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Effects of nutrient patchiness and N:P supply ratios on the ecology and physiology of freshwater phytoplankton Suttle, Curtis Arnold 1987

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EFFECTS OF NUTRIENT PATCHINESS AND N:P SUPPLY RATIOS ON T H E ECOLOGY AND PHYSIOLOGY OF FRESHWATER PHYTOPLANKTON by CURTIS ARNOLD SUTTLE B.Sc, The University of British Columbia, 1978 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY m THE FACULTY OF GRADUATE STUDIES (Departments of Botany and Oceanography) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA January 1987 ° Curtis Arnold Suttle, 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 1956 Main Mall Vancouver, Canada DE-fin/ft-n ii ABSTRACT Laboratory and field experiments examined several aspects of the interaction of freshwater phytoplankton species and plankton communities with nitrogen and phosphorus nutrient resources. The laboratory studies focused on the following three main areas: 1) effects of nutrient o 'patchiness' on phytoplankton community structure; 2) kinetics of phosphate (PO4 ) and ammonium (NH4 +) uptake of phytoplankton grown under non-steady-state but limiting rates of nutrient supply; 3) the effect of different N:P supply ratios on phytoplankton NH^ "1" and PO^*^ uptake kinetics and community structure. Nutrient 'patchiness' was simulated by altering the frequency of nutrient addition to cultures. Under conditions of infrequent addition (once per 18 days) dominance shifted to a larger species, and the average cell size of another species increased. Observations of PO4 uptake kinetics were not consistent with most other o studies where kinetics were determined under steady-state conditions. With respect to PO4 , the duration over which maximum uptake rates were sustained was species specific. There was a short lag before maximum uptake rates were realized, and whether maximum uptake rates occurred at the lowest or at intermediate dilution rates depended on the time scale over which the uptake measurements were made. NH^ + uptake rates were found to be greatly enhanced during the first few minutes of uptake. When natural plankton assemblages were grown under N:P supply ratios of 5:1, 15:1 and 45:1 (by atoms), the treatments selected for different competitive dominants. An N:P ratio of 45:1 resulted in total dominance by Synechococcus sp.; cultures grown under 5:1 and 15:1 supply ratios were dominated by Synedra radians, Nitzschia holsatica and Scenedesmus sp. NH4 and PO4 uptake kinetics were not the same in cultures grown under different supply ratios, and ratios of saturated PO4 to NH4 uptake rates were a good indicator of the N:P supply ratio under which the cultures were grown. This relationship was used to derive an index termed the Relative Uptake Ratio (RUR) which can be used to estimate N:P supply ratios in situ. iii Field investigations were conducted on an oligotrophic coastal lake. N H ^ + and PO^"^ uptake rates of size fractionated plankton (< and > 3 um), at a range of substrate concentrations, revealed that a large portion of the total uptake (50-90 % and 65-85 % for NH^ and PO4 , respectively) was attributable to cells in the < 3 um fraction. In addition, saturating PO4 uptake rates of the > 3 um cells were less sensitive to incubation time than smaller cells. The ratio of saturated PO4 to NH^ uptake rates were consistent with nutrient bioassay experiments, and indicated that N:P supply ratios in the lake were in the range where both N and P could be limiting to phytoplankton growth. iv T A B L E OF CONTENTS ABSTRACT ii LIST OF TABLES viii LIST OF FIGURES xi ACKNOWLEDGEMENTS xv INTRODUCTION 1 Community structure 2 Evidence of resource limitation 2 Role of nutrient ratios 3 Role of nutrient patchiness V Objectives and brief overview 8 CHAPTER 1: EFFECTS OF NUTRIENT PATCHINESS ON COMMUNITY STRUCTURE AND CELL SIZE OF A FRESHWATER PHYTOPLANKTON ASSEMBLAGE 11 Background 11 Materials and Methods 12 Culturing 12 Phytoplankton, bacteria and nutrient analyses 14 Results 15 Discussion 26 Community structure 26 Cell size 30 CHAPTER 2: PHOSPHATE UPTAKE RATES OF PHYTOPLANKTON GROWN AT DIFFERENT DILUTION RATES: TOWARDS A PHYSIOLOGICAL DEFINITION OF NON-STEADY-STATE CULTURE 33 V Background 33 Materials and Methods 34 Sampling and culturing 34 Uptake experiments 37 Results 39 Experiment I: OsctfZaforia-dominated cultures 39 Experiment II: Syraec/iococcus-dominated cultures 46 Discussion 51 Delayed maximum uptake rate 51 Changes in uptake rate with dilution rate 52 Sustainability of high uptake rates 53 CHAPTER 3: THE EFFECT OF N:P SUPPLY RATIO ON AMMONIUM UPTAKE IN FRESHWATER PHYTOPLANKTON: EVIDENCE FOR 'SURGE' UPTAKE 55 Background 55 Materials and Methods 56 Culturing 56 Uptake experiments 57 Results and Discussion 57 CHAPTER 4: THE EFFECT OF N:P SUPPLY RATIO ON THE RELATIVE UPTAKE RATIOS OF AMMONIUM AND PHOSPHATE: A METHOD TO ESTIMATE IN SITU N:P SUPPLY RATIOS 62 Background 62 Rationalization of the 'Relative Uptake Ratio' (RUR) 64 Materials and Methods 65 Collection and culturing 65 Uptake experiments 66 Physiological characteristics of competitive dominants 68 Nutrient chemistry 68 vi Results 69 Discussion 75 Relative Uptake Ratios 75 Competitive interactions 77 CHAPTER 5: CO-LIMITATION BY NITROGEN AND PHOSPHORUS IN AN OLIGOTROPHIC FRESHWATER L A K E 80 Background 80 Materials and Methods 81 Description of study site 81 Water collection and nutrient chemistry 81 Bioassays 84 Uptake experiments 84 Results 85 Discussion 98 Particulate nutrient ratios 98 Bioassays 99 RUR experiments 100 Nitrogen limitation 101 CHAPTER 6: TIME-COURSE OF SIZE-FRACTIONATED PHOSPHATE UPTAKE: ARE LARGER CELLS BETTER COMPETITORS FOR PULSES OF PHOSPHATE THAN SMALLER CELLS? 105 Background 105 Materials and Methods 106 Results 108 Discussion 117 Kennedy Lake 117 Sproat Lake 118 Do phosphate pulses favour larger cells? 119 vii CHAPTER 7: SIZE-FRACTIONATED AMMONIUM AND PHOSPHATE UPTAKE RATES OF PHYTOPLANKTON FROM AN OLIGOTROPHY FRESH-WATER L A K E 122 Background 122 Materials and Methods 123 Study site and sample collection 123 Dissolved and particulate nutrient analysis 123 *^N uptake experiments 124 3 2 P uptake experiments 124 Calculation of nutrient uptake kinetic parameters 125 Results 126 Discussion 144 Size-fractionated N H 4 + uptake 144 Pmax a n d ks f o r N H 4 + uptake 144 N H 4 + turnover times 146 P0 4 * 3 turnover times 147 Isotope ( 3 2P0 4* 3) distribution 149 Size-fractionated P 0 4 " 3 uptake 153 Ecological considerations 155 SUMMARY AND CONCLUSIONS 157 REFERENCES 159 viii LIST OF TABLES Table 1: Concentrations of nutrients (ug«l*^ ) in the water collected from Great Central Lake 13 Table 2: *^C-bicarbonate uptake rates (ug d'^h'^ug Chi"*), numbers of bacteria (xl(Aml"*) and glucose turnover times (hours) for each nutrient addition frequency. Each value is the mean from duplicate cultures; estimates of one standard deviation are shown in parentheses 29 Table 3: Temperatures, and concentrations of dissolved and particulate nutrients, at 1 m, in Kennedy Lake on the sampling dates 35 Table 4: Literature values of 'critical' ('optimum') N:P supply ratios for phytoplankton (by atoms) 63 Table 5: The effect of six N:P supply ratios (with replication) on the concentrations of particulate nutrients (PC, PN and PP), particulate nutrient ratios (C:N:P), saturated P 0 4 and NH 4 uptake rates (measured over 120 minutes), and on the Relative Uptake Ratio (RUR; see text), for cultures initiated with natural assemblages of phytoplankton. Nutrient specific uptake rates (V) are expressed as h'*, and volume specific rates (P) as umoM'^h"1 70 Table 6: Final species composition of replicate (Repl) phytoplankton cultures initiated with Kennedy Lake water and grown at different N:P supply ratios 73 Table 7: N H 4 + and P0 4" 3 uptake rates (umoU"1^"1) and RURs of clonal cultures grown semi-continuously under different N:P supply ratios. Synechococcus was isolated from, and was the competitive dominant in cultures of Kennedy Lake phytoplankton grown at 45:1; Synedra was a dominant in, and was isolated from cultures grown at 5:1. Estimates of i l s.d. are shown where replicate determinations of uptake rate were made. Standard deviations about RURs were determined as described in Yates (1981) 74 ix Table 8: Saturated P 0 4 * 3 and N H 4 + uptake rates (nmoH'^h'1) and the ratio of those uptake rates (RUR) can be used to yield an estimate of the in situ N:P supply ratio (Chapter 4). The estimate of one standard deviation (s.d.) about RUR was estimated as described in Yates (1981) 91 Table 9: Cellular uptake rates (Tmax; fmol'cell'^h"1), half-saturation constants (kg; uM) for P0 4" 3, minimum P cell quotas (qQ; fmol»ceU"*) and specific uptake rates ( V m a x ; h"*), in P-limited phytoplankton. V m c a / k s is the affinity for P 0 4 " 3 at low substrate concentrations. T m a x , kg, and q0 are from Smith and Kalff(1982) 102 Table 10: Dissolved nutrient (uM) and size-fractionated particulate nutrient (uM) and chlorophyll (ug«l"*) concentrations (21 s.d.) Maximum specific uptake rates (h"1) for N H 4 + (V N) and P 0 4 " 3 (V p) are shown for the less than and greater than 3 um, and unfractionated plankton community in Kennedy Lake, on three sampling dates 127 Table 11: The proportion of the total N H 4 + or P 0 4 ' 3 uptake in the < 3.0 um size fraction, relative to the amount of chlorophyll (Chi) in that fraction (i.e. ( P < 3 u m I Ptotai) I ( C h l < 3 u m I ChltQtal)). A value of unity indicates that the saturated chlorophyll specific uptake rates for the two size fractions, for a given nutrient, are the same. A value greater than one indicates that a disproportionate amount is entering the smaller size class 131 Table 12: Calculated kinetic data (Pmax, nmoM*1^"1; kg, uM) from the N H 4 + and P 0 4 * 3 uptake experiments for the less than and greater than 3 um size fractions. Data were derived by fitting least-squares regression lines to plots of S/V vs S (Woolf plots), for all substrate concentrations. Coefficient of determination (r^) and the probabilities (p) of the slopes being equal for Woolf plots of each size fraction are shown; p determined by analysis of covariance 139 X Table 13: Kinetic constants C^^, nmol*r1,h"i; kg, uM) and turnover times (TT, h) calculated either by using data for all but the highest two substrate concentrations (Lo), or using only the two highest additions (Hi). Lo values were derived by fitting least-squares regression lines to Woolf plots of the data; Hi values were calculated from the linear equation defined by the points associated with the two highest concentrations. P0 4" 3 turnover times calculated using Woolf plots were greatly in excess of values estimated from incorporation of carrier-free ^PO^" 3 (see text). Missing data were either the result of too few substrate concentrations to warrant the calculations (May), or because the data were not adequately described by a Michaelis-Menten fit (Sept) 142 q Table 14: Probabilities (p) of slopes being equal for Woolf plots of July P0 4 uptake data taken over different time intervals (minutes), as determined by analysis of covariance. Data are for unfractionated plankton samples, and do not include the 2.0 uM PCs concentration 143 xi LIST OF FIGURES Figure 1: Changes in the concentrations of P as soluble reactive phosphorus (SRP), N as ammonium (NH 4 + ) and nitrate + nitrite ( N O 3 " ) , and Si as reactive silicate (Si0 4"*), in cultures to which nutrients were added every 4 days; (solid symbols), replicate I; (open symbols), replicate U. Points are for single determinations. Dashed lines indicate an assumed relationship when the occasional intervening value was unreasonable. Arrows indicate the detection limit for each nutrient. Where data points fall on each other, replicate I values have been offset slightly to the right 16 Figure 2: As for Figure 1; data are for cultures to which nutrients were added every 8 days 18 Figure 3: As for Figure 1; data are for cultures to which nutrients were added every 16 days 20 Figure 4: Proportion of total phytoplankton cell volume made up by each cell type, for each replicate (I and II). (A), nutrients added once every 4 days; (B), once every 8 days; (C), once every 16 days. Arrows indicate time of nutrient additions 22 Figure 5: Average cell volume as a function of nutrient addition frequency for replicate cultures (I and II) at the end of the experiment 24 Figure 6: Frequency distributions of cell length for Synedra radians as a function of nutrient addition frequency, at the end of the experiment. Culture designation (A, B and C) as in Figure 4; (shaded), replicate I; (unshaded), replicate II 27 Figure 7: C:P and N:P ratios (by atoms) for cultures grown at different dilution rates. Each data point represents a single culture. The particulate P concentration is constant and independent of dilution rate. Curves are fitted by eye 40 xii Figure 8: Total cell volumes for cultures grown at different dilution rates. Each point represents a single culture. Curve is fitted by eye 42 q q Figure 9: Changes in perturbation P 0 4 uptake rates and P0 4 concentrations with time, for cultures grown at different dilution rates. Arrows show the o q first P 0 4 concentration measured after additional P 0 4 was added to some cultures. Phosphate concentrations: replicates A O, and B £ ) phosphate uptake replicates A M, and B (*). Dashed lines join data collected from replicate experiments run on the same cultures on different days 44 Figure 10: Perturbation P0 4* 3 uptake rates for different uptake intervals and dilution rates. Only rates over intervals where the P0 4" 3 concentration remained above 1.5 uM are shown. Vertical lines join data collected from replicate experiments run on the same cultures on different days. Curves are fitted by eye. W"^ A, v5-15 W ) v15-30 w > v30-45 Q > y50-60 ^  y60-90 p T h e d a s h e d l i n e Fitted for V^"^ represents a conservative interpretation and is not meant to imply a specific relationship 47 q q Figure 11: Perturbation P0 4 uptake rates and P0 4 concentrations for Synechococcus dominated cultures grown at 0.50 d"1. Phosphate concentrations: replicates A Q , B O, and C Phosphate uptake rates: replicates A B and C ft) 49 Figure 12: N H 4 + uptake rates (solid symbols) and N H 4 + concentrations (open symbols) for perturbation experiments on triplicate phytoplankton cultures, grown at N:P supply ratios of 5:1, 15:1 and 45:1 59 Figure 13: The relationship between N:P supply ratio and the Relative Uptake Ratio (RUR) of saturated P0 4" 3 uptake rates to saturated N H 4 + uptake rates, for cultures initiated with natural assemblages of fresh-water phytoplankton tt, and for unialgal cultures of Synechococcus sp. @), and Synedra radians Q. The regression equation is for the experiments initiated with lake water and xiii does not include RUR values estimated from the unialgal cultures or for one outlier © 71 Figure 14: Map showing Kennedy and Sproat Lakes and the location of the sampling stations (solid symbols) 82 Figure 15: Concentrations of N O 3 " + N O 2 " at 1 m in Kennedy Lake during 1983 and 1984 87 Figure 16: Particulate nutrient ratios (by atoms) at 1 m in Kennedy Lake during 1983 and 1984 89 Figure 17: Changes in relative fluorescence in nutrient enrichment bioassay experiments initiated with water collected at Kennedy Lake during May 1984. Different symbols represent individual fluorescence measurements taken on triplicate cultures 92 Figure 18: Changes in relative fluorescence in nutrient enrichment bioassay experiments initiated with water collected at Kennedy Lake during July 1984. Different symbols represent individual fluorescence measurements taken on triplicate cultures 94 Figure 19: Changes in relative fluorescence in nutrient enrichment bioassay experiments initiated with water collected at Kennedy Lake during September 1984. Different symbols represent individual fluorescence measurements taken on triplicate cultures 96 Figure 20: P0 4 " 3 uptake rates for the 0.2-3.0 um size fraction for the three time intervals 0-60, 60-120, and 170-240 minutes. Data are from Kennedy Lake in May 1984. Curves were fitted by eye 109 Figure 21: As for Figure 20, but for the > 3.0 um size fraction I l l Figure 22: P0 4 " 3 turnover times as a function of the P 0 4 * 3 concentration added; data are from Kennedy and Sproat Lakes 113 Figure 23: P0 4 " 3 turnover times and proportion of 3 2 P uptake into the 0.2-3.0 um xiv size fraction, preceding and following the addition of nutrients to Sproat Lake 115 Figure 24: Estimates of size-fractionated N H 4 + uptake rates for Kennedy Lake phytoplankton on each of three sampling dates. Rates are plotted against the N H 4 + concentration added, as measured values were apparently overestimates (see text). Incubations were for 2 hours 129 Figure 25: Percent of 3 2 P in 0.2 um filtrate over time, for a range of P0 4 ' 3 additions to Kennedy Lake plankton, in July. Curves from May and September were similar. Duplicates of the 0.1 uM addition were conducted. Note that the y-axis is a logarithmic scale 132 Figure 26: Size fractionated P0 4 ' 3 uptake rates for Kennedy Lake phytoplankton on each of three sampling dates. The uptake rates shown were estimated over 1 hour of incubation. The uptake rates measured in September may not have q been saturated by the 2.0 uM P0 4 addition. Rigler bioassays estimated ambient P0 4" 3 concentrations to be less than 0.01 uM on all dates 134 q O Figure 27: Percentage of total P taken up which entered the > 3.0 um fraction, q as a function of the added P0 4 concentration. Values are averages of the isotope distribution for all sampling periods. The error bars indicate 11 s.d. of the means 136 Figure 28: Woolf plot of the July P0 4 * 3 uptake data for the less than and greater than 3 um size fractions. Notice the deviation from linearity in the large size fraction, at the 2.0 uM concentration 140 Figure 29: Percent of total 3 2 P added remaining in solution when an asymptote is O reached, as related to measured P0 4 turnover times. Data were taken from the literature and are for a variety of lakes. Kennedy Lake data were not included, as they represented conservative estimates of turnover times (see text) 151 XV ACKNOWLEDGEMENTS I am greatly indebted to many whose help made the completion of this work possible. My academic supervisor P.J. Harrison provided laboratory space and financial support, and was always available for advice when needed. He was also very gracious in allowing me to pursue my own research goals. The other members of my research committee, W.E. Neill and R.E. DeWreede were also generous with their time and advice. All the members of the lab provided an atmosphere for discussion and critisism and I am sure these exchanges influenced much of the work undertaken in this thesis; however, J.S. Parslow, N.M. Price and P.A. Thompson deserve special mention in this regard. The field and laboratory work could not have been completed without an enormous amount of help and I am especially greatful to those who gave valuable time to assist, namely, W.P. Cochlan, N.M. Price, C.A. MacDougall, P.A. Thompson, P. and Y. DeSouza, and N. Demalig. The figures in the thesis were drafted by W.P. Cochlan. The greatest thanks are due to A.M. Chan who helped in all aspects of the field and laboratory studies, and provided support beyond that which could be expected of anyone, in every part of the work. The staff of the Lake Enrichment Program of the Department of Fisheries and Oceans were generous in lending the use of equipment and providing logistical support on occasion. Finally, financial support was provided by a NSERC post-graduate scholarship, a B.C. Science Council GREAT award, a MacMillan Fellowship, and teaching assistantships. The reaserch was supported by funds from a Department of Fisheries and Oceans Science Subvention Grant, and NSERC operating grant 58-0128. 1 I N T R O D U C T I O N Community structure The factors that are responsible for maintaining or changing structure in communities have long been a central theme of debate among ecologists. Terrestrial plant ecologists have gradually placed less importance on the view expounded by Clements (1916) that species change is primarily self-directed, and each species in an 'association' modifies the environment, allowing others to follow. The paradigm replacing it is a more individualistic species concept. Proposed in North America by Gleason (1926), and eloquently argued for by Drury and Nisbet (1973), it stresses that observed community structure is greatly influenced by stochastic phenomona and is largely unpredictable. Perceptions of phytoplankton community dynamics have in some respects been less contentious. The year to year reoccurrence of successional sequences in many temperate lakes (e.g. Moss 1980) and coastal marine habitats (e.g. Margalef 1958, Smayda 1980) has led to the view that species replacement is primarily deterministic. As well, the highly variable nature of the environment, and the success with which certain models (Riley 1942, Sverdrup 1953, Pingree et al. 1978) have been able to predict phytoplankton 'blooms' has emphasized the greater importance of physical processes in successional events in aquatic communities. Yet, it remains disconcerting that high species richness is a consistent feature of apparently relatively unstructured pelagic habitats. Ten to twenty species are typically found in what would amount to a thimble-full of water, in environments which are considered to be limited by at most a few resources. This has led to suggestions (Hutchinson 1961) that phytoplankton are an exception to the 'competitive exclusion principle' widely attributed to Gause (1934). Perhaps because phytoplankton represent nearly ideal organisms with which to study ecological processes because of their short generation times, ease of culture and high diversity, considerable progress has been made towards elucidating factors which potentially affect species composition in nature. A partial list of such mechanisms would include selective 2 predation by zooplankton (Ganf 1983), bacteria (Fitzgerald 1969, Burnham et al. 1984), viruses (Mayer and Taylor 1979), and fungi (Gaertner 1979), allelopathy (Keating 1977, Gleason and Paulson 1984), differential mortality in predator digestive tracts (Porter 1975, Johnson et al. 1982), differential sinking rates (Walsby and Reynolds 1980, Harrison et al. 1986), changes in light quantity and quality (Wall and Briand 1979), utilization of alternative sources of the same nutrient (Antia et al. 1980), auxotrophic requirements (Bonin et al. 1981), facultative phagotrophy (Daley et al. 1973, Bird and Kalff 1986), inherent spatial and temporal variability in aquatic environments (Richerson et al. 1970), ability of some phytoplankton to produce metal binding substances (Murphy et al. 1976), and limitation of different species by different resources (Tilman 1977, Kilham 1986). Evidence of resource limitation Despite the large number of factors which in concert dictate species composition, aspects of resource supply are thought to be particularly important in this regard. Although by no means a unanimous view (Hulbert 1970, Goldman et al. 1979) the concensus remains that oligotrophic aquatic habitats represent nutrient-limited systems (Schindler 1977, Moss 1980, Maestrini and Bonin 1981). In lakes the evidence for nutrient-limitation is convincing. A strong relationship has been demonstrated (e.g. Sakamoto 1966, Dillon and Rigler 1974) between the concentrations of spring overturn phosphorus (P) and mean summer chlorophyll a. Vollenweider (1976) extended these and other data to demonstrate that the trophic status of many lakes was predictable essentially from P loading criteria. In a literature far too large to review here, bioassay studies in bottles and bags using water from a spectrum of lakes ranging from marl to monomictic-montane (e.g. Wetzel 1972, Lane and Goldman 1984), repeatedly show increased algal biomass or growth rate in response to nutrient addition, and as long as the experiments have been designed carefully such results should be representative of 'field' conditions (Schelske 1984). Experiments in eutrophication control, where decreases in nutrient inputs resulted in declines in algal biomass (Edmondson 1972, Holmgren 1985), 3 provide additional examples of resource levels controlling phytoplankton standing-stock. Finally, uncontestable evidence of resource limitation comes from whole-lake fertilization studies (Schindler et al. 1973, Schindler and Fee 1974, Stockner and Shortreed 1985) where increased phytoplankton biomass can be directly attributed to inorganic nutrient enrichment. Clearly, as a general rule, phytoplankton production is nutrient limited in most freshwater systems; however, this in itself does not provide a means where aspects of resource supply may dictate species composition. The realization that resources could have a role in maintaining phytoplankton diversity has evolved from the concept that algal cells are not bathed in a homogeneous dilute solution in which a single nutrient is limiting. Rather, the contemporary viewpoint portrays potentially limiting nutrients being pulsed to the euphotic zone of oligotrophic areas, on a continuum of time and space scales ranging from very small perturbations delivered by individual zooplankton (Lehman and Scavia 1982a, b, Scavia et al. 1984) to large scale mixing events. In the first instance the 'patch' will disappear in seconds (Jackson 1980) while in the latter case the nutrient may remain detectable for days. The quality of such nutrient pulses will also vary. 'Deep' water mixed into the epilimnion typically contains high levels of nitrate (NOg") and silicate (SiO^" )^, and in most temperate oligotrophic lakes has large amounts of nitrogen (N) relative to P. In contrast, regenerated N from zooplankton is released as ammonium (NH 4 + ) or organic N at a wide range of N:P ratios (Ikeda et al. 1982, Madeira et al. 1982). Therefore the scenario exists whereby phytoplankton are continuously exposed to patches of resources of varying quantity and quality. It is a small subset of this problem which has provided the impetus for the research outlined in this dissertation. Role of nutrient ratios • Evidence that nutrient ratios may influence phytoplankton community structure comes from theoretical and laboratory studies and field observations. The first two supply a mechanistic approach for understanding the potential role of nutrient supply ratios in 4 structuring phytoplankton communities. The latter provide general, empirical observations which appear to corroborate the theoretical models in many instances. I will briefly consider both below. Peterson (1975) demonstrated theoretically that co-existence of two species should be possible when each is limited by a different resource. He proposed a partial solution to Hutchinson's (1961) 'paradox of the plankton' with a model formulated on familiar Monod (1942) growth kinetics, rmax' S *s + S where, r is the growth rate, rmax is the maximum growth rate, S is the concentration of the growth limiting resource and kg is the resource concentration at which r is one half rmax-Briefly, the model allowed the co-existence of several species of phytoplankton, and simply required that several resources be in short supply, and that the needs and uptake abilities of individual species for those nutrients be different. The assumption that as each species grows it sequesters nutrients in fixed proportions was unrealistic. Tilman (1977) used similar reasoning and Monod kinetics to develop a model which was based on the ratio of nutrient supply, and not on the ratio of nutrient removal by the phytoplankton. The model makes specific predictions of domains of competitive dominance between two phytoplankton species along continua of supply ratios for any two resources, provided that the growth kinetics for those resources have been defined. The essence of the model states that phytoplankton require resources in specific ratios that can be approximated as follows: given that growth on either of two nutrients (Sj and S2) is adequately described by Monod kinetics and that maximum growth rates ( r m a x ) on the nutrients are the same, then at any stated growth rate (r), Sj ^ s2 kj + Sj k2 + S2 5 which can be rearranged, S_l S2 The ratio of kj/k2 represents the switching point between limitation by Sj and S2 (Titman 1976). When the nutrient supply ratio is lower than kj/k2, limitation by Sj occurs, and when the ratio is higher than kj/k2, S2 is limiting. This threshold has been termed the 'optimum ratio' (Rhee 1974). If two species with different optimum ratios are grown along a continuum of supply ratios, there is a region where each species will be limited by a different resource and co-existence can occur. Outside of that region both species would be limited by the same resource and competitive exclusion would occur. Culture experiments subjecting Asterionella formosa, Cyclotella meneghiniana (Titman 1976, Tilman 1977) and three other diatoms (Tilman 1981) to a wide range of silicate SiO^"* to phosphate (PO^"3) supply ratios, largely confirmed the ability of the model to accurately predict such competitive interactions. As well, other studies were conducted (Holm and Armstrong 1981, Sommer 1983a, 1983b, Kilham 1986) which indicated that SiO^"^ to PO^"3 supply ratios could influence the structure of phytoplankton communities. Speculation that the model should hold for other nutrients encouraged the determination of optimum N:P (Rhee and Gotham 1980, Terry 1980) and C:P (Turpin 1986) ratios for several species. In addition, laboratory experiments were carried out examining the effect of N:P supply ratios on the structure of natural phytoplankton communities (Tilman and Kiesling 1984, Tilman et al. 1986); however, explicit testing of the model for nutrient pairs other than P 0 4 and SiO^ has not been done to date. Besides the laboratory studies there is also field evidence which indicates that nutrient supply ratios may be important structuring elements of phytoplankton communities. In a widely cited paper, Tilman (1977) argued that the relative distribution of the diatoms Asterionella formosa and Cyclotella meneghiniana, along a natural silicate-phosphate gradient 6 was consistent with predictions of his resource competition model. However, less emphasis should probably be placed on these observations because of sharp criticism (Sell et al. 1984) of the data base and some of Tilman's interpretations. Nonetheless, many observations of shifts to non- or low silicon (Si) requiring species, in conjunction with decreases in S i O ^ concentration historically (Carney 1982), seasonally (Kilham 1971, Bierman and Dolan 1981, Ferguson and Harper 1982, Sommer and Stabel 1983, Sommer et al. 1986) and in enclosure experiments (Lund 1972, Schelske and Stoermer 1972) suggests, as would be intuitively expected, that declining SiO^*^ availability relative to other resources is detrimental to diatoms. As well, nutrient manipulation studies indicate that changes in N:P supply ratios can select for, or against, algae capable of fixing atmospheric N. Schindler (1977) demonstrated that addition of low N:P ratio fertilizers (12:1, by atoms) to lakes in the Experimental Lakes Area of northwestern Ontario resulted in domination by N-fixing blue-green algae, whereas when a higher N:P ratio (32:1) was added, green algae dominated. Complementary results were obtained by Barica et al. (1980) who were able to suppress blooms of N-fixing algae by the addition of inorganic N. Indirect evidence that N:P ratios effect algal species composition comes from a literature compilation of information from 17 lakes. Smith (1983) reports "a dramatic tendency for blue-green algal blooms to occur when epilimnetic N:P ratios fall below about 29:1 by weight (64:1, by atoms), and for blue-green algae to be rare when the N:P ratio exceeds this value". The N:P ratios mentioned in this study are the ratios of total N to total P (particulate plus dissolved) in the water. The above examples show that there is considerable support for the hypothesis that nutrient ratios are important in influencing community composition of the phytoplankton. However, a weak link in attempting to extrapolate results from laboratory competition experiments to unperturbed natural environments is the inability to measure resource supply ratios in situ. Unlike experimental systems where the supply ratio is known, evidence that supply ratios are important in influencing community composition in nature relies on the tenuous supposition that measured concentrations reflect supply ratios. Another major n I weakness in using the Tilman model to explain species distributions is that the model was conceived for steady-state systems. Under such conditions there is no need to discriminate between uptake rate and growth rate as the two are equal (Burmaster 1979); hence the success of a model based on Monod kinetics, where growth rate is related to external nutrient concentration. In contrast, when resource levels fluctuate it is no longer possible to predict growth rate from nutrient concentration as the processes of uptake and growth become uncoupled. Clearly, successful prediction of competitive outcome in situations where limiting resources fluctuate, requires a much better understanding of the physiological response of species to non-steady-state resource supplies. Role of nutrient patchiness Empirical evidence indicates that resource fluctuations may also be an important factor affecting phytoplankton community structure. The studies of Sommer (1984, 1985) have shown that under regimes where the limiting nutrient is delivered in pulses that it is possible to maintain a more complex species composition than when it is added continuously. This is consistent with theoretical models (Grenney et al. 1973, Crowley 1975) which indicate that it is possible for more than one species to exist on a single, fluctuating, limiting resource. Other investigations have found that the degree of nutrient 'patchiness' is also able to affect phytoplankton community structure. Turpin and Harrison (1979) reported that when the frequency of addition of the limiting resource was changed from continuous to a once per day, competitive dominance shifted from diatoms of the genus Chaetoceros to the diatom Skeletonema costatum. Less frequent additions resulted in increases in cell size and further changes in community structure (Turpin and Harrison 1980). Similarly, Robinson and Sandgren (1983) found that the composition of multispecies assemblages was dependent on whether nutrients were added daily, weekly or at four week intervals, and others (Scavia et al. 1984, Sakshaug and Olsen 1986) have produced dominance changes in culture as a result of altering nutrient addition frequencies. Prior to this thesis little work had been undertaken which examined the effects of 'large-scale' nutrient patchiness on community composition. As well, minor consideration had been given to the role that patchiness might play in maintaining diverse size structure in phytoplankton communities. Arguments based on theory (Laws 1975, Smith and Kalff 1983) suggest that small cells should be superior competitors for resources when ambient q concentrations are low. Frequent observations that most of the P 0 4 flux in marine (Berman 1983), estuarine (Faust and Correll 1976, Friebele et al. 1978, Berman 1983) and freshwater (Schindler et al. 1979, Lean and White 1983) systems goes into the smaller size classes would seem to support this reasoning. In addition, thorough studies by Currie and co-workers (Currie and Kalff 1984a, b, Currie 1986, Currie et al. 1986) indicate that larger phytoplankton q (> 3.0 um) in P-limited systems are not able to meet their requirements by uptake of P 0 4 at in situ concentrations. However, despite trends of decreasing cell size with decreasing nutrient availability in both marine (Hulbert 1970, Herbland et al. 1985) and freshwater habitats (Watson and Kalff 1981), populations of larger cells are frequently found in nutrient depleted areas. The findings of Turpin and Harrison (1980) that less frequent nutrient additions select for larger cells, and the observations of Lean and White (1983) that at higher q concentrations more P 0 4 flux goes into larger size fractions indicates that bigger cells may sequester their P requirements from pulses of elevated concentration. Objectives and brief overview There were four main objectives of the thesis research. The first was to examine the effect of non-steady-state nutrient supplies on phytoplankton cell size, community composition, and uptake kinetics. The second was to ascertain the effects of different N:P supply ratios on phytoplankton community composition and uptake kinetics. A third objective was to determine if N:P supply ratios were potentially important in affecting community composition in oligotrophic coastal lakes. The final objective was to document the changes in demand for I q N H 4 and P 0 4 in small and large phytoplankton at different times of the year. 9 The effect of nutrient addition frequency on community composition was investigated in experiments described in Chapter 1. Changes in the frequency of nutrient addition affected community composition and cell size. The observation that less frequent nutrient additions resulted in increases in cell size indicated that larger cells were seqestering more of the limiting resource under rarer enrichment regimes. This result inspired the experiments q detailed in Chapter 6, which examined PO4 partitioning between large and small size fractions, of natural assemblages of freshwater plankton. As the nutrient addition frequency was only a major structuring element under very infrequent nutrient addition regimes, further studies were conducted under a single frequency of nutrient addition. As very little published data exist on the uptake characteristics of phytoplankton grown under non-steady-state nutrient supplies, the experiments in Chapters 2 and 3 were conducted. The results presented in these chapters provided unique information, and indicated that phytoplankton responded to pulsed nutrient supplies in a more complex fashion than had been previously realized. The effect of N:P supply ratios on community structure and phytoplankton uptake kinetics was studied in experiments outlined in Chapter 4. Different N:P supply ratios resulted in assemblages dominated by different species. As well, uptake rates of NH4 + decreased and q rates of PO4 increased in response to increases in the N:P supply ratio. This relationship provided the basis for the Relative Uptake Ratio (RUR) index described in this chapter. The RUR index, along with more traditional bioassay procedures, was used to provide evidence that N and P are potentially co-limiting resources in some west coast lakes (Chapter 5). The change in demand for PO4 and NH4 was investigated in a series of N H 4 and PO4 uptake experiments on size fractionated plankton, described in Chapter 7. If N and P co-limit phytoplankton growth in some oligotrophic west coast lakes, it is important to have a good understanding of how the dynamics of these resources change seasonally; these studies are a first attempt in this respect. Considerable effort was also spent in evaluating estimates of turnover times for the two nutrients in light of their possible role as co-limiting resources. 11 CHAPTER 1: EFFECTS OF NUTRIENT PATCHINESS ON COMMUNITY  STRUCTURE AND C E L L SIZE OF A FRESHWATER PHYTOPLANKTON ASSEMBLAGE BACKGROUND Empirical evidence indicates that resource fluctuations may be an important factor affecting phytoplankton community structure. Studies have shown that the frequency of nutrient addition can effect species richness (Robinson and Sandgren 1983, Sommer 1984, 1985), competitive outcome (Turpin and Harrison 1979, Scavia et al. 1984, Sakshaug and Olsen 1986) and cell size (Turpin and Harrison 1980). In nature, limiting resources are thought to be injected into nutrient deplete surface waters by a variety of mechanisms, ranging in scale from the excretion 'plumes' of individual zooplankton to major mixing events caused by upwelling or storm activity. However, the understanding of how such events may affect phytoplankton assemblages is still very limited. The experiments in this chapter were designed to simulate large scale nutrient 'patchiness' by subjecting a natural community of freshwater phytoplankton to a range of nutrient addition frequencies. The primary purpose was to examine the influence that such patchiness had on commmunity structure and cell size. As well, inorganic carbon uptake, glucose turnover times, and changes in bacterial numbers were monitored to ascertain if changes in the frequency of nutrient addition affected these parameters. 12 MATERIALS AND METHODS Culturing Water was collected in February, 1980, from Great Central Lake, a moderately large (55 km ) oligotrophic lake on Vancouver Island in southwestern British Columbia, Canada (49°22'N, 125°15'W). Limnological details are provided in LeBrasseur et al. (1978) and Stockner et al. (1980). Two hundred liters of water were collected from the 1 m depth and immediately filtered through 120 um screening to remove large grazers. Temperature at the time of collection was 3.6 °C; nutrient concentrations are shown in Table 1. Twenty liters of this water were placed in an incubator under continuous irradiance and over the next 6 days the temperature gradually increased to 10 °C, and the irradiance from 25 uE'm'^s"* to 120 uE«m «s . The remaining 180 liters of water were filtered through 0.45 um nitrocellulose filters, and stored in the dark at 4 °C. Cultures were initiated using 1 liter of 120 um filtered lake water and 1 liter of 0.45 um filtered lake water added to each of eight, 2.8 liter Fernbach flasks. Every two days the cultures were thoroughly mixed and diluted by pouring off 1 liter of culture and replacing it with the same volume of 10 °C, 0.45 um filtered lake water. This results in a dilution rate (D) of 0.35 d"* (D = (-In f)/2 where f is the fraction of original volume remaining subsequent to a dilution). Each culture was supplied with additional N and P either once per 4 days, once per 8 days or once per 16 days, with all treatments beginning on Day 3 of the experiment. Pulses were administered by supplementing the filtered lake water with ammonium-nitrate and ammonium-phosphate at an N:P ratio of 15:1 (by atoms). Concentrations of the pulses were varied such that each culture received the same nutrient flux over 16 days (40.2 ug P-P0 4" 3, 117.6 ug N-NOg" and 154.0 ug N-NH 4 + ). The positions of the flasks in the incubator were initially randomly assigned and then switched when diluted to minimize any possible light effects. Table 1: Concentrations of nutrients (ug*!"1) in the water collected from Great Central Lake. Total P 4 Total dissolved P 1 Soluble reactive P < 1 Silicate 2460 Nitrate + nitrite 36 Ammonium 4 Dissolved organic N 50 14 Phytoplankton, bacteria and nutrient analyses Samples were enumerated for species composition and cell size on five occasions during the experiment. Cell numbers were estimated by counting a minimum of 200 cells of each species, except where species were so rare to make this impractical. Counts were made on 10 ml settled samples, under 400x magnification (Lund et al. 1958). Cell volumes were calculated for a minimum of 35 cells of each species, for each sampling date, using formulae for similar geometric shapes. Samples were taken prior to major pulsing events and at the termination of the experiment to alleviate problems caused by species which oscillate with the pulsing frequency. In addition, ^Cj-bicarbonate accumulation (Steemann Nielsen 1952) was used to estimate dissolved inorganic carbon (DIC) uptake rates. Alkalinity was estimated indirectly using potentiometric titration; DIC was calculated from pH, temperature, total dissolved solids and alkalinity (Amer. Pub. Health Assoc. et al. 1976). Two-hour incubations were carried out in duplicate in 125 ml Pyrex B.O.D. bottles, using the same conditions under which the algae were grown. Values of isotope uptake in dark bottles were subtracted from values obtained for light bottles. Chlorophyll a measurements for standardization of bicarbonate uptakes were done by acetone extraction and measured fluorometrically as described in Strickland and Parsons (1972). Numbers of bacteria were determined on four dates using the Acridine Orange Direct Count (AODC) method of Hobbie et al. (1977). As well, bacterial productivities were estimated at the same times and on one additional occasion by measuring the incorporation of tritiated glucose over 2 hours, and calculating turnover times (Azam and Holm-Hansen 1973). Modifications which were made to the methods are outlined in detail in Maclsaac et al. (1981). Productivity estimates for phytoplankton and bacteria were originally designed to coincide with the 'once per 8 day* nutrient addition frequency; however, logistical problems resulted in deviation from this scheme for the last two sampling dates. 15 Samples for nutrients were taken each time immediately prior to when the cultures were diluted. Soluble reactive P (SRP) was analyzed by the chemical technique of Murphy and Riley (1962), as outlined in Stainton et al. (1977). Analysis for ammonium (NH 4 + ) was by the Solorzano (1969) technique, for nitrate (NOg") by the method of Morris and Riley (1963), for total P and total dissolved P by the method of Traversy (1971), and for silicate (Si0 4" 4) as proposed by Armstrong (1951); these techniques were automated in the manner described in Stephens and Brandstaetter (1983). Dissolved organic N was analyzed as in Stephens and Brandstaetter (1983). R E S U L T S Changes in the concentrations of SRP, NH 4 , NOg", and Si0 4 over the course of the experiment are shown in Figures 1 through 3. Low concentrations of SRP in the initial stages of the experiment suggest that P likely limited algal growth at the beginning of the experiment; however, by the end of the experiment low NOg", NH 4 + and Si0 4" 4 concentrations under the most frequent nutrient addition regimes, may have resulted in different species being limited b}' different resources. The replacement of Rhizosolenia sp. by Synedra radians was characteristic of the initial stages of all three treatments when expressed either as a proportion of total cell volume (Fig. 4), or as a proportion of total cell number (data not shown). S. radians remained dominant under the more frequently pulsed regimes, while Tabellaria fenestrate became dominant under conditions where nutrients were added least often. The pulsing frequency also affected the phytoplankton size distribution; the largest cells occurred when nutrients were added least often (Fig. 5). This was mainly because of a shift in species composition from S. radians to the larger T. fenestrata. Under the 'once per 16 days' nutrient addition regime the mean cell volumes of S. radians and T. fenestrata were 880 um 3 (s.d. = 288, n = 71) and 3466 um3 (s.d. = 820, n = 55), respectively. However, it 16 Figure 1: Changes in the concentrations of P as soluble reactive phosphorus (SRP), N as ammonium (NH 4 + ) and nitrate + nitrite (NOg"), and Si as reactive silicate ( S i 0 4 " 4 ) , in cultures to which nutrients were added every 4 days; (solid symbols), replicate I; (open symbols), replicate II. Points are for single determinations. Dashed lines indicate an assumed relationship when the occasional intervening value was unreasonable. Arrows indicate the detection limit for each nutrient. Where data points fall on each other, replicate I values have been offset slightly to the right. 17 10 20 TIME (d) 30 18 Figure 2: As for Figure 1; data are for cultures to which nutrients were added every 8 days. 19 20 Figure 3: As for Figure 1; data are for cultures to which nutrients were added every 16 days. TIME (d) 22 Figure 4: Proportion of total phytoplankton cell volume made up by each cell type, for each replicate (I and Jl). (A), nutrients added once every 4 days; (B), once every 8 days; (C), once every 16 days. Arrows indicate time of nutrient additions. o o 83 24 Figure 5: Average cell volume as a function of nutrient addition frequency for replicate cultures (I and II) at the end of the experiment. I LT I LT I LT once per 4 days once per 8 days once per 16 days NUTRIENT ADDITION FREQUENCY to was also in part the result of a significant (p < 0.001; Mann-Whitney U test) increase in the length distribution (diameter was constant) of S. radians (Fig. 6). When nutrients were added most frequently, 37 and 39 % of the cells were greater than 69 um in length for replicates I and II, respectively. In contrast, under the least frequent nutrient addition regime, 81 and 75 % of S. radians were longer than 69 um, for the two replicates, respectively. There were no pronounced treatment effects on chlorophyll specific *4C-bicarbonate incorporation rates (Table 2). Similarily, numbers of bacteria and glucose turnover times were not strongly influenced by the frequency with which nutrients were added (Table 2). DISCUSSION Community structure Competitive exclusion of all but a single species did not occur under any treatment (Fig. 4), which is consistent with theoretical models (Grenney et al. 1973, Crowley 1975) and empirical evidence (Robinson and Sandgren 1983, Sommer 1984, 1985) that fluctuations of a limiting resource allow for maintenance of more complex community structure in the phytoplankton. However, besides undetectable concentrations of SRP, low levels of NH 4 + , NOg" and Si0 4" 4 in the most frequently pulsed cultures may have allowed for the coexistence of several species with different relative resource requirements (e.g. Peterson 1975, Tilman 1977). Consequently, it is impossible to state categorically that continued species richness in all cultures was solely the result of fluctuations in a single limiting resource. Yet the nutrient addition regime appeared to be responsible for the greater importance of Tabellaria when nutrients were added least often. Previous studies have shown that the genus Synedra is almost invariably the best P competitor at temperatures less than 18 °C when nutrients are added continuously (Sommer 1983b, 1985, Smith and Kalff 1983, Tilman and Kiesling 1984, Kilham 1986), or even when pulsed at weekly intervals (Sommer 1984, 1985). In my study, 27 Figure 6: Frequency distributions of cell length for Synedra radians as a function of nutrient addition frequency, at the end of the experiment. Culture designation (A, B and C) as in Figure 4; (shaded), replicate I; (unshaded), replicate II. 28 Table 2: C-bicarbonate uptake rates (ug C T »h »ug Chi ), numbers of bacteria (xlO »ml ) and glucose turnover times (hours) for each nutrient addition frequency. Each value is the mean from duplicate cultures; estimates of one standard deviation are shown in parentheses. TREATMENT DAY 1 4C-UPTAKE BACTERIA GLUCOSE TURNOVER Once/4 d 3 0.87 (.06) 0.45 (.01) 3.65 (.06) • i 11 46.0 (52.5) 1.41 (.28) 0.74 (.06) t t 19 2.16 (.08) 0.66 (.06) 1.95 (.25) t t 25 3.39 (.91) n.d. n.d. t t 31 6.95 (.23) 0.78 (.05) 2.22 (.03) Once/8 d 3 0.83 (.33) 0.46 (.00) 3.49 (.21) • t 11 20.1 (*) 0.97 (.07) 1.46 (.37) t t 19 2.08 (*) 0.56 (.00) 2.74 (.02) t t 25 3.43 (.61) n.d. n.d. t t 31 7.22 (2.32) 0.68 (.04) 1.95 (.13) Once/16 d 3 0.46 (.14) 0.46 (.01) 3.47 (.42) t t 11 2.42 (*) 1.13 (.01) 1.08 (.18) t t 19 1.98 (.45) 0.49 (.06) 2.96 (.10) I t 25 2.28 (1.01) n.d. n.d. t t 31 9.30 (.13) 0.64 (.16) 2.70 (.13) data not obtained for duplicate culture • data not collected 30 Tabellaria only came into prominence when additional nutrients were administered at 16 day intervals. Under this treatment, concentrations of NOg" and S i 0 4 " 4 were highest and therefore presumably least limiting. These conditions would be expected to confer dominance to Synedra where P was added more often. In addition to theoretical studies (Grenney et al. 1973), empirical data have also shown that the frequency of addition of a limiting resource can affect the outcome of competition in phytoplankton cultures. Using N-limited chemostats initiated with natural assemblages of marine phytoplankton, Turpin and Harrison (1979, 1980) found that competitive dominance could be shifted between different diatom genera, depending on the schedule of N H 4 + addition. As well, Scavia et al. (1984) and Sakshaug and Olsen (1986) were able to demonstrate that short-term nutrient patchiness can affect competitive outcome in P-limited freshwater phytoplankton cultures. My results extend these findings to demonstrate that patchiness on the scale of weeks is potentially an important process influencing community structure, and that the presence of T. fenestrata in P-limited field populations may be indicative of infrequent pulses of P 0 4 " 3 into the epilimnion. Results of Stockner and Shortreed (1975) showing T. fenestrata to be associated with periodic upwelling events would seem to support this hypothesis. Differences among treatments in bicarbonate uptake, bacterial numbers, and glucose turnover times were not evident (Table 2). The results have been included, as comparable data are not available in the literature. The high bicarbonate rates obtained on Day 11 indicate that these values may be in error. If these values are ignored it suggests that there was an increase in photosynthetic efficiency as the experiment progressed, regardless of treatment. Cell size I also observed an increase in cell volume under conditions where nutrients were added least often. Similar results were reported by Turpin and Harrison (1980) who found that decreases in the frequency of nutrient additions from continuous to once weekly, resulted in larger cells, although their observations were made on NH^"1" limited cultures of marine phytoplankton. In contrast, I did not observe changes in cell size in response to pulsing frequencies of less than two weeks. As well, I believe that this is the first report of a change in nutrient addition frequency resulting in a shift in the size distribution of a species. The mechanism whereby nutrient pulsing frequency is able to dictate cell size and community composition is unclear. It has been suggested by Turpin and Harrison (1980) that because smaller cells have a higher specific respiration rate they may be less able to survive periods of nutrient deprivation. Therefore, they would be rarer when resource supplies were infrequent. An alternative explanation that has been invoked to account for changes in community structure in response to nutrient fluctuations is that individual species possess uptake kinetics that specialize on different aspects of a fluctuating resource. Phytoplankton uptake kinetics are generally approximated by a rectangular hyperbola of the form, T »S max T = ks + S where T is the uptake rate per cell, Tmax is the maximum uptake rate, S is the resource concentration, and kg is the resource concentration at which half the maximum uptake rate occurs. It is envisaged that species with a high Tmax for the limiting resource can take advantage of nutrient pulses, while species with a low kg sequester the resource when the ambient concentration is low (Eppley et al. 1969, Turpin and Harrison 1979). In fact it is the initial slope of the uptake curve (Tmax/kg) and not kg per se which should dictate competitive advantage at low substrate concentrations (Healey 1980). As well, although the value of Tmax has influence on competitive advantage, a more important factor when considering large nutrient pulses is the duration over which Tmax is sustainable. As demonstrated in Chapter 2 q very large differences exist in the ability of P-limited species to maintain elevated P 0 4 uptake rates. The capacity to sustain elevated uptake rates might be expected to be related to cell size; larger cells with their greater cell volumes and presumably greater storage capacities may sustain elevated uptake rates for longer periods. Some evidence consistent with this 32 q expectation was found in the field studies of P 0 4 uptake by size fractionated plankton presented in Chapter 6. However, contradictory data obtained in laboratory studies (Chapter 2), revealed that some small species can maintain enhanced uptake rates in excess of an hour. Consequently, a convincing relationship between the ability to sustain high uptake rates and cell size has yet to be demonstrated. Related to the maximum uptake rate, and what dictates the success with which a species will capitalize on a saturating pulse of a limiting nutrient, is its ability to sequester the resource relative to its minimum requirement. Strictly speaking the ratio of the maximum amount of nutrient that can be accumulated to the minimum amount required to maintain a positive net growth rate should be a good measure of the competitive ability of a species when nutrient patches are large. It is an indirect measure of the number of daughter cells which can be produced from a given nutrient patch assuming the nutrients once stored are recoverable and that losses by excretion are negligible. Intuitively, bigger cells would be expected to have an advantage because of their potentially larger storage capacity; however, whether this is adequate to offset their larger minimum requirements (c.f. Rhee and Gotham 1980) is not certain. Results from my experiments suggest that it is. It is also evident that species are able to alter their physiology in response to particular patchiness regimes (Quarmby et al. 1982, Chapter 2), and consequently there is no a priori reason why a single species should not be a good competitor over a range of patchiness regimes. In fact, an increase in Tmax without a concomitant increase in kg would not only enable a species to sequester more nutrient during an elevated pulse, but would also increase its ability to sequester more nutrient at low ambient concentrations by virtue of the effect that an increase in Tmax has on initial slope (Parslow et al. 1984a, b). Although N H 4 + limited cultures of Thalassiosira pseudonana were not found to possess a steeper initial slope for uptake, despite an elevated Tmax (Parslow et al. 1985a), the possibility remains that some species may be capable of such a strategy. Perhaps this is why some species such as Synedra acus are able to dominate under P-limited conditons over a wide variety of nutrient addition regimes (Smith and Kalff 1983, Sommer 1983a, b, 1984). 33 C H A P T E R 2: PHOSPHATE U P T A K E RATES OF PHYTOPLANKTON GROWN AT  DIFFERENT DILUTION RATES; TOWARDS A PHYSIOLOGICAL DEFINITION OF NON-STEADY-STATE CULTURE BACKGROUND Laboratory studies (Chapter 1 and references cited within) have demonstrated that competitive outcome in assemblages of phytoplankton can depend on whether the concentration of the limiting nutrient fluctuates, or is constant. As well, considerable discussion has ensued on the significance of such patchiness in the aquatic environment {e.g. McCarthy and Goldman 1979; Jackson 1980; Turpin et al. 1981; Lehman and Scavia 1982a, 1984; Currie 1984), with some of the data for this dialogue arising from experiments on chemostat-grown cultures. In these experiments the chemostat is perturbed with a known concentration of the limiting nutrient. The uptake of the nutrient is then followed, either by its disappearance from solution, or by incorporation of isotope into the phytoplankton. This technique has revealed that cells grown in chemostats have specific uptake rates for the limiting nutrient far in excess of maximal growth rates (Conway et al. 1976; Conway and Harrison 1977; McCarthy and Goldman 1979; Goldman and Glibert 1982). The obvious shortcoming of this approach is that chemostats are not an adequate representation of growth conditions if patchiness is important; by definition, in chemostats cells are being grown under conditions of constant nutrient supply (Rhee 1980). As Quarmby et al. (1982) have shown, the physiological state of phytoplankton in chemostats differs from those grown in a patchy nutrient environment. Clearly, in order to understand processes structuring the aquatic environment more studies of non-steady-state systems are needed; semi-continuous culturing provides a method to study such systems. Depending on the concentration of the limiting nutrient in the dilution 34 medium, the culture can approximate a chemostat or a batch culture. If the concentration added is low, then the ambient concentration will also be low, and selective pressure will be for high affinity, similar to conditions in a chemostat. Alternatively, if the concentration added is high then the ambient concentration will fluctuate considerably, and selection will occur initially for high maximum uptake rate, and subsequently for high affinity, similar to conditions encountered in a batch culture. The purpose of this study was to examine the maximum phosphate uptake rates of natural phytoplankton assemblages, grown at different nutrient supply rates, in phosphorus-limited, semi-continuous cultures. Such information is required if models capable of predicting competitive outcome in non-steady-state environments are to be constructed. MATERIALS AND METHODS Sampling and culturing Water for the experiments was obtained at oligotrophic Kennedy Lake, on Vancouver Island in southwestern British Columbia. Limnological features of the lake are described in Chapter 5. Samples (20 liters each) were collected from 1 m at the sampling site shown on Fig. 14, and gently filtered through 120 um screening to remove large grazers. Water was collected for Experiment I (Exper I) on 15 May 1983 and for Experiment II (Exper II) on 16 August 1983. Water temperatures and concentrations of particulate and dissolved nutrients, at the time of collection, are presented in Table 3. After collection, the water was transported to the laboratory within 8 hours, diluted to 40 liters with WC medium (Guillard and Lorenzen 1972) as modified for each experiment (see below), and placed in a 20 °C incubator at 100 uE»m"^'s"* continuous irradiance. When the community was in vigorous growth, as 35 Table 3: Temperatures, and concentrations of dissolved and particulate nutrients, at 1 m, in Kennedy Lake on the sampling dates. May 15 August 16 (Exper I) (Exper II) Temperature (°C) 14 21 Chlorophyll a (ug'l"1) 0.75 n.d. Soluble reactive P (uM) < 0.05 < 0.05 Ammonium (uM) 0.08 < 0.04 Nitrate + nitrite (uM) 0.36 < 0.04 Particulate P (uM) 0.05 n.d. Particulate N (uM) 1.14 0.79 Particulate C (uM) 3.58 12.71 • data not collected 36 determined by increasing in vivo fluorescence, the water was used to initiate the experimental cultures. The medium used was slightly different for the two experiments. In Exper I, Tris (2-amino-2 hydroxymenthy-1,3 propandiol) and phosphate (K^HPO^) were reduced to 0.125 g*r* and 1.67 uM, respectively. The Tris concentration was decreased as lower values were q adequate to stabilize pH; P 0 4 was decreased to maintain biomass within acceptable levels and ensure that it was the nutrient limiting growth. Bicarbonate and Si0 4 " 4 concentrations were doubled to 300 uM and 200 uM respectively, to keep concentrations above growth-rate limiting levels. KCI (98 uM) was added to replace the potassium lost when the phosphate concentration was decreased. In Exper II the medium was further modified by replacing Tris 1 q with equimolar MOPS, a buffer which does not interfere with N H 4 analysis; P 0 4 was reduced to 0.50 uM and 1.0 mM NaN0 3 was replaced with 22.5 uM NH 4C1. Residual N H 4 + (> 0.3 uM) in the cultures, prior to the daily dilutions, confirmed that P was limiting. Each 1.5 liter culture was grown in a Plexigla s® tube (62 mm i.d.), sealed on the bottom with a Nalgene® funnel. A length of silicon tubing was attached to the funnels, through which the cultures were drained. Plexiglas® was not found to decrease the growth rate of two test species, Asterionella formosa (clone EvD) and Chlamydomonas sp. Banks of Westinghouse 'Super-Hi' Output, Daylight fluorescent bulbs (F72T12/D/SHO) provided illumination from each side. Neutral-density screening was utilized to adjust the o 1 photon flux density within the tubes between 150 and 250 uE*m «s as measured using a quantum meter (Biospherical QSL-100) equipped with a submersible 4 collector. These levels are within the range where they should be saturating to growth but not inhibitory, for most species. Treatments were randomized among tubes to eliminate light effects; however, at the end of experiments the replicates agreed closely indicating irradiance effects were minimal. At a set time each day the cultures were thoroughly mixed by stirring, and a portion removed; new medium was then added and the cultures mixed again. The amount of culture removed was designed to achieve certain specific growth or dilution rates (D), which were 37 calculated as -In f, where f is the fraction of the original volume remaining subsequent to a dilution. Specific growth rates are expressed as growth constants (reciprocal of division time), and have units of d"1 (Guillard 1973). In Exper I each of 21 cultures were diluted at one of seven rates (0.10, 0.25, 0.33, 0.50, 0.75, 1.0 and 1.5 d**), with treatments prescribed in triplicate. In Exper II the three cultures were diluted at 0.50 d"*. In vivo fluorescence of cultures was monitored daily and community composition regularly. A minimum of 200 cells (or filaments) of each of the dominant species were counted using settled samples, to achieve an accuracy of approximately 85 % (Lund et al. 1958). When small cells were abundant counts were also made using a haemocytometer. Biomass was expressed in terms of cell volume and was estimated using formulae for similar geometric shapes. Uptake experiments q PO^ uptake experiments were conducted when the community composition was stable as indicated by fluorescence varying less than 10 % for 3 dajrs, and greater than 80 % of the individuals being made up of a single species or the relative proportion of the most abundant species changing less than 20 % over 7 days. Cultures were diluted for a minimum of 31 days. In Exper I uptake experiments were chosen so replicate treatments were not run on the same day; as well, experiments were repeated on some cultures on different days, to ensure they were physiologically stable. In Exper II, however, the three experiments were run simultaneously. Uptake measurements were not run on all replicate cultures, and data from the 1.0 d"* treatment were lost because of technical problems. Uptake measurements were made on subsamples taken immediately before the normal dilution time and spiked with phosphate (K^HPO^) to a concentration near 9.0 uM. In Exper II 5.0 uM N H 4 + was also added as part of a parallel study on N H 4 + uptake; less than 0.5 uM of it was taken up over the course of an experiment. All other nutrients were assumed to be in excess based on their supply ratio in the medium. The disappearance of P0 4 " 3 was followed using a Technicon® 38 Auto- Analyser® and the chemical technique of Murphy and Riley (1962) as outlined by Stain ton et al. (1977). Following the initial perturbation, P0 4" 3 was re-added to some samples to maintain a high, saturating concentration. Uptake experiments were carried out at 20 °C O "1 and 100 uE«m «s . Cultures were agitated frequently during experiments. At predetermined time intervals 15 ml were removed from the subsample and filtered, at a vacuum of less than 120 mm Hg, using a Millipore® multiple filtration unit modified to hold larger sample cups. Whatman GF/C filters (mean retention size of 1.2 um) were used in Exper I; GF/F filters (mean retention size of 0.7 um) were used in Exper II, because of the smaller cell sizes. q Filtered standards confirmed that no exchange of PO4 occurred on the filters or filtration apparatus. The filters (24 mm) were pre-treated by washing with 20 ml of 3.7 % HCl (by wt.), followed by 400 ml of deionized, distilled water, and drying at 60 °C. Uptake rates (uM*h**) were divided by the initial particulate P concentration (P in the phytoplankton, uM) to yield P-specific uptake rates (V where V is the specific uptake rate, T is the interval over which V is calculated, and D is the dilution rate from which the subsample was drawn. As these are P-limited cultures, the P in the phytoplankton is the minimum amount required for the cells to maintain the specified growth rate. Consequently, the P-specific uptake rate permits an evaluation of uptake relative to the amount of P that the cells need in order to undergo division. Particulate P was assumed to equal the PO4"3 concentration in the dilution medium, as soluble reactive P was below detection limits in the culture subsamples (implying that the cells had sequestered it all). This assumption is not valid if the algae release the P as non-reactive organic P04"3. Lean and Nalewajko (1976) q have shown that the assumption is reasonable as more than 97 % of the P 0 4 taken up by P-limited phytoplankton is retained in the cells. Particulate C and particulate N were measured using a Carlo Erba 1106 elemental analyser. 39 RESULTS Experiment I: Oscillatoria-dominated cultures The (C:P) and (N:P) ratios of the phytoplankton (Fig. 7) decrease as the dilution rate increases. This is consistent with data from P-limited chemostat cultures (Fuhs et al. 1972; Goldman 1979) and shows that daily-dilution, semi-continuous culturing can be used to achieve different degrees of P-limitation. In a similar manner biomass (expressed as total cell volume) decreases as dilution rate increases (Fig. 8). The data (Fig. 9) show that cultures grown at s 0.75 d ~\ demonstrate an acceleration in uptake rate from the 1-5 to the 5-15 minute interval (i.e. V^'^Q J.Q jg < V^'^Q J.Q 75), q following a PO4 perturbation. After the 5-15 minute interval there is a gradual decline in uptake rate. This pattern is the same if uptake rate is expressed (as is sometimes done) on a particulate C basis, as particulate C changed less than 10 %, on average, over the course of the 2 hour incubations. It appears that this decline is the result of feedback inhibition and not q decreasing substrate concentration, as further addition of PO4 to some cultures (Fig. 9) did not enhance the uptake rate. Also, experiments conducted on a 0.50 d"* culture filtered through a 3.0 um filter, showed no measurable uptake, indicating that bacteria were not influencing the pattern observed. The time-courses of uptake rate in the 1.5 d"* cultures are strikingly different; W'^j g is much higher than in following intervals, but is still much less than V^'^Q J.Q 75. The difference may be because of the high PO4"3 supply rate, although community composition may also be important. The 0.1 to 0.75 d"* cultures were dominated by Oscillatoria sp. (93 % of total cell volume; range: 74 % to 99 %); whereas, in both 1.5 d"* cultures only 19 % of the total cell volume consisted of Oscillatoria sp. Small colonial algae, probably chrysophytes, and a heterotrophic microflagellate, probably Paraphysomonas sp., made up most of the remaining cell volume at the 1.5 d** dilution rate. The community composition of these cultures was 40 Figure 7: C:P and N:P ratios (by atoms) for cultures grown at different dilution rates. Each data point represents a single culture. The particulate P concentration is constant and independent of dilution rate. Curves are fitted by eye. 41 350 42 Figure 8: Total cell volumes for cultures grown at different dilution rates. Each point represents a single culture. Curve is fitted by eye. T O T A L C E L L V O L U M E ( m m 3 - L ' 1 ) 44 Figure 9: Changes in perturbation P O 4 uptake rates and P O 4 concentrations with time, for cultures grown at different dilution rates. Arrows show the first P O 4 " 3 q concentration measured after additional P O 4 was added to some cultures. Phosphate concentrations: replicates A Q, and B phosphate uptake replicates A tt, and B (*). Dashed lines join data collected from replicate experiments run on the same cultures on different days. U P T A K E R A T E ( h " ) PHOSPHATE (J L /M) 46 likely affected by the presence of the heterotrophic microflagellate, which was present at all dilution rates, but was relatively much more abundant at 1.5 d"^. Fig. 10 illustrates that as the dilution rate increases from 0.10 to 0.50 d"* there is also an increase in a n ( j yl5-30 However, the length of time that the high uptake rate is sustained decreases as the dilution rate increases, causing a more rapid decline in uptake rate with increasing dilution rate. As a result, at later intervals ( V 3 0 " 4 5 , V 5 0 " 6 0 , and V60'90) the highest uptake rate occurs at the lowest dilution rate. It is not possible to discern trends in W with dilution rate because of the high variability in W This variability is more likely the result of the error associated with measuring small changes in concentration over short time intervals, and is probably not physiological variabilty. Experiment II: Synechococcus-dominated cultures An enhancement of V^'^ relative to V^"^ was also characteristic of Exper II (Fig. 11). In other respects the results differed from Exper I as a further increase in uptake rate was observed in the interval subsequent to V^'^' and the uptake rate remained higher for a longer period. Unlike Exper I these cultures were dominated by a chroococcoid cyanobacterium, probably Synechococcus sp.; no other cell types were observed to be present. This is consistent with the view that cells with high surface area to volume ratios should be superior nutrient competitors (Smith and Kalff 1982, 1983). The prominence of larger cells in Exper I may have been due to the presence of heterotrophic flagellates. In Exper II cells were counted at the beginning and the end of the uptake period; however, no change in numbers was discernible. 47 Figure 10: Perturbation PO4 uptake rates for different uptake intervals and dilution rates. Only rates over intervals where the PO^ concentration remained above 1.5 uM are shown. Vertical lines join data collected from replicate experiments run on the same cultures on different days. Curves are fitted by eye. V1'5 ft, V 5 " 2 5 ft, V 7 5 " 3 0 (a), v30-45 Q v50-60 ^ y60-90 p T h e d a s h e d i i n e fitted f o r VJ-5 r e p resents a conservative interpretation and is not meant to imply a specific relationship. 48 49 Figure 11: Perturbation PO4 uptake rates and PO4 concentrations for Synechococcus dominated cultures grown at 0.50 d'*. Phosphate concentrations: replicates A Q , B O, and C (4). Phosphate uptake rates: replicates A B M, and C (if). U P T A K E R A T E 51 DISCUSSION Three major observations were made in these experiments. Firstly, there was a delay q before the maximum uptake rate (Vmax) occurred, subsequent to P0 4 addition to P-limited cultures. Secondly, the highest uptake rates over the first 30 minutes of incubation were associated with intermediate dilution rates. Thirdly, the manner in which the uptake rate q changed, following a P0 4 addition, appears to be species specific. Delayed maximum uptake rate The observation of an increasing uptake rate subsequent to a nutrient pulse has been observed for NO3" (e.g. Collos 1980; Dortch et al. 1982; Parslow et al. 1984b) and Si0 4" 4 starved (Conway and Harrison 1977) cultures, but has seldom been reported in response to q q P 0 4 addition. Chisholm and Stross (1976a) found such a delay when P0 4 was added to nutrient-replete cultures of Euglena gracilis, which were synchronized to a 14:10 Iight:dark cycle. The time-scale of the response (2 hours) was inconsistent with my results; as well, the uptake rate was constant when P-limitation was also imposed on the synchronized division cycles (Chisholm and Stross 1976b). Similarly, perturbations of P-limited continuous cultures (Burmaster and Chisholm 1979; Gotham and Rhee 1981) and P-starved batch cultures (Simonis et al. 1974; Brown et al. 1978; Senft et al. 1981; Parslow et al. 1984b) did not result in lags before Vmax occurred. It is not clear why the conditions of this study permitted the observation of an increase q in saturated uptake rates with time in the initial intervals following a P 0 4 addition, while q experiments where cells were starved and then pulsed with P0 4 have not. The most reasonable explanation seems to be that Vmax is in some way tied to the cell cycle, and that repeated pulsing is required to entrain the cells. This may reveal cellular processes which are normally obscured in a randomly dividing population. Although there was no evidence of an increase in cell number over the course of the uptake experiments, could be out of 52 phase, but associated with synchronous division outside of the uptake period. The absence of a delay in Vmax in the cultures diluted at 1.5 d"* is consistent with this reasoning, as cells whose average generation times are shorter than 1 day are unable to entrain to a rhythm with a circadian periodicity (Chisholm 1981). Cell division in phytoplankton can be entrained to N pulses (Yoder et al. 1982; Chisholm et al. 1984) and Si pulses (Busby and Lewin 1967; Sullivan 1977), but to my knowledge has not been reported for P pulses. In contrast, liquid q cultures of higher plant cells can entrained to PO^ pulses (Amino et al. 1983), consequently, phytoplankton may respond similarly under certain conditions. q The observation of enhanced uptake capability for P O 4 when cells are grown under q P O 4 limited conditions is well described for bacteria (e.g. Rosenberg et al. 1969), cyanobacteria (e.g. Falkner et al. 1980; 1984; Riegman and Mur 1984), fungi (e.g. Burns and Beever 1979) and microalgae (for review see Cembella et al. 1984). This could be an adaptation to take advantage of ephemeral, elevated concentrations (sensu: McCarthy and Goldman 1979), or a mechanism to increase growth rates at low homogeneous concentrations (Parslow et al. 1984a, 1984b). It is fairly well agreed (cf. Burns and Beever 1979; Gotham and Rhee 1981) that the high capacity uptake system is regulated by being derepressed when P O 4 " 3 is limiting to growth. My data do not contradict this model, but suggest that under the experimental conditions I used, an allosteric activator may also be important. This activator q would increase the affinity of the transport system for P O 4 and be responsible for the enhancement of uptake from V*'^ to V^'^. A final point is that although V^'^ is, on average, enhanced three-fold over V^'^, the uptake rate in the initial period is not low. W"^  is enhanced at least 100 times more than required to sustain the specific growth rate averaged over 24 hours, except in those cultures diluted at 1.5 d-1. Changes in uptake rate with dilution rate Although the culturing technique was able to generate a variety of P-limited states, as 53 shown by the range of C:P and N:P ratios observed, the uptake pattern was not consistent with most of the data obtained from chemostats and batch cultures. In this study the highest q uptake rates over the 30 minutes following a PO4 perturbation were associated with cultures maintained at intermediate dilution rates (Fig. 10). Standardizing the data to particulate C or cell volume would only exaggerate this relationship because of the high values of these parameters at low dilution rates (Figs. 7 and 8). Although the highest uptake rates have occasionally been found to be associated with intermediate nutrient supply rates in phytoplankton (Burmaster and Chisholm 1979; Healey 1985) and higher plants (Schjorring and Jensen 1984), they are typically associated with the lowest dilution rates (Rhee 1973; Gotham and Rhee 1981; Falkner et al. 1984; Riegman and Mur 1984) at which cell viability is retained (Rhee 1980). (Although the uptake rates in these studies are expressed on a per cell or per biomass basis, as opposed to a P-specific basis, the same would be true for specific uptake rates, as cell quota decreases as dilution rate decreases). Similarly, in batch culture the specific uptake rate (Parslow et al. 1984b), the uptake rate per cell (Aitchison and Butt 1973) and the uptake rate per chlorophyll a (Falkner et al. 1984) typically increases with increasing q P 0 4 starvation. However, maximum uptake rates have also been found to be constant over a range of dilution rates when expressed on a biomass basis (Healey and Hendzel 1975; Nyholm 1977), and over a range of starvation times when expressed on a per cell basis (Parslow et al. 1984b). Sustainabilify of high uptake rates The duration over which high specific uptake rates can be sustained differs between Exper I and Exper II. In the Oscillatoria sp. dominated cultures of Exper I, uptake rate declined with each successive interval from V^'^ (Fig. 9), with the rate of this decline increasing with dilution rate (Fig. 10). Parslow et al. (1984b) also found the uptake rate declined rapidly following a PO4"3 spike to starved cells. Declines in uptake rate with short-q term accumulation of cellular P suggest that PO4 uptake in Oscillatoria sp. is regulated by 54 feedback on the uptake system by an internal P pool, as has been proposed for seven species of cyanobacteria (Riegman and Mur 1984; Riegman 1985). My data indicate that such an internal pool is not directly coupled to the total P content of the cells (i.e. the internal pool is a subset of the total P pool), as the uptake rate initially increases and then decreases as cellular P rises. In the Synechococcus sp. dominated cultures of Exper II there was no reduction in uptake rate (Fig. 11), or in the uptake rate per cell, in response to increasing cellular P over the short term. This pattern of a sustainable high uptake rate per cell, while cell quota is rapidly increasing, has also been observed for Monochrysis lutheri (Burmaster and Chisholm 1979). The ability to sustain high uptake rates for an extended period would be of competitive importance in areas where ephemeral pulses of elevated nutrient concentration exist. As yet, patches of nutrients saturating to uptake, have not been demonstrated to exist for sufficient time to have a selective role, although solitons (internal waves which reach the surface) may provide such a mechanism (Holligan et al. 1985). 55 CHAPTER 3; THE EFFECT OF N;P SUPPLY RATIO ON AMMONIUM UPTAKE IN FRESHWATER PHYTOPLANKTON; EVIDENCE FOR 'SURGE' UPTAKE BACKGROUND The observation that N-limited phytoplankton display a several-fold increase in uptake rate for N H 4 + , when exposed to a saturating concentration of the nutrient, was first reported by Syrett (1953) for the freshwater chlorophyte Chlorella vulgaris. Fitzgerald (1968) noted that these rates were not sustainable and decreased quite rapidly once the N deficit was overcome. Conway et al. (1976) documented the response in considerably more detail in marine diatoms and recognized that the enhanced uptake rates consisted of two phases, a short-lived period of very high uptake which was termed 'surge uptake' and a longer, sustainable phase which was characterized as 'internally' controlled. In a subsequent paper, Conway and Harrison (1977) were able to demonstrate that the magnitude of these responses were species specific, and suggested that these differences might be important in dictating competitive advantage in oligotrophic areas of the ocean. McCarthy and Goldman (1979) emphasized that these highly elevated transients could be a mechanism whereby phytoplankton in nutrient depleted areas could sequester N H 4 + that was distributed in ephemeral micropatches, and although the existence of such patches has been criticized on theoretical grounds (Jackson 1980), empirical data showing that phytoplankton can utilize P 0 4 patches produced by zooplankton (Lehman and Scavia 1982b) lends some credence to the hypothesis. Possibly because of the dogma that oligotrophic freshwater lakes represent P-limited systems, little effort has gone into discerning whether freshwater phytoplankton also possess the transient rapid uptake phenomenon associated with marine species. Indications that N limitation may be more common than previously thought in oligotrophic freshwater systems (Goldman 1960, Lane and Goldman 1984, Priscu and Priscu 1984, Priscu et al. 1985, Chapter 56 5) make this an ecologically, as well as a physiologically, relevant question. As well, much of the data on which arguments for the importance of nutrient 'patchiness' to phytoplankton ecology have been based, were obtained from continuous cultures where by definition 'patchiness' does not exist (Rhee 1980). The data in this chapter more fully characterize 'surge' uptake in freshwater phytoplankton grown under non-steady-state, N-limited conditions. M A T E R I A L S A N D M E T H O D S Culturing A 20 liter water sample containing a natural assemblage of phytoplankton was removed from oligotrophic Kennedy Lake on 16 August 1983. Limnological features of the lake are described in Chapter 5. Water temperature at the time of collection was 21 °C. The water was collected and treated precisely as explained in Chapter 2. As previously described, the water was transported to the laboratory and diluted 1:1 with artificial medium, modified by increasing the bicarbonate and S i0 4 " 4 concentrations, to 300 and 200 uM, respectively, and replacing Tris buffer with MOPS buffer. SiO^* 4 was added as recommended in Suttle et al. (1986) to prevent polymerization. Inorganic N was decreased to 20 uM and was supplied as NH^Cl; inorganic P was supplied as K2HPO4 at a concentration of 2.0 uM. When the phytoplankton were in exponential growth as indicated by measurements of in vivo fluorescence they were used to initiate nine, 1.5 liter cultures. Each culture was diluted daily to achieve a specific growth rate of 0.5 d"1 and was subjected to one of three N:P supply ratios (5:1, 15:1, and 45:1; by atoms). Concentrations (uM) of N H 4 and P0 4 "° were varied to maintain similar biomasses under each treatment and were as follows: (N:P, N H 4 , P0 4), 5:1, 10.0, 2.0; 15:1, 15.0, 1.0; 45:1, 22.5, 0.5. (10 uM N H 4 + under N limitation gives q approximately the same cell yield as 0.5 uM PO4 under P limitation). Cultures were exposed to continuous light and grown under the conditions outlined in Chapter 2. 57 The cultures were diluted at a set time each day. Following thorough mixing 39.3 % of the volume was removed; replacement medium was then added and the cultures mixed again. The dilution procedure achieved a specific growth or dilution rate of 0.50 d**. As shown in Chapter 2 this technique can be used to realize a variety of nutrient limited states. Uptake experiments After the cultures had been diluted for a minimum of 31 days, and community composition had stabilized, NH 4 + uptake rates for each culture were determined. A stable community was defined as one in which the abundance of the dominant species and in vivo fluorescence varied less than 10 % over 3 days. Uptake measurements were made on samples of culture taken immediately before the normal dilution time. Samples were inoculated with saturating concentrations of NH^Cl (ca. 20 uM, except 45:1 cultures in which less was added because of residual NH 4 + ), and the disappearance of the nutrient followed from solution. Subsamples (15 ml) were taken immediately upon nutrient addition, and at predetermined intervals for 2 hours. They were promptly filtered through Whatman GF/F filters which had been pre-treated by washing with 20 ml of 3.7 % HCI (by weight), followed by 400 ml of deionized, distilled water and oven drying at 60 °C. Filtered standards confirmed that no measurable exchange of NH 4 + occurred on the filters or filtration apparatus. The filtrate was analysed chemically for NH 4 + , and N-specific uptake rates (h"*) were calculated as described in Chapter 2 using particulate N measured using a Carlo Erba 1106 elemental analyser. R E S U L T S A N D DISCUSSION After 31 days the two lowest N:P ratios (5:1 and 15:1) were dominated by a mixed assemblage of species consisting of two diatoms, probably Synedra radians and Nitzschia holsatica (E.F. Stoermer, pers. comm.), a green alga Scenedesmus sp., and a chroococcoid blue-green alga, probably Synechococcus sp. In the 45:1 (N:P ratio) cultures, only Synechococcus 58 was observed. Evidence that the different N:P ratios resulted in different degrees of N and P limitation and the reasons that a more complex community structure could be maintained in the lower N:P ratio cultures are discussed in the following chapter. The nutrient disappearance traces and resultant uptake rates calculated from them are depicted in Fig. 12. As would be expected from previous work on marine species (McCarthy and Goldman 1979, Goldman and Glibert 1982, Dortch et al. 1982) the elevated uptake rates are only associated with those cultures grown under N limitation, or in this case at the two lowest N:P ratios (Fig. 12). Measured uptake rates in these cultures, over the 1-5 minute interval following perturbation, were between 25 and 71 times the rate required to maintain their average growth rate over 24 hours. Consequently, cells exposed to a patch of nutrient, saturating to uptake, could sequester between 7 and 21 % of their daily N requirement in less than 5 minutes. These rates are of the same order as those reported by others (McCarthy and Goldman 1979; Parslow et al. 1984a, 1984b, 1985a, 1985b) for N deplete cultures of Thalassiosira pseudonana (clone 3H), measured over similar time intervals. The observation that rapid initial uptake rates of N H 4 + were only associated with low N:P supply ratios suggests that this should also be a feature of N-limited natural freshwater phytoplankton communities, as it is for marine assemblages (Glibert and Goldman 1981). Priscu and Priscu (1984) added ^ N-NH^"1" to lakewater samples and found that the rate of isotope enrichment was considerably elevated over the first several minutes, relative to subsequent intervals. As they and others (Dugdale and Goering 1967, Price et al. 1985) have pointed out, such experiments can be influenced by isotope regeneration. The initial uptake rates observed are measurements of gross uptake; however, later declines in the rate can be attributed to efflux of the label. The results I report confirm that the rapid decline in the rate of isotope accumulation observed by Priscu and Priscu (1984) was probably not the result of isotope efflux, but was due to a decrease in the net rate of accumulation. A significant, and to my knowledge previously unreported finding, is the appearance of a rapid, short-term shutdown immediately following surge uptake (Fig. 12). There are several mechanisms which could account for this pattern. The simplest explanation is that there was a 59 Figure 12: N H 4 uptake rates (solid symbols) and N H 4 concentrations (open symbols) for perturbation experiments on triplicate phytoplankton cultures, grown at N:P supply ratios of 5:1, 15:1 and 45:1. 60 T I M E Cmins) 6 1 short lag before the N H 4 could be processed into amino acids, resulting in a temporary reduction in net uptake. Alternatively, the decrease could be the result of a sudden loss of membrane potential due to influx of cations. Ullrich et al. (1984) found the membrane potential in Lemna gibba (duck weed) was reduced drastically when N-starved plants were pulsed with NH4 + . This was not accompanied by a concurrent reduction in uptake rate, although this could be attributable to the less frequent sampling regime used by them, or perhaps it is not a feature associated with multicellular plants. The reason why this response has not been observed in previous culture studies of rapid time course N H 4 + uptake in marine phytoplankton (Goldman and Gilbert 1982, Parslow et al. 1984b, 1985a) is unknown; however, such a pattern may only be a feature of ion-dilute environments. Perhaps the most likely possibility is that the short-term shutdown may be apparent only in synchronously dividing populations. Work has shown that the cell cycle of certain marine phytoplankton can be strongly entrained to N H 4 + pulses (Chisholm et al. 1984), especially under continuous irradiance; yet short time-course studies of N H 4 + uptake in cultures grown under such conditions have not been reported. Although not explicitly examined, it is reasonable to expect freshwater species to also synchronize to N H 4 + pulses. If the shutdown is a reproducible feature of N H 4 + entrained cultures then such cultures may prove useful for studying regulatory aspects of N H 4 + uptake in phytoplankton. Following shutdown the uptake rates increase again to a level about 12-fold greater than required to sustain the average growth rate over 24 hours. This rate is probably equivalent to the 'internally' controlled phase of uptake recognized by Conway et al. (1976). The teleological nature of arguments regarding adaptive significance dictates that they should not be pursued with pertinacity. Suffice to say that any of the transient phenomena that have been recognized as being integral with uptake processes for limiting nutrients, could confer competitive advantage under particular regimes of nutrient patchiness. The onus must now be on identifying the temporal and spatial scales of such patchiness in aquatic ecosystems. 62 C H A P T E R 4; THE EF F E C T OF N;P S U P P L Y RATIO ON THE R E L A T I V E U P T A K E  RATIOS OF AMMONIUM AND PHOSPHATE; A METHOD TO ESTIMATE IN SITU N:P S U P P L Y RATIOS BACKGROUND There is clear evidence from nutrient manipulation studies in lakes (e.g. Schindler 1974, Barica et al. 1980) that large alterations in the relative rates of supply of N and P can select for, or against, algae capable of fixing atmospheric N (Schindler 1977, Barica et al. 1980). Similarly, data collated by Smith (1983) suggest that in unperturbed systems low levels of N relative to P result in dominance by N-fixing, blue-green algae. In theory, more subtle changes in N:P supply ratios should also be capable of causing shifts in community structure. It is known that the critical supply ratio at which different phytoplankton species switch from N to P limitation can range from approximately 7:1 to 45:1, by atoms (Table 4). However, in order for N:P supply ratios to affect species composition it is necessary for the two nutrients to co-occur as limiting resources, that is some species must be limited by N and others by P. This would be expected when the in situ supply ratio is within the range of critical supply ratios found in phytoplankton. Unfortunately, the hypothesis that N:P supply ratios may be important in influencing phytoplankton community structure in natural systems has remained largely untestable, as methods for estimating supply ratios in situ have been lacking. As the N:P ratio of phytoplankton growing at less than maximum rates is approximatly the same as the N:P ratio of the inflow medium, over a wide range of ratios (Rhee 1978, Goldman er al. 1979, Terry 1980, Ahlgren 1985), this suggests that it may be possible to estimate N:P supply ratios from the chemical composition of particulate matter in nature. However, this does not represent a satisfactory solution because of the variable contribution of detrital material to the particulate pool. Table 4: Literature values of 'critical' ('optimum') N:P supply ratios for phytoplankton (by atoms). Species Critical N:P Melosira binderana 7 a Microcystis sp. 9 a Synedra ulna 10a Asterionella formosa 12a Selanastrum minutum 15-22^ Ankistrodesmus falcatus 21 a Pavlova lutheri 21-45c Selanastrum capricornutum 22 a Fragilaria crotonensis 25 a Synechococcus linearis 25-45d Scenedesmus obliquus 30 a aRhee and Gotham 1980 bElrifi and Turpin 1985 cTerry 1980 dHealey 1985 64 Rationalization of the 'Relative Uptake Ratio' (RUR) It has been recognized for many years that the physiological state of phytoplankton has considerable influence on their ability to sequester nutrients which are in short supply. Decades ago it was demonstrated that algae possessed enhanced uptake rates for N H 4 + when N-starved (Syrett, 1953), and for P 0 4 + (Blum 1966) when P-starved. It is also known that the degree to which the maximum cellular uptake rates for N H 4 or P 0 4 are enhanced, is influenced by the extent to which N or P, respectively, are limiting to growth. The most often observed patterns for both nutrients are increases in uptake rates as the degree of limitation increases (e.g. Fuhs et al. 1972, Rhee 1973, Riegman and Mur 1984, Eppley and Renger 1974, McCarthy and Goldman 1979), or the highest uptake rates associated with an intermediate degree of limitation (Burmaster and Chisholm 1979, Terry 1983, Healey 1985, Chapter 2). The pattern observed is likely influenced by the time over which measurements were taken (Chapter 2, Fig. 10). Irrespective of the shape of the curve it is clear that uptake rates for N H 4 or P 0 4 _ o are typically greatly enhanced in P or N deficient cells with respect to P or N sufficient ones (e.g. Aitchison and Butt 1973, Healey and Hendzel 1975, Healey 1977, Grillo and Gibson 1979, Dortch et al. 1982, Goldman and Glibert 1982, Parslow et al. 1984a, b). The usefulness of this relationship has not escaped the scrutiny of researchers attempting to identify the form of nutrient limitation in a particular area, and it has served as the basis for many bioassay tests (Fitzgerald 1969, Healey 1979, Vincent 1981a, b, White et al. 1985). Similarly, it serves as the starting point for the index proposed here. In natural phytoplankton communities there are a large number of species present at any given time, presumably representing a reasonably wide spectrum of individual requirements for N and P. Consequently, over a rather broad range of N:P supply ratios, roughly from 10:1 to 40:1, one would expect some species to be N-limited and others to be P-i q limited, some species to possess enhanced uptake kinetics for N H 4 and others for P 0 4 . The proportion of cells limited by each nutrient will largely be a function of the N:P supply 65 ratio; at low ratios most cells will be limited by N, and at high ratios by P. Therefore, at low ratios one would expect to observe elevated uptake rates for N H 4 relative to PO4 , and the q converse at high supply ratios. Intuitively, determining the ratio of the maximum PO4 uptake rate to the maximum NH^"*" uptake rate (RUR) for any phytoplankton community should provide a realistic estimate of their relative demand for N and P, and hence an index of the in situ N:P supply ratio. Nutrient supply rates and the time interval over which the uptake measurements are made could clearly influence this estimate; these concerns are addressed in the discussion. The goal of this chapter is to demonstrate that knowledge of phytoplankton nutrient uptake kinetics can be utilized to make realistic estimates of in situ N:P supply ratios. Such a capacity would permit examination of the relationship between supply ratios and species composition in natural populations. It might have potential management applications, as well. Knowledge of the effect that N and P additions are having on the relative supply ratios of the two nutrients in a system should permit adjustments to be made to loading regimes to prevent the occurrence of blue-green algal blooms. In addition, data are presented in this chapter which suggest that good competitors for one nutrient may be poor competitors for others, a requisite if N:P supply ratios are to be important in dictating competitive outcome. MATERIALS AND METHODS Collection and culturing Phytoplankton were collected for the first experiment from Kennedy Lake on 16 August 1983, and for the second from Sproat Lake on 8 July 1986. Limnological details of the lakes are described in the following chapter. Water was collected from 1 m at stations at least 1 km from shore, and immediately filtered through 120 um screening. Water temperatures at the times of collection were 21 t in Kennedy Lake and 19 °C, in Sproat Lake. Following collection, the water was transported to the laboratory and treated in the identical manner as 66 outlined in the preceding chapter. When the phytoplankton were in vigorous growth they were used to initiate a series of experimental cultures. Each culture was subjected to one of several N:P supply ratios with the objective of determining how the treatments affected phytoplankton physiology and community structure. The concentrations (uM) of NH 4 and P0 4 which were added to the modified medium were dependent on the N:P ratio desired, and were as follows: (N:P, N, P); 5:1, 10.0, 2.0; 10:1, 15.0, 1.5; 15:1, 15.0, 1.0; 25:1, 25.0, 1.0; 35:1, 35.0, 1.0; 45:1, 22.5, 0.5. Water from Kennedy Lake was used for the 5:1, 15:1, and 45:1 cultures; the 10:1, 25:1, and 35:1 cultures were started using Sproat Lake water. The culturing methods and apparatus used for the first experiment were described previously (Chapter 3). In the second experiment in lieu of using large volumes (1.5 liters), cultures were reduced to 200 ml and were grown in 500 ml polycarbonate Erlenmeyer flasks. Mixing was accomplished by a shaker table which agitated (200 rpm) the flasks for 2 minutes every hour. Treatments were run in triplicate for the first experiment and in duplicate for the second. In both experiments the dilution regime previously described was followed and the amount of culture removed achieved a specific growth rate of 0.50 d"*. Uptake experiments q _L PO4 and NH4 uptake experiments were conducted after the cultures had been diluted for a minimum of 31 days. In the first study this represented a stable community composition in which in vivo fluorescence and abundance of the dominant species, over 3 days, varied less than 10 %. However, in the second experiment fluorescence remained unstable and species composition continued to change. Uptake measurements were made on subsamples of culture taken immediately before the normal dilution time. Concentrations of NH^Cl and K2HPO4, saturating to uptake, were added to the subsamples either simultaneously (first experiment) or the subsamples were split and the nutrients added separately (second q experiment). The protocol was changed to alleviate any possible interactions between P0 4 and N H 4 + uptake as has been observed in Lemna gibba (duckweed) by Ullrich et al. (1984). Similar interactions have been identified for NOg" and P 0 4 by Terry (1982), and for NOg" and N H 4 by numerous workers (McCarthy 1981). The amount of P 0 4 and N H 4 added was 5.0 uM and 20.0 uM, respectively. At the extreme ratios residual concentrations of one of the nutrients frequently remained; in such instances smaller perturbations of the nutrient in excess were administered. Uptake of P 0 4 and N H 4 were determined by following their q disappearance from solution over 2 hours. At low N:P ratios the rates of P 0 4 disappearance were slow and the calculated uptake rates less precise; therefore, P-orthophosphate was 09 q used as a tracer in Exp II. I confirmed that P traced P 0 4 disappearance by measuring both isotope incorporation and SRP concentration in several cultures. Uptake experiments were carried out under the conditions described in Chapter 3. Immediately after nutrient addition, and subsequently at predetermined time intervals, 15 ml was removed from the subsample and filtered at a vacuum of less than 120 mm Hg, for nutrient analysis. In the first experiment pretreated (Chapter 3) Whatman GF/F filters, were q _i_ used. Filtered standards confirmed that no measurable exchange of P 0 4 or N H 4 occurred 00 q on the filters or filtration apparatus. For the second experiment and all °^PO^ uptake 09 q studies, Nuclepore, 0.2 um polycarbonate filters were used. Carrier-free P 0 4 in dilute HCI was diluted with autoclaved, deionized, distilled water and filtered twice through a rinsed 0.2 um polycarbonate filter, to remove any labelled particulate material. Uptake rates were obtained by inoculating samples with K2HP0 4 , to the desired concentration, and immediately adding carrier-free 3 2 P 0 4 " 3 to achieve ca. 30,000 dpm.ml" .^ At the beginning and end of the incubation duplicate 25 ml samples were filtered; uptake rates were calculated from the isotope incorporation into particulate matter over the course of the incubation. Filters were placed in scintillation vials and dissolved in a 10:1 mixture of methylene chloride and ethanolamine (Lean and White 1983), before adding scintillation cocktail and counting. The specific activities of the samples were determined by removing replicate 1 ml subsamples of the labelled solution, evaporating the water, and counting the radioactivity. Nutrient specific uptake rates (h ) were calculated as detailed in Chapter 2. The Relative q - i i Uptake Ratio (RUR) is defined as the volume specific P 0 4 uptake rate (umoMiter 'hr"*) divided by the volume specific N H 4 + uptake rate, as determined over a 2 hour incubation. Physiological characteristics of competitive dominants Species composition was monitored regularly during the first experiment using microscope counts (Chapter 1), and at the end of the study clones of the competitive dominants from each treatment were isolated into unialgal culture. A dominant from the lowest N:P ratio, Synedra radians, and the dominant from the highest ratio, Synechococcus sp., were q grown individually at the three N:P supply ratios (5:1, 15:1, and 45:1) and their P 0 4 and N H 4 + uptake kinetics determined. Cultures were grown semi-continuously in polycarbonate 1 q flasks for a minimum of 31 days, and their uptake kinetics for N H 4 and P 0 4 determined as described above (second experiment). Nutrient Chemistry Nutrient concentrations were determined using automated analysis and the techniques of Murphy and Riley (1962) for P0 4 " 3 and Solorzano (1969) for N H 4 + , as outlined by Stainton et al. (1977). Particulate C and N were measured using a Carlo Erba 1106 elemental anatyser. Particulate P, Si0 4" 4 , and dissolved inorganic C (DIC) were analysed by the methods of Stainton et al. (1977), with the exception that CO2 was determined using an infra-red gas analyser (Horiba PIR-2000), equipped with an integrator to measure peak area. Nutrients not measured were assumed to be in excess based on their supply ratio in the medium. R E S U L T S Measurements of particulate nutrient ratios, and P 0 4 and N H 4 uptake rates (Table 5), were consistent with a change in the relative importance of N and P as limiting resources, in response to changes in the N:P supply ratio. As well, DIC measurements on cultures (data not shown) were all greater than 120 uM (mean of triplicate samples), which is in excess of requirements needed to achieve maximum growth rates(Goldman and Graham 1981, Miller et al. 1984, Turpin 1986). Consequently, C-limitation should not have been a factor. S i 0 4 " 4 concentrations also remained well in excess of growth requirements. q Changes in the N:P supply ratio affected the relative uptake rates of P 0 4 and N H 4 + , in the manner expected (Table 5), resulting in a close relationship between RUR values and N:P supply ratios (Fig. 13). Volume specific uptake rates (umoM"**h"*) did not q follow a consistent pattern; elevated P 0 4 uptake rates were found only at the highest N:P supply ratio, and were similar at others, while high N H 4 + uptake rates occurred under both 5:1 and 15:1 supply ratios. The change in community composition over time was followed in Exp I (Table 6). The 45:1 N:P supply ratio resulted in complete dominance by Synechococcus sp.; no other species were observed. In all cultures grown under the two lower N:P supply ratios two diatom species, probably Synedra radians and Nitzschia holsatica (E.F. Stoermer, pers. comm.), and a green alga Scenedesmus sp. were most abundant in terms of cell volume. RUR values for Synechococcus and Synedra (Table 7), when grown under different N:P supply ratios did not lie on the line established with experiments initiated with natural phytoplankton assemblages (Fig. 13). This was because Synechococcus demonstrated a low maximum uptake rate for N H 4 + with respect to Synedra, under N-limited conditions, whereas Synedra showed low q P 0 4 uptake rates, with respect to Synechococcus under P-limitation (Table 7). Table 5: The effect of six N:P supply ratios (with replication) on the concentrations of particulate nutrients (PC, PN and PP), particulate nutrient ratios (C:N:P), saturated PO^ and NH4 uptake rates (measured over 120 minutes), and on the Relative Uptake Ratio (RUR; see text), for cultures initiated with natural assemblages of phytoplankton. Nutrient specific uptake rates (V) are expressed as h"\ and volume specific rates (P) as umoM"*«h"*. Supply PC PN PP C:N:P Phosphate Ammonium RUR Ratio (umoMiter"1) (by atoms) P V P V 5:1 A 202 13.2 2.5 80.8: 5.3:1 0.12 0.05 4.03 0.31 0.03 B 241 15.2 2.9 83.1: 5.2:1 0.19 0.07 4.44 0.29 0.04 C 186 13.4 2.6 71.5: 5.2:1 0.28 0.11 3.93 0.29 0.07 10:1 A 0.11 1.10 0.10 B 0.22 1.79 0.12 15:1 A 233 18.8 1.3 179:14.5:1 0.55 0.42 5.23 0.29 0.11 B 267 17.8 1.2 223:14.8:1 0.71 0.59 5.10 0.29 0.14 C 221 19.3 1.3 170:14.8:1 0.54 0.42 4.81 0.25 0.11 25:1 A 0.75 1.03 0.73 B 0.38 0.58 0.66 35:1 A 0.84 0.69 1.2 B 0.44 1.03 0.43 45:1 A 164 22.1 * 0.5 328:44.2:1 3.49 6.98 <.10# >35, B 162 20.3 * 0.5 324:40.6:1 3.73 7.46 0.12 0.01 31. C 156 21.0 * 0.5 312:42.0:1 3.83 7.66 0.20 0.01 19. * no detectable uptake; however, a maximum rate can be estimated, not measured; assumed to equal concentration in supply medium as undetectable in culture. 71 Figure 13: The relationship between N:P supply ratio and the Relative Uptake Ratio (RUR) of q I saturated PO4 uptake rates to saturated NH^ uptake rates, for cultures initiated with natural assemblages of freshwater phytoplankton tt, and for unialgal cultures of Synechococcus sp. (^ ), and Synedra radians Q. The regression equation is for the experiments initiated with lake water and does not include RUR values estimated from the unialgal cultures or for one outlier tt-72 N = P SUPPLY RATIO (by atoms) Table 6: Final species composition of replicate (Repl) phytoplankton cultures initiated with Kennedy Lake water and grown at different N:P supply ratios. Proportion of Total Cell Volume N:P Supply Synedra and Ratio (Repl) Synechococcus Scenedesmus Nitzschia 5:1 (A) 0.09 0.85 0.07 (B) 0.09 0.67 0.24 (C) 0.09 0.37 0.54 15:1 (A) 0.12 0.44 0.44 (B) 0.08 0.82 0.10 (C) 0.06 0.13 0.80 45:1 (A) 1.00 0.00 0.00 (B) 1.00 0.00 0.00 (C) 1.00 0.00 0.00 Table 7: N H 4 and P 0 4 uptake rates (umoM'Mf1) and RURs of clonal cultures grown semi-continuously under different N:P supply ratios. Synechococcus was isolated from, and was the competitive dominant in cultures of Kennedy Lake phytoplankton grown at 45:1; Synedra was a dominant in, and was isolated from cultures grown at 5:1. Estimates of ±1 s.d. are shown where replicate determinations of uptake rate were made. Standard deviations about RURs were determined as described in Yates (1981). Supply Synechococcus Synedra Ratio P 0 4 * 3 N H 4 + RUR P 0 4 ' 3 N H 4 + RUR 5:1 0.10 (0.06) 0.50 (0.20) 0.20 (0.14) 0.032 1.24 (0.16) 0.03 15:1 1.46 (0.46) 0.41 (0.13) 3.56 (1.61) 0.062 2.03 (0.40) 0.03 45:1 1.82 (0.68) 0.03 60.7 0.32 0.11 2.91 75 DISCUSSION There are two points that are important consequences of the data presented here and which will be emphasized below. Firstly, Relative Uptake Ratios (RURs) should be useful for estimating N:P supply ratios under at least some conditions. Secondly, the competitive dominant under P-limited conditions demonstrated poor ability to sequester N H 4 + when grown under N limitation, while a co-dominant from a N-limited culture showed very low q uptake rates of P 0 4 under P-limitation; this implies good competitors for one nutrient are poor competitors for others, a necessity if N:P supply ratios are to be important in dictating species composition. Relative Uptake Ratios It was not unexpected that laboratory studies showed a relationship between N:P supply ratios and RURs. Frequent reference is made to elevated P0 4 " 3 uptake rates being associated with P-limited conditions (for review see Cembella et al. 1984), and enhanced N H 4 + uptake rates with N-limited growth (for review see Goldman and Glibert 1983). The influence of between species variability in initial uptake rates (Conway and Harrison 1977, Goldman and Glibert 1982, Quarmby et al. 1982, Chapter 2) on the relationship is reduced by employing relatively long incubation times. Exposure to saturating nutrient concentrations for 2 hours should be long enough for most species to replenish internal stores and reduce uptake rates (Goldman and Glibert 1982, Lehman and Sandgren 1982, Parslow et al. 1984a, b), although some species may require longer (Dortch et al. 1984, Chapter 2). Therefore, the rate measured is largely a reflection of how full internal nutrient pools were at the beginning of the incubation, and less a measure of the short-term physiological response of a specific phytoplankton assemblage to nutrient-limited growth. The incubation time was also chosen so that results would be compatible with field measurements, where longer incubation times may 76 be required to ensure that measurable uptake has occurred, particularly in oligotrophic habitats. Uptake rates of individual nutrients are not only a function of physiological status, and therefore may not be sensitive enough to ascertain the nutritional condition of a phytoplankton community. For example, perhaps because of differences in biomass and species composition, N H 4 + uptake rates in the cultures grown at a N:P supply ratio of 15:1 are considerably higher than those cultures grown at 10:1 (Table 5), although the latter are slightly more N-limited. However, when the RURs are examined it is apparent that the relative degree of N q and P limitation of the two treatments is similar. Also, the only large increase in PO^ uptake rate is at the highest N:P supply ratio, which might lead one to assume that the P status of the other cultures is similar to one another, yet the RUR values correctly indicate that there is a gradation of P-limited states among the treatments. The influence of algal growth rate (dilution rate) on the RUR was not examined in this study and should be the subject of further investigation. Growth rate effects would be most pronounced in very productive areas, where members of the algal community may be growing at near maximum rates. Under such conditions cellular nutrient pools would be nearly replete; consequently, capacity for uptake would be limited. As well, at higher growth rates the critical N:P ratio at which an alga shifts between N and P limitation, decreases (Terry 1980). Therefore, the relative demand for N and P is both a function of the N:P supply ratio and the supply rate of N and P with respect to the algal growth rate. However, changes in growth rate when growth rates are low will influence the RUR less, because cellular pools of N when N is limiting and of P when P is limiting, are generally close to minimum up to ca. 50 % of the maximum growth rate (Fuhs 1969, Rhee 1973, Droop 1974, Harrison et al. 1976, Goldman and McCarthy 1978). As uptake rates are thought to be influenced by the size of internal nutrient pools, small changes in growth rate should have little effect on uptake rate under such conditions. Growth rate effects on RURs should only be important when N and P are approaching non-limiting supply rates, when small changes in supply rate can cause large 77 changes in internal nutrient pools and hence in uptake rates. However, under such conditions growth rates are less limited by nutrients and as a result supply ratios are of less interest. It is important to note that N H 4 + rather than NOg" should be used as the N source for uptake experiments, because N-limited or N-starved cells retain the capability of incorporating NH^ + irrespective of the N source on which they were grown; the same is not necessarily true for NOg" grown cells (Dortch et al. 1982, Horrigan and McCarthy 1982, Parslow et al. 1984a, Nakamura 1985). This consideration is important for field incubations where the N sources supporting growth are not known; consequently, NOg" uptake could be more a reflection of the N source supporting growth than the nutritional status of the community. As well, the presence of any ambient N H 4 + during field incubations could strongly inhibit uptake of NOg". It should be emphasized that in field studies RURs will only yield estimates of supply ratio when N, P, or both nutrients are limiting to algal growth; this should be confirmed using alternate means. Under such conditions accurate estimates would be expected within the range of supply ratios where both nutrients are growth limiting. RURs that yield supply ratio estimates of less than 5:1 or greater than 45:1 should only be interpreted as evidence that P or N is not limiting, respectively. In this manner the index could be used to monitor the effect of nutrient loading on aquatic systems, as RUR values are essentially 'instantaneous' measures of the relative demand for N and P, and hence the N:P supply ratio. The ability to estimate in situ N:P supply ratios will also provide a method to examine the influence that these ratios have on the distribution of phytoplankton species. It is well known that low ratios select for species capable of fixing their own N (Schindler 1977, Barica et al. 1980, Tilman and Kiesling 1984), but it is not known if finer levels of taxonomic distribution can be correlated with N:P supply ratios. Competitive interactions In the experiment where the change in community composition was followed, it was found that different N:P supply ratios selected for different competitive dominants (Table 6). 78 Unlike most other studies using natural freshwater phytoplankton communities (Smith and Kalff 1983, Sommer 1983b, Kilham 1986, Tilman et al. 1986, Chapter 2) the most P-limited conditions produced complete dominance by a cyanobacterium, Synechococcus sp., while under the more N-limited conditions the diatoms Synedra radians and Nitzschia holsatica, and the green alga Scenedesmus sp. were most abundant, in terms of cell volume. Dominance by Synechococcus is consistent with the arguments of several workers (cf. Smith and Kalff 1982) that cells with the highest surface area to volume ratios should be the best competitors for limiting nutrients; however, empirical evidence has generally demonstrated that diatoms are able to outcompete other taxa under conditions of P-limited growth. The discrepancy may stem from the fact that experiments in which diatoms consistently dominated under P-limited conditions were all run at 18 °C or less, whereas the studies reported here were carried out at 20 °C. Other workers (Tilman and Kiesling 1984, Tilman et al. 1986) have shown that the ability of diatoms to compete for P decreases at temperatures greater than 17 °C, while the ability of greens and blue-greens to compete increases. An alternate explanation may be that competitive outcome is influenced by the N source provided. In the work cited above, N was provided as NOg" whereas, in my experiments NH^"1" was used. Studies have shown that some phytoplankton achieve lower maximum growth rates on NOg" than on N H 4 + (Ward and Wetzel 1980, Rhee and Lederman 1983), while a few may not be able to grow at all on NOg" (Antia et al. 1975). If differences in the abilities and efficiencies with which various algal types utilize NOg" and N H 4 + are widespread, this could be of major selective importance, especially in N-deplete systems where N H 4 + rather than NOg" will probably be the N source supporting most production (Dugdale and Goering 1967, Axler et al. 1981). It is suggested that in further studies examining the effect of N:P supply ratios on phytoplankton that N H 4 + is the more realistic source to use. Under the lower N:P supply ratios Scenedesmus, Synedra, Nitzchia and Synechococcus persisted. The apparent co-existence of several species at the lower supply ratios is likely because of the fluctuating resource supply imposed as the result of the daily dilution regime; Sommer (1984, 1985) has found that pulses of a limiting resource allow for more complex phytoplankton communities. This presumably occurs by alternatively selecting 79 for a sustainable high maximum uptake rate when the resource is abundant, and high affinity when it is scarce. Such fluctuations would occur in the levels of NH^"*" because of its relatively high concentration in the dilution medium, and consequently would be of selective importance in cultures where N was a limiting resource. In contrast, changes in the concentration of PO4" 3 in the cultures are relatively slight following dilution, resulting in selection only for high affinity in P-limited cultures; in essence, mimicking a chemostat and making co-existence impossible. The observation that diatoms were good competitors for N is unusual for freshwater species grown under NOg" limitation (Tilman and Kiesling 1984, Tilman et al. 1986), but is commonly observed for N H 4 + -limited marine species (Harrison and Davis 1979, Turpin and Harrison 1979); possibly diatoms are better competitors for N H 4 + than NOg". An important observation is that RURs determined for Synechococcus and Synedra, grown at different N:P supply ratios, fell considerably outside of the relationship established using natural phytoplankton assemblages, especially at ratios other than those from which they were isolated (Fig. 13). Synechococcus was found to possess poor ability to sequester N H 4 + whenN was limiting, compared to Synedra, while Synedra showed little enhanced q uptake for P 0 4 under P-limited conditions, when compared to Synechococcus (Table 7). This provides evidence that superior competitors for one nutrient are inferior at assimilating other nutrient; this is essential if resource supply ratios are to dictate community composition. Although the experiments presented here demonstrate that RURs should be fairly robust indicators of in situ N:P supply ratios, it should be remembered that current techniques do not allow us to determine N and P flux rates in nature; consequently, the method lacks corroboration from independent measurements of N:P supply ratios, and results should be interpreted cautiously. It is hoped that the index will be of use to investigators examining the effects of N:P supply ratios on phytoplankton community structure, and to those concerned 80 C H A P T E R 5: CO-LIMITATION BY NITROGEN AND PHOSPHORUS IN A N OLIGOTROPHIC FRESHWATER L A K E B A C K G ROUND Models and laboratory studies have successfully shown that both N:P and Si:P supply ratios can direct the outcome of competition among phytoplankton species (Tilman et al. 1986). However, demonstration of the importance of resource supply ratios in nature has been elusive. Tilman's observation (1977) that the relative abundance of two diatom species was distributed along ambient Si:P concentration gradients in much the same manner as along supply ratio gradients in laboratory studies, has been the subject of some contention (Sell et al. 1984, Tilman et al. 1984). Although studies have shown that it is possible to select for or against phytoplankton capable of fixing gaseous N by perturbing systems with low or high N:P ratio fertilizers, respectively, there is little evidence to suggest that in situ N:P supply ratios direct community composition. Two major obstacles have made extrapolation of results from the laboratory to natural systems difficult. The most serious problem has been the lack of a method to estimate nutrient supply ratios in situ. The second, has been the difficulty in demonstrating that more than one resource simultaneously limits growth in a phytoplankton community. In the previous chapter I proposed that it is possible to estimate in situ N:P supply q _i_ ratios from the Relative Uptake Ratio (RUR) of saturated P 0 4 to saturated N H 4 uptake rates. In this chapter I have utilized this index, along with a more traditional bioassay approach, in an effort to demonstrate that N and P are coupled as limiting nutrients in some oligotrophic, limnetic systems. 81 MATERIALS AND METHODS Description of study site The most intensive field studies were conducted on Kennedy Lake (49°04'N, 125°30'W), although some work was also carried out on Sproat Lake (49°14'N, 125°06'W). Kennedy Lake is a medium-sized (64 km ), oligotrophic lake located on Vancouver Island in southwestern British Columbia (Fig. 14). It is a coastal, warm-water, monomictic lake mixing completely from about November to March. Studies were conducted in the main basin of the lake (47 km2) at a station about 1 km from shore; a smaller arm (17 km2) was fertilized with inorganic nutrients during the study for the purpose of enhancing fish productivity (Stockner 1981), but these additions have little effect on the region of the lake where my experiments were conducted. The lake is rapidly flushed, the entire volume of the lake turning over approximately annually. The mean depth of the main arm is 27 m, although in the region of sampling the depth was in excess of 50 m. Concentrations of soluble reactive phosphorus (SRP) and N H 4 + are at or near detection limits throughout the year. Sproat Lake is very similar to Kennedy Lake in size (41 km ) and in most other respects, however, it is somewhat deeper (mean depth 59 m) and slower flushing (8 years). Both lakes are circumneutral (ca. pH 7), stratify strongly at about 10 m, and are quite clear, with Secchi depths typicalty greater than 5 m and 10 m in Kennedy and Sproat lakes, respectively. Further limnological details of the lakes can be found in Nidle et al. (1984) and Stockner et al. (1980). Water collection and nutrient chemistry At Kennedy Lake, water for bioassays, nutrient analysis, and RUR uptake experiments was collected from 1 m at the sampling station shown in Fig. 14. Immediately subsequent to collection, the water was filtered through 120 um screening, and transported to shore where uptake experiments were conducted, and samples were processed for nutrient analysis. Kennedy Lake was sampled on 15 May and 13 August in 1983, and on 12 May, 15 82 Figure 14: Map showing Kennedy and Sproat Lakes and the location of the sampling stations (solid symbols). 83 84 July and 22 September in 1984. Sampling at Sproat Lake was carried out on 14 and 15 May, 1985, in the same manner as at Kennedy Lake. Samples for dissolved nutrients were filtered through ashed (450 °C for 6 hours) Whatman GF7F filters which were prerinsed with 100 ml of lake water; samples were frozen immediately on dry ice. Chlorophylls were filtered onto GF/F filters and analyzed as recommended by Strickland and Parsons (1972). Nitrate (NOg') + nitrite (subsequently referred to as NOg"), N H 4 + , and SRP were analysed as described in Stainton et al. (1977). All meaurements were based on duplicate or triplicate samples. Bioassays Bioassay experiments, designed to identify potentially limiting nutrients, were conducted each time Kennedy Lake was sampled. Water for these was removed from the lake, taken to the laboratory, and used to initiate cultures within 5 hours of collection. To each of 21-250 ml Erlenmeyer flasks, 225 ml of lake water was added. Each flask was either assigned as a control to which no nutrients were added or subjected to one of the following six nutrient additions: P0 4 " 3 (22 uM), NOg" (556 uM), vitamins (Vit), trace metals (TM) + iron (Fe), P0 4 " 3 + NOg", or combined nutrients; concentrations of Vit and TM were as described in Guillard and Lorenzen (1972). Treatments and controls were replicated in triplicate. The response of the cultures to the additions was followed by monitoring in vivo fluorescence over a q minimum of 18 days. The design of the assay was improved in 1984 by omitting the P 0 4 + q NOg" treatment, and adding the following single omission treatments: combined - P 0 4 , combined - NOg", combined - TM, and combined - Vit. Uptake experiments Experiments to determine the RUR were conducted each time the lakes were sampled. Saturated P0 4 " 3 uptake rates were determined as described in the preceding chapter, with the following changes: For experiments conducted at Kennedy Lake, two-500 ml samples of pre-85 screened (120 um) lake water, each contained in 1-liter flat-bottomed boiling flasks, were inoculated with K^HPO^ to a concentration of 2.0 uM, and promptly labelled with ° P 0 4 " . Flasks were wrapped with neutral density screening to prevent photoinhibition, and incubated in running water at lake temperature for 2 hours. For the Sproat Lake studies the water was placed in 500 ml borosilicate bottles and incubated in situ at the 1 m depth. Water for N H 4 + uptake determinations was incubated in the presence of 10 uM ^ N H ^ C l as described for the PO^ uptake experiments, except in Kennedy Lake where 2.5-liter samples in 3-liter flasks were used. At the end of the incubations, samples were filtered onto precombusted GF/F filters. The * enrichment of the particulate material on the filters was determined by emission spectrometry (Jasco Model N-150) as described in La Roche (1983), and the N H 4 + uptake rates calculated by the equations of Dugdale and Goering (1967). Details of the method are described in Harrison (1983). Concurrent, uptake versus substrate concentration q j_ curves confirmed that 2.0 uM PO4 and 10.0 uM N H 4 were saturating to uptake except q during September 1984, when PO4 may not have been (see Chapter 7). During this month q o unexpectedly high uptake rates also resulted in greater than 30 % of the P being removed from solution; on all other occasions less than 10 % of the initial addition was removed. The amount of incorporated was always less than 2 % of that added. Estimates of in situ N:P supply ratios in Kennedy and Sproat lakes were calculated from the data in Table 5 using an inverse linear regression of x on y (Pagano 1981), where x is the N:P supply ratio and y is log RUR (x = 14.67y + 26.65). RUR values in the lakes were obtained by dividing the uptake rates (umoW" «h ) of PO4 by those of N H 4 , as determined over a 2 hour interval. RESULTS Dissolved inorganic nutrient concentrations in Kennedy Lake were consistent with the hypothesis that N and P might be co-occurring as limiting nutrients. Concentrations of N H 4 + 86 and total P were near or below detection limits (0.3 and 0.03 uM, respectively) on the sampling dates and throughout much of the year (Nidle et al. 1984, Nidle and Shortreed 1985). As well, levels of NO3" were below detection limits from June to September, 1983, and in late summer in 1984 (Fig. 15), indicating that N was potentially in short supply. Ratios of particulate nutrients (Fig. 16) showed no consistent seasonal trends although C:N ratios tended to be highest in summer. NOg" values from dates other than those shown in Table 8 and particulate nutrient data are from Nidle et al. (1984) and Nidle and Shortreed (1985); these data are based on single determinations. Results from the 1984 bioassay experiments (Figs. 17, 18 and 19) show major increases in biomass as measured by in vivo fluorescence in response to the treatment where all nutrients were added, but no increase in treatments where only P was added or where N was omitted. A large increase in response to these treatments would indicate clear P-limitation and would be inconsistent with the hypothesis of co-limitation by N and P. Some increases were noted in treatments where N alone was added or where all nutrients except P were added, indicating some synthesis of chlorophyll, increase in biomass, or change in physiological state in response to N addition. This is consistent with N being in short supply; if N were in excess further additions should not produce a response. Bioassay studies from 1983 were congruent with those from 1984 and the data have not been reported. However, they did supply further evidence of close coupling between N and P as limiting resources in Kennedy Lake, as the increases in fluorescence in response to the N + P treatment were similar to the increases observed when all nutrients were added. There was no indication from the bioassays that vitamins or trace metals were limiting algal biomass, therefore results from these treatments are not shown. Estimates of RUR values for Kennedy Lake were between 3.1 and 44.0 (Table 8), which corresponds to estimated in situ N:P supply ratios of 35 to 52, respectively. In contrast, data from uptake experiments conducted at Sproat Lake resulted in RURs of 0.21 and 0.54, corresponding to N:P supply ratios of 17 and 23, respectively. Figure 15: Concentrations of NO3" + NO2" at 1 m in Kennedy Lake during 1983 and 1984. Figure 16: Particulate nutrient ratios (by atoms) at 1 m in Kennedy Lake during 1983 and 1984. 90 91 Table 8: Saturated PC^"** and N H 4 uptake rates (nmoM*1*"1) and the ratio of those uptake rates (RUR) can be used to yield an estimate of the in situ N:P supply ratio (Chapter 4). The estimate of one standard deviation (s.d.) about RUR was estimated as described in Yates (1981). Date Saturated Uptake Rates RUR (s.d.) Estimated N:P (d/m/y) P0 4 _ 3(s.d.) N H 4 + (s.d.) Supply Ratio Kennedy Lake: 05/05/83 13.6 (2.5) 3.5 (0.8) 3.9 (1.1) 35 13/08/83 21.2 (5.0) 6.1 (0.6) 3.5 (0.9) 35 12/05/84 16.6 1.5 11. 42 15/07/84 38.7 12.2 3.1 34 22/09/84 294.6 6.7 44. 51 Sproat Lake: 14/05/85 12.3 (0.6) 59.6 (9.3) 0.21 (0.03) 17 15/05/85 21.8(13.1) 40.7 (2.8) 0.54 (0.32) 23 92 Figure 17: Changes in relative fluorescence in nutrient enrichment bioassay experiments initiated with water collected at Kennedy Lake during May 1984. Different symbols represent individual fluorescence measurements taken on triplicate cultures. RELATIVE FLUORESCENCE Figure 18: Changes in relative fluorescence in nutrient enrichment bioassay experiments initiated with water collected at Kennedy Lake during July 1984. Different symbols represent individual fluorescence measurements taken on triplicate cultures. 5.00 1.00 0.50 Control 0.10 0.05 5 1.00 z UJ 6 0 .50 LU or O 3 £ 0.10 UJ or 0.05 1.00 0.50 r 0.10 0.05 All Nutrients ' ' ' ' ' ' ' ' ' ' ' ' ' ' • i i i i All -N J i i i i i i i i i i i i i i i i i i All - P 0 0 1 ' ' ' ' ' 1 1 1 ' 1 ' ' ' ' ' ' ' ' ' ' 1 ' 1 ' ' ' ' ' ' ' ' 1 1 1 1 • 0 5 10 15 0 5 10 15 2 0 TIME (days) Figure 19: Changes in relative fluorescence in nutrient enrichment bioassay experiments initiated with water collected at Kennedy Lake during September 1984. Different symbols represent individual fluorescence measurements taken on triplicate cultures. 97 98 DISCUSSION Unfortunately, it is not a trivial problem to demonstrate that N and P are co-limiting resources in any given system; several lines of evidence are required. Nutrient concentrations are important and relatively easy data to obtain. It stands to reason that if a resource is growth limiting it will not be abundant; however, it can be scarce but not limiting to growth. One of the reasons that Kennedy Lake was chosen as a potential candidate to demonstrate co-limitation by N and P is that concentrations of NO3" (Fig. 15), SRP, total P, and N H 4 + are frequently below detection limits (0.07, 0.03, 0.03 and 0.30 uM, respectively) during the growing season (Nidle et al. 1984, Nidle and Shortreed 1985, Suttle unpublished data). Particulate nutrient ratios Particulate nutrient ratios are frequently used as indicators of N or P deficiency. Goldman et aZ.(1979) have argued that phytoplankton growing at close to maximum rates typically possess C:N:P ratios near 106:16:1, and that significant deviations from this ratio occur under P or N limitation. Specifically, under conditions of P-limited growth C:P and N:P ratios increase, while under conditions of N-limited growth the C:N ratio increases and the N:P ratio decreases (Healey 1973, 1975, Sakshaug and Holm-Hansen 1977, Goldman et al. 1979). This is because the quantity of the limiting resource in cells is generally reduced with respect to other nutrients. The C:N ratios at Kennedy Lake ranged from 6.3 to 12.1 in 1983 and from 4.7 to 12.3 in 1984 (Fig. 16), with several values greater than 8.5 and in the range associated with moderate N deficiency (Healey 1975, Healey and Hendzel 1980). C:P and N:P ratios were much more variable and ranged over an order of magnitude; many approximated the physiological extreme achievable by algae under the most P-limited conditions (Fuhs et al. 1972, Healey 1975, Goldman et al. 1979, Chapter 2). The extreme variability in C:P and N:P ratios likely arose from analytical errors in the particulate P determinations. This is supported by the fact that variations in N:P and C:P estimates paralelled changes in the estimates of the 99 particulate P concentrations (Nidle et al. 1984, Nidle and Shortreed 1985). Estimates of the particulate P concentrations reported by Nidle and co-workers were based on single determinations, and ranged nearly three-fold and ten-fold between May and September, in 1983 and 1984, respectively. In contrast, my 1984 measurements (Table 10) were made in triplicate and showed less than 25 % variation over the same interval. In addition, because an unknown and likely variable proportion of the particulate material is detrital in origin, it is difficult to interpret particulate nutrient ratios in terms of the form of nutrient limitation. Bioassays Nutrient bioassays using either natural phytoplankton assemblages or test organisms have frequently been used to assay the form of nutrient limitation in freshwater and marine situations (for review see Schelske 1984). Although such assays can be criticized on the grounds that containment causes potential artifacts by separating cells from sources of nutrient cycling, such assays appear to be reasonable predictors of the form of nutrient limitation in natural systems (O'Brien and deNoyelles 1976, Schelske et al. 1978, Schelske 1984). In vivo fluorescence was chosen as a measure of algal response because of its sensitivity and simplicity. Despite being influenced by the physiological state of the cells (Kiefer 1973, Loftus and Seliger 1975), fluorescence can and has been used as a rough estimate of chlorophyll a in enrichment bioassay experiments (e.g. Thomas 1969). If N and P are tightly coupled as limiting nutrients little or no increase in fluorescence would be expected in response to an addition of N or P alone, as the addition of one nutrient would drive the other into limitation for all species. Similarly, if a combination of nutrients are added except for either N or P little change in fluorescence should be observed. In response to P addition alone, or when all nutrients except N were added, there were slight if any changes in fluorescence relative to the controls (Figs. 17, 18, 19). If N had been in excess, a several-fold increase would have been expected on the basis of the size of internal N reserves alone (Dauta 1982, Dortch et al. 1984). In contrast, increases in fluorescence were noted on all dates in response to N addition alone, or when all nutrients except P were added, suggesting some synthesis of chlorophyll in response to increased N availability. Kiefer's (1973) observation that fluorescence per unit chlorophyll increases under N deficiency indicates that chlorophyll synthesis had occurred, as increases in fluorescence per chlorophyll would not be expected in response to a N addition. The increases, however, were much less than the fifty-fold observed when the combined nutrients were added. The decline in fluorescence frequently observed over the first few days of the bioassays was likely due to a decrease in chlorophyll in response to higher irradiances in the growth chamber than in the field. The bioassay data provide strong support for the contention that N and P are co-limiting nutrients in Kennedy Lake. RUR experiments Further evidence for co-limitation by N and P in Kennedy Lake was obtained by conducting RUR experiments on two dates in 1983 and three dates in 1984. It was rationalized that if N and P were co-limiting, estimates of the in situ supply ratio should lie between 5:1 and 45:1, roughly the range of critical (optimum) ratios that have been obtained for phytoplankton (Table 4). Results of the experiments (Table 8) indicated that supply ratios were generally in the range that would be expected for co-limitation to occur. As a further test of the utility of the index, RUR experiments were carried out on Sproat Lake during May 1985. Sproat Lake is similar to Kennedy Lake in many respects; however, dissolved inorganic N tends to disappear sooner and remain below detection limits longer (Nidle et al. 1984, Nidle and Shortreed 1985), suggesting that it is a more N deplete system. The RUR data supported this reasoning and yielded supply ratio estimates of 17:1 and 23:1. Although simultaneous experiments were not conducted on Kennedy Lake, the similarity of results between 1983 and 1984, when compared to the 1985 data from Sproat, indicate that N:P supply ratios are probably lower in Sproat Lake. The interpretation of the results presented in this chapter leads one to conclude that Kennedy Lake and probably Sproat Lake are systems in which N and P are coupled as limiting nutrients. Other investigations of oligotrophic coastal Pacific lakes may reveal that co limitation by N and P is the rule rather than the exception as levels of dissolved inorganic N 101 typically drop to near or below detection limits in the majority of them during summer. For example, in 12 of 14 coastal lakes regularly monitored in 1983 as part of the British Columbia Lake Enrichment Program (Stockner and Shortreed 1985) concentrations of NOg" were at times 0.14 uM or less (Nidle et al. 1984). The paradigm that N limitation in oligotrophic freshwater lakes is rare is probably incorrect. Nitrogen limitation Although N has occasionally been recognized as a limiting resource in some oligotrophic limnetic systems (e.g. Goldman 1960, Goldman and Armstrong 1969), it is generally associated with eutrophy. Part of the reluctance to accept N as being a nutrient limiting algal growth in pristine lakes, is undoubtedly the logical assertion that N deficits can be overcome through fixation of atmospheric N by cyanobacteria (Schindler 1977). The results presented here do not contradict those arguments, but imply that supplies of inorganic P can be inadequate to support growth of N-fixers. As emphasized below, evidence suggests that N-fixers are not good competitors for P, and would be poor at sequestering it when ambient concentrations are low. Intuitively, in order for a cell to divide it must be able to double the internal reserves of the nutrient which limits its growth; therefore, its growth rate will depend not only on the uptake rate of the resource but also on the minimum size of this internal pool. Healey (1980) has pointed out that for uptake that is described by a rectangular hyperbola, the substrate affinity at low nutrient concentrations is Vma3Jks (c.f. Chapter 1). Calculations (Table 9) made from data presented in Smith and Kalff (1982) demonstrate that the two N-fixers they examined, Anabaena planctonica and Aphanizomenon flos-aquae, displayed lower affinities for P 0 4 * 3 than the other algae observed. In contrast, Synedra, a genus which has been found to be the best P-competitor in numerous studies (Smith and Kalff 1982, Sommer 1983b, 1986, Chapter 1), displayed the highest. Further evidence that N-fixers are poor competitors for P can be found in the studies of Schindler et al. (1979). Carrier free additions of ^P-PO^" 3 to a P-limited algal community dominated by Anabaena resulted in nearly all of the label being incorporated into smaller cells, as determined using autoradiography. Although Table 9: Cellular uptake rates (Tmax; fmohcell'1*'1), half-saturation constants (kg; uM) for in P-limited phytoplankton. Vmaxlks is the affinity for PO4 at low substrate concentrations. Tmax, kg, and q0 are from Smith and Kalff (1982). Species Tmax kg qQ Vmax Vmax/kg Synedra acus 15.6 0.06 6.67 2.34 39 Asterionella formosa 18.0 0.06 9.13 1.97 33 Fragilaria crotonensis 132.3 0.03 159.35 0.83 28 Anabaena planctonica 73.5 0.06 53.87 1.36 23 Aphanizomenon flos-aquae 57.1 0.06 35.81 1.59 27 Oscillatoria tenuis 332.3 0.04 290.00 1.15 29 103 Anabaena was unsuccessful at removing P0 4 at ambient concentrations in the presence of competitors, when separated from smaller cells by filtration they were able to take up the label. Finally, the fact that in numerous studies (Smith and Kalff 1982, Sommer 1983b, 1986, Chapters 1 and 4) employing natural assemblages of phytoplankton grown in P-limited continuous or semi-continuous cultures, N-fixers have not dominated, intimates that they are not good P competitors. Therefore, it seems reasonable to hypothesize that although N is a limiting resource in these lakes, N-fixers remain essentially absent because of their inability to obtain adequate P. There has been no attempt made in this study to ascertain the utility of the RUR index in lakes of different trophic status. As discussed in the preceding chapter RUR values should be relatively insensitive to algal growth rate except under conditions where they are approaching maximum rates. As well, there is no reason to expect RURs to be influenced by biomass, as long as adequate nutrients are added to ensure that uptake rates are not substrate limited during the course of incubation. More serious problems may arise in communities dominated by N-fixing cyanobacteria. Vincent (1981a) found that N H 4 + uptake rates decreased in N-depleted cultures of heterocystous blue-green algae, relative to N-replete ones, and that uptake capacities were lower in field populations dominated by N-fixers. This could cause an underestimate of the N:P supply ratio if there was also not a concomitant reduction in P uptake rates. The occasional observations of enhanced N H 4 + uptake rates in the presence of significant concentrations of DIN (White et al. 1985, 1986), suggests that under such conditions RURs may indicate N-limited growth, and hence not reflect supply ratios. This is not necessarily the case as freshwater phytoplankton may not be as efficient at sequestering N H 4 + as their marine counterparts, and consequently N:P supply ratios may be low even when N H 4 + levels are detectable. For example, the occurrence of heterocystous blue-green algal populations, suggestive of N-limited conditions, in the presence of substantial concentrations of N H 4 + is not uncommon (e.g. Ward and Wetzel 1980); furthermore, there are numerous observations of detectable N H 4 concentrations remaining in lakes where NO3" has dropped to below detection limits (e.g. Schindler et al. 1973, White et al. 1985, 1986). Published data on residual N H 4 + concentrations in N H 4 + limited cultures of freshwater phytoplankton are scarce, however, Giddings (1977) found residual concentrations of 0.2 to 0.4 uM for Scenedesmus, a genus which was found to be a good N competitor in the experiments reported in Chapter 4. In addition, residual levels of N H 4 + in cultures of Synechococcus and Synedra grown at an N:P supply ratio of 5:1, during the RUR experiments, were found to be about 1 uM, consistent with the view that measurable N H ^ + concentrations can still be limiting to the growth of freshwater algae. 105 C H A P T E R 6: TIME-COURSES OF SIZE-FRACTIONATED PHOSPHATE UPTAKE:  A R E L A R G E R C E L L S BETTER COMPETITORS FOR PULSES OF PHOSPHATE T H A N S M A L L E R CELLS? BACKGROUND Previous studies have found that at low ambient concentrations most of the P0 4 " 3 flux is directed into smaller cells (Schindler et al. 1979, Lean and White 1983, Currie and Kalff 1984a), which agrees with arguments that small cells should be superior competitors for limiting nutrients (Laws 1975, Smith and Kalff 1983). Nonetheless, populations of large cells are also frequently observed in nutrient deplete areas. If such larger cells are unable to obtain adequate P to meet their growth requirements by taking up P0 4 " 3 (Currie 1986) an obvious question is, "How do they?" ln Chapter 1, consistent with the observations of Turpin and Harrison (1980) I found that infrequent nutrient addition selected for larger cells. Such results, in conjunction with reports that at increasing phosphate concentrations a greater proportion is taken up into larger size fractions (Schindler et al. 1979, Lean and White 1983), and that zooplankton are able to produce patches of P utilizable by phytoplankton (Lehman and Scavia 1982a), caused me to form the hypothesis that larger cells may obtain their P from patches of elevated concentration. However, contradicting data of Lehman and Sandgren (1982) indicated q that cells which pass through a 5.0 um filter have much higher chlorophyll specific P 0 4 uptake rates than larger cells. Yet their data also showed that the smaller cells sustained the greatly elevated uptake rates for only a short period of time; uptake rates in the 60-90 min interval were about 30 % of those in the 0-5 min period. In Chapter 1, I suggested that under q P-limited conditions larger cells may be able to sequester adequate P 0 4 to overcome their larger cellular requirements (Shuter 1978) by sustaining elevated uptake rates, during nutrient pulses, for a longer time than their smaller counterparts. 106 The purpose of this study was to examine time courses of P0 4 " 3 partitioning between the less than and greater than 3.0 um size fractions from two oligotrophic lakes, in order to test the hypothesis that larger cells can sequester sufficient P during an elevated pulse to meet their growth requirements. Experiments were carried out in one instance by enriching samples oo q of lake water with various levels of ° ^ P - P 0 4 and following incorporation into the two size fractions. In the other instance carrier- free 3 2 P - P O V 3 was added to lake water before and after the addition of inorganic nutrients to a lake from an aircraft. Nutrients are routinely added to several western Canadian lakes in this manner for the purpose of enhancing growth rates of juvenile anadromous salmon (Stockner 1981, Stockner and Shortreed 1985). This provided an unusual opportunity to quantify, in situ, the partitioning of a P0 4 " 3 pulse into small and large phytoplankton fractions. MATERIALS AND METHODS Experiments were carried out on Kennedy and Sproat Lakes; the lakes and locations of the sampling stations are described in the preceding chapter. P0 4 " 3 uptake experiments at Kennedy Lake were conducted in May 1984, and those at Sproat Lake in May 1985. Water was collected from the 1 m depth at the stations shown in Fig. 14, and filtered through 120 um screening. At Kennedy Lake, water was dispensed into 1- or 2- liter flat-bottomed boiling flasks and, 3 2 P - P 0 4 (ca. 30,000 dpm»ml"^) was used to determine P0 4 " 3 uptake rates at ambient (carrier-free addition) and five added concentrations (0.05, 0.10, 0.25, 0.50, and 2.0 uM); the 0.1 uM treatments were duplicated. Samples were incubated in flowing water, under natural light, at lake temperature. Screening was used to decrease light to non-photoinhibitory levels. At the end of 60, 120, 170 and 240 minutes, two 25 ml subsamples were removed from the 500 ml lakewater samples and filtered through either a 0.2 um or 3.0 um pore size, polycarbonate filter. Radioactivity on the filters was determined by liquid scintillation counting and uptake rates were calculated from the amount of P incorporated 107 into the particulate material in each size fraction, as outlined in Chapter 4. The amount of uptake in the 0.2 to 3.0 uM fraction was estimated by subtraction of the isotope uptake on the 3.0 um filter from that on the 0.2 um filter. At Sproat Lake carrier-free additions of 3 2 P - P 0 4 were added to samples of lakewater preceding and at several times following the aerial fertilization of the lake with ammonium nitrate and ammonium phosphate (3.0 mg m"2 of P O 4 " 3 ) . At times of 0, 2, 5, 10, 15, 20 and q o 30 minutes after the addition of P to lakewater samples, two-25 ml subsamples were filtered and processed as described for Kennedy Lake. These data were used to estimate the proportion of P O 4 " 3 incorporated into each size fraction. q P O 4 turnover times in the lakewater were estimated as described in Lean and White 0 0 q (1983). Briefly, turnover time is derived by following the incorporation of a P-PO4 q o q addition into particulate material over time. The percentage of the initial P-PO4 addition which remains in solution at each sampling time is then calculated, and plotted on semi-logarithmic graph paper, against time (e.g. Fig 25). The initial slope of this line multiplied by 2.303 is an estimate of the uptake rate constant (h"*), the reciprocal is the turnover time. In q o Q many instances the percentage of o*P-P04 remaining in solution will approach an asymptote with time; in such cases it is necessary to subtract the asymptote from the measured values to yield a straight line, before calculating the slope. The above analysis q o q q assumes P-PO4 exchanges with only two compartments, a P O 4 pool and a particulate pool (plankton). Lean (1973a, b) has shown this assumption is not always valid under.very P-deplete conditions when a third compartment can be involved. There was no indication that the qp q P-PO4 kinetics in Sproat Lake could be resolved into three components, although the sampling frequency was probably inadequate to permit resolution of such a pool. q P O 4 concentrations in Kennedy Lake and in Sproat Lake prior to fertilization were q o q estimated using the Rigler (1966) bioassay. In this method a range of ° * P - P 0 4 concentrations are added to lakewater and the incorporation of isotope into particulate matter followed over time. The typical pattern observed is that at the lowest concentrations the most q o rapid rates of isotope uptake are measured, because there is more P per unit substrate at 108 the lowest concentrations (the specific activity is higher). If the assumption is made that q PO4 uptake rates should increase in a hyperbolic manner as substrate concentration increases (approximate a Michaelis-Menten relationship), then an estimate can be made of the maximum PO4" 3 concentration. As the rate of isotope uptake is usually much higher in the q carrier-free additions, even when PO4 concentrations are assumed to be at detection limits q the calculated rates of PO4 assimilation are much higher at the lowest substrate additions. However, by assuming lower and lower ambient levels the calculated rates of uptake at the q lowest concentrations will gradually swing towards zero, and the uptake rate versus PO4 concentration curve will approximate a hyperbola. The maximum assumed concentration which generates an acceptable hyperbola is the Rigler estimate. RESULTS Ambient PO4 concentrations were estimated to be less than 0.01 uM in both lakes. Uptake rates in the 0.2-3.0 um fraction were verj' sensitive to the time which they were exposed to elevated concentrations of PO4" 3 (Fig. 20). Rates determined over the 60-120 minute interval were less than 30 % of those recorded over the 0-60 minute interval, and those measured over 170-240 minutes were only half those for the 60-120 minute interval, ln contrast, there was little difference in uptake rates measured over the first two time intervals for the larger size fraction (Fig. 21); however, in the 170-240 minute interval uptake rates had declined to less than 20 % of the rate over the previous time period, except at the highest substrate level where they declined only slightly. q Fig. 22 summarizes the relationship between the PO4 turnover time in lakewater and the concentration of substrate added, as determined for several dates. As would be intuitively expected when the concentration of the external pool is high, turnover times are q longer. The effect of the aerial fertilization on PO4 turnover time is illustrated in Fig. 23. 109 Figure 20: PO4 uptake rates for the 0.2-3.0 um size fraction for the three time intervals 0-60, 60-120, and 170-240 minutes. Data are from Kennedy Lake in May 1984. Curves were fitted by eye. O i l I l l Figure 21: As for Figure 20, but for the > 3.0 um size fraction. 113 Figure 22: P 0 4 " 3 turnover times as a function of the PO4 concentration added; data from Kennedy and Sproat Lakes. 114 10.000 _ 1,000 L -c 1 100 1 ^ rA UJ o h-" 8 Ld • SPROAT L A K E - MAY > o A o KENNEDY LK. - MAY z rr A KENNEDY LK. - JULY 10 I I I I I I I I I I I I I I I I I I I I I 0 0.5 1.0 1.5 P0 4 3 ADDED (iM) 2.0 115 Figure 23: P 0 4 turnover times and proportion of P uptake into the 0.2-3.0 um size fraction, preceding and following the addition of nutrients to Sproat Lake. 117 Immediately following nutrient addition the turnover time increased approximately two orders of magnitude; it remained elevated for at least 3 hours before decreasing to about what it was before. If these turnover times are usedas an index of PO^ concentration (c.f. Fig. 22), it is apparent that partitioning between the size fractions was dependent on the concentration of q PO4 . The proportion going into the 0.2-3.0 um plankton was almost the mirror image of the turnover times; when concentrations were high uptake into the large size-fraction was strongly favoured. DISCUSSION Kennedy Lake The Kennedy Lake data support the hypothesis that larger cells are able to maintain elevated uptake rates for longer than smaller cells. Uptake rates in the small size fraction were approximately 70 % lower in the second hour than in the first, whereas they remained virtually unchanged in the larger size class (Figs. 20 and 21). Closer scrutiny of the data indicate, however, that despite the ability to sustain elevated uptake rates for longer, the q larger size class may not be able to sequester enough PO4 to realize the same P-specific growth rate as the smaller cells. In Kennedy Lake, 55 % of the chlorophyll a was in the >3.0 um fraction and a similar proportion of the particulate P, assuming particulate P and chlorophyll are distributed similarly between the fractions (data from Table 10 indicate that this is a reasonable approximation). Therefore, if the particulate P is in the plankton and the q larger cells are able to obtain more than 55 % of a PO4 pulse, they should be able to realize a higher P specific growth rate than smaller competitors. In fact after 4 hours only 28 % of the isotope was incorporated into the larger fraction. As the data show, maximum uptake rates per liter (and approximately per chlorophyll) are initially much higher in the small size class than in the larger one. It is only in the last time interval (170-240 minutes) that the 118 uptake rates of the smaller cells decrease to the level where they are similar to the 'elevated' uptake rates associated with the larger cells. Consequently, the smaller cells benefit more from the nutrient pulse than do the larger ones. The above analysis rests on the assumption that the proportion of particulate P in cells is the same for the two size fractions. This may not be true as significant amounts of P have been found to occur in colloids and fibrils in lakewater (Lean 1976, Paerl and Lean 1976). If such colloids or fibrils were present in Kennedy Lake they would likely pass a 3.0 um filter, but may be trapped on the Whatman GF/F glass-fiber filters used for particulate P analysis. This would result in an overestimate of the particulate P in the small fraction and, therefore, cause an underestimate of their P specific uptake rates. Hence the advantage of the smaller cells may be even larger than indicated here. Sproat Lake Data from Sproat Lake (Fig. 23) also show that cells in the larger size fraction are able to sustain elevated uptake rates for longer than small cells. Two hours following nutrient q addition the proportion of P 0 4 entering the > 3.0 um fraction had increased from 35 % to 60 % of the total, and another hour later had further increased to 85 %. When the nutrient concentration decreased, as indicated by the shorter turnover times, the proportion entering the large size class declined to 40 %, similar to what it was before the fertilization. However, when biomass is taken into consideration the results from the experiments conducted at Sproat Lake are quite different from those at Kennedy Lake. Prior to nutrient addition the proportion of PO^"3 entering each size fraction was similar when expressed on a chlorophyll basis (ca. 65 % of the chlorophyll was in the small fraction), but after fertilization the principle flux was into the larger cells (Fig. 23). Two hours after the nutrients were added about 40 % of the uptake was into the small fraction; this had decreased to 15 % after 3 hours. Subsequent to 5 hours, when the turnover time had returned to pre-fertilization rates, the amounts of isotope going into each size-class were again similar on a chlorophyll specific basis. Clearly, in this instance the large fraction was getting most of the pulse. 119 Do phosphate pulses favour larger cells ? There are two arguments that can be used to resolve the discrepant results from the two experiments. The first is that because the time scales over which the experiments were conducted are different, it is conceivable that the uptake rates in the 0.2-3.0 um fraction would have continued to decline in the Kennedy Lake samples, such that after 3 hours most of the q PO4 would be entering the large fraction, as was the case in Sproat Lake. This implies that larger cells would have to be exposed to elevated levels of P 0 4 " 3 for several hours in order to obtain enough to maintain a growth rate similar to the small cells. Such a strategy would not permit large cells to meet their P requirements through utilizing nutrient patches produced by individual zooplankton, but perhaps could be of some significance when large amounts of nutrients enter the euphotic zone via upwelling or storm events. An alternative explanation is that the two size fractions may have been competing for different resources in Sproat Lake, but not in Kennedy Lake. The rationalization behind this rests on two observations. The first is that estimates of in situ N:P supply ratios (by atoms), derived from experiments examining the ratio of saturated P 0 4 " 3 to saturated N H 4 + uptake rates, range from 34:1 to greater than 45:1 in Kennedy Lake, and from 17:1 to 23:1 in Sproat Lake (Chapter 5). Secondly, most of the cell volume in the larger size fraction consists of diatoms, while in the smaller range it consists largely of small cyanobacteria, probably Synechococcus (Shortreed and Stockner unpubl. data). The 'critical' supply ratios at which phytoplankton shift between P and N limitation range between 7:1 and 25:1 for diatoms and 25:1 and 45:1 for Synechococcus (Table 4). Consequently, in Kennedy Lake both size fractions q may have been limited by P 0 4 , whereas, in Sproat Lake Synechococcus may have been N q limited and the diatoms P limited. Therefore in Sproat Lake, most of the P 0 4 during a large pulse went into the larger cells not because they were better competitors, but because the small cells were not P-limited. The greater than 3 um fraction from Kennedy Lake was able to sustain elevated PO4"3 uptake rates for longer than the smaller fraction, when both size classes were likely limited by P. It appears unlikely, however, that the larger cells would have been able to 120 sequester enough P to realize the same specific growth rate as the smaller cells, in pulses of less than 2 hours duration. Interpretation of the results is complicated because one cannot be certain that the physiological status of cells in the two size fractions was equivalent. In Chapter 2, I demonstrated that a unicellular cyanophyte, probably Synechococcus, isolated q from Kennedy Lake was able to sustain greatly elevated maximum uptake rates for P 0 4 , for at least 2 hours. Therefore, the possibility remains that in Kennedy Lake internal pools of P were less depleted in the smaller cells and, consequently, high uptake rates could not be q sustained for as long. This would be consistent with the smaller cells being better P 0 4 competitors and is supported by observations that Synechococcus can outcompete other organisms in P-limited cultures (Chapter 4). The experiments presented here do little towards elucidating a mechanism whereby larger cells are able to obtain adequate resources under nutrient limited conditions. Conceivably, some large cells may be 'physiologically small'. Certain diatoms seem most likely to fit this criterion; their large vacuoles (Strathmann 1967) result in a relatively small cytoplasm relative to their surface area. This is in agreement with many competition experiments (Smith and Kalff 1983, Sommer 1986, Kilham 1986, Tilman et al. 1986, Chapter q 1) which have found species of Synedra to be good P 0 4 competitors. Yet Smith and Kalff (1983) maintain that even correcting for cytoplasmic volume certain species of Synedm were q better competitors for P 0 4 than expected. Another point to consider is that it may be presumptuous to assume that organisms of different sizes are competing for the same resources. Nutrient bioassay experiments have suggested that multiple resource limitation may occur in some oligotrophic areas (Menzel et al. 1963, Thomas 1969, de Haan et al. 1982, Lane and Goldman 1984, Wurtsbaugh et al. 1985). If this is true it is possible that smaller cells may not be the best competitors for all nutrients. Finally, it must be remembered that in nature different size classes may not need to achieve the same specific growth rates. If loss rates are less in the larger cells a lower uptake rate may support a higher net population growth rate. 121 In conclusion, the results from this study do not support the contention that larger cells are able to meet their P0 4" 3 requirements by sequestering their P from pulses of elevated concentration. In Kennedy Lake the maximum uptake rates of larger cells did not decrease as rapidly as those in the smaller fraction, however, the degree of enhancement on a P specific basis was much higher for the smaller cells. Consequently, the P doubling time was much q shorter in the smaller size fraction. In Sproat Lake most of the P0 4 uptake was in to the larger size class; however, the estimated N:P supply ratios in the lake are such that the smaller cells may be N limited. As a result the greater uptake by the large cells was likely not an indication of better competitive ability at elevated concentrations, but rather, evidence of q P0 4 sufficiency in the smaller cells. 122 C H A P T E R 7; SIZE-FRACTIONATED AMMONIUM AND PHOSPHATE U P T A K E  RATES OF P H Y T O P L A N K T O N FROM A N OLIGOTROPHIC FRESH-WATER L A K E BACKGROUND Numerous studies have characterized uptake kinetics of P0 4 " 3 (e.g. Rigler 1964, Lean 1973a, Lean and White 1983, Tarapchak and Herche 1986) and N H 4 + (e.g. Dugdale and Dugdale 1965, Liao and Lean 1978, Murphy 1980, Axler et al. 1982, Priscu et al. 1985, Whalen and Alexander 1986) in field populations of freshwater phytoplankton, yet seldom have both processes been examined in the same body of water at the same time. There is now some impetous to do so with the realization that co-limitation by N and P may be more common in aquatic systems than previously thought (Lane and Goldman 1984, Priscu and Priscu 1984, Tamminen et al. 1985, Chapter 5). As well, the discovery of a ubiquitous biomass of picoplankton in lakes and oceans (for review see: Stockner and Antia 1986) raises questions with respect to the partitioning of N and P demand between different size fractions of the plankton community. In addition, the competition and nutrient uptake experiments in Chapter 4 indicated that different sized cells may have different relative requirements for N and P. A very small cyanobacterium (Synechococcus) was found to outcompete other species at high N:P ratios, and q _L also demonstrated high P 0 4 uptake rates but low N H 4 uptake rates. In contrast, much larger species were found under lower supply ratios; the one dominant tested, Synedra, displayed poor ability to sequester P 0 4 but high uptake rates for N H 4 . These results q j_ suggest that the partitioning of P 0 4 and N H 4 demand between small and large size fractions may be different in nature. This chapter documents several experiments designed to characterize in detail the q i P 0 4 " ° and N H 4 uptake kinetics of the less and greater than 3 um size fractions in Kennedy 123 Lake. This information can be used to make inferences about the relative demands for N and P in these two size classes. MATERIALS AND METHODS Study site and sample collection Experiments were conducted at Kennedy Lake. The limnology of the lake and a map showing the location of the lake and sampling station (Fig. 14) has been detailed in Chapter 5. Samples for nutrient analysis and uptake experiments were collected from the 1 m depth and filtered through 120 um screening and taken to the shore in 20-liter carboys. Dissolved and particulate nutrient analyses q Dissolved nutrient concentrations (except P 0 4 ) were determined for lakewater which had been filtered through combusted (450 °C for 6 hours), rinsed (100 ml deionized-distilled water, followed by 100 ml lakewater), Whatman GF/F glass fiber filters. N H 4 + , NO3" and soluble reactive phosphorus (SRP) concentrations were determined using automated analysis and the techniques of Solorzano (1969), Woods et al. (1967), and Murphy and Riley (1962), respectively, as outlined by Stainton et al. (1977). Maximum P0 4 " 3 concentrations were estimated by the bioassay method of Rigler (1966); details of the technique were presented in Chapter 6. Particulate nutrient and chlorophyll a concentrations were determined for total and 3.0 um filtered lakewater. Particulate material was trapped on GF/F filters and promptly frozen at -78 °C on dry ice; measurements were generally in triplicate. Particulate C and N were ascertained using a Carlo Erba elemental analyser, and particulate P by acid digestion as described in Stainton et al. (1977). Chlorophyll was extracted in 90 % acetone and analysed fluorometrically (Turner Model 10) by the method of Strickland and Parsons (1972), 124 uptake experiments On three sampling dates, 2.5-liter samples of lakewater were dispensed into 3-liter flat-bottomed boiling flasks. Each flask was inoculated with one of five concentrations (0.1, 0.2, 0.5, 1.0, or 5.0 uM) of 99 atom % 1 5 N H 4 C 1 , except on the May sampling date when only the three lowest concentrations were used. The flasks were wrapped with screening to reduce the incident irradiance by 60 % and prevent photoinhibition; incubations were at lake temperature. The flasks were frequently agitated during incubation and sampled after 2 hours. Each sample was split; half was filtered through a 3.0 um polycarbonate filter (to obtain the less than 3 um fraction) before being filtered onto a combusted GF/F filter (effective retention, 0.7 um). The other half was filtered directly onto a GF/F filter to obtain the uptake by the total lake plankton. The enrichment of the particulate material on the filters was determined by emission spectrometry (Jasco Model N-150) as described in La Roche (1983), and the N H 4 + uptake rates were calculated using the equations of Dugdale and Goering (1967). Corrections for changes in particulate N over the course of the incubation (Dugdale and Wilkerson 1986) proved to be unnecessary because of the slow uptake rates and short incubation times used. As has been found in other studies of N H 4 + uptake in freshwater (Liao and Lean 1978, Murphy 1980, Priscu and Priscu 1984), uptake rates were observed to decrease at higher concentrations, suggesting that the chemical method overestimated the ambient N H 4 + at the beginning of the experiment. The expected rectangular hyperbola was only evident when ambient N H 4 + concentrations were assumed to be near zero; therefore, uptake rates are plotted against the N H 4 + concentration added. Uptake rates in the > 3 um size range were calculated by subtracting uptake rates determined for the < 3 um fraction from those measured for the total sample. Details of the method can be found in Fiedler and Proksch (1975) and Harrison (1983). P uptake experiments These studies were carried out concurrently with the experiments. Immediately prior to use, carrier-free 3 2 p Q -3 m dilute HCl was diluted with autoclaved, deionized, distilled 125 water and filtered twice through a rinsed 0.2 um polycarbonate filter to remove any particulate material in the ampoule which might be labelled. The isotope was added to samples of lakewater (500 ml), contained in 1 liter flat-bottomed boiling flasks, to which one of several q concentrations of I^HPO^ had been added seconds before. The concentrations of P 0 4 added to the May samples were 0.00, 0.05, 0.10, 0.25, 0.50, and 2.00 uM; in the July and September samples the 0.25 and 0.50 uM additions were replaced with a 0.30 uM addition. Because of the large number of samples and concurrent incubations, it was only possible to replicate the 0.1 uM addition. Immediately after the isotope was added (ca. 30,000 dpm'ml" )^ and at predetermined time intervals up to a maximum of 4 hours, replicate 25 ml aliquots of the samples were removed. Each aliquot was then filtered through either a 0.2 um or a 3.0 um polycarbonate filter, to permit uptake rates to be calculated for the two size fractions. The filters were placed in scintillation vials and dissolved in 200 ul of a 10:1 mixture of methylene chloride and ethanolamine (Lean and White 1983), before adding scintillation fluor and counting. Rates of P 0 4 " 3 uptake were calculated from the isotope incorporated into the particulate matter in each of the size fractions. The amount of uptake in the 0.2-3.0 um fraction was estimated by subtraction of the isotope uptake on the 3.0 um filter from that on 1 q the 0.2 um filter. To allow calculation of specific activities (dpm»umol of P 0 4 ) for each of the samples, replicate 1 ml subsamples from each of the treatments were removed and placed in scintillation vials. The water from each subsample was then evaporated, fluor added and the activity counted with a scintillation counter. Calculation of nutrient uptake kinetic parameters i q N H 4 and P 0 4 uptake rates in phytoplankton are generally described by a rectangular hyperbola analagous to the Michaelis-Menten model used to explain simple enzyme-substrate reactions. In this model, the uptake rate (T) is related to the nutrient concentration (S) by the equation, T^(TmtjS)/(ks+S) 126 where, T m a x is the maximum uptake rate per cell and kg is the nutrient concentration at which the uptake rate is one-half maximum (c f. Chapter 1). When T is divided by the cellular concentration of S (S can be 'free' or combined to form larger molecules) it is referred to as the nutrient specific or specific uptake rate and is designated V. Uptake rate can also be expressed on a per volume basis (umoW'^ h" )^. This is typically done with 'field' measurements, where the nutrient concentration per cell is unknown; it is designated P in this thesis. The kinetic constants (Pmax and kg) for the uptake data described in this study were estimated using a least-squares regression on the following linear transformation of the above equation (replacing T with P): (SIP) = (l/Pmax)S + (ksiPmax) When S/P is plotted against S (Woolf plot) the reciprocal of the slope is Pmax, the x-intercept is -kg, and the y-intercept is the uptake rate constant. The reciprocal of the uptake rate constant yields the turnover time of the ambient nutrient pool. If the substrate concentration cannot be measured, the x-intercept provides an estimate of kg plus the ambient substrate concentration. As the ambient concentration is unknown in these experiments, calculated values of kg represent an overestimate of the true value. This linearization was chosen over the more commonly used Lineweaver-Burk transformation as it provides more reliable estimates of P m a x and kg (Dowd and Riggs 1965, Robinson 1985). Significant differences in the Woolf plots were tested by using a t-test for comparison of slopes. R E S U L T S q Maximum concentrations of PO4 for each month were estimated to be less than 0.01 uM, using the Rigler assay. The concentrations of other nutrients in the Kennedy Lake water samples used for uptake experiments are given in Table 10. The proportion of chlorophyll in the < 3 um fraction ranged from ca. 40 to ca. 64 % of the total. Concentrations Table 10: Dissolved nutrient (uM) and size-fractionated particulate nutrient (uM) and chlorophyll (ug*\'h concentrations (-1 s.d.) Maximum specific uptake rates (h"^ ) for NH 4 (Vjv^ a n (* ^ 4 (Vp) are shown for the less than and greater than 3 um, and unfractionated plankton community in Kennedy Lake, on three sampling dates. 12 May 14 July 22 Sept Chi (total) 0.69 (0.02) 0.35 (0.05) 0.55 (0.15) (< 3.0 um) 0.31 0.14 (0.05) 0.35 (0.03) PC (total) 7.3 (0.8) 15.6 (1.2) 12.8 (1.0) (< 3.0 um) 8.6 11.7 (1.7) 6.1 (0.3) PN (total) 0.82 (0.03) 1.8 (0.3) 1.6 (0.1) (< 3.0 um) 0.88 1.5 (0.3) 0.96 (0.09) PP (total) 0.054 (0.003) 0.041 (0.011) 0.046 (0.002) (< 3.0 um) n.d. 0.011 (0.004) 0.025 (0.011) Ammonium 0.07 0.28 (0.01) 0.14 (0.04) Nitrate 1.37 0.66 (0.01) 0.12 (0.07) SRP < 0.03 < 0.03 < 0.03 VN (total) 0.0018 0.0068 0.0067 (< 3.0 um) 0.0017 0.0065 0.0030 (> 3.0 um) 0.0080 0.0053 Vp (total) 0.61 1.74 22.5 (< 3.0 um) 4.21 31.8 (> 3.0 um) 0.84 11.5 • datum not collected 128 of particulates C, N and P were quite variable between sampling dates, although a significant proportion was always associated with the smaller size class. Maximum NH4 + uptake rates were highest in July whether expressed on a per volume (Fig. 24), or nutrient specific (Table 10) basis. This was coincident with the lowest algal biomass, measured as chlorophyll. Conversely, the lowest uptake rates occurred in May when chlorophyll concentrations were highest. Most of the N H 4 + uptake in May and July was associated with cells passing through a 3.0 um filter, while uptake rates in September were very similar for both size classes. When uptake was standardized to the amount of chlorophyll in each size fraction (Table 11), a disproportionate amount of N H 4 + was taken up by smaller cells in May and July; however, in September the opposite was true, when uptake was highest in the larger size fraction. qo q An example of the P-PO4 isotope disappearance curves for the > 3 um July lakewater samples are shown in Fig. 25. Trajectories of the curves were similar among months, and isotope incorporation into particulate matter was quite slow. Asymptotic values qo for the carrier-free additions were high and only about 60, 30 and 38 % of the P was taken up over the course of the incubations in May, July and September, respectively. Such isotope disappearance traces yielded saturated uptake rates for P 0 4 " 3 , for both size fractions, which were lowest in May and highest in September (Fig. 26). On the latter date, a hyperbolic relationship between concentration and uptake rate was not obtained, and the uptake rate may not have been saturated at a PO4" 3 concentration of 2.0 uM. As well, proportionately more PO4" 3 was removed by the small size fraction on all sampling dates, whether expressed on a volume (Fig. 26), chlorophyll (Table 11), or nutrient specific (Table 10) basis, although chlorophyll specific rates were more similar later in the year (Table 11). The proportion of PO4" 3 entering the two size fractions was also dependent on concentration (Fig. 27). On all dates, the amount of P 0 4 " 3 entering the > 3 um size fraction, relative to that taken up by the q smaller size class was decreased by the addition of P 0 4 ; however, at still higher concentrations of PO4* 3 the proportion of isotope taken up by the larger cells increased. 129 Figure 24: Estimates of size-fractionated N H 4 uptake rates for Kennedy Lake phytoplankton on each of three sampling dates. Rates are plotted against the N H 4 " concentration added, as measured values were apparently overestimates (see text). Incubations were for 2 hours. 081 131 Table 11: The proportion of the total N H 4 or P0 4 uptake in the < 3.0 um size fraction, relative to the amount of chlorophyll (Chi) in that fraction (i.e. (P<:3um ^ ^total^ ^ (Chl<3um I Chltotaj)). A value of unity indicates that the saturated chlorophyll specific uptake rates for the two size fractions, for a given nutrient, are the same. A value greater than one indicates that a disproportionate amount is entering the smaller size class. 12 May 14 July 22 Sept N H 4 + 2.0 2.0 0.7 P O / 3 1.8 1.6 1.2 132 Figure 25: Percent of a P in 0.2 um filtrate over time, for a range of P 0 4 additions to Kennedy Lake plankton, in July. Curves from May and September were similar. Duplicates of the 0.1 uM addition were conducted. Note that the y-axis is a logarithmic scale. 133 134 Figure 26: Size fractionated PO^ uptake rates for Kennedy Lake phytoplankton on each of three sampling dates. The uptake rates shown were estimated over 1 hour of incubation. The uptake rates measured in September may not have been saturated by the 2.0 uM P0 4 " 3 addition. Rigler bioassays estimated ambient P0 4 " 3 concentrations to be less than 0.01 uM on all dates. 135 136 Figure 27: Percentage of total P taken up which entered the > 3.0 um fraction, as a q function of the added PO^ concentration. Values are averages of the isotope distribution for all sampling periods. The error bars indicate * 1 s.d. of the means. 40. MAY 20 0_ o < ce 801 S 60 , I 40J rO A CL Pi 20_ JULY O 01 cr UJ CL PHOSPHATE (^M) 138 Estimates of the kinetic constants P m a x and ^ s for the < 3 um and > 3 um size fractions are presented in Table 12. Probabilities of the slopes of the curves tf/Pmax) being equal for the two size fractions were less than 0.05 for all comparisons except the July P 0 4 uptake rates. As can be seen from Fig. 28 this is the result of a deviation from linearity at the two highest substrate concentrations, for uptake in the large size fraction. The probability of the slopes being equal when the datum for the 2.0 um P 0 4 concentration is deleted from the regression analysis is < 0.001. Decreases in the slope of Woolf plots at higher substrate concentrations have the effect of causing overestimates of kg and the turnover time of the ambient nutrient pool. However, when the data from the two size fractions are pooled (Table 13), there is little apparent difference in estimates of kinetic constants obtained from back-q extrapolation from the high or low substrate portions of the curve for P 0 4 uptake data. The same was not true for N H 4 + uptake data, and estimates of kg and turnover time were higher when calculated using the uptake data from the two highest substrate concentrations. Although estimated values of kg were frequently different for the small and large size fractions (Table 12), there was general^ little difference in Pmax/ks for N H 4 + uptake for the two size classes. Pmax/kg is an estimate of the initial slope of an uptake versus concentration curve, and hence is a measure of nutrient affinity at low concentrations (Healey 1980). In contrast, initial slopes for the PC*4"3 uptake curves were considerably higher for those cells which passed through a 3.0 um filter. The time over which incubations were conducted also had an effect on the measured kinetic parameters. As shown in Table 14, there were significant q changes in the slope of Woolf plots for July P 0 4 uptake data over the course of the incubation. 139 Table 12: Calculated kinetic data (Pmax, nmoH" 1*" 1; kg, uM) from the N H 4 + and P0 4 " 3 uptake experiments for the less than and greater than 3 um size fractions. Data were derived by fitting least-squares regression lines to plots of S/V vs S (Woolf plots), for all substrate concentrations. Coefficient of determination (r £ ) and the probabilities (p) of the slopes being equal for Woolf plots of each size fraction are shown; p determined by analysis of covariance. AMMONIUM UPTAKE PHOSPHATE UPTAKE Date Size P max ks Pmaxlks r2 P P max ks Pma^ks r2 P 12/5 <3um 1.7 0.08 0.02 0.98 <0.001 28.9 0.10 0.29 1.00 <0.05 >3um 0.5 • •0.01 -0.02 0.94 5.6 0.13 0.04 0.99 14/7 <3um 10.4 0.36 0.03 0.99 <0.001 47.9 0.07 0.72 1.00 <0.10 >3um 2.4 0.06 0.04 1.00 30.5 0.48 0.06 0.94 22/9 <3um 3.0 0.10 0.03 1.00 <0.02 794.* - - ->3um 3.9 0.24 0.02 1.00 * 241. _ values not derived from Woolf plots; uptake rate may not have been saturated. * data not described by Michaelis-Menten kinetics. 140 Figure 28: Woolf plot of the July PO4 uptake data for the less than and greater than 3 um size fractions. Notice the deviation from linearity in the large size fraction, at the 2.0 uM concentration. 141 142 Table 13: Kinetic constants (Pmax, nmoH *h ; kg, uM) and turnover times (TT, h) calculated either by using data for all but the highest two substrate concentrations (Lo), or using only the two highest additions (Hi). Lo values were derived by fitting least-squares regression lines to Woolf plots of the data; Hi values were calculated from the linear equation defined by the points associated with the two highest q concentrations. PO4 turnover times calculated using Woolf plots were greatly in excess of values estimated from incorporation of carrier-free ^ P O ^ " 3 (see text). Missing data were either the result of too few substrate concentrations to warrant the calculations (May), or because the data were not adequately described by a Michaelis-Menten fit (Sept). AMMONIUM UPTAKE PHOSPHATE UPTAKE Date Cone P max TT P max TT 12 May Lo 38.4 0.13 3.3 Hi 33.8 0.06 1.8 14 July Lo 9.3 0.08 8.6 64.9 0.08 1.3 Hi 13.6 0.58 43. 76.6 0.14 1.9 22 Sept Lo 5.3 0.03 6.1 Hi 7.3 0.43 59. 143 Table 14: Probabilities (p) of slopes being equal for Woolf plots of July PO4 uptake data taken over different time intervals (minutes), as determined by analysis of covariance. q Data are for unfractionated plankton samples, and do not include the 2.0 uM P0 4" concentration. Time intervals p 0-10 vs 0-60 0-10 vs 0-180 0-60 vs 0-180 < 0.10 < 0.05 < 0.01 144 DISCUSSION Size-fractionated NH^ + uptake To my knowledge previous studies characterizing size fractionated NH^"*" uptake have not been carried out for freshwater lakes. I found that cells which passed through a 3.0 um filter were responsible for between approximately 90 and 50 % of the total uptake measured on the three sampling dates, when presented with a saturating addition of NH^"1". This is consistent with uptake studies conducted in marine systems. Glibert et al. (1982) and Furnas (1983) found that a large proportion of N H 4 + uptake was associated with cells in the < 10 um size class in coastal regions. Smaller cells were found to be less significant contributors to measured uptake rates in the Benguela upwelling region off southwest Africa (Probyn 1985), but cells less than 10 um in size were still responsible for between 36 and 51 % of the uptake observed, with organisms less than 1 um accounting for up to 27 % of the nitrogen assimilation. High rates of N H 4 + incorporation by small-sized cells are congruent with other investigations which have found that the smallest components of planktonic communities are frequently major primary producers in limnetic (Tison and Wilde 1981, Rai 1982, Tamminen et al. 1985, Fahnenstiel et al. 1986) and marine (Li and Dickie 1985, Glover et al. 1986) environments. P m a x and kg for NH^ + uptake Seasonal changes were apparent in the maximum uptake rate for N H 4 + in the < 3 um fraction. The maximum chlorophyll specific uptake rate in the < 3 um size class declined to less than that in the > 3 um cells, in September; it had been about two-fold greater in May and July (Table 11). This shift may have been an indirect result of a change in species composition. In May and July the < 3 um cells consisted of approximately 20 % and 50 % eukaryotic cells, respectively, the remainder were unicellular Synechococcus-\ike cyanobacteria. In September, 20 % of the < 3 um cells were eukaryotes and the rest were mainly small 145 colonies (< 5 cells) of GZoeorTiece-like cells, 2 to 3 um in greatest dimension. One explanation for the smaller contribution of the < 3 um fraction to total N H 4 + uptake is that the sheath associated with the colonies caused them to be trapped on the 3.0 um filters. The large proportion of chlorophyll that transgressed the filter (Table 10) and the observation that uptake rates for N H 4 declined about 65 % from July to September, while those for P 0 4 only decreased 25 %, suggests that this was not the sole reason. An alternate postulate is that the colonial bluegreen algae were able to reduce their N demand by fixation of atmospheric N. Gloeothece and other sheath-producing, non-heterocystous cyanobacteria are capable of aerobic N-fixation, while sheath-less forms are not, although the sheath per se does not appear to play a role (Kallas et al. 1983). The possibility exists that the colonial forms observed in Kennedy Lake were able to supplement their N requirements by N-fixation whereas, the non-sheathed unicells were not. Besides the obvious and significant differences in J > m a x for N H 4 + uptake, between the size fractions, there were also differences in estimated values of ke (Table 12). However, the significance of these differences cannot be ascertained from Woolf plot data, as values of kg are obtained by extrapolation beyond the range of data used to derive the regression estimate. Values of kg did not appear to be related to cell size. This contrasts with data obtained by Sherr et al. (1982) who found that the large freshwater dinoflagellate Peridinum cinctwn (> 120 um) had an estimated * s approximately 10-fold greater than cells which were able to pass through 20 um screening. As well, Eppley et al. (1969) found a relationship between kg for N H 4 + uptake and cell size, in N-depleted cultures of marine phytoplankton. One would not necessarily expect close agreement between size related values of kg obtained in lab studies with those from field studies, because kg appears to be strongty influenced by the physiological state of cells (e.g. Parslow et al. 1985a). Therefore unless the cells in the two size fractions were limited to the same extent by N, differences in physiological status would likely mask any size related effects. The range in * s values obtained for N H 4 + uptake at Kennedy Lake (0.06-0.36; the single negative value is not included and resulted from very low measured uptake rates) were similar to those obtained by others investigating N H 4 + uptake in 146 freshwater (Murphy 1980, Axler et al. 1982, McCarthy et al. 1982, Priscu et al. 1985, Whalen and Alexander 1984, 1986) and marine systems (Maclssac and Dugdale 1969, Sharp et al. 1980, Fisher et al. 1981, Kanda et al. 1985). Despite some apparent differences in kg, the initial slopes of the uptake curves (^*maA/ s^) were in agreement, indicating that the affinities for N H 4 + were very similar for the two size classes (Table 12). NH^+ turnover times There were some deviations from linearity in the Woolf plots at high N H 4 + concentrations. Different kinetic constants were obtained when either the uptake rates from the two highest concentrations, or uptake rates from all but the highest concentration were used for calculations (Table 13). Williams (1973) used a mathematical model to show that heterogenous communities of species possessing a range of values for kg, would not be anticipated to conform to a simple Michaelis-Menten relationship; estimates of ^ s and turnover time would be exaggerated if concentrations greater than kg were used for computations. The observed non-linearity in my data is not altogether consistent with Williams' predictions. The model predicts that deviations from linearity will be most acute at substrate concentrations less than kg, and essentially linear at greater values. For unknown reasons the data presented here are apparently most non-linear at concentrations considerably above kg Although the calculated turnover times are likely overestimates (6.1 and 8.6 hours), they are comparable to data obtained by Murphy (1980) in Lake Ontario and Lake Erie, and by Paasche and Kristiansen (1982) in the Oslofjord, Norway, but are considerably more rapid than found by Axler et al. (1981) in a subalpine lake and b3' Whalen and Alexander (1986) in Toolik Lake, Alaska. The extremely short turnover times reported by McCarthy et al. (1982) for Lake Kinneret, Israel, were not obtained from Woolf plots; rather a single 'trace' addition was made to each water sample and the observed uptake rate divided by the ambient concentration to calculate a turnover time. As the 'trace' addition frequently resulted in concentrations considerably elevated above ambient, uptake rates would be enhanced and the calculated turnover times underestimated. The relatively rapid turnover time of the NH^"1" 147 pool in Kennedy Lake suggests that NH4 supply and demand are relatively tightly coupled, as has been argued for subalpine Castle Lake (Axler et al. 1981, 1982) and demonstrated in a coastal marine environment (Gilbert 1982). q POj~ turnover times The sampling intervals used in the PO^"3 uptake experiments were not frequent enough to allow accurate turnover times to be calculated by following disappearance of carrier-oo q free o^P04 from the lakewater; however, a conservative estimate can be made assuming q PO^ is partitioned into only two compartments, a dissolved and a particulate pool. The time scale on which sampling took place prevented the resolution of a third P compartment (Lean 1973a, b), if present, and therefore a two compartment model was adopted. qo q If the disappearance from lakewater of a carrier-free addition of PO4 is plotted against time on semi-log graph paper, it will generally approach an asymptote (e.g. Fig 25). If the asymptote is subtracted from the measured values then the slope of the resulting straight line is a measure of the uptake rate constant in units of time"*, the reciprocal of which is the turnover time of the ambient pool (Lean and Rigler 1974, Peters 1978, Chapter 6). Because of the infrequent sampling regime followed in these experiments, at most one point was obtained before an apparent asymptote was approached, consequently, turnover time will be overestimated. Using this approach turnover times were estimated to be less than 14,27 and 6 minutes on the May, July and September sampling dates, respectively. These values are much less than those obtained using Woolf plots (Table 13), suggesting that the latter has overestimated the true turnover time by at least 800 % in one instance. q Brown et al. (1978) reported that PO4 turnover times in algal cultures were overestimated when calculated by back-extrapolation from high substrate regions of Woolf plots, however, the inconsistencies were not as large as reported here. Similarily, eight-fold overestimates of turnover time are in excess of the amount of error which Williams (1973) predicted could occur due to extrapolation from high substrate regions of Woolf plots. Such large errors are consistent with the empirical data of Tarapchak and Herche (1986) who found 148 discrepancies as large as 3000 % in turnover times, calculated from low and high substrate regions of Woolf plots. Results of their simulation analysis revealed that large deviations were apt to occur in situations where there were large ranges in the value of kg, and in the abundances of individual species, and when those species with the lowest kg values were the most prevalent. It seems likely that these conditions would have been met at Kennedy Lake. There are generally greater than ten common species at a given time, ranging in size from q ^ 9 approximately 4 um° for small cyanobacteria and flagellates to 1500 um° and 3000 um for Rhizosolenia sp. and Peridinium sp., respectively, with the small cells being much more abundant. Therefore, if the small cells have lower k. values than the larger cells, as would be expected from arguments based on the ratio of surface area to volume (Malone 1980), this should lead to significant overestimates of turnover time. If true, it seems prudent to dismiss the turnover times calculated from the Woolf plots, and accept that the rates calculated from oo q incorporation of carrier- free " P O / ° are closer to the correct values. The calculated turnover times for PO^"3 in Kennedy Lake are generally slower than those reported for some Canadian lakes (Rigler 1964, Levine 1975, Peters 1979, White et al. 1981, Chow-Fraser and Duthie 1983 and Planas and Hecky 1984), and for Lake Balaton in Hungary (Istvanovics and Herodek 1985), but are similar to observations made on several central European lakes (Peters 1975). The rates are somewhat faster than observed for Lake Ontario (Lean and Nalewajko 1979), Lake Kinneret in Israel (Halmann and Stiller 1974, Berman 1985) and for most New Zealand lakes (White et al. 1981); they are much quicker than found in shallow productive prairie lakes (Prepas 1983) and northern Manitoba resevoirs (Planas and Hecky 1984). If the turnover times obtained in this study approximate true rates it is evident that the values obtained for Kennedy Lake are intermediate when compared to those above. Lean and Nalewajko (1979) proposed that PO4" 3 turnover times could be used as an index of P-deficiency; however more recently, Lean et al. (1983) have pointed out that turnover time is not only a function of PO4 ' 3 demand by the plankton, but it is also influenced by the ambient q q concentration of PO4 and plankton biomass. Using an index which relates maximum PO4 149 uptake rate to an 'optimum' photosynthetic rate, Lean and Pick (1981) argue that even when PO^"3 turnover times were in excess of 14 minutes, Lake Superior phytoplankton were severely P-limited. Despite this limitation in using turnover time as an index of P deficiency, times in excess of an hour probably indicate P-sufficiency, while shorter times suggest an indeterminant degree of deficiency (Lean et al. 1983, c.f. Fig. 3 in Nalewajko and Voltolina 1986). Consequently, Kennedy Lake would fall into the P-deficient category. Another note of caution should be added regarding the use of linear transformations for calculating uptake kinetic parameters. Although it is well known that both PO4 and NH^ uptake kinetics can change rapidly over short incubation times (e.g. Chapters 2, 3 and 6), this is frequently not taken into consideration when linearization techniques are used (e.g. Axler et al. 1982, Priscu et al. 1985). The data in Table 14 illustrate that significant differences in estimates of kinetic parameters can result from the period over which uptake is measured. Most often the linearized equations are used to estimate turnover times of nutrients when ambient concentrations are not known. Fortunately, radioisotopes of P negate the necessity of q using linear transformations to estimate PO4 turnover times; however, the problem remains for nitrogenous nutrients. Consequently, until short time-course studies are done using NH4 and NOg" it will not be known if current estimates of the turnover times of dissolved inorganic N are realistic. Isotope (32P04~3) distribution o n A somewhat unusual feature of the data presented here is the high proportion of P which remains in solution (40, 70 and 62 % for May, July and September, respectively), when 'apparent' isotopic equilibrium has been reached. Similar data have been described for a series of central European lakes (Peters 1975), where asymptotic values were generally observed to range between 22 and 70 %, although values as low as 1 % were recorded. Some data indicate q that higher asymptotes are partially the result of higher PO4 levels (Peters 1979). Intuitively, this suggests lower P demand under such circumstances, and is consistent with observations that asymptotes of a few percent or less are frequently found in lakes with very 150 rapid turnover times (Lean and White 1983, Chow-Fraser and Duthie 1983, Istvanovics and Herodek 1985). However, if data from numerous sources are pooled (Fig. 29), there is only a very weak correlation between asymptotic value and turnover time (r 2 = 0.13); if data with turnover times longer than 120 min are included, the correlation is further weakened (r* = 0.03). The probabilities of the slopes being equal to zero are greater than 0.20 (t-test) in both cases. Clearly, asymptote position is dictated by factors other than P O 4 " 3 demand, as measured by turnover time. qo q The °*P that remains in solution is potentially present as P O 4 , low molecular weight 'X-P', or high molecular weight 'colloidal-P' (Lean 1973a). A fourth possibility, that the qp q PO4 is abiotically bound to substances in the filtrate has been examined, but not found to be a significant process (Lean 1973a, Lean and Rigler 1974, Tarapchak et al. 1981). Rapid qp production of P-labelled 'dissolved' organics are thought to be characteristic of extemely P-deplete environments, with very rapid P O 4 turnover times (Lean and Nalewajko 1979), and consequently would not be expected in Kennedy Lake. It is possible that the Kennedy Lake isotope disappearance curves represent 'transitional' curves which can occur between low and moderate degrees of P-limitation (Chow-Fraser and Duthie 1983). Such curves have high 'apparent' asymptotes, but are associated with longer turnover times than estimated for Kennedy Lake. The reasons for the high asymptote values in this lake are unknown, and further work should be done to elucidate the mechanisms which control isotope partitioning into the 'soluble' and particulate phases. Another recurring feature of the isotope depletion curves that has previously been described (Lean and White 1983) was that in samples to which P O 4 " 3 and 3 2 P 0 4 * 3 were qp q added, the asymptotic values were lower than in samples to which "^PO^ alone had been q added (Fig. 25). One reason for this observation could be that the plankton reduce the P O 4 to a threshold level. Because there is less ^ T O ^ relative to P O 4 " 3 as the amount of P O 4 " 3 in solution is increased, there will be less 3 2 P in solution if the P O 4 * 3 is reduced to a threshold level. One would have to speculate that when higher concentrations were added the asymptotes did not decrease so far because internal nutrient stores in the cells were filled, 151 Figure 29: Percent of total P added remaining in solution when an asymptote is reached, as q related to measured PO4 turnover times. Data were taken from the literature and are for a variety of lakes. Kennedy Lake data were not included, as they represented conservative estimates of turnover times (see text). UJ < or 70 r 60 r-50 G" 4 0 o z < UJ or 0. CM ro 30 20 10 I-0 Jl L X 20 40 60 80 TURNOVER TIME (min) 100 120 153 which caused the uptake rate to slow. An alternate explanation offered by Lean and White (1983) is that in the absence of additional substrate more 'colloidal' P is produced, which would be trapped on the filter. This seems less likely for the Kennedy Lake data for the reasons described above, but does remain a viable alternative for other systems. Size-fractionated POj~ uptake q The < 3 um fraction was also responsible for a major portion of the observed P 0 4 uptake, contributing to 84, 65 and 77 % of the saturated uptake rates observed for May, July and September, respectively (Fig. 25). The observed deviation from Michaelis-Menten kinetics in September was probably the result of significant substrate depletion at the lower PO4 additions, because of very high uptake rates. This is evidenced by the observation that over 22 % of the 0.1 uM addition was taken up over the first hour of the incubation. The average saturated specific uptake rate in the small size fraction (Table 10), over the same time interval, was an astounding 31.8 h"*. In other words, in one hour, assuming that all the particulate P is intracellular (a conservative estimate), enough P has been accumulated by each cell on average to meet the requirements of 45 new cells. Also consistent with the N H 4 + uptake data were the temporal changes in chlorophyll specific (Table 11) and volume specific (Table 12) maximum P 0 4 " 3 uptake rates. The data in Table 12 also suggest that there were large size related differences in the values of kg on the July sampling date. These apparent differences emphasize the importance of checking for linearity in transformed uptake data. At the highest concentration the value of SIP for the large size fraction falls well off the line. If this point is excluded from the regression analysis the correlation coefficient improves (r* = 1.00) and the estimates of P m a x (nmoM'h*-1) and ks (uM) change markedly from 30.5 and 0.48 to 9.3 and 0.05, respectively. The estimate of affinity ^Prrualks, h"*) also changes from 0.06 to 0.19, although the increase is not enough to equal the value of 0.72 obtained for the smaller cells. If the affinity of the smaller size fraction is higher than that of the larger cells, it is in agreement with data collected by Smith and Kalff 154 (1982) for individual species of phytoplankton, and is in agreement with the paradigm that smaller cells should be better nutrient competitors when concentrations are low (Malone 1980). Unlike the results of several other studies (Faust and Correll 1976, Friebele et al. 1978, Lean and White 1983, Berman 1983, Currie and Kalff 1984a, b), the observed flux of qp q carrier-free PO^ was not consistently into the small size fraction. In May and September qp less than 50 % of the carrier-free P was incorporated into larger cells while in July approximately 80 % of the uptake was into the > 3 um size class (Fig. 27). These data were qp derived by averaging the proportion of the total P taken up by the > 3 um size class over all sampling intervals {i.e. 0, 15, 30, 60, 120, 240 minutes), and may not agree exactly with the data in Fig. 26. Currie et al. (1986) also occasionally found that a large proportion of P O 4 was taken up by cells trapped on a 3.0 um filter; these instances were restricted to times when P O 4 " 3 demand was lower, as indicated by slower P O 4 " 3 turnover times and lower alkaline phosphatase activities. The Kennedy Lake data did not contradict their findings as the greatest uptake into larger cells also occurred when the estimated P O 4 * 3 turnover time was least. As well, July was the month in which the 'relative uptake ratio' of saturated P O 4 " 3 to saturated NH^"1" uptake rates were lowest (Chapter 5), suggesting that a decrease in the N:P q supply ratio reduced demand for P O 4 An apparently previously unreported observation was the manner in which q p q partitioning of P between the size classes was affected by the concentration of P O 4 that was added (Fig. 27). In contrast to the findings of Lean and White (1983), that even slight q increases in the concentration of P O 4 added to lakewater shifted most of the uptake into the q larger sized cells or aggregates, my results indicated the opposite. Small increases in P O 4 favoured uptake into the < 3 um fraction; however, at still higher concentrations more of the PO^"3 uptake was again associated with larger material. I have no explanation for this, but it does indicate that concentration can effect P cycling in unexpected and complex ways. 155 Ecological considerations From the data presented here it is apparent that organisms which were capable of passing a 3 um filter generated a significant portion of the demand for N H 4 and PO4 in Kennedy Lake. Whether or not bacterial or algal cells were responsible for the demand cannot be stated with certainty. In recent years there has been considerable'evidence put forth that most of the ambient P0 4" 3 flux in oligotrophic freshwater habitats (Currie and Kalff 1982b, Lean and White 1983, Currie 1986, Currie et al. 1986) and a considerable proportion in a range of marine situations (Faust and Correll 1976, Friebele et al. 1978, Krempin et al. 1981, Berman 1983) may be attributable to bacteria. As well, tracer studies of N H 4 + uptake in the marine environment using N (Probyn 1985) and N (Fuhrman pers. com.) have indicated that a large percentage of the in situ flux is directed into cells capable of passing through a 1.0 um filter. q In Kennedy Lake during the periods of highest PO4 demand (May and September), 45 and 64 % of the measured chlorophyll was in the < 3 um fraction. In contrast, typically less than 30 % of the chlorophyll passed through a 3.0 um filter in Lake Memphremagog, and the eleven other lakes studied by Currie and co-workers (Currie and Kalff 1984b, Currie et al. 1986). The low levels of total chlorophyll in Kennedy Lake (< 0.69 ug*!"*) suggest that it is also considerably more oligotrophic than the Quebec lakes where the lowest levels were twice as high; therefore, extrapolation of results between the two limnospheres may be premature. 1 q In addition, as maximum chlorophyll-specific N H 4 and P0 4 uptake rates were no more than two-fold different between the two size fractions (Table 11), it is unneccessary to hypothesize bacterial uptake to explain unrealistically high chlorophyll-specific uptake rates in the small size class. In other words, the observations are not inconsistent with algae being responsible for the majority of nutrient uptake by small cells in Kennedy Lake. Some inferences can also be made about the apparent competitive abilities of cells from the two size classes, assuming that they are competing for the same resources. As PmajJks is an estimate of the uptake rate constant as uptake rate approaches zero, it is an indicator of the ability of phytoplankton to sequester nutrients at low concentrations. The data in Table 12 156 indicate that Pm(UJks for N H 4 + uptake was similar for both size fractions, therefore, they should have similar uptake rates at in situ concentrations. However, unless they require the same quantity of resource to divide, equal uptake rates will not result in equal growth rates. If the amount of particulate nutrient in a size fraction indicates the amount of that resource the size fraction requires in order to double, then growth rate will depend on the nutrient specific uptake rate (i.e. the uptake rate divided by the particulate nutrient concentration). As well, the maximum specific uptake rate divided by kg should be a better indicator of competitive prowess. Such a calculation for N H 4 + uptake (Vpj/ks) yields values for the small and large size fractions, respectively, of 0.02 and 0.13 for July, and 0.03 and 0.02 for September. This implies that in July, when estimated N:P supply ratios were lowest (Chapter 5), the larger cells should have the highest nutrient specific uptake rates. The above analysis rests on the unreasonable assumption that all of the measured particulate nutrient is made up of living cells; however, as long as the ratio of living nutrient to detrital nutrient is similar for both size classes, the conclusion will be similar. As co. 50 % of the < 3 um cells consisted of unicellular cyanobacteria these results are of additional interest because they support my finding that some Sy/zec/iococcMs-like species are poor competitors for N H 4 + relative to some larger cells (Chapter 4). Long doubling times for particulate N in small size fractions is in agreement with the results of Furnas (1983) and Probyn (1985). A comparable analysis for P0 4 " 3 uptake can only be carried out for July because of missing data. In contrast to the above results, Vmax/ks was 60 for the < 3 um fraction and only 1.8 for the larger fraction. This also agrees with the finding of Chapter 4 that small cells are very efficient competitors for P0 4 " 3 q _L Finally, it is interesting to note that the ratio of saturated P 0 4 to N H 4 uptake rates was not the same between the two size groups. The ratios for the less than and greater than 3 um size classes, respectively, were approximately 17 and 11 in May, 4.6 and 12.7 in July, and 265 and 62 in September. Although, these differences may reflect changing size related resource requirements, or shifts among species with disparate physiological characteristics, it serves to emphasize that measurements of rate processes made on relatively 157 SUMMARY AND GENERAL CONCLUSIONS As emphasized in the Introduction, phytoplankton exist in an environment that is inherently variable and, intuitively, would be expected to possess adaptations to maximize their fitness under such conditions. It seems reasonable to assume that such variability would also extend to nutrient supply processes, and as oligotrophic waters are generally thought to be extremely nutrient limited systems, one would expect strong selection for physiological mechanisms to procure variable resource supplies. Resource supplies would be anticipated to vary in two main ways. They would be expected to be 'patchy' in time and space, and such patches would be qualitatively variable. As very little work had been done examining the effects of such variation on phytoplankton physiology and community structure, the work outlined in this thesis was undertaken. The results of my work indicate that phytoplankton are sensitive to pulsed nutrient addition regimes. Patchiness on the order of weeks not only affected species composition in culture, but also resulted in a shift in dominance to a larger species, and to an increase in cell size of a second species. Uptake kinetics in non-steady-state grown cultures were also inconsistent with most of the data collected from chemostats. Maximum uptake rates for PO^"3 were observed to be highest in cultures grown at intermediate nutrient supplj' rates, and rapid N H 4 + uptake was followed by a short-term shut-down in NH^"1" limited semi-continuous cultures. Changes in nutrient quality also affected competitive outcome. Different phytoplankton species dominated in cultures grown under different N:P supply ratios. As well, the nutrient uptake kinetics of phytoplankton were sensitive to the supply ratio under which they were grown; rapid rates of N H 4 + uptake occurred under more N limited conditions, and elevated PO4" 3 uptake rates were associated with cultures grown under more P-limited conditions. An index was developed based on the manner in which NH4 and P04"° uptake kinetics changed as a function of N:P supply ratio. This index provided evidence that N:P 158 supply ratios in some west coast lakes occur in the range where both N and P would be expected to limiting to phytoplankton growth. Consequently, community composition may be sensitive to changes in the N:P supply ratio in these lakes. Nutrient bioassay experiments confirmed that N and P were closely coupled as limiting nutrients in one of these lakes. Field studies were also conducted which examined N H 4 and P 0 4 uptake rates of size fractionated cells on several occasions. Larger cells were able to maintain elevated uptake q rates for longer than small cells when exposed to a pulse of P 0 4 . These experiments emphasized the importance of the < 3 um size fraction which was responsible for most of the N H 4 + and P 0 4 ' 3 uptake observed. The relative demand for N H 4 + and P0 4 " 3 by the two size fractions was not the same, implying that changes in the N:P supply ratio could affect the two size classes differently. It is apparent from these studies that patchy supplies of limiting resources, and alterations in N:P supply ratio affect the physiology of phytoplankton. As well, both of these processes affect community structure in laboratory experiments. The difficulty now lies in determining the significance of these processes in natural environments. The development of an index which can be used to estimate N:P supply ratios in situ is a first step towards elucidating the role of resource supply ratios in nature. 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