrected by the channels ratio method utilising a curve prepared from 14C-toluene. An isotope discrimination fac- tor of 1.05 was applied. Particulate and DOC production rates were corrected for dark bottle activity, which was always a small fraction of light bottle counts. In colony cultures, the abundance of colonies i n triplicate 50 ml samples was enumerated using a stereomicroscope. Cell abundance was calculated as (colony abundance) x (cells per colony), where cells per colony was determined as described in Verity et al. (1988a). In solitary cell cultures, cell abundance of triplicate subsamples was measured in Palmer-Maloney chambers using a Zeiss Photomicroscope 11. Cell growth rates were expressed as population doublings according to K(doub1ings . d-') = (l/t)logz (Nt/N"), where N, and No were abundances at time t and 0. Carbon (C) growth rates were also calculated by normalizing colony and cell photosynthesis rates to their C content according to K, (doubl. . d-l) = (l/t) log2 [(C + AC)/C], where C is the initial carbon content and AC is the production rate Results Colonies Chlorophyll a-specific photosynthesis increased asymptotically with irradiance in a temperature- dependent manner (Fig. 1). The slopes of the P- I curves increased with temperature (Table I ) . Saturation apparently occurred at 100-200 pmol photons m-2 s-l at 0" and 2°C; at > 150 pmol m-2 s-' at 5°C; at > 200 p o l photons m-2 s-l at 10°C; and at > 250 pmol m-2 s-l at 20°C. Maximum observed photosynthetic rates were also tem- perature-dependent, ranging from 2.2 (0°C) to 8.1 (20°C) g C (g Chla)-' h-I. Similar functional relationships are evident when photosynthesis is normalized t o colony carbon (Fig. 2). Carbon-specific incorporation increased asymptotically with irradiance, but saturation apparently occurred at lower light levels than that observed for pigment-specific photosynthesis. Maximum rates ranged from 0.25 to 1.15 pg C . pg colony-C-' . d-I. Table 1 . Slopes of the light-limited portion (aB. g C . g Chla-' . h-l ' p o l photons-' m-* s - ' ) of the P-I curves at each temperature. Bracketed values indicate 95% confidence intervals around slopes. Fits of these data to the exponential model yielded the following equations: as (of solitary cells) = 0.033e"'"4"cmp ' and as (of colonies) = 0.026e"fwN"'Emp LYE ( 2 95% CI) Temp. ("C) Solitary Cells Colonies 0 2 5 10 20 ,032 (.006) ,025 (.005) ,035 (.008) .02Y (.006) ,040 (.007) ,031 (.006) ,065 (.012) ,063 (.025) ,073 (.022) .077 (.012) 120 P. G. Verity, T . J . Smayda & E . Sakshaug 11) , hi1 1 I I(pnole * rn.'*s.') FIR. 1 . Chl u-spccific photosynthesis ( P ) of colonies as a function of irradiance ( I ) . Colonies were grown at 0" (0). 2 " (0). 5 O ( 0 ) . 10" ( m ) , and 20" (A,. Error bars represent 1 SD. I ? . 1 - _ I I I h 1im 200 3 0 0 I(pmole * m-* - 5.') Fig 2. Carbon-specific photosynthesis (P) of colonies as a function of irradiance ( I ) . Svmbols as in Fig. 1 . Carbon excretion was generally a small fraction of particulate fixation (Fig. 3). Percent extra- colonial release ( P E R ) was inversely related to temperature and ranged from 1-7% (20°C) to 6- Colony C (ng) Fig. 4 . Nitrogen ( N ) and carbon ( C ) contents of colonies grown at all temperatures and irradiances. Error bars represent 1 SD. Dashed lines illustrate 95% confidence intervals of regression slope Geometric mean regresaion: Colony N (ng) = 0.587 + 0 135 (Colony C . ng): r: = 0.991. n = 24. 19% (O'C). The highest relative release occurred at higher irradiances at all temperatures. Colony carbon and nitrogen were linearly related and independent of irradiance, tem- perature, and colony size (Fig. 4). The regression slope is equivalent to a mean C : N (by weight) of 7.41. Colonies ranged from an average of 12 to 385 ng C , 2 to 49 ng N, and 228 to 2,560 cells per colony (data not shown), w i t h the largest colonies occurring at low temperatures. Ratios of C:Chla (by weight) exhibited tem- perature-dependent asymptotic relationships with irradiance (Fig. 5 ) . C : Chla increased with irradiance, but this trend was most evident at low temperatures. Ratios ranged from 65-99 (20°C) to 109-159 ( O O C ) . N:Chl a ranged from %13 (20°C) to 15-20 (O'C) in colonies containing 0.1- 0.4 (20°C) and 1.9-2.4 (OOC) ng Chla per colony (data not shown). Fig. 3. Carbon cxcrction ( E ) of colonies exprcsscdas the pcrcent of total incorporation of "C into organic carbon ( P + E ) . S!mbols as In Fig. 1 FIR. 5 The ratio in colonies of carbon to chlorophyll (I (C :Chl u ) . bg Height. as a function of irradiance (1). Symbols as in Fig. I . Photosynthesis, excretion, and growth rates 121 3 . I 0 I00 200 3w 400 I(pno1e m I s.') Fig. 6. Chl n-specific photosynthesis (P) of solitary cells as a function of irradiance ( I ) . Symbols as in Fig. 1 . 0 I 2 3 P(gC - g Cell C1 d-') Fig. 8. Comparison between cell-based population growth ( K ) and carbon-based photosynthesis ( P ) of solitary cells. Symbols as in Fig. 1 . Geometric mean regression: K ( d i v . d - ' ) = -0.023 + 0.986 [P. gC (g Cell C ) - ' . d-'I; 1' = 0.944, n = 24, Solitary cells Chlorophyll a-specific photosynthetic rates were temperature-dependent and increased with irradiance in a quasi-asymptotic manner (Fig. 6). The slopes of the P-I curves (2) (symbols as in Platt et al. 1980) increased with temperature (Table 1). Saturation was not clearly observed at the irradiances used at 0", 2", and 5"C, but was approached at 200400 pmole photons . m-* s-l at 10" and 20°C. Maximum observed photo- synthetic rates increased from 2.7 (0°C) to 12.2 (20°C) gC (g Chla)-I h-I. Q l o values for these maximum rates were not calculated as they may not represent actual maximum rates (e.g. Pz). Population growth rates of cells were also tem- perature-dependent and exhibited asymptotic relationships with irradiance (Fig. 7). Saturation of cell division occurred at lower light levels than did photosynthesis, as observed for carbon- specific photosynthesis of colonies. Maximum growth rates increased from 0.3 (OOC) to 2.3 (20°C) doublings per day. Photosynthesis measured in 24-hour incubations represents net carbon incorporation and, when normalized to cell carbon, is a measure of carbon growth rate. A comparison of this parameter to independently determined cell division rates (Fig. 8) shows a slope not significantly different from 1.0 and an intercept not significantly different from 0, indi- cating that the cells were in balanced growth. As with colonies, carbon excretion was gen- erally a small fraction of particulate fixation (Fig. 9). Percent extra-cellular release (PER) was an inverse function of temperature, ranging from 2% (20°C) to 10-20% (0°C). PER increased with irradiance only at the colder temperatures. Cell carbon (C) and nitrogen (N) were linearly related independent of irradiance, temperature. 1 0 100 200 3 0 0 1 0 100 2w 300 I(pnole ni' s.I) Fig. 7. Population growth rates of solitary cells ( K ) as a function of irradiance (1). Symbols as in Fig. I . Fig. 9. Carbon excretion ( E ) of solitary cells expressed as the percent of total incorporation of I4C into organic carbon (P + E ) . Symbols as in Fig. I . 122 P. G . Verity, T . J . Smayda & E . Sakshaug and cell size (Fig. 10). The regression slope is equivalent to a mean C : N ratio of 6.6 (by weight). Cells ranged from an average of 10-1 10 pg C , 1- 16 pg N , and 0.1-1.9 pg Chla per cell (data not shown). Weight ratios of C : Chla exhibited tem- perature-dependent asymptotic increases with irradiance (Fig. 11). a trend particularly evident at lower temperatures. Ratios ranged from 39-70 (20°C) to 51-100 (OOC). N : Chla ranged from 6- 10 (20°C) to 9-16 (OOC) (data not shown). Discussion A major objective of this study was t o evaluate the comparative physiological potential of solitary cells and colonies of Phaeocystis. as modulated by environmental parameters, to aid in under- standing the bloom dynamics of this enigmatic 0 20 40 60 80 100 120 Cell C (pg) Fig. 10. Nitrogen ( N ) and carbon ( C ) contents of solitary cells grown at all temperatures and irradiances. Error bars represent 1 SD. Broken lines illustrate 95% confidence intervals of regression slope Geometric mean regression: Cell N ( p g ) = 0 012 + 0.152 (Cell C. pg); r? = 0.989. n = 24 I20 3 I I I I I I) I N ' 2 0 0 300 400 I(pnole. m.' S.'I f i g . /I. The ratio in solitary cells of carbon to chlorophyll a ( C : C h l a ) . by weight. as a function of irradiance ( I ) . Symbols as i n Fig. I . alga. Despite a centennial of investigation (Pou- chet 1892; Lagerheim 1893; Gran 1902), factors influencing the inception and regulation of Phae- ocysfis blooms are poorly understood and for several reasons: (1) taxonomic uncertainties and the question of physiological clones; (2) incom- plete description of the life cycle; (3) lack of physiological data on life cycle stages within a single clone; and (4) inability to distinguish soli- tary Phaeocysfis cells in natural assemblages of nanoplankton. The taxonomic confusion centers around species designation. Although nine species have been described (Sournia 1988), most records of blooms have been attributed to Phaeocystis cf. pouchefiii and P . globosa, based primarily on dif- ferences i n colony morphology. On the basis of culture experiments, Kornmann (1955) concluded that P . globosa is a young stage (= Jugend- stadium) of P . pouchetii. O n the basis of eco- logical data, Gran (1902) favored their taxonomic separation. He characterized P . pouchetii as a cold-water form very sensitive t o higher tem- peratures, and P . globosa as a hardier form which occurs during the warmest season. Guillard & Hellebust (1971) found that large colonies formed by their tropical clone resembled descriptions of P . globosa. This morphotype, absent in their cold . water clones, occurred in natural populations col- lected near Woods Hole. The Norwegian clone in the present study reproduced spherical colon- ies, as did a U.S. east coast isolate (Verity et al. 1988b), although very large, old colonies of both clones would occasionally show more elongate or lobed shapes (e.g. Batje & Michaelis 1986). We have insufficient evidence to definitively establish the identity of our clone or its relationship to the P . pouchetiilglobosa debate, and we accept the recommendation of Sournia (1988) to refer to it as Phaeocystis cf. pouchetii. Cell size, and hence carbon/nitrogen content, may also vary considerably among and within clones. Cells of Phaeocystis cf. pouchetii are typi- cally 3-8 pm in diameter (Kornmann 1955; Parke et al. 1971; Chang 1984; Jahnke 1989). Distinctly larger cells up to 10-12 vm, however, have been reported (Ostenfeld 1904; Hallegraeff 1983; Tande & Bimstedt 1987). Mean size of solitary cells in the present study ranged from 3 p m to 10 pm and was inversely related to temperature. Carbon content was 10-11Opg per cell. These directly measured values compare to those of 15- 35 pg per cell for 4-7 pm cells (Jahnke 1989), and to calculated carbon contents of 9.5 pg cell-' for 5 pm cells (Weisse & Scheffel-Moser 1990) and 258 pg cell-' for 6-10 vm cells (Tande & BAmstedt 1987). The uncertain relationship discussed above between systematics and morphological varia- bility is exacerbated by the possible occurrence of physiological clones, specifically thermal clones (Guillard & Hellebust 1971; Guillard & Kilham 1977). Their cold-water clones grew between 4°C (lowest level tested) and 13"C, but not above 1 6 T , while a tropical clone grew between 17°C and 27"C, but died at 14°C. Kayser's (1970) cold- water clone grew best at 15°C (highest tem- perature tested), but with difficulty at 5°C. Simi- larly, Phaeocystis colonies from the North Sea could not be induced to grow below 5°C (Grimrn & Weisse 1985; Weisse et al. 1986), and rapid multiplication was observed only above 7°C. Nat- ural populations, however, occur at considerably lower temperatures (e.g. Smayda 1958; Eilertsen & Taasen 1984; Verity et al. 1988a, b). The clone in the present study could be cultured as distinct morphotypes over a broad temperature range, whereas another arctic clone of P. cf. pouchetii could not (Jahnke 1989). These apparent con- tradictions between lab data and field obser- vations may reflect true physiological adaptations, inadequate culture methods or acclimation to growth conditions, or occurrence of multiple species. Resolution requires full description of the life cycle of Phaeocystis cf. pouchetii and definition of taxonomic status with respect to other species (Jahnke 1989). The present data illustrate that different mor- photypes of Phaeocystis vary in their physiological performance even within a single clone. While colonies and solitary cells exhibited similar func- tional responses to variations in irradiance and temperature, they differed substantially in mag- nitude. At 0-5"C, solitary cells were always more photosynthetically efficient than colonies at a given irradiance (Fig. 12). The slope of the light- limited portion of the P-I curve for solitary cells exceeded that for colonies at 0-5"C, although the differences were not significant at p > 0.05 (Table 1). This suggests that solitary cells were somewhat better adapted than colonies t o low light and temperature. However, chlorophyll a-specific excretion by solitary cells also exceeded that of colonies (Fig. 13), such that solitary cells actually released larger portions of their photosynthate (PER), especially at 0°C and 2°C. The PER of Photosynthesis, excretion, and gro wrh rates 123 6 5 A A 8 A 0 8 A 0 0 0 I I I 0 IW 200 I ( p n o l e - m." 5.') Fig. 12. Comparison of chl o-specific photosynthesis ( P ) of colonies (open symbols) and solitary cells (filled symbols). as a function or irradiance ( I ) . Cultures were grown at 0" (0). 2" (0). and 5°C ( A ) . Data from Figs. 1 and 6. (1 100 2 0 0 I ( p o l e m.' * 5.') Fig. 13. Comparison of pigment-specific excretion (E) of col- onies (open symbols) and solitary cells (filled symbols), as a function of irradiance ( I ) . Cultures were grown at 0" (0). 2" (0). and 5°C ( A ) . both morphotypes were indistinguishable at 5°C. The mB values were similar t o those of natural Phaeocystis populations advected beneath sea ice (Palmisano et al. 1986) and support those authors' conclusion that this alga is capable of adapting to a broad range of irradiances at low temperatures. The responses at higher temperatures were dif- ferent. Photosynthesis by solitary cells still exceeded that of colonies at 10-20°C (Fig. 14), although the distinction was less than at Q-5"C. Values of aB were indistinguishable at 10-20°C (Table 1). The most salient physiological shift was observed in carbon excretion rates (Fig. 15). Solitary cells excreted less carbon per unit of chlorophyll a than did colonies, particularly at higher irradiances. The net effect was that PER of colonies exceeded that of solitary cells at 10- 20°C. 124 P . G. Verity, T . J. Smayda & E . Sakshaug 11 l l Y l ? l Y I 302 400 11 punole * m.' * s.' t F I ~ . 14. As in Fig. I ? . but cultures grown at 10" (0) and 20°C (0). -k O h r 3 0 0 a 100 2 0 0 3 0 4 0 I(pnde * m-* - s-') h g IS. As i n Fig. 13. but cultures grown at 10" (0) and 20°C (0). The major structural difference between these morphotypes is the large gelatinous matrix of the colonies (Chang 1984) which apparently serves as a storage depot for labile organic carbon (Lan- celot & Mathot 1985; Veldhuis & Admiraal 1985; Lancelot et al. 1986). Release of DOC external to the colonies was generally only a small fraction of total incorporation. Previous high PER values for colonies of 1 6 4 4 % (Guillard & Hellebust 1971) were not confirmed in the present study. This difference may reflect that very dense cul- tures were grown at low nutrient levels and high irradiances in the prior study. Assuming that sub- stantial amounts of photosynthate are channeled into the gelatinous matrix of the colonies (Lan- celot & Mathot 1985; Veldhuis & Admiraal1985). the low rates of DOC release measured here indicate that organic carbon associated with col- ony mucus is retained on the glass fiber filters during low pressure filtration. This implies that biochemical parameters measured using filter-col- lected colonies (Figs. 4 and 5, 10 and 11; see also Verity et al. 1988a) accurately represent the carbon, nitrogen. and chlorophyll a contents of the entire cell-matrix complex. The relatively low and constant C : N ratio of colonies (7.4) in the present study implies that most of the colony carbon is in the form of cells, or that organic nitrogen is also being deposited in the matrix, as suggested elsewhere for field populations (Verity et al. 1988a). Calculations imply that colonies with an overall C : N ratio of 7.4, in which cells ( C : N = 6.6: Fig. 10) contribute < 50% of total colony carbon, should have an average C : N com- position of the gelatinous matrix of < 9.0 (Fig. 16). Comparison of photosynthetic performances of colonies and solitary cells may offer insights into their latitudinal distributional patterns. From the perspective of light utilization and the gross par- titioning of photosynthate, low temperatures and irradiances favor colonies. Solitary cells are more efficient at utilizing light for photosynthesis, but they release a larger fraction of it as DOC than do colonies. In contrast, high temperatures and irradiances favor solitary cells, and colonies appear to be more stressed. This stress may be reflected in the relatively low carbon growth rates of colonies at 10-20°C (Fig. 2 ) . In addition to excreting more cabon, the colonies may be respir- ing more, perhaps associated with the incipient stages of a life cycle transition from non-motile colony cells to motile swarmers. These general conclusions may, in part, explain why blooms of colonies apparently occur almost exclusively in colder waters, or at lower temperatures in tem- D Cell C I Colony C (%) Fig. 16. The C : N ratios of colony gelatinous matrix required to produce an overall colony C : N ratio of 7 . 4 when cells within the colonies have a C : N ratio of 6.6. Colony matrix C : N ratio< are plotted against the '3- contribution of cell carbon to tofal colon) carbon Photosynthesis, excretion, and growth rates 125 diatoms. A high Chl a:C ratio and a low P: for the large diatoms, however, counteract each other such that the maximum carbon-normalized pho- tosynthetic rate ( P k ) , which expresses the maxi- mum hourly growth rate, becomes more similar for the three groups than individual Chl a:C ratio and P:. The similarity between the three groups in terms of the maximum growth becomes even more evident when growth rate at optimum light While the three groups of phytoplankton thus are not very different in terms of growth in strong light, the diatoms appear t o be more efficient than Phaeocystis in low light. This can be inferred from the very high carbon-normalized photosynthetic efficiency d which again is due to the very high Chl a:C ratio. There is thus no support for the notion that Phaeocystis might dominate in cold is considered. perate regions. Similarly, solitary Phaeocystis cells are a ubiquitous component of tropical oceans, whereas colonies are rarely observed, and seldom in abundance. A comparison of our data for Phaeocystis with data for cultures of large diatoms and Phaeocystis- dominated Barents Sea communities which con- tain diatoms (Table 2) reveals that all three differ significantly in terms of the Chl a:C ratio and photosynthetic parameters. The Barents Sea com- munities, not unexpectedly, exhibit properties somewhere between the two extremes: Phaeo- cystis colonies and shade-adapted large diatoms. Above all, large diatoms and Phaeocystis differ in that diatoms have a much higher Chl a:C ratio, which has been emphasized previously (see Saks- haug 1989 for review). On the other hand, P t is 3 4 times higher for Phaeocystis than for large Table2. Some properties of shade-adapted Phaeocysris (15-45 ymol m-* SKI) relative to other algae; 14 h day length. Chlorophyll- normalized (aB, P i ) and carbon-normalized (&. P$) photosynthetic parameters pertain to filtered samples. Values in brackets represent light-adapted cells. Temp Solitary' Colonies' Large diatoms2 Barents Sea' Chla:C 0" 2" 5 " a0 0" 2" 5" &(lo-') 0" 2" 5" P i 0" 2" 5" P6 0" 2" 5 " Ik 00 2" 5 " Clm(d-7 0" (obs.) 2" 5 " 0.014 0.013 0.015 0.032 0.035 0.040 0.45 0.46 0.60 3.4 4.5 5.5 0.048 0.059 0.083 106 129 138 [0.28] [0.44] (0.55j 0.008 0.048 [0.028] 0.031 [0.013] 0.011 0.014 0.025 0.023 [0.025] 0.020 [0.026] 0.029 0.031 0.20 0.32 0.42 2.2 2.5 3.1 0.018 0.028 0.043 1.1 0.8 0.038 [0.70] 0.62 10.341 11.71 0.9 11.61 [0.048] 0.028 [0.021] I This study Thalnssiosiru nordenskioeldii and Chuetoceros furcellarus; 25 and (brackets) 400 pmol m-L s-' (Sakshaug et al. 1991). Phaeocystis-dominated populations (viscous water) with diatoms, Barents Sea. Based on data from F. Rey presented in Calculated as P: . 14 Sakshaug & Slagstad (1991 this volume): average for populations deeper than 70 m and (brackets) in the upper 20 m . 126 P. G. Verity, T. J . Smayda & E . Sakshaug waters due to its particularly efficient utilization of low light. Rather, the frequent dominance of Phaeocysris in cold waters might be due to a high frequency of large initial stocks, or perhaps sedimentation and grazing rates may be par- ticularly low for Phaeocystis during initial phases of blooms. Inception of Phaeocystis blooms: a hypothesis Temperature- or irradiance-dependent shifts i n performance, however, do not explain the incep- tion of colony blooms, for which two mechanisms are known. One, only recently documented (Ver- ity et al. 1988b), is multiplication and enlargement of small colonies into larger ones, which then cleave into two daughter colonies of similar size and cell number. This process, however, is aug- mented by or secondary in importance to the formation of new colonies from single cells (Kor- mann 1955; Kayser 1970; Parke et al. 1971). Since the latter transformation occurs in nature at tem- perature and light conditions under which either morphotype can be maintained in culture, it is likely that additional factors are implicated. Numerous environmental factors have been invoked as bloom triggers. including temperature (Jones & Haq 1963); decreased concentrations of silicate and phosphate (Jones & Spencer 1970; Gieskes & Kraay 1975; van Bennekom et al. 1975); trace metals (Morns 1971; Davidson & Marchant 1987); and edaphic effects (Jones & Haq 1963). Conclusive evidence in support of these hypotheses is lacking (Cadee & Hegeman 1986; Weisse et al. 1986). Nutrient concentrations per se do not appear to solely regulate dominance by or transitions among life cycle stages. For example. tropical waters, which tend to be oligo- trophic, rarely show prodigious colony blooms. and chronic nutrient deprivation can also induce emigration of cells out of colonies, which suggests that low nutrient conditions favor solitary cells. However, colonies are inhibited and solitary cells are predominant when excess nutrients are added to cultures (see citations in Methods). A key, consistent observation is that Phaeo- cystis colony blooms usually follow the spring diatom maximum, implying that perhaps chemical modification of seawater via biological con- ditioning or secretion of allelochemic substances (Smayda 1980) is a prerequisite for colony for- mation. In this regard, there is the provocative suggestion that some species of the diatom genus Chaetoceros produce a chemical compound which initiates the change from the motile to non-motile stage (Boalch 1984). Free-living Phaeocystis cells can attach to surfaces by means of their flagella (not haptonema) (Kornmann 1955; Parke et al. 1971). and 8-cell colonies are found attached to chain-forming diatoms, frequently Chaetoceros (Smayda & Verity unpubl. data). We propose that Phaeocystis blooms develop through a two-step process requiring the sequen- tial involvement of both stages in the life cycle. Solitary cells increase in abundance due to their rapid growth rates and small size which prevents efficient retention by suspension-feeding met- azoan herbivores (Verity & Smayda 1989). Col- onies develop from these cells, perhaps facilitated by the presence of diatoms, and in this stage Phaeocysris blooms accumulate gradually from slow-growing populations subjected t o minimal population losses to predation. 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D.C.