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APPLIED AND ENVIRONMENTAL MICROBIOLOGY. June 1989. p. 160(5-1611 Vol. 55. No. 6 0099-2"40/89/061605-07S02.00/0 Copyright t 1989. American Society for Microbiology

Experimental Tests of Nutrient Limitation in Freshwater Picoplankton JOHN D. WEHR Ca/lder E(ology Center. Ford/han Unih-ersitv. Bo.x 273, Armslomik. Newt- Yo,-k 10504 Received 27 Januac-ry 1989/Accepted 20 March 1989

On the basis of correlative studies, picoplankton in Calder Lake, New York, are apparently unaffected by seasonal fluxes in nutrient (N and P) levels. In this small eutrophic lake, picoplankton (<2.0- to 0.2-,um size) and nanoplankton (<20 to >2 ,um) predominate. Microplankton (>20 ,um) are typically least important. Experiments were conducted in situ to test whether N, P or N/P ratios affect the predominance of these smaller organisms. Manipulations were run in 4-liter microcosms during June, July, and August 1988, corresponding to periods of increasing stratification and nutrient depletion. Following nutrient additions, were harvested and fractionated into three size classes. Microplankton and nanoplankton were significantly stimulated by both N (2.5 to 50 ,uM) and P (I to 20 ,uM) additions. The severity of nutrient limitation was greatest during July. Picoplankton responded less strongly to N additions and were never P limited. These field data support laboratory studies which indicate that bacterium-sized phytoplankton use nutrients more efficiently and are superior competitors within mixed commtinities.

Bacterium-sized phytoplankton are widespread and abun- (J. D. Wehr. Hydrobiologia. in press). The phytoplankton dant in the world's oceans and in many lakes (40. 43). This community consisted predominantly of nanoplankton and has caused a major reevaluation of microbial ecology in picoplankton, about 74% of the total as measured aquatic ecosystems. These microorganisms, termed pico- by chlorophyll ai (Chl ti). Despite surface blooms of colonial , include autotrophic cells from 0.2 to 2 p.m in size (e.g.. Microcvstis and Anabaiernai spp.) and (38). Algal picoplankton may predominate in certain North subsurface blooms of large (Ceraditiiiii hirii d- American Great Lakes (6, 13) and in Lake Zurich. Switzer- ielali). picoplankton represented the largest (P < 0.001) land (7). Studies of smaller eutrophic. oligotrophic. and proportion of phytoplankton biomass in 1987. In 1988. meromictic lakes indicate the importance of these microor- nanoplankton predominated (P < 0.001). This contradicts ganisms more widely (8, 10, 24. 37). Remarkably. both somewhat the findings of other studies (e.g., reference 44). marine and freshwater studies report that the predominant Past Calder Lake data suggest that midsummer nutrient taxon is the cyanobacterial genus Svnechlococcis. The role limitation, when N/P ratios are either >30 (= P limitation), that picoplankton play in aquatic and or <15 (= N limitation, 32. 35). may affect only the largest within the is now thought to be considerable species and favor picoplankton dominance. For the present (26, 39). study, it was decided that direct experiments would be Less has been said in this expanding literature about what conducted in situ to test these observations. The following four were tested. Nutrient favors a role these tiny may play in nutrient cycles. hypotheses (i) loading shift from picoplankton- to nanoplankton- and microplank- Marine picoplankton apparently dominate N uptake in south ton-dominated communities. (ii) A specific N/P ratio (or Atlantic and Antarctic waters (27, 28). These organisms range) can be identified which may be used to predict a were found to have marked preferences for NH4-N over predominant phytoplankton size class. (iii) Microplankton N03-N. In contrast, a picoplankter from Lake 302-S (a (large cyanobacteria in particular) are primarily limited by P. Nannochloris sp.) was efficient with both forms. equally while nanoplankton will be both N and P limited. (iv) while a co-occurring nanoplankter (a Cliir .sochronuiilina sp.) Picoplankton will never be P limited, despite temporally may be unable to metabolize NO-N (47). Laboratory stud- changing nutrient conditions over the course of a summer. ies suggest that some strains of Sv'zllChococcits may be quite adept at metabolizing a variety of N forms (23) and that MATERIALS AND METHODS uptake rates may be quite rapid (30). Previous studies suggest that freshwater algae are unable Study site. Experiments were conducted in Calder Lake. to compete with bacteria for inorganic phosphate (9. 32). yet which is located in southeastern New York. Chemical and smaller picoplankton may still prove to be equal competi- physical features were detailed previously (Wehr, in press). tors. Short-term. very rapid P uptake r-ates by nano- plus but a brief description is provided here. Calder is a 3.9-ha picoplankton in Lake Michigan support this idea (19). In dimictic lake with an average depth of 2.8 m and a maximum times of nutrient surplus. picoplankton may not. however. depth of 6.7 m. The lake stratifies in late spring (usuallv late be superior competitors. For example. there is strong evi- May). and autumn turnover usually occurs in early October. dence which suggests that smaller-sized cells lose their Typically. ice cover forms on the lake in December and competitive advantage when P levels increase (18). Another breaks up in mid-March. The lake is spring fed and is located study found that smaller cells are relatively more abundant within a relatively undisturbed deciduous forest. The Ford- in lakes with lower levels of total P (44). although nano- and ham University Limnology Laboratory is located on the picoplankton were not differentiated. southern shore of Calder Lake. Samples are processed and Recently. I completed a 2-year study of picoplankton analyzed there. in a small eutrophic system. Calder Lake. N.Y. Field procedures. Experiments were run on 3 June. 7 July.

1605 1606 WEHR APPL. ENVIRON. MICROBIOL.

NITROGEN ADDITIONS lected on 0.2-pLm-pore-size Nuclepore filters (0.1 to 0.2 liter per sample, 2-p.m-pore-size filters retained <1% of cells of (,amol/L) Svnellcococciis sp. laboratory cultures). Filters were placed in extraction vials containing 3 to 4-ml of neutral (with 2.5 5.0 12.5 25 50 MgCO3) 90% acetone (1), ground by using a glass pestle and extracted cold (4°C) in darkness for 18 h. Extracts were made to 5 ml and centrifuged, and Chl a was measured 2.5 5.0 12.5 25 50 1 -u spectrophotometrically (Shimadzu UV-160) and corrected I 0 for pheophytin a (21). Water chemistry samples for compar- n ative collections were filtered (0.45-p.m pore size) prior to I i- 12.5 25 2 _s analysis to remove organisms and approximate dissolved fractions Soluble reactive was by r_0c only. phosphate analyzed 3 mu the antimony-molybdate method; the same procedures were 12.5 0 D followed for total dissolved P after persulfate-H,S04 diges- tion (11). NH44-N was determined via phenol-hypochlorite, was determined the sulfanilamide- = N: P RATIO 5.0 and NO3- by using 10 - Cd reduction to NO,- LI NNED method, following (22). 0 Phytoplankton from the water column and experimental z bags were routinely identified by using an inverted micro- 2.5 20 U) scope, after Lugol iodine for preservation and sedimentation (42). Not all picoplankton cells were counted by this method, FIG. 1. Experimental design of nutrient additions for microcosm and numbers were verified after in Lake. Concentriations are levels added to occasionally being experiments Callder onto experimental bags (each treatment wals run in triplicate). filtered (unpreserved) 0.2-pLm-pore-size prestained black Nuclepore filters and examined by epifluorescence microscopy (43). Biomass is presented strictly as Chl a, and 26 August 1988, each for 4 days. On each starting date, primarily because many treatments and replicates and sev- a complete set of water chemistry and phytoplankton sam- eral sampling dates could be processed much more effi- ples were collected for comparison with the microcosm ciently. However, spot comparisons found a reasonable study. For comparative samples and experiments, water was correspondence between cell numbers and Chl a concen- collected by peristaltic pump, fitted with prewashed silicone trations. Data were compiled and analyzed by using the tubing. Experimental microcosms were 4-liter Cubitainers SYSTAT statistical program (47). Formal hypothesis tests attached to floats which were anchored to a location near the were performed by using regression models with nutrient center of the lake. Water was collected from a 1-m depth, levels or N/P ratios as independent variables; the signifi- prefiltered with 250-p.m-pore-size Nitex mesh (with in-line cance of the fit was determined by least-squares analysis filter units) to remove large grazers, and directly added to the (mathematical solutions via analysis of variance [ANOVA]). microcosms. Smaller mesh sizes proved to remove larger phytoplankton species. Each treatment was replicated three RESULTS times. The overall design for each date was the same (Fig. 1). This provided several total loading regimens where [N], [P], Experiment 1. In June, Calder Lake had begun to stratify or the N/P ratio was held constant. Experimental bags were and water clarity was near its maximum (Fig. 2). Soluble fertilized by additions of NH4CI (stock [N] = 10 pM), reactive and total soluble P levels declined slightly since KH2PO4 (stock [P] = 4 p.M), or both by using syringes fitted spring turnover (cf. March results: 3 pLg of soluble reactive with 0.2-p.m-pore-size sterile filter units (Gelman Acrodisc). phosphate per liter, 7 pg of total P per liter in epilimnion). KCI was added to bags to equalize K additions to all Inorganic N in the epilimnion was essentially unchanged treatments. They were mixed and attached to the flotation since turnover. Chlorophyll densities were similar among the anchors. Bags were positioned so that light penetration three size fractions, with little difference with respect to approximated levels measured in situ at a 1-m depth. The depth. Control microcosms and (surface) reference lake lake water is fairly well buffered (ca. 500 p.eq/liter), and water samples contained phytoplankton in the following measurements at the end of each experiment indicated that proportions: picoplankton > nanoplankton > microplankton algal and microbial activity did not appreciably shift HCO- (Fig. 3). Total phytoplankton biomass increased in response or CO, levels in the bags. At the end of each experiment, to nutrient additions, both N and P loading caused a shift to microplankton were collected in situ by using a peristaltic nanoplankton-dominated communities. Microplankton re- pump and 25-mm in-line filter units fitted with 20-p.m- mained the size fraction with the lowest biomass levels, pore-size Nitex mesh disks. Water was then taken to the regardless of nutrient regimen. ANOVA tests indicate that laboratory for further fractionation (<30 min later). microplankton, while present at lower levels, did not re- Laboratory procedures. Phytoplankton fractions follow spond significantly to either nutrient (Table 1). Nanoplank- the definitions of Sieburth et al. (38); microplankton = cells ton were very strongly N limited and also significantly P or colonies of <200 to >20 p.m: nanoplankton = <20 to >2 limited. Picoplankton responded significantly only to N pLm; and picoplankton = <2 to >0.2 p.m. Following mi- additions. No significant effects of N/P ratio were observed. croplankton removal, filtrate was filtered twice more under Communities in unfertilized bags and in surface waters in low vacuum pressure (<25 to 100 mm Hg; 20) in the the lake in early June consisted primarily of a Synechococ- laboratory. A series of 1,000-ml Nalgene polysulfone filter (ciS sp. and also CvNptoinonas erosa, Ci-yptoinonas oVata, units were linked in series to process several sample depths and the chrysophyte Diniobrvon cylindricuin. Following N simultaneously. Nanoplankton were collected on 2.0-p.m- additions (N/P ratios .25), many phytoplankton species pore-size Nuclepore filters (ca. 0.1 to 0.3 liter per sample or increased, including a Mailloniona.s sp. 2, C. ovata, Eiinotia replicate); picoplankton passed through and were later col- cf. c(Irl'nt(i, and 1). cylill1ricitn. abundance Vol. SE. 1989 NUTRIENT LIMITATION IN PICOPLANKTON 1607

PHOSPHORUS (sg / L) NITROGEN (Cg / L) 5 10 15 20 0 50 100 150 200

1. o * 0-0 SRP 1- . q 0-0N03-No o / / *-S TOT-P 0-S 2 2c-2-7 t * NH4-N E 3. 0Q ci) 4. _ i 4. /0 \ 5 6

_Z TEMPERATURE ( °C ) Chlorophyll a (,t.g/L) 0 5 10 15 20 25 34 o 2 3 4 0~ 0 0-0_0 Micro 1.rp* * *0= Nano 24-\A = Pico 2 o

0-c4i) 3 0' 4- 0 :1 5 0 A 6 / FIG. 2. Physical and chemical conditions in Calder Lake on 3 June 1988. when the first microcosm experiment was run. Water clarity is expressed as Secchi depth (m).

-11" V.u cline (Fig. 4). Surface temperatures increased, and stratifi-

- MICRO N11 2 - NANO cation was more obvious. Also, a noticeable decline in CP am PICO, nitrogen and, to some extent, phosphorus occurred in the oo epilimnion. Nitrogen additions resulted in increases in total -i~~~~~~~~~~~~~~~~~~~~- phytoplankton biomass (Fig. 5). P additions also stimulated >- 4.0- phytoplankton growth, but concentrations >2 F.M had little X~~~~~~~~~~~~~~~~ or no effect on biomass. As in June, picoplankton predomi- nated without fertilization (micrograms of Chl a per liter: Ofn2.0 FI picoplankton, 0.52; microplankton, 0.29; nanoplankton, C. 0.16). but nanoplankton became the largest proportion of the total biomass with either N or P addition. At this time, 0 2.5 5.0 12.5 25 50 microplankton also increased with both N (ca. fourfold NITROGEN ADDED (jAM) greater) and P (ca. sevenfold greater) manipulations. These large biomass differences were very highly significant, espe- 12.0- -J r2.0- MICRO cially in response to N fertilization (Table 1). Unlike the ez- NANO larger two size classes, picoplankton was not P limited. 0' L - PICO 9.0- Phytoplankton species composition in July within the 0 -J -J 6.0 TABLE 1. Tests of nutrient limitation of phytoplankton IC" biomass in three size fractions within experimental a. 0 microcosms in Calder Lake" 3.0- 0 -j Expt (date) Size fraction N addition P addition N/P riatio 2: C. 0.0 rl nn 1 (June 1988) Microplankton NS"' NS NS C 0 1.0 2.0 4.0 10 20 Nanoplankton + +" NS Picoplankton NS NS PHOSPHORUS ADDED (IM) +"d FIG. 3. Effects of N aind P additions on phvtoplankton biomaiss 2 (Julv 1988) Microplankton +' +' NS in microplankton. nainoplankton. and picoplankton size classes Nanoplankton +' +" +" within Calder Lake. June 1988. Error bars indicate ±1 standard Picoplankton + ' N S N S error (/ = 3). 3 (August 1988) Microplankton +' +" + Nanoplankton +- +d NS in massive increases increased slightly. P additions resulted Picoplankton +d NS +' in cryptomonads (C. ero.sa plus C. ovatti were >50% of total cell numbers). which were much less abundant in control Indiependent variables were classified on the basis of N and P addition aind bags. There were also increases in the Fagilarii N/P r-aitio aind analyzed by ANOVA. NS. Not significant. (r-iotonlesis (rare in control bags). ) .())1. Experiment 2. By July. water clarity had declined and d P < 0.)5 phytoplankton biomass increased greatly near the thermo- P ().()1. 1608 WEHR AppI ENVIRON. MICROBIOL.

PHOSPHORUS (Cg / L) NITROGEN (jg / L) 5 10 15 0 50 100 150 2001-

1 . 0 0-0 N03-N _E 2- T / **- NH4-N 2- 0 3.

a)Q) 4- 5.

_ TEMPERATURE ( °C ) Chlorophyll a (,ug/L) 5 10 15 20 25 0 1 2 3 4 °0 1k 1 T o- - Micro 1 *-*=0 Nano ^-A= Pico E 2 Q

Q) 0

FIG. 4. Physical and chemical conditions in Calder Lake on 7 July 1988. when the second microcosm experiment was run. Water clarity is expressed as Secchi depth (m). Chi a value at 6-m depth for microplankton. 30.5 jig/liter. epilimnion and in control bags was fairly diverse. It was erosva. In contrast to results of the June experiment. N numerically dominated by a Svinechococcus sp. and included additions resulted in large increases (more than 10-fold several greens (especially Pedfiastrum borvinl/n, Oocv,stis greater) in Svnechococcus numbers and also in the numbers bo)rgei, Stichococcis bacillaris, Scenedesmnus quadlricaudla of several chlorophytes observed in control treatments, Anikistr-odes nniis.ficlkatis), the chrysophytes MAillonwnoaitis sp. especially A. fii1catits and Scenedesinus quadricalda, P 8 (7.5 to 9 rim) and D. cvlindricuim, and the cryptomonad C. fertilization had a very different response, because of the greater stimulation of larger phytoplankton, and little effect on Svneclhococcis abundance. Greens dominated, particu- 8.0 - larly A. JalcatitIs and P. boryalinimn. Another taxon seen la IJ~MICRO rarely in control and N treatments, Coelastritm mnicropormn, Cm Ea N.ANO I 6.0 O was also abundant. Chrysophytes were less abundant in 0 P-fertilizing treatments. -J Experiment 3. In August, chlorophyll densities were at 4.0- their greatest levels in Calder Lake, both in surface and Ia- 0 subsurface communities (Fig. 6). The former consisted of a:: 2.0- colonial cyanobacteria and the deepwater community of CeradinlltiraIndlltiditiella and several smaller taxa (especially 0.0-- C. erosa). Secchi depth was shallower, and inorganic N and P were near their summer minima. Experiments revealed 0-1 0 2.5 5.0 12.5 25 50 NITROGEN ADDED that the entire community (all size classes) responded posi- 0 (/hM) tively to N additions, but there was an apparent threshold 12.0 near 12.5 pLmol/liter (Fig. 7). There were also very large -J - MICRO increases in nanoplankton and microplankton with only I-J - NANO 1-[.mol/liter additions of P, with no further enhancement at 9.0 PICO greater levels. These were the largest biomass levels mea- 0 -4 sured in any experiment and were more than an order of 6.0 magnitude greater than controls. No consistent response 0- was seen in the picoplankton. ANOVA indicates that nitro- 0 3.0- gen alone stimulated all fractions most strongly and that 0 -4 picoplankton was again unaffected by P fertilization (Table I 0- 1). Even though the lake most strongly stratified in August, P limitation was apparently less severe for nanoplankton and 0 1.0 2.0 4.0 10 20 microplankton than in July. PHOSPHORUS ADDED (,uM) Control communities were considerably different from FIG. 5. Effects of N and P additions on phytoplankton biomass those observed earlier, consisting of colonial cyanobacteria in microplankton. nanoplankton. and picoplankton size classes A t(iabenai linnetiuca, Microcvstis (lrl-lginoSoI, and a Syn- within Calder Lake. July 1988. Error bar s indicate + 1 standard error echococcus sp., as well as Malloinonatis cf. caudlata. This (n = 3). result was quite similar to that observed in the epilimnion VOL. 55. 1989 NUTRIENT LIMITATION IN PICOPLANKTON 1609

PHOSPHORUS (Cg / L) NITROGEN (.g / L) 5 10 15 50 100 150

_Ce-c 0.- Q) 0

TEMPERATURE ( °C ) Chlorophyll a (Ag/L) 5 10 15 20 25 30 0 1 2 3 f% .

1 i\\/ o0-0- Micro I 2- '~~~~~~ ~~ 0~-0= Nano

~-4- 64- '

5-a 5 __

FIG. 6. Physical and chemical conditions in Calder Lake on 26 August 1988. when the third microcosm experiment was run. Water clarity is expressed as Secchi depth (m). Chi a values at 0.1- and 6-m depths for nanoplankton. 5.4 and 27.8 p.g/liter. respectively: for microplankton at 6 m, 18.2 ,Lg/liter.

during August. In response to nitrogen addition, species cyst per 26 vegetative cells in control bags to 1 in 130 after N composition shifted slightly, with increases in numbers of fertilization. P-enriched treatments resulted in greater num- most taxa but especially of the colonial green Gonizuii bers of most taxa, although Aniabaenba and Microcvstis spp. pect'orale, which was not common in control treatments. had only modest (<20%) increases. An unidentified Pyraini- There was however, a very noticeable decline in heterocyst inonas species, rare in other treatments, increased by more production by Anabaenia spp., from an average of 1 hetero- than 10-fold following P additions.

DISCUSSION -J Calder Lake is a small, eutrophic lake, typical of many regions; its phytoplank- -J which are found in north temperate 0 ton composition and biomass are comparable with those of well-studied eutrophic systems elsewhere in the world (e.g., 0 ItL Lunzer Untersee, Windermere, Mendota; cf. references 16, 31. and 46). As such, it may be seen as a fair place to test the idea that smaller cells, and picoplankton in particular, are -J I relatively unimportant in eutrophic systems (15, 40, 44). -C Biomass estimates over a period of 2 years in Calder Lake 0 2.5 5.0 12.5 25 50 indicate that picoplankton may predominate over nano- 0 NITROGEN ADDED plankton and microplankton despite the presence of large. (/iM) colonial cyanobacteria (45). a. 0 Direct experiments reported here support the first stated 0 hypothesis that nutrient enrichment favors a nanoplankton- -J dominated community. This only partly supports earlier hypotheses, however, as no enrichment treatments or N/P la ratios favored microplankton dominance. In June, there were large numbers of the large colonial chrysophyte Dinio- IsI br-yon sp., and in August. colonial cyanobacteria Anabaenia and Microcystis spp. were common, yet none of these 0 microplankters surpassed smaller taxa as the largest propor- tion of the total phytoplankton biomass. This is somewhat surprising, as Microcystis spp. have a much lower critical 0 1.0 2.0 4.0 10 20 N/P ratio (ratio = 9) than many chlorophytes, such as PHOSPHORUS ADDED (14M) Scenedesinuis spp. (ratio = 30) or Antkistrodesitulis spp. (ratio = would that under P 7. Effects of N and P additions on biomass 21: 33). One expect increasing loading, FIG. phytoplankton no in microplankton, nanoplankton, and picoplankton size classes N-fixing cyanobacteria should predominate (34). Thus, within Calder Lake, August 1988. Error bars indicate ±1 standard strong evidence was found to identify N/P ratios that can error (n = 3). predict a predominant phytoplankton size class. Greater 1610 WEHR APPL. ENVIRON. MICROBIOL. experimental N/P ratios in Calder Lake did not favor the ACKNOWLEDGMENTS dominance of Synechococucis spp., as has been shown in Financial support for this study was provided by an endowed oligotrophic lakes (41). An artificial reduction in phosphorus grant from the J. P. Routh Foundation and a Faculty Research Grant loading within eutrophic Lake Trummen, Sweden, caused a from the Research Office of Fordham University. shift from a "net plankton" (>20 pm) to a nanoplankton Technical assistance was provided by T. Toolan. (<20 p.m) community (14); however, picoplankton were not LITERATURE CITED differentiated from nanoplankton. In the absence of further 1. American Public Health Association. 1985. Standard methods for nutrient increases in Calder Lake, picoplankton frequently the analysis of water and wastewater. 16th ed. American Public represent the largest proportion of phytoplankton biomass. Health Association, Washington, D.C. The present study also indicated that nanoplankton and 2. Andersen, 0. K., J. C. Goldman, D. A. Caron, and M. R. not to nutrient limitation Dennett. 1986. Nutrient cycling in a microflagellate food chain. picoplankton do respond similarty 111. Phosphorus dynamics. Mar. Ecol. Progr. Ser. 31:47-55. and should be considered separately. 3. Bird, D. F., and J. Kalff. 1987. Algal phagotrophy: regulating An alternative explanation for these results would be that factors and importance relative to in Dinobryon and protozoan grazing during the 4-day experiments (Chrysophyceae). Limnol. Oceanogr. 32:277-284. would likely have been most intense on the smallest phyto- 4. Bloem, J., C. Albert, M.-J. B. Gillissen, T. Berman, and T. E. plankton size fractions, thus causing a microplankton-dom- Cappenberg. 1989. Nutrient cycling through phytoplankton, tests from bacteria and , in selectively filtered Lake Vechten inated community. Preliminary of grazer removal water. J. Plankton Res. 11:119-131. Calder Lake assemblages by using various pore sizes of 5. Buechler, D. G., and R. D. Dillon. 1974. Phosphorus regenera- Nitex mesh found that pore sizes finer than 250 pLm collect tion in freshwater paramecia. J. Protozool. 21:339-343. significant numbers of algal cells, which were the principal 6. Caron, D. A., F. R. Pick, and D. R. S. Lean. 1985. Chroococcoid focus of this study. Some of these, such as D. cvlindricu(m cyanobacteria in Lake Ontario: vertical and seasonal distribu- and Cryptornonas ovata, which were abundant during the tions during 1982. J. Phycol. 21:171-175. also act as on smaller and 7. Chang, V. T.-P. 1980. Zwei neue Synechococcits-Arten aus dem spring, may phagotrophs algal Zurichsee. Schweiz. Z. Hydrol. 42:247-254. bacterial cells (3, 25). Planktonic protozoans consume sig- 8. Craig, S. R. 1984. Productivity of algal picoplankton in a small nificant quantities of heterotrophic and autotrophic pico- meromictic lake. Int. Ver. Limnol. Verhandl. 22:351-354. plankton (26, 36), which may favor larger size fractions in 9. Currie, D. J., E. Bentzen, and J. Kalff. 1986. Does algal- these experiments. Their activity may also have resulted in bacterial phosphorus partitioning vary among lakes? A compar- nutrient regeneration within the bags (4, 5). Some protozo- ative study of orthophosphate uptake and alkaline phosphatase ans, such as have been active in activity in freshwater. Can. J. Fish. Aquat. Sci. 43:311-318. tintinnids, may consuming 10. Drews, G., H. Prauser, and D. Uhlmann. 1961. Massenvorkom- nanoplankton (29), which nonetheless experienced marked men von SYnechococcus planktonicus nov. spec., einer soli- increases in biomass in response to both N and P additions. taren, planktischen Cyanophyceae, in einem Abwasserteich. The hypothesis that specific nutrients limit different size Beitrag zur Kenntnis der sogenannten -pL-Algen.' Arch. Mi- classes seems clear. Experiments indicate that microplank- krobiol. 39:101-115. ton and nanoplankton were limited both by N and P, with P 11. Eisenreich, S. J., R. T. Bannerman, and D. E. Armstrong. 1975. limitation more A simplified phosphorus analysis technique. Environ. Lett. becoming intense by midsummer (July). This 9:43-53. dual limitation is known to occur in oligotrophic systems 12. Ejsmont-Karabin, J. 1983. Ammonia nitrogen and inorganic (41). In contrast, Calder Lake picoplankton were N limited, phosphorus excretion by the planktonic . Hydrobiologia although apparently less severely than larger size fractions. 104:231-236. Finally, experiments support the hypothesis that picoplank- 13. Fahnenstiel, G. L., L. Sicko-Goad, D. Scavia, and E. F. Sto- ton were never P limited, even though concentrations of ermer. 1986. Importance of picoplankton in Lake Superior. soluble reactive in the were Can. J. Fish. Aquat. Sci. 43:235-240. phosphate epilimnion usually 14. Gelin, C., and W. Ripl. 1978. Nutrient decrease and response of <0.05 p.M during the summer. This would be expected, as various phytoplankton size fractions following the restoration of greater surface-area-to-volume ratios can affect nutrient Lake Trummen, Sweden. Arch. Hydrobiol. 81:339-367. absorption rates and result in intrinsically thinner unstirred 15. Glover, H. E., D. A. Phinney, and C. S. Yentsch. 1985. Photo- layers at the cell surface (30). Protozoans may have in- synthetic characteristics of picoplankton compared with those creased the regeneration of P (2, 4) within the bags, although of larger phytoplankton populations, in various water masses in this did not alleviate P limitation in larger size the Gulf of Maine. Biol. Oceanogr. 3:223-248. apparently 16. Hutchinson, G. E. 1967. A treatise on limnology, vol. 2. Intro- fractions. From this study, one may conclude that pico- duction to lake biology and the limnoplankton. John Wiley & plankton may still dominate phytoplankton communities in Sons, Inc., New York. eutrophic lakes, particularly as seasonal periods of nutrient 17. Lean, D. R. S. 1973. Phosphorus dynamics in lake water. depletion may limit the growth of larger species. Science 179:678-679. Phytoplankton have considerable effects on nutrient cy- 18. Lean, D. R. S., and E. White. 1983. Chemical and radiotracer cles through utilization and storage of key elements (17, 35). measurements of phosphorus uptake by lake plankton. Can. J. Fish. Aquat. Sci. 40:147-155. The relative importance of heterotrophic and autotrophic 19. Lehman, J. T., and C. D. Sandgren. 1982. Phosphorus dynamics picoplankton within the microbial loop as sinks or sources of of the procaryotic nanoplankton in a Michigan lake. Limnol. N and P has become better known. Rapidly growing rotifers, Oceanogr. 27:828-838. microflagellates, and other protozoans which graze on these 20. Li, W. K. W. 1986. Experimental approaches to field measure- cells can accelerate the cycling of nutrients within the loop, ments: methods and interpretation. Can. Bull. Fish. Aquat. Sci. which reduces losses of nutrients from the pelagic zone (2, 4, 214:251-286. 21. Lorezen, C. J. 1967. Determination of chlorophyll and pheo- 12). This study suggests that algal picoplankton may play a pigments: spectrophotometric equations. Limnol. Oceanogr. central role not only in microbial food webs but also as 12:343-346. nutrient recyclers, through superior competitive abilities and 22. Mackereth, F. J. H., J. Heron, and J. F. Tailing. 1978. Water subsequent consumption by micrograzers. analysis: some revised methods for limnologists, no. 36. Fresh- VOL. 55, 1989 NUTRIENT LIMITATION IN PICOPLANKTON 1611

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