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Nitrogenous nutrition of phytoplankton from the Northeastern subarctic Pacific Ocean Varela, Diana Esther 1998

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NITROGENOUS NUTRITION OF PHYTOPLANKTON FROM THE NORTHEASTERN SUBARCTIC PACIFIC OCEAN by D I A N A E S T H E R V A R E L A "Licenciatura" (Hons.) in Oceanography, Universidad Nacional del Sur, Argentina, 1985 M . A . in Marine Biology, Boston University, U S A , 1991 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y in T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Department of Earth and Ocean Sciences) We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A December 1997 © Diana Esther Varela, 1997 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 rhy 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 € firth Unci Oc&lSl $tt&lce S The University of British Columbia Vancouver, Canada Date i e g ^ l W I DE-6 (2/88) ABSTRACT T h e goals o f this thesis were to investigate the uptake rates o f inorganic and organic forms of nitrogen, the relative proportions o f new and regenerated pr imary product ion, and the interactions between uptake o f a m m o n i u m and nitrate by phytoplankton f rom the N E subarctic Paci f ic Ocean. Ni t rogen uptake rates, and physica l , chemical and b io logica l characteristics o f the euphotic zone were studied dur ing winter, spring and late summer for two years a long a transect extending from 4 9 ° N , 1 2 7 ° W to 5 0 ° N , 1 4 5 ° W . N e w and regenerated pr imary product ion were estimated by means o f 1 S N isotopes. A m m o n i u m ( N H 4 + ) was usual ly taken up at the highest rates throughout the euphotic zone dur ing a l l seasons. U r e a uptake rates were lower than those o f N H 4 + , but higher values were occas ional ly observed, part icularly dur ing the spring o f 1993. Nitrate (N0 3 ~) uptake rates represented on average 2 1 % o f the depth integrated total nitrogen uptake, both longi tudinal ly and seasonally. T h e / - r a t i o was overestimated on average by 3 6 % when urea was excluded from the calculat ion. T h e order o f preference for nitrogen by the entire phytoplankton assemblage was N H 4 + > urea > N 0 3 \ T h i s system functioned ma in ly on regenerated nitrogen forms year round, despite the avai labi l i ty o f N 0 3 " i n most o f the region. S m a l l (< 2 pirn) and large (> 2 pim) phytoplankton took up N H 4 + at higher rates than urea and N 0 3 \ Dep th integrated/-ratios were 0.16 for smal l cel ls and 0.25 for large cells . The order o f preference for the nitrogen sources by smal l cells was N H 4 + > urea > N 0 3 " , and by larger cells it was N H 4 + = urea > N 0 3 \ P icoplankton were responsible for the greatest proport ion o f new and regenerated pr imary product ion. T h e presence o f N H 4 + inhibi ted N 0 3 " uptake i n the coccol i thophore, Emiliania huxleyi. Nitrate uptake rates were reduced to half the m a x i m u m value at 0.24 piM N H 4 + , and m a x i m u m inhib i t ion was « 100% at 2.2 N H 4 + . Extrapolat ing this laboratory result to f ie ld condit ions, the inhib i t ion o f N 0 3 " uptake rates for the smal l size class o f phytoplankton is predicted to be 37 to 6 9 % for the range o f ambient N H 4 + concentrations found i n the N E subarctic Pac i f ic . T A B L E OF CONTENTS Abst rac t ii Tab le of Contents iii L i s t of Tables vi L i s t of Figures viii Acknowledgmen t s xiv GENERAL INTRODUCTION 1 Nitrogen availability and metabolism in phytoplankton 1 Regenerated, new and export production 3 The importance of new production 6 The Northeastern Subarctic Pacif ic 8 General features of the physical oceanography 8 E c o l o g i c a l dynamics 11 Thesis goals and organization 12 CHAPTER 1: Seasonal variability of physical, chemical and biological characteristics of the euphotic zone in the N E subarctic Pacific Ocean 15 In t roduct ion 15 Mater ia ls and Methods 16 Phys ica l measurements 19 Chemical and biological measurements 19 Statistical analyses of chemical and biological data 21 Replication of water samples for chemical and biological analyses 21 Precision of analytical techniques 22 Resul ts 22 Phys ica l characteristics 22 Incident irradiance 22 Temperature, salinity and a, of the surface mixed layer 24 Chemical and biological characteristics 27 Dissolved nutrient concentrations 27 Chlorophyll a, and particulate nitrogen and carbon concentrations 37 Stat ion P26 39 Phytoplankton assemblages 44 D i s c u s s i o n 48 Nutrient distributions and sources 48 Nitrate, s i l ic ic acid and phosphate 48 Urea and ammonium 50 Chlorophyll a d is t r ibut ion 53 Phytoplankton assemblages 56 Summary 58 IV CHAPTER 2: Seasonal variability in nitrogenous nutrition of natural phytoplankton assemblages in the N E subarctic Pacific Ocean 59 Int roduct ion 59 Mater ia ls and Methods 60 Nit rogen tracer experiments 60 Measurement of atom % 1 S N and calculation of nitrogen uptake rates 62 Calculation of indices of nitrogen nutrition 64 Stat is t ical analyses 66 Replication of water samples for 1 S N uptake rate experiments 66 Precision of the 1 S N tracer technique 66 Resul ts 68 Depth profiles of nitrogen uptake rates a n d / -ratios 68 Depth integrated nitrogen uptake rates a n d / -ratios 75 Uptake rates of N 0 3 " , urea and N H 4 + 75 / -ratios, new and regenerated primary production 75 New and regenerated production at P26 80 Total nitrogen uptake rates 82 Nitrogen uptake rates and ambient nitrogen concentrations 84 Relat ive preference indices 84 / -ratios vs. nutrient ratios 84 N-specific uptake rates a n d / -ratios vs. nutrient concentrations 87 D i s c u s s i o n 90 New and regenerated production in the N E subarctic Pacific 90 Comparison with previous studies in the N E subarctic Pacific 92 Comparison of the N E subarctic Pacific with other H N L C regions 93 Comparison of the N E subarctic Pacific with other North Pacific ecosystems ... 94 Preference of regenerated nitrogen over N 0 3 " 95 Factors affecting nitrogen uptake in the N E subarctic Pacific 97 Limitations of the 1 S N tracer technique 101 Problems associated with sample confinement 101 Contribution of heterotrophic bacteria to nitrogen uptake 103 Summary 105 CHAPTER 3: Nitrogen uptake by size-fractionated phytoplankton assemblages from the oceanic N E subarctic Pacif ic Ocean 106 In t roduct ion 106 Mater ia ls and Methods 107 Chemical and biological measurements 107 Nit rogen uptake experiments 108 Resul ts 109 Init ial environmental conditions 109 Contribution of each size class to nitrogen uptake by the entire assemblage 109 Partitioning of the nitrogen forms used by each size class 113 Preference of nitrogen sources by each size class 117 D i s c u s s i o n 120 Summary 123 CHAPTER 4: Effect of ammonium on nitrate utilization by Emiliania huxleyi, a coccolithophore from the oceanic N E Pacific, grown on a light:dark cycle 124 In t roduct ion 124 Mater ia ls and Methods 127 Laboratory experiments 127 Cul ture condit ions 127 V Maintenance cultures 127 Experimental cultures 128 A n a l y t i c a l methods 130 M o d e l s used 131 F i e l d experiments 132 Resul ts 134 Labora tory experiments 134 Physiological characteristics for Emiliania huxleyi 134 Diel periodicity of N 0 3 " uptake rate and other physiological parameters for Emiliania huxleyi 134 Effect of increasing N H 4 + concentrations on N 0 3 " uptake rate in Emiliania huxleyi 137 F i e l d experiments 141 Effect of increasing N H 4 + concentrations on N0 3 ~ uptake rate by natural assemblages of phytoplankton from station P26 141 D i s c u s s i o n 143 Physiological characteristics for Emiliania huxleyi 143 Diel periodicity of N0 3 ~ uptake rate and other physiological parameters in Emiliania huxleyi 145 Effect of increasing NH 4 +concentrations on N 0 3 " uptake rate 147 E c o l o g i c a l implicat ions 152 Summary 154 G E N E R A L C O N C L U S I O N S 155 F U T U R E S T U D I E S 159 L I T E R A T U R E C I T E D 160 A P P E N D I C E S 182 A: Sampl ing stations 182 B: Precis ion of analytical techniques 183 C: Vertical profiles of temperature, salinity and a, 186 D: Incident surface irradiance during 1 S N experiments 190 E: 1 S N uptake experiments with pre-filtered and unfiltered water 192 F: Effect of irradiance on nitrogen uptake rate 195 v i LIST OF TABLES Table 1.1. Table 1.2. Table 1.3. Table 1.4. Table 2.1. Table 2.2. Cruise dates for C T D profiles and water sampling along Line P, and seasons the cruises represent. Station P26 is also known as Papa or P. During February 1994, station P26 could not be reached, thus P23A was the most offshore station sampled (see text and F ig . 1.1) 18 Mixed layer characteristics for all Line P stations and cruises. Temperature, salinity and ot for the mixed layer were calculated as the mean values from surface to the calculated mixed layer depth. See text for details on calculation of mixed layer depth. Values shown for P26 in February 1994 correspond to P23A. During September 1992 and May 1994, two C T D profiles were obtained for P26. Dashed line (-) indicates that data are not available 25 Depth integrated (100-1% IG) nutrient, chlorophyll a, and particulate nitrogen and carbon concentrations for all Line P stations and cruises. During February 1994, vertical profiles for those parameters were not measured at P20, thus integrated values could not be calculated, and values shown for P26 correspond to P23A. Seasonal means for winter and spring were obtained by averaging the two winter and the two spring cruises, respectively. Dashed line (-) indicates that data are not available 31 List of phytoplankton species identified from samples collected along Line P from September 1992 to M a y 1994 46 Natural variability in absolute, and N - and Chi a-specific uptake rates for nitrate, urea and ammonium. Replicate tracer experiments were performed on samples taken from different bottle casts obtained in a 1 to 4 day period mainly at P26 during every cruise. Errors are expressed as the mean coefficient of variation (C .V. , %) and mean standard deviation (S.D.) of groups (n) of replicates (2 to 7). Units for mean S.D. are: ng-at N L" 1 d"1 for p N , d"1 for V N , and /^g-at N (}4g Chi a)'1 d"1 for p N / C h l a 67 Depth integrated (100-1% I o) absolute uptake rates (mg-at N m"2 d"1) of nitrate, urea and ammonium, and depth integrated/-ratios for Line P stations during all cruises. Values shown for P26 in February 1994 correspond to P23A. Seasonal means for winter and spring were obtained by averaging the two winter and the two spring cruises, respectively. Dashed line (-) indicates that data are not available 77 Table 2.3. Depth integrated (100-1% I 0) N-specific (d"1) and Chi a-specific uptake rates (pig-at (pig Chi a)'1 d"1) for nitrate, urea and ammonium for Line P stations during all cruises. Values shown for P26 in February 1994 correspond to P23A. Seasonal means for winter and spring were obtained by averaging the two winter and the two spring cruises, respectively 78 Table 3.1. Depth profiles of the initial environmental conditions of seawater collected at P26 for size fractionated nitrogen uptake rate experiments. Particulate nitrogen and chlorophyll a for the > 2 yim size fraction were calculated as the difference between > 0.7 pirn and 0.7-2 pirn size fractions. Dashed line (-): no vii data are available 110 Table 3.2. Contribution of the 0.7-2 pim size fraction to the depth integrated N-specific and absolute uptake rates of nitrate, ammonium, urea and total nitrogen by the entire assemblage (> 0.7 pirn) 114 Table 3.3. Depth integrated/-ratios for the entire assemblage (> 0.7 pirn), and the 0.7-2 pirn and > 2 ftm size fractions for both experiments. During M a y 1994,/-ratios were calculated including and excluding urea 118 Table 4.1. Physiological parameters for Emiliania huxleyi cultures grown in modified E S A W with 30 ]AM nitrate under saturating irradiance on a 14:10 L : D cycle at 10.5°C 135 Table A . 1. Location and water depth of sampling stations in the N E subarctic Pacific 182 Table B . 1. Precision of the analytical techniques for dissolved nutrient, chlorophyll a, and particulate nitrogen and carbon concentrations employed during the field component of this thesis. Units for mean S.D. are: j4g L" 1 for Chi a and pig-at L _ 1 f o r a l l other parameters 184 Table B.2. Precision of the 1 S N tracer technique employed during the field component of this thesis. Errors are shown for absolute, and N - and Chi a-specific uptake rates. Units for mean S.D. are: ng-at N L" 1 d"1 for p N , d"1 for V N , and pig-at N (j*g Chi a)"1 d ' 1 for p ^ C h l a. 185 Table F. 1. Estimated parameters ( a n d K L T ) from the least squares Michaelis-Menten fit to the uptake vs. irradiance data for P16 during February 1994 and P26 during May 1994. Errors for the parameters are given as ± 1 S.E. (n = 6). K L T is given in % of surface incident irradiance 198 Vll l LIST OF FIGURES Figure 1. Diagram of the major fluxes for new ( ^ ^ ^ * ) and regenerated ( primary production in the oceans. The support for regenerated production is nitrogen recycled within the euphotic zone (mainly as N H 4 + and urea). New production is mainly based on the use of N 0 3 " supplied from below the euphotic zone. The export (= sedimentation) balances the upward fluxes of N 0 3 " . Other new nitrogen inputs are also possible from terrestrial runoff, atmospheric sources and horizontal advection 4 Figure 2. Diagram of the N E Subarctic Pacific Ocean indicating the extent of domains and current systems. The sampling transect Line P, as well as Station P (*), are shown. (Adapted from Dodimead etal., 1963 and Favorite etal., 1976) 10 Figure 1.1. Location of sampling stations along Line P in the N E subarctic Pacific Ocean. Station P26 is also known as Papa or P 17 Figure 1.2. Incident surface irradiance for (A) March 1993, (B) M a y 1993 and (C) M a y 1994. Irradiance data are not available for the other cruises. Data are represented from the day of arrival at P4 to the day of departure from P26. Stations on top of each figure are placed on day of arrival at that station 23 Figure 1.3. Mixed layer and 1% I o depths for Line P stations during (A) March 1993, (B) February 1994, (C) May 1993, (D) May 1994, (E) September 1992 and (F) September 1994. The © symbol represents estimated 1% I o depths (see text for details). The 1% ID depth was not available for P20 during February 1994. When two symbols for mixed layer or 1% IG depth appear at P26, they indicate separate measurements (derived from separate vertical profiles of a t or underwater irradiance) 26 Figure 1.4. Vertical profiles of nitrate, urea and ammonium concentrations for Line P stations during the winter cruises: (A) March 1993 and (B) February 1994. When urea concentration was below detection (< 0.05 /*g-at L 1 ) , a value of zero was used in the figures. During February 1994 at P20, only data at ca. 3 m were available (see text for details) 28 F i gure 1.5. Vertical profiles of nitrate, urea and ammonium concentrations for Line P stations during the spring cruises: (A) May 1993 and (B) M a y 1994. When urea or nitrate concentration was below detection (< 0.05 /<g-at L" 1 ) , a value of zero was used in the figures. During May 1993 at P26, nitrate and ammonium concentrations were measured on May 21, and urea on May 24. 29 Figure 1.6. Vertical profiles of nitrate, urea and ammonium concentrations for the late summer cruises, at (A) Line P stations during September 1992 and (B) station P26 during September 1994. When urea or nitrate concentration was below detection (< 0.05 /<g-at L" 1 ) , a value of zero was used in the figures 30 IX Figure 1.7. Contour plots of nitrate concentration along the Line P transect for (A) March 1993, (B) February 1994, (C) May 1993, (D) May 1994 and (E) September 1992. Solid lines indicate contour intervals of 1 pig-a.t L" 1 . Solid circles (•) indicate the sampling depths 32 Figure 1.8. Contour plots of silicic acid concentration along the Line P transect for (A) March 1993, (B) February 1994, (C) May 1993, (D) M a y 1994 and (E) September 1992. Solid lines indicate contour intervals of 2 ftg-at L " 1 ; dotted lines show additional contours. Solid circles (•) indicate the sampling depths 33 Figure 1.9. Contour plots of phosphate concentration along the Line P transect for (A) March 1993, (B) February 1994, (C) May 1993 and (D) M a y 1994. Solid lines indicate contour intervals of 0.1 /^g-at L" 1 . Solid circles (•) indicate the sampling depths 34 Figure 1.10. (A) Sil icic acid to nitrate, and (B) nitrate to phosphate ratios for all stations along Line P and all cruises. Each bar represents the mean of the three surface values (100, 55 and 30% of ID). Error bars represent ± 1 S.E. of the mean. A t station P20 during February 1994, bar represents the ratio of single values. N o S i (OH) 4 : N 0 3 " ratios are shown for P4 during September 1992 and May 1994, and for P12 during September 1992, since surface nitrate concentrations were undetectable 36 Figure 1.11. Contour plots of chlorophyll a concentration along the Line P transect for (A) March 1993, (B) February 1994, (C) May 1993, (D) M a y 1994 and (E) September 1992. Solid lines indicate contour intervals of 0.1 pig L " 1 ; dotted lines show additional contours. Solid circles (•) indicate the sampling depths 38 Figure 1.12. Contour plots of particulate nitrogen concentration along the Line P transect for (A) March 1993, (B) February 1994, (C) May 1993, (D) M a y 1994 and (E) September 1992. Solid lines indicate contour intervals of 0.2 pig-at L" 1 . Solid circles (•) indicate the sampling depths 40 Figure 1.13. Surface (0-10 m) concentrations of (A) nitrate, (B) phosphate, (C) silicic acid, (D) urea, and (E) ammonium at P26 for all cruises. Each bar represents the mean of replicates + 1 S.E. Horizontal lines of the same type join cruises that are not significantly different from each other. When lines overlap, it implies that the Tukey test was not able to detect differences at the P level considered. In all cases A N O V A s were tested at P = 0.05, and Tukey tests at (A) P = 0.05, (B) P = 0.05, (C) P = 0.05, (D) P = 0.20 and (E) P = 0.05 41 Figure 1.14. Surface (0-10 m) concentrations of (A) chlorophyll a, (B) particulate nitrogen and (C) particulate carbon at P26 for all cruises. Each bar represents the mean of replicates ± 1 S.E. Horizontal lines of the same type join cruises that are not significantly different from each other. When lines overlap, it implies that the Tukey test was not able to detect differences at the P level considered. In all cases A N O V A s were tested at P = 0.05, and Tukey tests at (A) P = 0.1, (B) P = 0.05 and (C) P = 0.1 42 Figure 1.15. Vertical profiles of diatoms and photosynthetic flagellates for Line P X stations during (A) March 1993, (B) February 1994, (C) May 1993, (D) M a y 1994, and (E) September 1992. The categories 'diatoms' and 'photo flagellates' include all species listed on the left and right hand side of Table 1.4, respectively. Only a few data points are available throughout the euphotic zone for stations P4 to P20 during February 1994. N o data are available for September 1994 45 Figure 1.16. Contribution of each phytoplankton group to the total integrated cell number for Line P stations during (A) March 1993, (B) February 1994, (C) M a y 1993, (D) M a y 1994 and (E) September 1992. During February 1994, integrations at P4 and P16 were done to the deepest light depth available, and only one depth was used for stations P12 artd P20 (see Fig. 1.15). Groups contributing < 1% to the total were not included 47 Figure 2.1. Vertical profiles of absolute uptake rates of nitrate, urea and ammonium for Line P stations during the winter cruises: (A) March 1993 and (B) February 1994. Rates of urea uptake at P4 during March 1993 are approximate values, since they were calculated from a single mixed layer value of urea concentration. Data are not available for the 1% I o depth at P23A during February 1994 69 Figure 2.2. Vertical profiles of absolute uptake rates of nitrate, urea and ammonium for Line P stations during the spring cruises: (A) May 1993 and (B) May 1994. During M a y 1993 at P26, nitrate and ammonium uptake rates were measured on 21/5, and urea uptake rates on 24/5. Note the different scale for P4 on M a y 1994 70 Figure 2.3. Vertical profiles of absolute uptake rates of nitrate, urea and ammonium for the late summer cruises for (A) Line P stations during September 1992 and (B) station P26 during September 1994 71 Figure 2.4. Contour plots showing absolute uptake rates of (A, B) nitrate, (C, D) urea, and (E, F) ammonium during March and May 1993. Solid lines indicate contour intervals of 40 ng-at N L" 1 d"1; dotted lines are intervals of 20 ng-at N L" 1 d"1. Solid circles (•) indicate sampling depths 72 Figure 2.5. Contour plots showing absolute uptake rates of (A, B) nitrate, (C, D) urea, and (E, F) ammonium during February and M a y 1994. Solid lines indicate contour intervals of 40 ng-at N L" 1 d 1 ; dotted lines are intervals of 20 ng-at N L" 1 d 1 . Solid circles (•) indicate sampling depths 73 Figure 2.6. Contour plots showing absolute uptake rates of (A) nitrate, (B) urea, and (C) ammonium during September 1992. Solid lines indicate contour intervals of 40 ng-at N L d"1; dotted lines are intervals of 20 ng-at N L" 1 d"1. Solid circles (•) indicate sampling depths 74 Figure 2.7. Vertical profiles of/-ratios for Line P stations during (A) March 1993, (B) February 1994, (C) M a y 1993, (D) May 1994, (E) September 1992, and (F) September 1994 76 Figure 2.8. Contribution of the depth integrated absolute uptake rate of different nitrogen sources to the total nitrogen absolute uptake rate along Line P for (A) March 1993, (B) February 1994, (C) May 1993, (D) M a y 1994, (E) September 1992 and (F) September 1994 79 XI Figure 2.9. Depth integrated/-ratios calculated as p N 0 3 " / (pN0 3 ~ + p N H 4 + ) and p N 0 3 / (pN0 3 ~ + purea + p N H 4 + ) for all Line P stations and cruises plotted against each other. The 1:1 line is shown for reference 81 Figure 2.10. Depth integrated (A) absolute uptake rates, and averages for (B) absolute and (C) N-specific uptake rates of total nitrogen for Line P stations during all cruises. Depth integrated averages (B and C) were calculated by dividing the depth integrated absolute and specific uptake rates by the depth of integration (i.e. depth of the euphotic zone) 83 Figure 2.11. Relative preferences indices (RPIs) for nitrate, urea and ammonium calculated at all depths, stations and cruises. Total nitrogen concentration includes tracer additions 85 Figure 2.12. /-ratio vs. nutrient ratio for all depths, stations and cruises. Nutrient ratio includes tracer additions. (A) Ammonium and urea, (B) only ammonium, and (C) only urea are included in the calculation. Open symbols represent data from 100, 55 and 30% I 0 , and solid symbols represent data from 10, 3.5 and 1% I o . Sol id line is the least squares fit to the data 86 Figure 2.13. N-specific nitrate and urea uptake rates vs. ambient ammonium concentration for all depths, stations and cruises. Open symbols represent data from 100, 55 and 3% IG, and solid symbols represents data from 10, 3 . 5 a n d l % I o 88 Figure 2.14. /-ratio vs. ambient nitrate concentration for all depths, stations and cruises. Nitrate concentration includes tracer additions. Open symbols represent data from 100, 55 and 30% I o , and solid symbols represent data from 10, 3.5 and 1% I o . The exponential model (Piatt and Harrison, 1985, and Harrison etal., 1987) applied to the data as well as the fitted parameters are i nc luded 89 Figure 3.1. Depth profiles of N-specific uptake rates of (A) nitrate and (C) ammonium, and absolute uptake rates of (B) nitrate and (D) ammonium for the entire assemblage of phytoplankton (> 0.7 pim) and for the 0.7-2 pirn and > 2 pirn size fractions at P26 during September 1992 I l l Figure 3.2. Depth profiles of N-specific uptake rates of (A) nitrate, (C) ammonium, and (E) urea, and absolute uptake rates of (B) nitrate, (D) ammonium, and (F) urea for the entire assemblage of phytoplankton (> 0.7 yim) and for the 0.7-2 yim and > 2 pirn size fractions at P26 during M a y 1994 112 Figure 3.3. Depth integrated (A) N-specific and (C) absolute uptake rates of nitrate and ammonium for September 1992, and (B) N-specific and (D) absolute uptake rates of nitrate, ammonium and urea for May 1994 for the 0.7-2 pim and > 2 yim size fractions. The numbers on top of the bars indicate the percent contribution of that nitrogen source to the total nitrogen requirements for each size fraction 115 Figure 3.4. Depth profiles of/-ratios for (A) September 1992 (no urea), and (B) May 1994 excluding urea and (C) May 1994 including urea for the entire assemblage of phytoplankton (> 0.7 yim), and for the 0.7-2 yim and > 2 pim size fractions 116 X l l Figure 3.5. Nitrate, ammonium and urea relative preference indices (RPIs) for the (A) 0.7-2 pirn and (C) > 2 pirn size fractions for September 1992, and (B) 0.7-2 pirn and (D) > 2 pcm size fractions for May 1994. RPIs were calculated without urea ('no urea') for September 1992, and with ('all') and without urea ('no urea') during May 1994. See text for details on RPI calculation. . 119 Figure 4.1. Changes in (A) nitrate concentration and (B) nitrate uptake rate (calculated for each 3 h period, starting at 6 h; see text for details), for Emiliania huxleyi cultures during the 14:10 L : D experiment. The gray area (14 to 24 h) indicates the dark period. Each symbol represents the mean of triplicate cultures ± 1 S.E. If no error bars are visible, they are smaller than the s y m b o l 136 Figure 4.2. Changes in (A) culture density, (B) culture fluorescence, and (C) fluorescence per cell for Emiliania huxleyi during the 14:10 L : D experiment. The gray area (14 to 24 h) indicates the dark period. Each symbol represents the average of triplicate cultures ± 1 S.E. If no error bars are visible, they are smaller than the symbol 138 Figure 4.3. Changes in (A) nitrogen quota, (B) total carbon quota (including coccoliths), (C) chlorophyll a quota, and (D) cell volume for Emiliania huxleyi during a 14:10 L : D cycle. The gray area (14 to 24 h) indicates the dark period. Each symbol represents the average of triplicate cultures ± 1 S.E. If no error bars are visible, they are smaller than the symbol 139 Figure 4.4. (A) Nitrate uptake rate and (B) relative nitrate uptake rate at increasing ammonium concentrations for cultures of Emiliania huxleyi grown in a 14:10 L : D cycle. Each symbol represents a determination from a single culture (n = 56). The models applied to the data as well as the fitted parameters (+ S.E.) are included 140 Figure 4.5. N-specific nitrate uptake rate vs. ambient ammonium concentration for the natural assemblage of phytoplankton in surface waters at station P26 in the N E Pacific. Each symbol represents a single determination (n = 40). Fil led symbols ( • ) are for the N H 4 + addition experiment (+ N H 4 + ) done at P26 during May 1994. The models applied to the data as well as the fitted parameters (+ S.E.) are included 142 Figure 4.6. Percent inhibition of nitrate uptake rate at increasing ammonium concentrations for cultures of Emiliania huxleyi grown in a 14:10 lightdark cycle. Same data as used in Figure 4.4 but transformed to percentages. Each symbol represents a determination from a single culture (n = 56). The three models used to fit the data are also included. Arrows point to minimum and maximum surface ammonium concentrations measured at station P26 in the N E Pacific during all cruises 153 Figure C. 1. Vertical profiles of ot, temperature and salinity for the upper 200 m at Line P stations for the winter cruises: (A) March 1993 and (B) February 1994. .. 187 Figure C.2. Vertical profiles of a t , temperature and salinity for the upper 200 m at Line P stations for the spring cruises: (A) May 1993 and (B) May 1994 188 Figure C.3. Vertical profiles a t , temperature and salinity for the upper 200 m for the late xiii summer cruises, at (A) Line P stations for September 1992 and (B) P26 for September 1994 189 Figure D1. Incident surface irradiance during 1 S N incubation experiments on Line P during (A) March 1993, (B) May 1993 and (C) M a y 1994. The date on the top left corner of each figure denotes the start day. Vertical dotted line symbolizes midnight. A t P26 during May 1993, the irradiance curve on 21/5 corresponds to 1 5N-nitrate and ammonium incubations, and the irradiance curve on 24/5 corresponds to the 1 5 N-urea incubation experiment. A t P26 during May 1994, irradiance curve on 19/5 corresponds to the experiment presented in Chapter 2, and the irradiance curve on 21/5 corresponds to the incubation experiment presented in Chapter 3 191 Figure E . 1. Vertical profiles of absolute uptake rates of l sN-nitrate and 1 5N-ammonium measured on pre-filtered (116 pirn) and unfiltered water at station P26 during September 1992 194 Figure F. 1. N-specific uptake rate of nitrate, urea and ammonium as a function of % I 0 for (A) P16 during February 1994 and (B) P26 during M a y 1994. The least squares Michaelis-Menten fit to the data is shown in dotted lines 197 xiv ACKNOWLEDGMENTS I would like to extend my gratitude to the many people who have, in one way or another, helped me in this research. M y deepest appreciation goes to my research supervisor, Dr. Paul J. Harrison. He gave me all the opportunities to excel in research and teaching, and has been a kind and supportive supervisor (morally and financially). I have always been impressed by his good humor and patience. I particularly appreciate that he made me feel more like a colleague than a graduate student. It has been a great pleasure and honor to work with him. Everyone in my supervisory committee (Drs. Timothy Parsons, Stephen Calvert and Chi-Shen Wong) has been helpful and always approachable. I am also thankful to the additional members of my examining committee (Drs. Robert Guy, Krist in Orians and Wil l iam Neill) and my external examiner (Dr. Yves Collos) for challenging questions and insightful comments. Other faculty members of Oceanography (Drs. A l a n Lewis, F.J.R. Taylor) and Botany (Dr. Tony Glass) have also been at hand for the odd question or discussion throughout my Ph.D. I am grateful to Dr. Alan Lewis for encouraging me into teaching. Dr. Wil l iam Cochlan taught me the nitrogen-15 technique and kindly answered my many 'urgent' email messages. Drs. Catriona Hurd and Kedong Y i n showed me the tricks of our scary-looking nutrient autoanalyser. Dr. Deborah Muggl i was a great colleague and friend with whom I spent many days under the rain, 'enjoying' the outdoors. She isolated the phytoplankton species that I used in my research. Dr. John Berges was inspirational; his dedication and love for science and his kind nature made him a great colleague. David Jones saved me from many frustrating moments with the emission spectrometer. I would like to thank Frank Whitney for being an incredibly patient and accommodating chief scientist; he also ran the nutrient autoanalyser in the Line P cruises, and gave me some helpful tips on the early and final stages of my thesis. Many thanks are extended to all the scientists that participated in the cruises, and to all the officers and crew members of the C.S.S. "John P. Tul ly" . There was never a shortage of U B C people who provided support during these cruises;' thanks are extended to Brad McKelvey , Mark Wen, Sean Doherty, Steven Ruskey, Jim Powlik, Sarah Thornton, Dr. Philip Boyd, and particularly to Hugh McLean who was always kind and positive even in the roughest seas. Maureen Soon gladly analysed hundreds of C N S samples, and Jeanette Ramirez did the phytoplankton identification and enumeration. I am grateful to Dr. Dave Crawford for his kind disposition in reading every line of my thesis and providing excellent revisions. I would also like to extend my thanks to Tony Larson who provided insightful comments in my lab experiment. I enormously appreciate the help provided by Shannon Harris during this last year. She has been a kind friend and colleague, who encouraged and supported me through the last stages of my thesis. Dr. Philip Boyd had a noticeable influence on my career. Shawn Chen, Anne Fisher, Maude Lecourt, Robert Goldblatt, Linda Greenway, Mike St. John, Maggie Kasekende, M i n g Guo, Al len Mil l igan, Anthony Fielding, Nelson Sherry, Joe Arva i , Joe Needoba, Beth Borahold, Bente Nielsen and all my office mates have been helpful or supportive in many ways. I express my appreciation to all the office staff of Oceanography (especially Carol Leven and Chris Mewis). People in the Botany workshop have been of enormous help when faced with seemingly impossible deadlines. M y many thanks go to everyone in P.J. Harrison's lab and in the ex-Oceanography Department. Financial support was provided by U B C University Graduate and Chevron Fellowships, by the Natural Sciences and Engineering Research Council (through the Canadian JGOFS Project) and by the Department of Fisheries and Oceans of Canada. I would like to express my deepest gratitude to my parents, E lv i a and Pedro, who have been my strongest supporters at every stage of my life, and they must share my success. I extend my warmest thanks to my brother and grandmother. I cannot adequately express my gratitude to my husband, Kerry, whose support, love and companionship have been invaluable all along. He taught me that i f I can climb a mountain, I can get through my thesis. It gives me the greatest pleasure to dedicate this thesis to my family. -1 GENERAL INTRODUCTION Nitrogen availability and metabolism in phytoplankton Nitrogen plays an important role in primary productivity since it is one of the most abundant elements (after carbon, hydrogen and oxygen) in the organic matter of algal cells, and hence, an essential component of live phytoplankton (Syrett, 1981, 1988). Nitrogen has been frequently cited as the nutrient that limits phytoplankton growth in marine environments (e.g. Ryther and Dunstan, 1971; McCarthy and Carpenter, 1983; Codispoti, 1989) because it is generally found at very low concentrations in surface waters of vast regions of the open ocean (McCarthy, 1980; Sharp, 1983). However, it is not universally accepted that nitrogen is the single most limiting nutrient of primary productivity in the oceans (e.g. Hecky and Ki lham, 1988; Howarth, 1988). There is evidence that, among the macronutrients, phosphorus can also limit oceanic productivity in certain coastal regions (e.g. Smith, 1984; Harrison etal., 1990; Fisher et al., 1992). Sil icon limitation also occurs in the oceans (e.g. Nelson and Brzezinski, 1990; Nelson and Treguer, 1992, Dugdale etal, 1995), but this element is only required by organisms with siliceous frustules such as diatoms, and thus, silicon limitation should have a greater effect on changing the species composition of a region than on primary productivity. Furthermore, among the micronutrients, iron and other metals may at times limit phytoplankton productivity in oceanic waters (e.g. Morel etal, 1991; Martin, 1992). Although several different nutrients may be low enough to limit phytoplankton productivity or alter ecosystem structure at times, nitrogen is still recognized as the major controlling factor. Phytoplankton cells utilize dissolved inorganic nitrogen (DIN) mainly as nitrate (N0 3 "), nitrite ( N 0 2 ) and ammonium ( N H 4 + ) , and dissolved organic nitrogen (DON) mainly as urea and some free amino acids (see reviews by McCarthy, 1980; Syrett, 1981; Paul, 1983; Wheeler, 1983; Antia etal., 1991). There is also evidence that other D O N compounds can be taken up by 2 some microalgae, such as combined amino acids, amino sugars, purines and pyrimidines (Paul, 1983; Antia etal., 1991). Dissolved dinitrogen gas (N 2 ) is the most abundant nitrogen form in the oceans (Scranton, 1983), however, autotrophic utilization of N 2 is limited to cyanobacteriaand the rate of N 2 fixation seems to be generally low in the pelagic environment (Howarth etal., 1988), although temporally important in some areas (e.g. Kar l etal., 1992, 1997). Nevertheless, the contribution of biological fixation of N 2 to the total nitrogen inputs to the surface waters of oligotrophic oceans requires further assessment (e.g. Carpenter, 1983; Kar l etal., 1992, 1997). A t present, of all the available nitrogen forms, N0 3 ~, N H 4 + , and urea are considered the main sources for phytoplankton nitrogenous nutrition. The metabolism of nitrogen in phytoplankton comprises four basic steps, as outlined by Wheeler (1983): (a) uptake or transport of a particular form of nitrogen across the cell membrane, (b) assimilation into small organic metabolites (amino acids), (c) incorporation into macromolecules (proteins), and (d) catabolism or breakdown of macromolecules into small metabolites. Although uptake strictly refers to transport across the membrane, the word "uptake" has been generally used to imply a more general process that also includes assimilation and incorporation. The early study of Dugdale (1967) proposed that the rate of nutrient uptake was related to nutrient availability by a rectangular hyperbola of the Michaelis-Menten type. Dugdale's (1967) model has been confirmed in numerous laboratory and field studies of nitrogen dynamics. The shape of the relationship between nitrogen uptake rate and availability is indicative of carrier-mediated transport of nitrogen through the cellular membrane (i.e. active transport; see reviews by Collos and Slawyk, 1980; McCarthy, 1981; Falkowski, 1983; Wheeler, 1983; Syrett, 1988). Once inside the cell, N 0 3 " is reduced to N 0 2 " and then to N H 4 + , by N 0 3 " reductase and N 0 2 " reductase, respectively, while urease is involved in the conversion of urea to N H 4 + in most algae (McCarthy, 1980; Syrett, 1981, 1988; Falkowski, 1983; Wheeler, 1983). Intracellular N H 4 + (derived from N 0 3 " reduction or urea breakdown, or taken up directly from outside the cell) is then assimilated into amino acids by one of two pathways: glutamic dehydrogenase (GDH) or glutamine synthetase (GS)/glutamate synthetase ( G O G A T ) (Collos and Slawyk, 1980; McCarthy, 3 1980; Syrett, 1981, 1988; Falkowski, 1983; Wheeler, 1983). Many studies suggest that N H 4 + assimilation is predominately via the G S / G O G A T pathway (Falkowski, 1983; Syrett, 1988) and the first product of assimilation is glutamine. The next series of steps are between amino acids and proteins and represent cellular growth (Collos and Slawyk, 1980; Goldman and Glibert, 1983). The rate of growth, however, is not always coupled with the rate of uptake, and thus these two rates are sometimes quite different (Collos and Slawyk, 1980; McCarthy, 1981). Many other physiological processes can occur at different stages of nitrogen metabolism. For example, the suppression of N 0 3 " transport and/or assimilation by N H 4 + has been well documented (see reviews by McCarthy, 1981; Dortch, 1990). Regenerated, new and export production In an ideal closed euphotic zone, all the nutrients required by phytoplankton would be provided by remineralization of the in situ produced organic matter. In this way, the closely coupled system of remineralization-assimilation could indefinitely maintain itself. In the real ocean, however, this cycle has losses (e.g. sinking of organic matter, grazing and feeding by transient heterotrophs) and the system would run down if those losses were not balanced by inputs of nutrients from outside the euphotic zone. Phytoplankton production resulting from nitrogen forms that are recycled within the euphotic zone (mainly N H 4 + and urea) and from nitrogen forms that enter the system from external sources (mainly N0 3 ") are referred to as "regenerated" and "new" primary production, respectively (Dugdale and Goering, 1967; Fig . 1). Other sources of new nitrogen can also be regionally and temporally important, such as terrestrial runoff, atmospheric inputs, and horizontal advection (Eppley and Peterson, 1979). However, in most of the open ocean, new nitrogen is assumed to come mainly from upwelling or upward diffusion from deeper waters (Lewis etal., 1986; Piatt etal., 1989). In a steady-state ocean, the upward flux of N 0 3 " , which fuels new production, would be balanced by the vertical flux of particulate organic nitrogen out of the euphotic zone (i.e. "export production", Eppley and Peterson, 1979; 4 other N Figure 1. Diagram of the major fluxes for new ("^••) and regenerated ( — primary production in the oceans. The support for regenerated production is nitrogen recycled within the euphotic zone (mainly as N H 4 4 " and urea). New production is mainly based on the use of N O 3 - supplied from below the euphotic zone. The export (= sedimentation) balances the upward fluxes of N 0 3 \ Other new nitrogen inputs are also possible from terrestrial runoff, atmospheric sources and horizontal advection. 5 Berger etal., 1989; Fig . 1) over significant spatio-temporal scales. However, there remains some controversy over the magnitudes of these two fluxes. Export production is of great importance for the biogeochemical cycles since it represents "losses" of nutrients towards regions far removed from the surface ocean, and it is the avenue for nutrient sequestration into deep waters or burial in sediments. "Biological carbon pumps" are responsible for such fluxes of carbon and other nutrients from the atmosphere into seawater, and then into organic and calcium carbonate particles, followed by export out of the euphotic zone (Volk and Hoffert, 1985; Longhurst and Harrison, 1989). Thus, "biological carbon pumps" have a major role in determining the distribution of carbon and other nutrients in the oceans (e.g. Sarmiento and Siegenthaler, 1992). However, there is no consensus on the role of the biology of surface waters in ameliorating the increase in atmospheric carbon dioxide ( C 0 2 ) due to anthropogenic inputs (e.g. Banse, 1991a, Broecker, 1991, Longhurst, 1991; Ritschard, 1992). It has been suggested, for example, that only large changes in the rates of uptake of C 0 2 by marine photosynthetic organisms wil l have a direct effect on anthropogenic C 0 2 , but these changes are unlikely (Sarmiento and Siegenthaler, 1992). However, there still are many uncertainties about the mechanisms of the "biological carbon pump" which need to be studied to obtain a better understanding of its role in the global carbon cycle. New production (and, hence, the export flux) has been difficult to estimate mostly because of limitations of the methodology and failure to consider other possible fluxes (besides the upward flux of N<D3" and downward flux of particles). Methods used to estimate new production include in vitro techniques (e.g. 1 S N 0 3 " assimilation by planktonic assemblages) and measurements of bulk properties (e.g. sedimentation rate of particles, consumption of oxygen below the euphotic zone, net oxygen accumulation in the euphotic zone, upward N 0 3 " flux, winter accumulation of N 0 3 " above the seasonal thermocline) (Eppley, 1989; Piatt etal, 1989). Each method has a restricted time scale over which the results are valid (Piatt and Harrison, 1985; Piatt etal, 1989). For example, while 1 5 N 0 3 " assimilation represents an "instantaneous" estimate (hours to a day), sedimentation rate below the euphotic zone is integrated over the time the sediment trap is deployed (Piatt etal, 1989). Care must be exercised when comparing rates estimated with different 6 methods; however, these difficulties do not invalidate any particular method as long as the appropriate time scales are taken into account. On the other hand, different methods may provide complementary insights. If in vitro techniques are performed frequently (e.g. seasonally), their results wi l l approximate more closely the fluxes measured by sediment traps. Inaccuracy in the estimation of, or even failure to consider, other fluxes can also account for inequities between new and export production. These fluxes include: (a) accumulation and export of dissolved organic matter produced in the euphotic zone (Legendre and Gosselin, 1989; Toggweiler, 1989; Carlson etal., 1994; Ducklow etal., 1995); (b) input of riverine and atmospheric sources of new nitrogen (e.g. N 0 3 " , N 2 , N H 3 , N H 4 + ) (e.g. Legendre and Gosselin, 1989; Piatt etal., 1989); (c) denitrification in the water column and sediments (Hattori, 1983; Codispoti, 1989); (d) nitrification at the bottom of the euphotic zone (Kaplan, 1983; Ward, 1987; Ward etal., 1989; Eppley etal., 1990); (e) vertical flux of D I N from the surface to deeper waters (or vice versa) by excretion due to diel migration of interzonal biota (Longhurst and Harrison, 1988; Longhurst etal., 1989, Longhurst, 1991); (0 vertical flux of carbon by consumption at the surface and respiration at depth (or vice versa) by diel migrant biota (Longhurst etal., 1990, Longhurst, 1991), and (g) vertical flux of carbon by consumption at the surface and respiration outside the ocean by birds and mammals (e.g. in the Antarctic ecosystem; Huntley etal., 1991). Reconsideration of these diverse fluxes is necessary to obtain a proper global budget of nitrogen and carbon in the oceans. The importance of new production Aside from providing an estimate of export production, new production is a very useful flux to quantify. The "/-ratio" is the fraction of total primary production due to new production (i.e. /-ratio = new nitrogen uptake / new nitrogen uptake + remineralized nitrogen uptake; Eppley and Peterson, 1979), and can be used as an indicator of the composition of the planktonic food 7 web, the physical stability of the upper water column, and the efficiency of the ecosystem function (e.g. Piatt etal., 1992; Legendre and Le Fevre, 1989, 1991). Abundant new production (i.e. a high export rate) is characteristic of ecosystems dominated by large autotrophic cells, while the reverse is true in the case when the standing stock is mainly comprised of small phytoplankton (Legendre and Le Fevre, 1989, 1991). Legendre and Le Fevre (1989, 1991) suggested that intermediate conditions between these extremes are also possible. For example, large cells may be grazed upon within the euphotic zone, and then exported, or even remain cycling within the surface waters. Small cells may also be aggregated by physical or biological processes which increases the likelihood of export over recycling (Goldman, 1988; Legendre and L e Fevre, 1989, 1991). Legendre and Le Fevre (1989, 1991) stressed the importance of hydrodynamical characteristics in determining the dominance of new over regenerative systems. In cases when the annual physical forcing is weak (i.e. high stability of the upper water column with low vertical flux of N0 3 ") , the structure of the ecosystem wi l l tend to favor a microbial food web, with low rates of new production and export. This is generally the case in oligotrophic oceans. However, these systems could also have episodic intrusions of N0 3 ~ resulting from physical disturbances (i.e. mixing events; e.g. Piatt and Harrison, 1985; Piatt etal., 1989; Falkowski etal., 1991), which may temporarily stimulate new production by large cells mainly at the base of the euphotic zone (e.g. Goldman, 1988). A t the other extreme are upwelling systems and regions with pronounced spring blooms where large cells dominate the phytoplankton throughout the euphotic zone and in which new production and export fluxes are high (Legendre and Le Fevre, 1989, 1991; Peinert et al., 1989). In all these regions, vertical fluxes of N 0 3 " are assumed to be driving the systems to one extreme or another. However, other factors are also important and may produce deviations from the "classic" views. For instance, the type and response of heterotrophs present in the euphotic zone are also critical in determining the likelihood of export over euphotic zone recycling (Peinert etal., 1989). Although /-ratios do not provide information on the pelagic processes per se, they are somewhat indicative of the trophic structure of the ecosystem. New production and 8 the /-ratios can also predict the efficiency of the pelagic ecosystem in supporting higher trophic levels of production in the pelagic and benthic ecosystems (Piatt etal., 1992). Factors that limit new production in the oceans wi l l impact upon the magnitude of export production that wi l l be available for consumption by higher trophic levels, sequestration in deep waters, or burial in sediments. New production in oligotrophic oceans may be limited by the availability of dissolved N 0 3 . However, new production in some other areas (high-nutrient, low-chlorophyll; H N L C regions) is low despite the persistence of high concentrations of N0 3 ~. H N L C regions are found in the equatorial Pacific, the Antarctic and the subarctic Pacific. In these three areas, the limitations of new production are still not well understood (Chisholm and Morel , 1991). Some of the factors limiting phytoplankton biomass and productivity in H N L C regions are common to all regions, but others may be specific to each area. Common limiting factors may include: dissolved iron, zooplankton grazing, and N H 4 + inhibition of N 0 3 " utilization, or some combination of these (see Cullen, 1991; Dugdale and Wilkerson, 1991, 1992). However, temperature and deep vertical mixing may be exclusive to the Antarctic region (Dugdale and Wilkerson, 1991, 1992). The following is a brief description of the case in the N E subarctic Pacific Ocean. The Northeastern Subarctic Pacific General features of the physical oceanography The Subarctic region of the North Pacific is separated from the Subtropical region by the Subarctic Boundary, at about 40°N, which nearly coincides with the area where annual mean precipitation equals evaporation (Dodimead etal, 1963; Tabata, 1976). The Subarctic Boundary is defined by the vertical 34 isohaline (Dodimead etal, 1963). The Subarctic Region has relatively cool water and low salinity (due to an excess in precipitation relative to evaporation; Tabata, 1976). The water column is characterized by a permanent halocline between 100-200 m, below which salinity increases only very gradually with depth (Dodimead etal., 1963; Tabata, 9 1976). During the summer, a secondary halocline develops between 30-50 m, which coincides with a seasonal thermocline that results from intensive warming (Dodimead etal., 1963; Tabata, 1976). In the Subarctic region, it is salinity, rather than temperature, that determines the vertical stability of the upper 500 m (Tabata, 1976). The surface circulation of the N E subarctic Pacific is dominated by the Subarctic Current which flows eastward (Fig. 2), originating in the N W Pacific from the mixing of the Kuroshio and Oyashio currents (Dodimead et al., 1963; Tabata, 1975). The Subarctic Current divides into two streams about 500 km offshore at 45-50°N: the north-flowing Alaska Current which forms a cyclonic gyre around the Gulf of Alaska, and the south-flowing California Current (Dodimead et al., 1963; Tabata, 1975; Fig. 2). The entire N E Subarctic region experiences a weak wind-forced upwelling, with the higher upward vertical velocities (on the order of 3 m yr"1) in the centre of the Gulf of Alaska (Talley, 1985; Gargett, 1991). The Ridge Domain is surrounded by the Alaska Current (Fig. 2), and is characterized by the upwelling of cold, saline, nutrient-rich, but oxygen-poor, deep water into the dilute surface layer of the region (Favorite etal, 1976). Upwelling also occurs during late spring, summer and early fall along the coasts of California, Oregon, and Washington ( U S A ) , and British Columbia (Canada) due to a seasonal reversal of the coastal flow. This area is known as the Upwelling Domain (Fig. 2), and is characterized by lower temperatures and higher salinities than in the offshore areas during the summer (Favorite etal, 1976). Other areas with consistent physical properties in the N E Subarctic Region are the Dilute and Transition Domains (Fig. 2). The Dilute Domain is located at the divergence of the eastward flowing Subarctic Current (Favorite etal, 1976), and is the result of high coastal freshwater discharge due to very high annual rates of precipitation over the continent (Favorite et al, 1976; Royer, 1982; Freeland etal, in press). Finally, the Transition Domain lies just northward of the Subarctic Boundary, and is characterized by marked north-south gradients of temperature and salinity. 10 Figure 2. Diagram of the NE Subarctic Pacific Ocean indicating the extent of domains and current systems. The sampling transect Line P, as well as Station P (*), are shown. (Adapted from Dodimead etal., 1963 and Favorite et al, 1976). 11 Ecological dynamics The biological oceanography of the oceanic region of the N E subarctic Pacific has been extensively studied since the 1950s, mainly at the site of the Canadian weathership station (P, Papa, or P26) at 50°N, 145°W (Fig. 2). Early results showed low levels of primary productivity and phytoplankton biomass (McAllister etal., 1960) despite high concentrations of N 0 3 " , silicic acid (Si(OFI) 4) and phosphate (HP0 4 2~) (McAllister etal., 1960; Anderson et al., 1969) and strong seasonal stratification (Dodimead et al., 1963, Favorite etal., 1976; Tabata, 1976). McAll is ter et al. (1960) concluded that the dominant small phytoplankton (< 10 pirn, in their study) were kept in check by grazing activity. More recent work during the 1980s by the S U P E R (Subarctic Pacific Ecosystem Research) program confirmed the suggestion that phytoplankton stocks were under grazer control (Miller and S U P E R group, 1988; Mi l le r etal., 1991a,b; Mi l le r , 1993). However, they rejected the hypothesis that copepods were responsible for such control, since their grazing capacity on phytoplankton during the spring was insufficient to control algal growth (Miller and S U P E R group, 1988). The S U P E R group concluded that microzooplankton were the main grazers maintaining a tight control on the dominant phytoplankton (< 2-5 pm; Mil ler and S U P E R group, 1988; Mi l le r etal., 1991a,b; Mil ler , 1993). They then postulated the " M i x i n g and Micrograzer Hypothesis" in which grazing by microzooplankton controlled the phytoplankton stocks throughout the year due to the lack of deep vertical mixing which would have reduced phytoplankton and microzooplankton numbers to a minimum (as seen in the North Atlantic; see Parsons and L a l l i , 1988). These microheterotrophs were also providing recycled nutrients for phytoplankton growth (Miller etal., 1991a,b; Mi l le r , 1993). Furthermore, it was suggested that N H 4 + recycling was limiting N0 3 ~ utilization (and, hence, new production) in this region (Wheeler and Kokkinakis , 1990). However, the N H 4 + inhibition of N 0 3 " uptake rates by phytoplankton required further experimental confirmation. The S U P E R group finally agreed with earlier V E R T E X (Vertical Transport and Exchange) studies (Martin and Fitzwater, 1988; Martin etal., 1989), that iron was limiting the growth of the large phytoplankton in the oceanic subarctic Pacific (Miller etal., 1991a,b, 1993). A t the end of the S U P E R program, the "Iron Hypothesis still 12 needed further investigation, and their results on the nitrogen dynamics of the subarctic Pacific opened new questions. In the 1990s, the Canadian J G O F S (Joint Global Ocean Flux Studies) set out to extend our understanding of the pelagic ecosystem processes in the N E subarctic Pacific. It was shown that iron additions increased the biomass, growth and N 0 3 " uptake of the generally rare diatoms, while there was little response to the iron additions by the small cells (Boyd etal., 1996). These experiments confirmed that iron limitation was responsible for the predominant presence of small cells, while grazing controlled their abundance. When the J G O F S studies started, the nitrogen dynamics of the N E subarctic Pacific were not well understood. The importance of urea as a nitrogen source was not known on a longitudinal or seasonal basis, and the effects of ambient N H 4 + on N 0 3 " utilization by phytoplankton had not been experimentally tested in this region. Thesis goals and organization The goals of this thesis were to evaluate the rates of uptake of different nitrogen sources, and the nitrogen uptake interactions between N H 4 + and N 0 3 " by phytoplankton from the N E subarctic Pacific Ocean. Three specific questions were addressed by this research: 1) What are the interannual, seasonal and longitudinal changes in the relative proportions of new and regenerated production by natural assemblages of phytoplankton from the N E subarctic Pacific? 2) Are the predominant small cells (< 2 ^m) responsible for most of the nitrogen uptake in this region? 3) Are the ambient N H 4 + concentrations reducing the uptake rate of N 0 3 " by a phytoplankton species representative of the most abundant size-class in the oceanic N E subarctic Pacific? 13 Field experiments were performed to solve the first two questions, while a laboratory experiment was the approach for answering the third question. Field studies were conducted as part of the Canadian J G O F S Project aboard the C.S.S. "John P. Tul ly" . Cruises to the N E subarctic Pacific were undertaken seasonally over a period of two years, from September 1992 to September 1994, along a transect (Line P) extending from the continental slope off the southwest corner of Vancouver Island (British Columbia) to open waters in the N E Pacific (Station P at 50°N, 145°W; Fig . 2). This thesis comprises four chapters, general conclusions and a series of appendices. Chapter 1 provides a general description of the physical, chemical and biological characteristics of the euphotic zone along Line P from winter to late summer during the two years. The goal was to characterize the study area in terms of water temperature, salinity and density, dissolved nutrient ( N 0 3 \ urea, N H 4 + , S i (OH) 4 , and HP0 4 2 " ) concentrations, phytoplankton biomass (chlorophyll a), particulate nitrogen and carbon concentrations, and phytoplankton species composition. Chapter 2 presents the nitrogen uptake rate experiments conducted along the transect for the different seasons during the two years. The main goals were: (1) to determine interannual, seasonal, longitudinal, and vertical variability in the rates of N 0 3 \ urea and N H 4 + uptake, (2) to estimate the rates of new and regenerated production, and (3) to evaluate the nitrogen preferences for phytoplankton from the N E subarctic Pacific. In Chapter 3, experiments on the size-fractionation of nitrogen uptake are shown for the oceanic end of the transect during two cruises. The primary goals were: (1) to determine the fraction of N 0 3 " , urea and N H 4 + uptake rate by intact phytoplankton assemblages due to phytoplankton < 2 and > 2 pirn, and (2) to evaluate the nitrogen preferences by each size-class. Chapter 4 presents the results of laboratory experiments on the effects of N H 4 + on N 0 3 " utilization by the coccolithophore, Emiliania huxleyi, isolated from the N E subarctic Pacific. The main goal was to determine the response of the uptake rate of N 0 3 " to N H 4 + concentrations in E. huxleyi under carefully controlled conditions that closely simulated the original oceanic 14 environment for the summer period. A n attempt is made to relate these results to the ecological environment in the subarctic Pacific. Additional laboratory data on diel periodicity of N 0 3 " uptake are also provided. Finally, the general conclusions from all chapters are presented after Chapter 4. 15 CHAPTER 1 S E A S O N A L V A R I A B I L I T Y O F P H Y S I C A L , C H E M I C A L A N D B I O L O G I C A L C H A R A C T E R I S T I C S O F T H E E U P H O T I C Z O N E IN T H E N E S U B A R C T I C P A C I F I C O C E A N I N T R O D U C T I O N The physical oceanography of the subarctic Pacific has been studied for decades, and a comprehensive characterization of the region has been obtained (see "General Introduction"; e.g. Dodimead et al; 1963, Favorite etal, 1976; Tabata, 1975, 1976). Chemical and biological descriptions of the upper 100 m mainly focused on oceanic sites (Station P; McAll is ter etal, 1960; review by Mil le r , 1993; Boyd etal, 1995; Wong etal, 1995), although other research teams extended their studies to coastal waters as well (Anderson etal, 1969; Anderson etal, 1977, reviews by Parsons, 1987, and Sambrotto and Lorenzen, 1987; Whitney etal, in prep.). Surface nitrate concentrations were observed to increase from coastal to oceanic regions in the N E subarctic Pacific (Anderson etal, 1969; Whitney etal, in prep). Surface nitrate was depleted during spring and summer only at the coastal sites, while nitrate concentrations at station P never fell below 5 pig-ai L" 1 even during the summer period (McAllister etal, 1960; Anderson etal, 1969; Whitney etal, in prep.). Despite the persistence of high nitrate levels in the oceanic area, chlorophyll a concentrations were low year round, with an annual average of 0.4pig L" 1 (Wong etal, 1995). Thus, the oceanic N E subarctic Pacific is considered an H N L C region (see "General Introduction"). Surveys of the physical characteristics, as well as dissolved nutrients, chlorophyll a and particulate nitrogen and carbon were carried out during the last decade along Line P (C.S. Wong and F. Whitney, pers. comm.). Despite the extensive studies done in the region, ambient 16 concentrations of regenerated nitrogen forms (urea and ammonium) were never measured, and the dominant phytoplankton species were not identified along this transect. The main objective of this chapter is to show interannual, seasonal and spatial variability of physical, chemical and biological parameters in the euphotic zone in the N E subarctic Pacific. Thus, data are presented for the following throughout the euphotic zone at several locations along Line P for different times of the year: (1) water temperature, salinity and density, (2) dissolved nitrogen (nitrate, urea, ammonium), silicon and phosphorus concentrations, (3) chlorophyll a, and particulate nitrogen and carbon concentrations, and (4) phytoplankton species composition. Records of surface irradiance are also shown for the periods when data were available. The description offered here is limited to the time span of this field study (2 years) and is not necessarily a reflection of the long term trends. M A T E R I A L S A N D M E T H O D S Studies were conducted at 5 stations (P4, P12, P16, P20 and P26) along Line P during September 1992, March and May 1993, and February and May 1994, and only at P26 during September 1994 (Fig. 1.1 and Table 1.1). Note that P26 wi l l be used to refer to station Papa during this thesis. Severe weather conditions during the February 1994 cruise did not allow tracking any further west than station P23A (Fig. 1.1); thus P23A was the most offshore station sampled during that cruise. Other station details such as latitude, longitude and water depth are presented in Appendix A (Table A . 1). 145° 135° 125°W 145° 135° 125°W Figure 1.1. Location of sampling stations along Line P in the N E subarctic Pacific Ocean. Station P26 is also known as Papa or P. 18 Table 1.1. Cruise dates for CTD profiles and water sampling along Line P, and seasons the cruises represent. Station P26 is also known as Papa or P. During February 1994, station P26 could not be reached, thus P23A was the most offshore station sampled (see text and Fig. 1.1). Cruise Station Date Season C T D profile Water sampling P4 10 Sep 92 10 Sep 92 P12 11 11 I. Sep 92 P16 12 12 Late summer P20 14 14 P26 17 & 22 16 P4 7 M a r 9 3 8 M a r 9 3 P12 9 10 II. Mar 93 P16 11 11 Late winter P20 11 12 P26 13 15 P4 15 May 93 16 May 93 P12 17 17 III. May 93 P16 18 18 Spring P20 20 19 P26 22 21 & 24 P4 8 Feb 94 8 Feb 94 P12 10 16 IV. Feb 94 P16 10 15 Winter P20 14 11 P23A 14 13 P4 12 May 94 13 May 94 P12 14 14 V. May 94 P16 15 15 Spring P20 16 17 P26 1 9 & 2 0 19 VI. Sep 94 P26 11 Sep 94 11 Sep 94 Late summer 19 Physical measurements Incident surface solar irradiance (i.e. incident surface photosynthetic active radiation; I G ) was continuously measured with a Licor LI-192SB Quantum sensor (calibrated for use in air), which was mounted in a shade-free area on the after-deck of the ship, approximately 7 m above the sea surface. The measurements were recorded on a Licor 1000 data logger, and data were available as 10 min averages. Light data are only available for March and May 1993, and May 1994. Vertical profiles of underwater irradiance were measured with a Licor L I - 192SB Quantum sensor (calibrated for use in water). In a few instances, the light profiles could not be obtained due to inclement weather conditions or because sampling commenced before dawn. In those cases, the light depths were estimated by using the most recent light profile, the incident irradiance and the transmissometer profile when available. Hence, slight differences between estimated and actual light depths are expected for those stations. Vertical profiles of conductivity, temperature and pressure were obtained with Guildline C T D Models 8705 and 8737 by personnel from the Institute of Ocean Sciences (Sidney, B.C. ) . Vertical profiles of sigma-t (o,) were derived from temperature and salinity data using the expression given by Mil lero and Poisson (1981). The mixed layer depth was identified as the depth where a 0.125 change in a t was first observed relative to the surface value (after Levitus, 1982). Temperature, salinity and a t for the mixed layer were calculated as the mean values from the surface to the calculated mixed layer depth. Chemical and biological measurements The time of the day when water sampling was done was variable among stations and cruises. The sampling time was dependent on the time of arrival on station and the order of priorities scheduled for each station. 20 Water samples for vertical profiles of chemical and biological parameters were taken with acid-cleaned Go-Flo bottles, Kevlar® line and Teflon® messengers. The use of such equipment permitted the collection of samples with minimum metal contamination (Bruland etal., 1979) for the other studies which were carried out from the same water samples, i.e. uptake rate experiments with 1 S N (Chapters 2, 3 and 4, this thesis) and 1 4 C (Boyd and Harrison, submitted). The 6 sampling depths corresponded to 100, 55, 30, 10, 3.5 and 1% of I D which were calculated from the vertical profile of underwater irradiance measured previously to obtaining water samples. A t station P20 during February 1994, weather conditions impeded the water sampling off the side of the ship, and thus samples were only collected from a clean seawater line that pumped water from 3-5 m depth (~ 55% I 0) to the ship's laboratory. Water samples were transferred to acid-clean carboys which were immediately sampled for dissolved nutrient, chlorophyll a, and particulate nitrogen and carbon concentrations as well as for phytoplankton identification. Water samples for dissolved nitrate + nitrite (N0 3 " + N0 2 ") , nitrite (N0 2~), silicic acid (Si(OH) 4 ) , phosphate (HP0 4 2 ") , and ammonium ( N H 4 + ) were processed immediately or kept at 4°C until analysis within a few hours using a Technicon® Autoanalyzer® II. Nitrate + N 0 2 " and N 0 2 " were measured following the automated procedure of Wood etal. (1967). Nitrite was only analyzed at P12 in March 1993, at all stations in May 1994 and at P26 in September 1994. Since N 0 2 " concentrations only comprised a small fraction of the N 0 3 " + N 0 2 " concentrations (see "Results"), N 0 3 " + N 0 2 " wi l l be referred to as N 0 3 " for the rest of this thesis. Sil icic acid and H P 0 4 2 " were measured following the procedures of Armstrong etal. (1967) and Hager etal. (1968), respectively. Minor modifications to the automated methods are described in Barwell-Clarke and Whitney (1996). Ammonium was analyzed either automatically (September 1992 to May 1994) as described by Slawyk and Maclsaac (1972) or manually (September 1994) as described by Solorzano (1969). Samples for urea (CO(NH 2 ) 2 ) were stored at -20°C until analysis ashore following the manual diacetyl monoxime method (Newell etal, 1967; Rahmatullah and Boyde, 1980) as recommended by Price and Harrison (1987). Water samples for dissolved nutrients were not filtered prior to analysis. No differences in nutrient concentrations were 21 observed between filtered (through pre-combusted Whatman® G F / F filters, ca. 0.7 / m i nominal porosity) and unfiltered samples as long as measurements were performed immediately or within a few hours after collection. Samples for chlorophyll a (Chi a; 0.5 L) were filtered onto Whatman® G F / F filters using a vacuum pressure of < 125 mm Hg , and stored at -20°C in a desiccator until analysis aboard ship or ashore. In May 1993, a sample from surface waters of P26 was also filtered onto a Poretics® 5 pirn polycarbonate filter. Chlorophyll a was extracted with 90% aqueous acetone, sonication and subsequent storage in the dark at -20° C for 20 to 24 h. Chlorophyll a concentrations were measured using in vitro fluorometry with a Turner Designs ™ Model 10 fluorometer (Yentsch and Menzel, 1963) as outlined by Parsons etal. (1984). Samples for particulate nitrogen and carbon ( P N and P C ; 2 L ) were filtered onto pre-combusted Whatman® G F / F filters using a vacuum pressure of < 125 mm Hg , and stored at -20°C until analysis ashore. After drying for 24 h at ca. 60°C, samples were analysed for P N and P C with a Carlo Erba Model NA-1500 Element Analyzer as described by Verardo et al. (1990). Samples for phytoplankton analysis were fixed with acidified Lugol ' s iodine solution (Parsons etal., 1984). Phytoplankton identification and quantification was performed using the inverted-microscope method (Utermohl, 1958). Statistical analyses of chemical and biological data Replication of water samples for chemical and biological analyses Due to time constraints in cruise schedules, replication of complete vertical profiles at every station and cruise was not possible. Routinely a single water sample was collected from each light depth from which chemical and biological analyses were done. During all cruises, however, additional time was allocated for sampling at P26, where replicate samples from surface waters (0-10 m) and occasionally from other depths were collected during a 1 to 4 day period. Therefore, the effect of cruise time on the surface concentrations of N 0 3 " , urea, N H 4 + , S i (OH) 4 , HPQ 4 2 " , Chi 22 a, P N and PC at P26 was investigated by using a one factor (cruise time) A N O V A for each one of those parameters. The concentration in surface waters was the dependent variable for each test. After each A N O V A , a multiple comparison test was utilized to determine where the differences in mean concentration were located among the six cruises. A Tukey test for unequal sampling sizes (Zar, 1996) was used for multiple comparisons because the number of replicates was not the same between cruises. Precision of analytical techniques Routinely, single samples were collected from the Go-Flo bottles for all analyses. However, during every cruise, subsamples (2 or 3) for nutrient, Chi a, P N and P C concentrations were drawn from the same Go-Flo bottles at selected stations and depths. The collection of multiple groups of subsamples allowed the determination of the precision of the analytical techniques. Appendix B presents a list of the parameters measured during this study, and the estimated errors are reported as the mean coefficient of variation and mean standard deviation (Table B . l ) . RESULTS Physical characteristics Incident irradiance The continuous recordings of incident irradiance are shown from the day of arrival on P4 to the day of departure from P26 for March and May 1993, and May 1994 (Fig. 1.2). Irradiance data are not available for September 1992, and February and September 1994. The lowest values were observed in March 1993 (Fig. 1.2 A ) , and the highest in May 1993 and 1994 (Figs. 1.2 B & C, respectively). During May 1994, however, incident irradiance showed more variability 23 !Z3 a o -»-> o ft 13 S <u o a • I—I ai a o • I—I o 1600 H P4 P12 P16 P20 P26 I I I I I I I I I 7 8 9 10 11 12 13 14 15 16 17 Day of March 1993 i I n i i i i i r 15 16 17 18 19 20 21 22 23 24 25 Day of M a y 1993 1 1 1 1 1 1 1 1 1—T 12 13 14 15 16 17 18 19 20 21 22 23 24 Day of M a y 1994 Figure 1.2. Incident surface irradiance for (A) March 1993, (B) M a y 1993 and (C) Mayl994 . Irradiance data are not available for the other cruises; Data are represented from the day of arrival at P4 to the day of departure from P26. Stations on top of each figure are placed on day of arrival at that station. 24 than during the previous May cruise, with irradiance levels comparable to winter values for a few days. Temperature, salinity and ot of the surface mixed layer A summary of the mixed-layer characteristics for all stations and cruises is presented in Table 1.2. The complete set of vertical profiles of temperature, salinity and a t is provided in Appendix C (Figs. C. 1, C.2 and C.3). The mixed layer was deeper during March 1993 and February 1994 than during the May 1993/1994 and September 1992/1994 cruises for all stations along the transect (Table 1.2). The depth of the mixed layer was compared to the depth of the 1% I D for all stations and cruises in Figure 1.3. During the winter cruises, the entire euphotic zone was within the mixed layer (i.e. 1% I G depth shallower than the calculated mixed layer depth; Figs. 1.3 A & B) . A t P4, the 1% IQ depth appeared slightly deeper than the mixed layer depth for both winter cruises; however, considering the errors involved in the determination of the light depths (e.g. inclination of the wire), those differences are most likely not significant. During the September cruises, instead, the euphotic zone of all stations was divided into a shallower mixed region and a deeper portion not affected by wind mixing (Figs. 1.3 E & F). The situation observed for the M a y cruises (mainly for 1993; Figs. 1.3 C & D) was intermediate between that of February/March and September, indicative of the progressive formation of a shallow mixed layer towards the summer. Mixed layer temperatures show the expected seasonal variability with the lowest values in winter, intermediate in spring and the highest in late summer for every station (Table 1.2). A decreasing trend of temperature was observed during every cruise from P4 to P26. Mixed layer salinity and a t slightly increased towards the open ocean. The most pronounced increase in salinity and a t was observed for the spring cruises, but especially from P4 to P12 during May 1994 which coincided with a very shallow mixed layer at P4 (Table 1.2). 25 Table 1.2. Mixed layer characteristics for all Line P stations and cruises. Temperature, salinity and a t for the mixed layer were calculated as the mean values from surface to the calculated mixed layer depth. See text for details on calculation of mixed layer depth. Values shown for P26 in February 1994 correspond to P23A. During September 1992 and May 1994, two C T D profiles were obtained for P26. Dashed line (-) indicates that data are not available. Characteristic Station Cruise winter spring late summer Mar 93 Feb 94 May 93 May 94 Sep 92 Sep 94 P4 77 63 40 16 24 -P12 97 83 58 33 21 -Depth P16 120 83 54 54 29 -(m) P20 108 91 44 61 32 -P26 106 92 42 69 43 -P26 - - - 83 40 38 P4 8.62 9.57 10.66 11.70 13.83 _ P12 7.91 9.33 9.16 10.17 16.12 -Temperature P16 8.05 9.14 9.11 9.80 15.20 -C Q P20 7.07 8.00 8.52 8.43 14.18 -P26 5.71 7.29 7.63 7.65 13.19 -P26 - - - 7.71 13.21 13.41 P4 32.53 32.37 32.31 31.37 32.09 P12 32.47 32.32 32.40 32.45 31.94 -Salinity P16 32.53 32.53 32.47 32.53 32.37 -P20 32.43 32.55 32.50 32.56 32.34 -P26 32.58 32.52 32.58 32.76 32.35 -P26 - - - 32.74 32.34 32.54 P4 25.24 24.97 24.74 23.83 23.98 _ P12 25.35 24.97 25.06 24.93 23.37 -P16 25.33 25.16 25.12 25.06 23.90 -P20 25.39 25.35 25.23 25.29 24.10 -P26 25.67 25.43 25.43 25.56 24.30 -P26 - - - 25.54 24.29 24.41 26 Station P26 P20 P16 P12 P4 P26 P20 P16 P12 P4 OH 40 H s o - a "•• 120-i — o-l A . Mar 93 o •© c . . . T -4—> 80-| 120 H OH 40 H 80-120 H 145 140 C. M a y 93 ^ o & T 135 T E . Sep 92 & o o -o" ..o 130 F. Sep 94 145 140 Longitude ( °W) - O - 1% Lj depth (measured) 1% ID depth (estimated) Mixed layer depth 135 130 Figure 1.3. M i x e d layer and 1% I 0depths for Line P stations during (A) March 1993, (B) February 1994, (C) M a y 1993, (D) M a y 1994, (E) September 1992 and (F) September 1994. The symbol © represents estimated 1% I Q depths (see text for details). The 1% I 0 depth was not available for P20 during February 1994. When two symbols for mixed layer or 1% I Q depth appear at P26, they indicate separate measurements (derived from separate vertical profiles of at or underwater irradiance). 27 Chemical and biological characteristics Dissolved nutrient concentrations The vertical, longitudinal and seasonal distribution patterns of urea and N H 4 + were different from those of N 0 3 \ Urea and N H 4 + concentrations did not show an evident trend with depth (Figs. 1.4, 1.5 and 1.6). Urea concentrations ranged from undetectable (< 0.05 /*g-at L" 1) to 1 /<g-at L" 1 in most cases, however higher values were also found principally during May 1993 when concentrations of up to 2 /•Jg-at L" 1 were measured. Ammonium concentrations varied from 0.1 to 1 /<g-at L " 1 ; however concentrations between 0.1 and 0.5 ><g-at L" 1 were mainly encountered throughout the euphotic zone. Depth integrated (100-1% I 0) concentrations of urea and N H 4 + did not show longitudinal or seasonal trends (Table 1.3). Thus, urea and N H 4 + concentrations exhibited a patchy distribution with depth, longitude and cruise time. One obvious feature of the distribution of N0 3 ~ along Line P was an increase in concentration from P4 to P26 for all cruises (Fig 1.7 and Table 1.3). Si l ic ic acid and H P 0 4 2 " concentrations also increased towards the open ocean during every cruise (Figs. 1.8 and 1.9, respectively, and Table 1.3). They all showed very similar patterns of constant concentrations with depth during the winter cruises (Figs. 1.7, 1.8 and 1.9, A & B) to a surface depletion relative to depth for M a y and September (Figs. 1.7, 1.8 and 1.9, C, D & E) which is most noticeable for the stations closer to the coast during May 1994 and September 1992. Note that there was no H P 0 4 2 " data available for September 1992. Winter concentrations of N 0 3 " varied from 4 /*g-at L" 1 at P4 to 12 /<g-at L " 1 at P26 during March 1993 (Figs. 1.4 A and 1.7 A ) . During the February 1994 cruise, the concentrations were within the same range; however, no data were obtained for P26 (only for P23A) so that the full range of variability from P4 to P26 was not available for February 1994, although an increasing trend towards oceanic waters was still evident (Fig. 1.7 B and Table 1.3). During the spring cruises, the surface depletion was mainly observed for P4 during May 1993 and extending up to P16 during May 1994 (Fig. 1.7 C & D , respectively). In May 1994, the surface depletion was PQ r- ^ I 1 I 1 I 1 I 1 I P20 © •© m-< •PI 1 1 1 1 1 1 1 1 1 ©©•• • •©• • ••© © P26 ® I" • v * K - n • <j-<r' 1 1 1 o o 1 . 1 1 1 o o 1 1 o 00 I 1 I 1 I 1 I 1 I ©•©••© ©•• < S3 _ c-l < — 00 • — I 1 I 1 I 1 I 1 I o o 00 (ui) qjdsQ £6 W W 1/6 H3H 00 o .2 o CO <D £ •S c •J o o +-» >> "3 o o" e •4—* o3 ON ON c 1 o fa c 3 o c O <D ON ON s •a s .o PL, 00 e •a 3 Q CO 2 oo (D T3 CQ 03 i2 O T3 T3 a C3 m S ON N ON <*-. . — I o Ul ^ <u . . > CO tu CO ^ '3 • U — i o <U u S <D 60 g u O o <u 3 > CO 0 0 ^ o £ ° a V PQ (X ©3®-©—®' I 1 I 1 I 1 I P12 P12 _ CM <•-. — 00 < • - . ...<... ::::0-: .•< — — o - _ O, i—1 ..® 00 — _ Ste-H H . < < — ^ .< "3 <I o 03 00 o o < • • • • • • • • < oi P2 98®" .•'©• • ® — . . . . © q <3CW d o d o o NO o 00 h- ^ Oo£ •C ^ •3 1 CO — O X> ' - ^ GO =3 ^ a o J fa - § o c o o o l-l c <u o o 8 '8 o c S ON 03 T - H 2 IT 3 2 CQ -a c c f , , 03 O CO co Q \ _<D ON 2 o? 03 ^ 8 '8 o 8 8 03 T3 e <D +-» 2 e -*-» 03 SO ON ON *—i >> a 00 c •c 3 Q XT c i 03 3 c ©o o c g .8 c 3 (N co ^» si 2 c o o N -a 3 co 03 <D 8 4> > e o s .8 00 E UH co 03 O t—I I—I CO 03 o >  03 O co o g (ui) incfea £ 6 A V W 176 A V W 30 o in d o d o c-4 d o d o c-4 d o d o I T ; d o d o c~4 d o d PQ 00 P20 ©•©••© © © • " ,...-0«::::i • o-<i-^-: : : : * : ! : ! " I 1 I 1 I 1 I 1 I P26 "©•©••© ® ©' • .<! <••••• • o O NO o 00 'J-_ <N i-H -u> 03 1 00 s. — 00 — Tt-© — O 2 03 CO oo '3 S-, O s c CO +2 ON o3 ON £ <u co O w CO 00 J3 X) <u co 3 CO 03 S Ii N C 0 NO Is 2 £ 1 m ia ON 3 ON oo O a 3 > 03 03 00 in o d c o •g S xt ° & l « s S - c CX 3 .8 a -t;.2 a CD ' ' > $ co co 03 3 O - i 3 3 ^ 03 PH c"6dHS (ui) qjdaa t76dHS 31 Table 1.3. Depth integrated (100-1% I o) nutrient, chlorophyll a, and particulate nitrogen and carbon concentrations for all Line P stations and cruises. During February 1994, vertical profiles for those parameters were not measured at P20, thus integrated values could not be calculated, and values shown for P26 correspond to P23A. Seasonal means for winter and spring were obtained by averaging the two winter and the two spring cruises, respectively. Dashed line (-) indicates that data are not available. Parameter Station Cruise Seasonal Means winter spring late summer winter spring Mar 93 Feb 94 May 93 May 94 Sep 92 Sep 94 P4 301 410 103 51 316 - 356 77 P12 525 513 187 167 370 - 519 177 N03" PI 6 449 467 265 317 296 - 458 291 (mg-atm2) P20 627 - 443 505 547 - 627 474 P26 957 724 435 724 678 478 841 579 P4 51.2 4.4 30.8 27.9 0.0 - 27.8 29.4 P12 33.9 37.0 34.8 38.6 15.2 - 35.5 36.7 Urea P16 53.9 48.7 42.9 19.7 68.6 - 51.3 31.3 (mg-at mJ) P20 2.1 - 53.6 0.2 69.4 - 2.1 26.9 P26 5.1 50.1 73.2 9.0 38.5 18.0 27.6 41.1 P4 38.7 23.7 15.1 22.1 22.9 - 31.2 18.6 P12 16.7 32.9 18.1 28.7 20.1 - 24.8 23.4 P16 16.3 22.8 10.9 42.8 39.9 - 19.5 26.8 (mg-at m2) P20 19.5 - 11.5 25.7 48.6 - 19.5 18.6 P26 22.7 41.7 14.6 14.6 25.7 25.2 32.2 14.6 P4 651 733 318 332 520 - 692 325 P12 833 978 453 447 631 - 905 450 Si(OH)4 P16 755 829 593 655 491 - 792 624 (mg-at m5) P20 878 - 757 879 1135 - 878 818 P26 1443 1179 706 1001 1253 790 1311 854 P4 74.5 61.5 26.3 22.9 - - 68.0 24.6 P12 65.7 66.4 29.4 39.7 - - 66.1 34.5 HPO/ P16 58.7 62.2 39.8 62.3 - - 60.5 51.0 (mg-at m2) P20 68.6 - 52.4 64.3 - - 68.6 58.4 P26 97.4 82.5 50.0 74.3 - 62.5 89.9 62.1 P4 26.9 8.8 20.1 14.5 17.0 17.9 17.3 P12 26.7 8.1 16.7 11.3 11.3 - 17.4 14.0 Chi a P16 14.3 9.2 12.7 20.0 35.2 - 11.8 16.4 (mg m2) P20 24.6 - 18.1 24.8 34.5 - 24.6 21.4 P26 14.6 12.4 23.2 14.2 35.1 19.7 13.5 18.7 P4 49.0 50.3 46.9 89.1 47.8 49.7 68.0 P12 31.8 32.8 41.7 68.1 49.5 - 32.3 54.9 PN P16 32.7 50.6 52.7 82.1 98.0 - 41.7 67.4 (mg-at m2) P20 40.8 - 51.2 85.3 108.3 - 40.8 68.3 P26 43.5 60.6 67.2 69.2 111.5 77.5 52.1 68.2 P4 397 389 307 524 238 - 393 416 PI 2 228 323 275 434 316 - 275 354 PC P16 218 393 351 521 625 - 305 436 (mg-at m2) P20 309 - 370 562 639 - 309 466 P26 341 493 550 422 777 529 417 486 32 A . Mar 93 B. Feb 94 145 140 135 130 Longitude (°W) Figure 1.7. Contour plots of nitrate concentration along the Line P transect for (A) March 1993, ( B ) February 1994, (C) M a y 1993, ( D ) M a y 1994 and (E) September 1992. Solid lines indicate contour intervals of 1 ^ g-at L " 1 . Solid circles (•) indicate the sampling depths. 3 3 A . Mar 93 B. Feb 94 145 140 135 Longitude (°W) Figure 1.8. Con tour plots o f s i l i c i c ac id concentration a long the L i n e P transect for ( A ) M a r c h 1993, (B) February 1994, (C) M a y 1993, (D) M a y 1994 and (E) September 1992. S o l i d l ines indicate contour intervals o f 2 ^g-at L - l ; dotted lines show addit ional contours. S o l i d c i rc les (•) indicate the sampl ing depths. 34 A . Mar 93 B. Feb 94 0 20 4 0 60 80 ft <u P 0 20 4 0 60 80 C. May 93 w i s p 7 T 1 r D. May 94 I "1-1 "» 107 • r • • —^->-«? T 145 140 135 130 145 140 Longitude (°W) 135 130 HPO4 ( ^g -a tL" 1 ) F igure 1.9. Contour plots o f phosphate concentration a long the L i n e P transect for ( A ) M a r c h 1993, (B) February 1994, (C) M a y 1993 and (D) M a y 1994. S o l i d lines indicate contour intervals o f 0.1 /<g-at L " 1 . S o l i d circles (•) indicate the sampl ing depths. 35 more intense than during M a y 1993 for the stations closer to shore, with undetectable levels of N03" (< 0.05 /ig-at L" 1) in the surface water at P4 (Figs. 1.5 A & B , and 1.7 C & D). While N03" concentrations at P26 during May 1993 decreased relative to March 1993 (Fig. 1.7 A & C), the same feature was not observed for the 1994 winter-spring transition (Fig. 1.7 B & D , and Table 1.3). Nitrate levels at P26 during May 1994 were close to winter values throughout the euphotic zone. During September 1992, the distribution of N03~ along the transect was similar to the one observed during May 1994 (Fig. 1.7 E & D, respectively). For this September cruise, surface N03" levels, which were close to the detection limit, extended to P12 and were still low at P16 (2 ^g-at l"1) and then increased to 6 and 8 pig-at L" 1 at P20 and P26, respectively (Fig. 1.7 E). During September 1994, surface N03" values were about 5/*g-at L" 1 at P26 (Fig 1.6 B) . Winter concentrations of S i (OH) 4 varied from 8 /^g-at L" 1 at P4 to 18 ptg-at L" 1 at P26 in March 1993, with a similar trend in February 1994 (Fig. 1.8 A & B). Surface S i (OH) 4 values were as low as 5.5-6.5 /^g-at L" 1 at P4, P12 and P16 during May 1994 and increased to 15-16 yig-atL" 1 at P26 during both M a y cruises (Fig. 1.8 C & D). During September 1992, the lowest S i (OH) 4 values were observed at the surface at P12 and P16, averaging 3.5 pig-at L" 1 . Phosphate concentrations ranged from 0.7 to 1.2 ]Ag-at L" 1 throughout the euphotic zone during winter and from 0.4 to 1.2 pig-at L" 1 in surface waters during spring, always increasing towards P26 (Fig 1.9 and Table 1.3). The overall averages (± 1 S.E.) for the S i ( O H ) 4 : N03" and N03": H P 0 4 2 " ratios for surface waters along Line P were 1.9 ± 0.04 (n = 139), and 6.9 ± 0.22 (n=120), respectively. The S i ( O H ) 4 : N03" ratios decreased from P4 to P26 during every cruise, with values ranging from 3.8 to 1.4 (Fig. 1.10 A ) . The N03": H P 0 4 2 " ratios increased with distance offshore during every cruise (Fig. 1.10 B) , with minimum and maximum values of 3.3 and 9.8, respectively. These trends were most likely the result of the lesser utilization of N03" in open ocean waters relative to more coastal waters, which was most pronounced during the May cruises. 36 Figure 1.10. (A) Silicic acid to nitrate, and (B) nitrate to phosphate ratios for all stations along Line P and all cruises. Each bar represents the mean of the three surface values (100, 55 and 30% of IQ). Error bars represent ± 1 S.E. of the mean. At station P20 during February 1994, bar represents the ratio of single values. No Si(OH) 4 : N0 3" ratios are shown for P4 during September 1992 and May 1994, and for P12 during September 1992, since surface nitrate concentrations were undetectable. 37 Nitrite, which was measured consistently during M a y 1994, represented only between 1-3% of the N 0 3 " + N 0 2 " values, and exhibited a slight increase with depth (data not shown). In summary, N 0 3 " , S i (OH) 4 and H P 0 4 2 " concentrations increased towards P26 during every season investigated, with lowest surface values during September and May and highest values during the winter throughout the euphotic zone. In contrast, urea and N H 4 + showed a more patchy distribution along the transect, with no seasonal variation. Chlorophyll a, and particulate nitrogen and carbon concentrations The lowest concentrations of Chi a were measured during February 1994, with < 0.18 pig L" 1 at every depth and station (Fig. 1.11 B , and see also Table 1.3). During March 1993, Chi a concentrations were higher than in February 1994 along the entire transect (Table 1.3), with values of 0.50-0.55 pig L " 1 at subsurface depths (3-10 m) at stations P4, P12 and P20, and 0.30 pig L" 1 deeper in the water column extending from P4 to P20 (Fig. 1.11 A ) . Surface Chi a concentrations, however, were 0.20 pig L" 1 during March 1993 at P26, similar to the values measured during February 1994 at P23A (Fig. 1.11 A & B). The two May cruises also showed different patterns in the distribution of Chi a During May 1993, the highest surface concentrations of Chi a were measured at P4 and P26, with values of 0.60 and 0.50 pig L" 1 , respectively (Fig. 1.11 C). May 1994 presented a more patchy distribution of Chi a, with maximum values of 0.4 pig L" 1 deeper in the water column, but with consistently low concentrations (0.20-0.30 pig L" 1) throughout most of the region (Fig. 1.11 D). September 1992 exhibited the highest Chi a concentrations for any cruise, with surface values as high as 0.66 pig L / 1 at P16, and decreasing towards both ends of the transect to ca. 0.50 pig L" 1 (Fig. 1.11 E). Very low values (< 0.20 pig L 4 ) were found at P12 during September 1992. Depth integrated Chi a concentrations were higher for the oceanic stations during September 1992 (Table 1.3). In summary, Chi a concentrations showed a patchy distribution along Line P. A seasonal trend was evident mainly for stations P4 to P20 from February to May in 1994, but not from March to May in 1993. Chlorophyll ^exhibited the highest values during the late summer period. 38 A . Mar 93 B. Feb 94 i 1 1 1 1 1 r C. May 93 D. May 94 145 140 135 Longitude (°W) Figure 1.11. Contour plots of chlorophyll a concentration along the Line P transect for (A) March 1993, (B) February 1994, (C) M a y 1993, (D) M a y 1994 and (E) September 1992. Solid lines indicate contour intervals of 0.1 ptg L" ; dotted lines show additional contours. Solid circles (•) indicate the sampling depths. 39 Particulate nitrogen presented the lowest values during the winter cruises (Fig. 1.12 A & B, and Table 1.3). Surface concentrations were elevated at P26 in May 1993, and at P4 and P12 in May 1994 (Fig. 1.12 C and D, respectively). The concentration of PN was higher in the entire water column from P16 to P26 in September 1992 (Table 1.3); however, the increase was most evident at the surface of P16 (Fig. 1.12 E). The highest concentration measured during this study was 2.6 pig-at L"1 at P4 during May 1994 and at P16 during September 1992. The distribution of PN did not show a consistent longitudinal pattern from year to year; PN also appears to be patchy along the transect (Table 1.3). Particulate carbon concentrations varied in a similar manner to PN (Table 1.3). The overall average (± 1 S.E.) for PC:PN (atomic C:N) ratios in this study was 7.1 ± 0.11 (n = 149). Some of the features observed on the distribution of Chi a were also evident for PN and PC. For example, September 1992 PN resembled Chi a with the highest concentrations located in surface waters at station P16, and very low values at the surface at station P12 (Figs. 1.11 E and 1.12 E). Depth integrated concentrations showed the highest Chi a, PN and PC at P16, P20 and P26 in September 1992 (Table 1.3). During May 1993, the highest values were observed at station P26 for Chi a, PN and PC (Table 1.3). The winter distributions also showed some similar patterns (e.g. Chi a as well as PN and PC concentrations increased slightly towards P26 during the February 1994 cruise; Figs. 1.11 B and 1.12 B, and Table 1.3). Despite some similarities, however, Chi a and PN/PC did not always seem to follow the same patterns. Station P26 Figures 1.13 and 1.14 summarize the results of the statistical comparisons of seasonal surface (0-10 m) concentrations of nutrients, Chi a, PN and PC at P26 during the six cruises. In all cases, ANOVAs detected significant differences among cruises (at P < 0.05). Horizontal lines on the top of Figures 1.13 and 1.14 indicate that the aposteriori Tukey test found no significant differences in mean concentrations between cruises. Lines of the same type join cruises that are not significantly different from each other. If lines overlap for one or more cruises, it means that 40 Figure 1.12. Contour plots of particulate nitrogen concentration along the Line P transect for (A) March 1993, (B) February 1994, (C) M a y 1993, (D) M a y 1994 and (E) September 1992. Solid lines indicate contour intervals of 0.2 /*g-at L~ r . Solid circles (•) indicate the sampling depths. 41 F igure 1.13. Surface (0-10 m) concentrations o f ( A ) nitrate, ( B ) phosphate, (C) s i l i c i c ac id , (D) urea, and (E) a m m o n i u m at P 2 6 for a l l cruises. E a c h bar represents the mean o f replicates ± 1 S .E . Hor i zon ta l l ines o f the same type j o i n cruises that are not s igni f icant ly different f rom each other. W h e n lines over lap, it impl i e s that the T u k e y test was not able to detect differences at the P level considered. In a l l cases A N O V A s were tested at P = 0.05, and T u k e y tests at ( A ) P = 0.05, (B) P = 0.05, (C) P = 0.05, (D) P = 0.20 and (E) P = 0.05. 42 Figure 1.14. Surface (0-10 m) concentrations of (A) chlorophyll a, (B) particulate nitrogen and (C) particulate carbon at P26 for all cruises. Each bar represents the mean of replicates ± 1 S.E. Horizontal lines of the same type join cruises that are not significantly different from each other. When lines overlap, it implies that the Tukey test was not able to detect differences at the P level considered. In all cases ANOVAs were tested at P = 0.05, and Tukey tests at (A) P = 0.1, (B) P = 0.05 and (C) P = 0.1. 43 the T u k e y test was not able to detect differences between those cruises at the level o f confidence u t i l ized for the test. T h e probabi l i ty (P) for those results is specified i n each case. Surface N 0 3 ~ concentrations at different times of the year were signif icantly different from each other (Tukey tests: P < 0.05) ( F i g . 1.13 A ) . Surface H P 0 4 2 " values were also signif icantly different among a l l cruises (Tukey tests: P < 0.05) (F ig . 1.13 B ) . Nitrate and H P 0 4 2 " concentrations were s ignif icant ly lower dur ing September 1994 (5 and 0.73 pig-at L " 1 , respectively) and higher dur ing the M a r c h 1993 cruise (12.2 and 1.21 pig-at L " 1 , respectively). Surface S i ( O H ) 4 concentrations also showed signif icantly higher values (18.7 pig-at L" 1 ) i n M a r c h 1993 and lower values (10 jUg -a t L" 1 ) i n September 1994 (Tukey tests: P < 0.05) (F ig . 1.13 C ) . Surface concentrations for the other cruises are intermediate between those two extremes; however, no significant differences were found among some of the other cruises (Tukey tests: P > 0.05) ( F i g . 1.13 C ) . Surface urea concentrations showed signif icant ly lower values (0.14-0.22 ^g-at L" 1 ) dur ing M a r c h 1993, and M a y and September 1994 ( T u k e y tests: P < 0.2) (F ig . 1.13 D ) . Highes t values were found dur ing M a y 1993 and September 1992 (0.70-0.88 pig-at L " 1 ) . Surface N H 4 + concentrations showed no significant differences between M a r c h 1993, M a y 1993, M a y 1994 and September 1994 (Tukey tests: P > 0.05) ( F i g . 1.13 E ) . M e a n N H 4 + concentrations for those 4 cruises ranged between 0.23 and 0.30 /*g-at L " 1 . M e a n N H 4 + concentration for February 1994 was s ignif icant ly higher (0.51 ^g-at L" 1 ) than the means for the other cruises ( T u k e y tests: P < 0.05), except for September 1992 where the test fai led to detect a significant difference. Surface C h i a concentrations were s ignif icant ly lower (0.16-0.23 ytg l"1) dur ing M a r c h 1993, and February and M a y 1994 (Tukey tests: P < 0.1) ( F i g . 1.14 A ) . T h e highest surface value (0.49 \ig L " 1 ) was observed for M a y 1993 (Tukey tests: P < 0.1). C h l o r o p h y l l a concentrations for the September cruises showed intermediate values (0.37-0.43 ptg L" 1 ) and were not s ignif icant ly different f rom one another (Tukey tests: P > 0.1). 44 Surface P N and P C concentrations were significantly higher (1.80 and 13.71 pig-aX I'1, respectively) for M a y 1993 and lower (0.56 and 4.24 pig l " 1 , respectively) for March 1993 (Tukey tests: P < 0.05 for P N and P < 0.1 for PC) (Fig. 1.14 B and C). In summary, the statistical comparisons detected significant seasonal differences for N 0 3 " , H P 0 4 2 " and S i (OH) 4 , but not for N H 4 + and urea, in surface waters at station P26. Surface Chi a was significantly higher for the spring of 1993, and lower for both winter cruises and the spring of 1994. Surface Chi a showed intermediate values for late summer. Particulate nitrogen and carbon showed a significant seasonal effect for the 1993 winter-spring transition, but the multiple range tests failed to find other significant differences. Phytoplankton assemblages The abundance of photosynthetic flagellates was always higher than the abundance of diatoms at every depth, station and cruise (Fig. 1.15). The categories 'Diatoms' and 'Photosynthetic Flagellates' used in Figure 1.15 include all species listed on the left and right hand side of Table 1.4, respectively. Only during May 1993 at P26 did the concentration of diatoms show an increase throughout the euphotic zone (Fig. 1.15 C). Phytoplankton composition data are not available for P26 during September 1994. The numerical contribution of each individual group to the total integrated cell number in the euphotic zone is shown in Figure 1.16. The average contribution of diatoms was 8 ± 2% (mean ± 1 S.E., n = 25) for the entire study. The highest values were observed on two occasions at P26 during M a y 1993 and February 1994, with a 38 and 22% contribution, respectively (Fig. 1.16 C & B , respectively). These higher concentrations of diatoms at P26 were due to an increase in the number of pennates which represented 90 and 95% of the total diatoms for May 1993 and February 1994, respectively. The average contribution of flagellates was 92 ± 2% (mean ± 1 S.E., n = 25) for the entire study. Flagellates from the classes Cryptophyceae, Prymnesiophyceae, and Prasinophyceae, and other unidentified species were cells < 10 pim and were responsible for 98 ± 1% (mean ± 1 S.E., n = 25) of the total flagellate contribution. The 45 0 1 2 3 0 - 8 % 9 f 40 -6 •' 80 - 6 • 0 - 1 1 1 1 <r>. o» 4 0 - « 8 0 - • P23A •S 40. CL <D Q 80-0 ' I I I o - r - a s : Of da 40 H 80 - \ I I I I I I Phytoplankton (x 1 0 6 ce l ls L " 1 ) J I I L I I I I °*. 6 • I I I I 0 1 2 3 1 1 1 1 0 1 2 3 1 1 1 1 0 1 1 2 3 1 1 1 8 8 8 * * O * O f 0 6 * o« 6 0 0 • 4 •' 6* di 6 • P 2 6 P 2 0 -(8 4 J I I L I I I I « o ••' o I 6 I I I I 8 0 - 9 •. — 9 • <5 9 • 6 - o • 40 - 4 *(. -<j > - 6 •> 8 0 - 6 »•' 6 •'" I I I I P 1 6 J I I L I I I I o • 9*' 6 • I I I I 9 o • 0 * 0 6 f 6«i nzn 9 i 9?' P 1 2 —o— diatoms photo flagellates 0 1 2 3 J I I L 9».. 9 > 9 •' 0 '* 6 i o • 9 8 i o 6 6 #' P 4 J I I L I I I I I I I I Figure 1.15. Ver t i ca l profiles o f diatoms and photosynthetic flagellates for L i n e P stations dur ing ( A ) M a r c h 1993, (B) February 1994, (C) M a y 1993, (D) M a y 1994, and (E) September 1992. T h e categories 'diatoms' and 'photo flagellates' inc lude a l l species l is ted on the left and right hand side o f Tab le 1.4, respectively. O n l y a few data points are avai lable throughout the euphotic zone for stations P 4 to P 2 0 dur ing February 1994. N o data are avai lable for September 1994. 46 Table 1.4. List of phytoplankton species identified from samples collected along Line P from September 1992 to M a y 1994. Bacillariophyceae (Diatoms) Parnate Grammatophora marina Navicula wawrikae Navicula spp. Pseudonitzschia spp. * % Pleurosigma spp. f Thalassionema nitzschioides Thalassiothrixfrauenfeldii Thalassiothrix longissima Tropidoneis lepidoptera unidentified spp. * Centric Asteromphalus heptactis Cerataulina pelagica Chaetoceros affinis Chaetoceros atlanticus Chaetoceros concavicornis Chaetoceros convolutus Chaetoceros danicus Chaetoceros spp. * Corethron criophilum Coscinodiscus spp. Cylindrotheca closterium * Cylindrotheca longissima Ditylum brightwellii Leptocylindrus danicus Leptocylindrus minimus Rhizosolenia alata Rhizosolenia delicatula Rhizosolenia fragilissima Rhizosolenia hebetata Rhizosolenia setigera Rhizosolenia stolterfothii Thalassiosira spp. unidentified spp. * Dinophyceae - autotrophic (Dinoflagellates) Ceratium fusus Ceratiumfurca Ceratium horridum Ceratium tripos Ceratium cf. pentagonum Ceratium cf. lineata Gonyaulax spinifera Gonyaulax spp. Gymnodinium spp. * Katodinium rotundatum Oxytoxum spp. Prorocentrum gracile Prorocentrum minimum unidentified athecate spp. unidentified thecate spp. * Chrysophyceae (Flagellates) Dictyochafibula Dictyocha speculum Cryptophyceae (Flagellates) Cryptomonas spp. Prymnesiophyceae (Flagellates) Chrysochromulina spp. * unidentified coccolithophorid spp. § Prasinophyceae (Flagellates) Micromonas pusilla Other autotrophic (Flagellates) unidentified flagellate spp. (< 5 pan) * Most abundant species in each group. The most abundant unidentified diatoms were all < 10 pan, and thecate dinoflagellates < 25 pan. $ Includes species identified as Nitzschia spp. in previous studies, t Possibly includes Gyrosigma spp. § Possibly includes Emiliania huxleyi. 47 A. Mar 93 B. Feb 94 100 80 60 40 20 0 C. May 93 100 E. Sep 92 P26 P20 P16 P12 P4 3 D. May 94 I 1 P26 P20 P16 P12 P4 d Other flagellates r~| Prasinophyceae jjj Prymnesiophyceae Cryptophyceae Chrysophyceae ] Dinophyceae | Centric Diatoms Pennate Diatoms Station Figure 1.16. Contribution of each phytoplankton group to the total integrated cell number for Line P stations during (A) March 1993, (B) February 1994, (C) May 1993, (D) M a y 1994 and (E) September 1992. During February 1994, integrations at P4 and P16 were done to the deepest light depth available, and only one depth was used for stations P12 and P20 (see Fig . 1.15). Groups contributing < 1% to the total were not included. 4 8 class Piymnesiophyceae possibly includes Emiliania huxleyi (under "unidentified coccolithophorid spp."). E. huxleyi was observed in live samples, but was not identified with certainty from fixed samples. The class Prasinophyceae (represented by Micromonaspusilla, < 2 pim) and unidentified flagellate species (< 5 pirn) were the most numerically dominant flagellates at most stations and seasons. In terms of biomass, surface Chi a for the 0.7-5 pim fraction represented 43% of the total biomass (> 0.7 pim) at P26 during May 1993. A t P26 during May 1994, Chi a for the 0.7-2 pim fraction (picoplankton) ranged between 44 to 81% of the total biomass in a vertical profile for the euphotic zone (see Chapter 3). These results suggest that the small phytoplankton cells were not only numerically abundant, but also accounted for a large portion of the biomass at P26. In summary, flagellates < 2-5 pirn dominated the entire area of study in terms of numbers, seasonally, vertically and longitudinally. Diatoms remained at very low levels, with only sporadically higher numbers due to an increase in pennates. DISCUSSION Nutrient distributions and sources Nitrate, silicic acid and phosphate Concentrations of N 0 3 " , S i (OH) 4 and H P 0 4 2 " in the euphotic zone increased towards the open ocean during all cruises. Very low surface concentrations of N 0 3 \ S i ( O H ) 4 and H P 0 4 2 " were observed at the stations closer to the coast during May and September. Nitrate, S i (OH) 4 and H P 0 4 2 " also decreased from winter to spring-summer in the oceanic sites; however complete depletion never occurred. The decrease in macronutrient concentrations along Line P from winter to spring-summer was the result of phytoplankton growth which was stimulated by the increase in solar irradiance and the shoaling of the mixed layer during spring-summer. The presence of transient thermoclines above the permanent halocline in the Line P region has been reported for the 49 warmer months of the year (Dodimead etal., 1963; Tabata, 1976; Denman and Gargett, 1988). Denman and Gargett (1988) showed that the shallow seasonal thermocline divides the euphotic zone in two areas of different bio-optical properties. Thus, under favorable physical conditions, phytoplankton utilized most of the available N03~, Si(OH)4 and HP0 4 2" at the stations closer to the coast, but only a portion of the ambient macronutrients at the oceanic sites. During May 1994, the difference in N03", Si(OH)4 and HP0 4 2" concentrations from one end of the transect to the other was more pronounced that during any other cruise. This may have been the result of the phytoplankton bloom already under way at the stations closer to the coast, but not yet occurring at the more oceanic sites. A delay in the phytoplankton blooms in the oceanic subarctic Pacific relative to the coastal areas has been reported by Parsons etal. (1966), and Parsons and LeBrasseur (1968). However, lack of complete depletion of macronutrients at the offshore end of the transect was observed during every cruise and is likely due to other factors limiting phytoplankton growth which will be discussed later in this chapter. Replenishment of N03", Si(OH)4 and HP0 4 2" comes from wind mixing mainly during the winter, but also from upwelling throughout the year (Favorite etal., 1976; Gargett, 1991). Anderson etal. (1969) claimed that the occurrence of an intensive entrainment of deep waters into the surface layers may have been responsible for the high N03" concentrations in the oceanic region. However, Gargett (1991) mentioned that the NE Pacific experiences weak upwelling due to the high stratification of the upper water column. Maximum upward vertical displacement in the NE subarctic Pacific is on the order of 3 m yr"1 (Talley, 1985; Gargett, 1991). Thus, entrainment is not that intense and the vertical supply of nutrients to the euphotic zone is slow. Although weak, the delivery of N0 3" into the surface seems to be sufficient to maintain a N03"-rich euphotic zone. The station closer to the coast, P4, is located at the edge between the Dilute and the Upwelling Domains, and may also receive occasional nutrient inputs from the seasonal coastal upwelling. 50 Urea and ammonium U r e a and N H 4 + showed spatial and temporal patchiness un l ike the patterns seen for N 0 3 " , S i ( O H ) 4 and H P 0 4 2 \ Whee le r (1993) also cou ld not f ind any seasonal or interannual patterns i n N H 4 + concentrations at station P26 . A l t h o u g h generally l o w , urea and N H 4 + concentrations showed sporadic h igh levels (e.g. urea was part icularly h igh dur ing M a y 1993). These results partly agree w i t h the findings o f Wheeler and K o k k i n a k i s (1990) for P 2 6 i n that surface N H 4 + concentrations were l o w and variable. Howeve r , their upper range for N H 4 + was 0.4 f<g-at L " 1 , wh i l e dur ing the present study a few values above 0.4 /<g-at L " 1 were also found. In a later publ icat ion, Whee le r (1993) reported N H 4 + concentrations o f up to 0.5 /<g-at L " 1 deeper i n the water c o l u m n w h i c h more c lose ly agree wi th the findings of this study. There seems to be a difference, however , i n the range o f urea concentrations measured dur ing this study and that o f Wheeler and K o k k i n a k i s (1990). T h e y mentioned that urea concentrations were generally lower than N H 4 + , but no values were presented. In the present study, urea concentrations were sometimes higher than N H 4 + , and showed wide var iabi l i ty . La rge var iab i l i ty i n urea concentrations has been documented for many marine systems. U r e a concentrations i n the open ocean, however, are generally lower than those closer to the continents (see reviews by Remsen etal., 1974 and A n t i a etal, 1991). F o r example , M i t a m u r a and Sai jo (1980) found concentrations ranging from 0.85 to 1.43 /<g-at L " 1 i n a coastal embayment i n Japan, and lower values i n the central N W Pac i f i c (0.19-1.02 /<g-at L " 1 ) and subarctic N W Pac i f ic waters (0.17-0.40 /-<g-at L" 1 ) f rom the surface to 50 m depth. R e m s e n (1971) showed that urea concentrations were patchy and variable (0.54-5.00 /<g-at L " 1 ) a long surface waters o f the Peruvian coast, and from coastal to offshore waters on the continental shelf o f the N E Uni t ed States (0.25-11.20 /<g-at L " 1 ) . H e also reported considerable fluctuations i n the vertical profiles o f urea concentrations i n both areas. Remsen (1971) also looked at a vert ical profi le i n the Sargasso Sea and found lower values compared to those of the other two regions; however , concentrations were s t i l l between 0.5-1.0 /<g-at L " 1 . E p p l e y etal. (1977) measured 0.1-0.6 /<g-at L " 1 o f urea i n the Central N o r t h Pac i f i c , w i th no evident seasonal var iabi l i ty . Har r i son etal. (1985) found urea 51 concentrations ranging from undetectable to > 2 pig-at L" 1 in the eastern Canadian Arctic. The concentrations of urea found in the present study were generally within the values reported by others for open waters, with a few occasional peaks. Local sources within the euphotic zone, such as bacterial degradation, phytoplankton secretion and zooplankton excretion, are generally responsible for the patchy distribution of urea and N H 4 + in seawater. The contribution of urea and N H 4 + to the total nitrogen pools by different compartments of the marine food web varies from one environment to another (Harrison, 1980, 1992). Bacterial contributions of organic and inorganic nutrients through heterotrophic activity have been recognized for some time (see Fenchel and Blackburn, 1979), although it has also been suggested that their contribution to net regeneration may be less than previously considered (Wheeler and Kirchman, 1986; Kirchman, 1994). Phytoplankton may also secrete N H 4 + and D O N (Hellebust, 1974; Sharp, 1977; Antia etal., 1991). In general, however, the contribution of phytoplankton to nitrogen recycling is assumed to be small, although this view has been recently challenged (Collos, 1992; Collos etal, 1992; Bronk and Glibert, 1993, 1994; Bronk etal., 1994). Nano- (2-20 ptm) and microzooplankton (20-200 /mi) seem to be more important agents for nutrient recycling than mesozooplankton (> 200 pm) (e.g. Johannes, 1968, Harrison, 1978; 1992; Caperon etal., 1979; Dagg etal., 1980; Glibert, 1982; Caron, 1991; Le Corre etal., 1996). Dagg etal. (1980) suggested that microzooplankton excretion was providing most of the recycled nitrogen to primary producers in the Peru upwelling system, since the copepod contribution was only 4.3% of the daily nitrogen requirements by phytoplankton. The excretion of nitrogen by these copepods was in the form of N H 4 + , urea and primary amines, with N H 4 + representing on average 70%, and urea 22% of the total nitrogen excretion (Dagg etal., 1980). Harrison etal. (1985) also showed that macrozooplankton excreted both N H 4 + and urea in northern Baffin Bay in the eastern Canadian Arctic. Rates of excretion of N H 4 + were again higher that those of urea, supplying ca. 40 and 3% of the N H 4 + and urea-N requirements, respectively (Harrison et al, 1985). Eppley etal. (1973) reported higher urea excretion (ca. 50% of total N excretion) in the 52 subtropical central gyre of the North Pacific. In general, however, zooplankton excretion is primarily in the form of N H 4 + , with urea and other D O N representing ca. 20% of the total nitrogen released (Bidigare, 1983). Nitrogen excretion by zooplankton has a larger impact on phytoplankton nitrogen requirements in open waters compared with coastal areas. Dagg etal. (1982) determined that about 13-16% of the daily N H 4 + requirements by phytoplankton in outer and mid-shelf sites in the Bering Sea were supplied by zooplankton excretion, while zooplankton contributions in coastal stations were only 2%. In summary, zooplankton excretion may account for up to 100% of the total N requirements for primary producers in oceanic environments in contrast to a contribution of < 20% in coastal regions (Billen, 1984). It is possible that urea could also come from other sources, such as macrofauna. Harrison etal. (1985) investigated alternative sources of urea in the Arctic, such as from seabird guano. They measured urea adjacent to a particularly large colony of seabirds, and although concentrations were not high, bacterial activity was twice as high compared to that of offshore sites suggesting the possibility of high rates of urea (and N H 4 + ) regeneration. Fish and mammals could also make important contributions of urea to the total nitrogen available for phytoplankton (e.g. McCarthy and Kamykowski , 1972; Whitledge and Packard, 1971; Whitledge and Dugdale, 1972, Remsen et al., 1974); however, in the open ocean these sources may be rather sporadic, but could explain episodic high values. In the oceanic subarctic Pacific, Wheeler etal. (1989) showed that most of the N H 4 + regeneration took place at night. They did not separate the remineralizing effects of mesozooplankton, microzooplankton and bacteria, but suggested that bacteria may not have contributed significantly to nighttime N H 4 + release since their production peaked during the day (Wheeler etal., 1989). Although bacteria could be important recyclers in other environments, in this region bacterial growth rates are limited by the supply of organic matter, and by temperature (Kirchman era/., 1989; Kirchman, 1990; Kirchman et al., 1993). Thus, their contribution to the recycled nitrogen in subarctic waters is likely to be small. In terms of the zooplankton contribution 53 to recycled nutrients, Wen (1995) calculated that copepods excreted 3.3% of the ambient N H 4 + concentrations in the mixed layer of P26 per day, which represented only 8% of the N H 4 + requirements by phytoplankton during May 1994. Therefore, other sources of recycled nutrients are expected, such as nitrogen excretion by nano- and microzooplankton (Frost, 1991, 1993). The abundance and distribution of heterotrophic nanoflagellates in the euphotic zone of the Line P stations were studied by Doherty (1995) during May 1993, February 1994 and May 1994. He found that heterotrophic (and also autotrophic) nanoflagellate abundance was an order of magnitude higher in May 1993 in comparison to May 1994 at most stations. These results may partly explain the higher levels of urea measured consistently along the transect during May 1993. Another possible contributing source could have been the presence of abundant concentrations of salps from P4 to P16 during May 1993, which were not present in 1994 (Wen, 1995). However, as shown in this chapter, N H 4 + concentrations were not unusually high, and considering that heterotrophs excrete both N H 4 + and urea, then there must have been an additional source of urea during M a y 1993. Comparisons between the abundance of nano and microzooplankton for February 1994 and M a y 1994 indicated that winter values were comparable to those from late spring (Boyd etal, 1995; Doherty, 1995). In contrast, abundance of mesozooplankton was lower in winter (March 1993 and February 1994) than during the M a y 1993/1994 cruises (Boyd etal., 1995; Wen, 1995). These results suggest that the year-round presence of nanozooplankton and the occasional high abundance of larger zooplankton in the N E subarctic Pacific are likely the most important sources of regenerated nutrients available for primary producers throughout the year. Chlorophyll a distribution Chlorophyll a concentrations were low along the entire transect in spite of sufficient levels of macronutrients for phytoplankton growth on most occasions. These findings confirm previous results (Parsons and LeBrasseur, 1968; Anderson et al, 1977; Wong etal:, 1995) which showed Chi a values rarely exceeding 1 pig L" 1 , and with very little seasonal variation. The present study 54 showed that, although low, phytoplankton biomass in surface waters of P26 was significantly different during winter, spring and late summer, with the highest values during May 1993. L o w phytoplankton standing stocks in the oceanic N E subarctic Pacific have been attributed to grazing impact on the phytoplankton assemblages, low availability of dissolved iron, and/or N H 4 + depression of N 0 3 " uptake rates by phytoplankton. The grazing impact of mesozooplankton (mainly copedods) in the subarctic Pacific has been determined to be high, but not sufficient to keep the phytoplankton stocks at low levels (Frost, 1987; Landry and Lehner-Fournier, 1988; Mi l l e r and S U P E R Group, 1988; Landry etal, 1993b). Dagg (1993) documented that the copepod community ingested between 6-15% of the daily total phytoplankton production at P26. However, Wen (1995) found higher values (33%) for M a y 1994, one of the periods covered in this thesis. Theoretical modelling and experimental work both point to microzooplankton as the major grazers of the dominant phytoplankton from the oceanic subarctic Pacific (Parsons and L a l l i , 1988; Frost, 1987, 1991, 1993; Mi l l e r and S U P E R Group, 1988; Mil ler etal., 1991a, b; Landry etal, 1993a; Fasham, 1995). It has been pointed out that the lack of sufficient levels of iron was responsible for the less than maximal growth rates of large phytoplankton cells, such as diatoms (Frost, 1991; Cullen, 1991; Boyd etal., 1996). Under these conditions, mesozooplankton grazing could keep pace with the growth of > 5 pim phytoplankton cells only because growth of these phytoplankton was depressed. However, upon iron addition, growth rate and total biomass of the large cells increased in in vitro experiments (Martin et al., 1989; Coale, 1991; Martin etal, 1991; Boyd et al., 1996) in the presence of mesozooplankton concentrations close to ambient oceanic values (Boyd etal, 1996). In contrast, smaller phytoplankton cells do not seem to be iron limited ( M a r t i n i al, 1989; Coale, 1991; Martin etal, 1991; Boyd etal, 1996). Boyd etal. (1996) indicated that phytoplankton cells < 5 pim showed little change in total biomass, production and net growth rates upon iron addition. Phytoplankton cells < 5 pim were growing at close to maximal specific growth rates under the ambient conditions (Booth etal, 1988; Boyd etal, 1996). Furthermore, results from laboratory studies on Emiliania huxleyi isolated from the oceanic 55 subarctic Pacific suggested than E. huxleyi may be adapted to the low ambient iron levels (Muggli and Harrison, 1996b). These results imply that, in the oceanic subarctic Pacific, the total biomass of the dominant small phytoplankton was maintained at low levels by microzooplankton grazing, but not by iron limitation (Landry etal., 1993a; Boyd etal, 1996). On the other hand, the growth rate of large cells seemed to be limited primarily by the availability of iron, and under this condition, they could not escape grazing control by mesozooplankton. Finally, inhibition of N 0 3 " uptake by N H 4 + may also be responsible for the persistently high N 0 3 " levels in the oceanic subarctic Pacific (Wheeler and Kokkinakis, 1990). The effect of N H 4 + on N 0 3 " uptake wi l l be discussed in Chapters 2 and 4 of this thesis. The combination of all these factors results in a system with low phytoplankton biomass year round. Another hypothesis put forward to explain the low phytoplankton biomass despite the persistence of high N 0 3 " in H N L C regions is S i (OH) 4 limitation. Dugdale and Wilkerson (1992), and Dugdale etal. (1995) proposed that the availability of dissolved silicon may reduce diatom productivity in the equatorial Pacific where S i (OH) 4 concentrations were low (Pefla etal., 1990). In the subarctic Pacific, S i (OH) 4 levels were higher than in equatorial Pacific waters, and S i (OH) 4 : N 0 3 " ratios were always higher than unity; hence S i (OH) 4 is probably not a limiting factor for diatom growth in subarctic waters. Most of the studies described above investigated the limiting factors of phytoplankton biomass in the vicinity of P26. However, because of the similarities in the distribution of Chi a along the transect, the same conclusions can probably be extended to at least P20, but a different scenario may be expected for stations P16 to P4. During September 1992 and May 1994, surface nitrogen depletion at the stations closer to the coast indicated high phytoplankton activity, but Chi a values were still low. These results may indicate that iron limitation was not the cause of the low phytoplankton biomass at P16, P12 and P4 (see L a Roche etal, 1996), but maybe a combination of N 0 3 " limitation and grazing by zooplankton. The presence of an active heterotrophic assemblage at the eastern end of the transect was most likely the reason for the higher P N concentrations measured in surface waters at stations P4 to P16 at least during May 1994. Doherty 56 (1995) found the highest depth integrated (100-1% I J abundance and biomass of heterotrophic nanoflagellates at P4 during May 1994, and Wen (1995) reported that at all stations, integrated (0-100 m) mesozooplankton biomass was higher in 1994 than in 1993. Thus, low Chi a levels were characteristic of the entire region; however, the limiting factors seem to be different along Line P. Chlorophyll a concentrations along Line P presented different patterns for 1993 and 1994. Chlorophyll a was lower in February 1994 than in March 1993. Based on observations of the weather conditions between the winter cruises, it was noticed that March 1993 had higher solar irradiance and calmer conditions compared to February 1994. The weather conditions encountered during February 1994 were probably more representative of winter in comparison to March 1993. A s for the differences between the May cruises, a simplistic explanation may be that May is a transitional month between winter and spring; thus variable environmental conditions (e.g. irradiance, wind mixing) from year to year may determine the early start or the delay of the 'phytoplankton bloom'. Marked interannual variability in phytoplankton biomass was also observed by Boyd and Harrison (submitted) for Line P during the period 1992-1997, part of which overlapped with the present study. Year-to-year variability was large at P4, and decreased with distance offshore (Boyd and Harrison, submitted). P h y t o p l a n k t o n a s s e m b l a g e s Phytoplankton cells < 5 pim numerically dominated the entire region along Line P. Results obtained by Doherty (1995) for the same cruises covered by this thesis showed that the 2-5 pim sized cells dominated the abundance and biomass of autotrophic nanoflagellates at all stations during winter and spring. His studies did not include the September period. During February 1994, the 2-5 pim cells represented > 90% of the total autotrophic nanoflagellate abundance and 70-80% of the biomass, while the 5-10 pim and 10-20 pim cells represented about 10 and 2% of the total abundance and 30 and < 10% of total biomass, respectively (Doherty, 1995). During May 1994, the 2-5 pim size class accounted for 47-87% of total autotrophic nanoflagellates 57 abundance and 10-90% o f the biomass; however , the 5-10 pirn cells also made substantial contributions at certain depths (4-70%; Doher ty , 1995). The abundance o f autotrophic nanoflagellates measured by Doher ty (1995) was not a lways consistent w i th the abundance data presented i n this chapter. The inconsistency between the two studies was most l i k e l y due to the different methodology employed for identif ication and enumeration o f groups and species: epifluorescence mic roscopy (Doherty , 1995) vs. l ight mic roscopy (this study). Phytoplankton assemblages dominated by smal l cells have already been reported by Parsons (1972) and A n d e r s o n etal. (1977) for different regions i n the N E subarctic Paci f ic . M o r e recently, B o o t h (1988) and B o o t h etal. (1988) showed that cel ls < 5 pirn averaged ca. 6 7 % , and cells < 2 pirn, 28 -34% o f phytoplankton biomass at P 2 6 dur ing M a y and Augus t o f 1984. Odate (1996) also found that cells < 2 pirn were dominant i n most o f the G u l f of A l a s k a . B o o t h (1988) and B o o t h etal. (1993) reported that the < 2 ]Am samples were composed almost exc lus ive ly o f Synechococcus spp . A m o n g the 2-5 pirn Prymnesiophyceae, Emiliania huxleyi was observed i n h igh concentrations (3 x 10 s cells L " 1 ; B o o t h etal., 1982); however , Phaeocystispouchetti was one order o f magnitude more abundant (Boo th etal., 1982; B o o t h etal., 1993). T a y l o r and Waters (1982) also reported h igh numbers o f E. huxleyi at P26 . S o m e o f the species identif ied by other researchers were not observed i n this study. B o o t h and her colleagues used not on ly l ight microscopy, but also epifluorescence, scanning electron microscopy and i n the most recent studies, t ransmission electron microscopy for species identif ication. Cons ider ing the l imitat ions o f l ight mic roscopy , the results o f this thesis are reasonably consistent w i t h previous studies. T h e slight increases in C h i a observed dur ing this study cou ld have been the result o f increases i n the contr ibut ion o f diatoms to the total assemblage o f phytoplankton. F o r example, C h i a values o f ca. 0.5 pig L " 1 ma in ly at P26 , but also at P4 , dur ing M a y 1993 co inc ided wi th a higher contr ibut ion o f pennate diatoms to the total phytoplankton assemblage. D u r i n g M a y 1994, stations P 4 and P 2 0 showed C h i a "patches" o f 0.3-0.5 pig L 1 and also a higher contribution o f pennates relative to other stations. S i m i l a r features are observed at P 1 6 dur ing September 1992, and at P 2 6 dur ing February 1994. Horner and B o o t h (1990) also found that among the diatoms, 58 pennates were more abundant than centric diatoms in the N E subarctic Pacific. Booth etal. (1993) showed that the large diatoms (> 20 pim) only represented 6% of the total phytoplankton biomass, but could sporadically contribute significantly to the phytoplankton stocks in this region (Clemons and Mil ler , 1984; Takahashi, 1986; Takahashi etal, 1990). Results from the N W subarctic Pacific also showed that increases in the typically low total Chi a (< 1 pig L" 1) were attributed to higher contributions by diatoms (> 10 pim; Odate, 1996; Odate and Maita, 1988/1989). In a review of the literature on size fractionated chlorophyll, Chisholm (1992) indicated that the contribution of diatoms to the total chlorophyll of a system which is normally characterized by low total phytoplankton biomass was very important. However, the higher autotrophic biomass observed here during May 1993, may not have been entirely due to slightly higher diatom contributions, since Doherty (1995) documented a higher abundance and biomass of autotrophic nanoflagellates at most stations during May 1993 than during May 1994. S U M M A R Y The euphotic zone along Line P showed similar chemical and biological characteristics. However, i f a distinction is to be made between 'more' or 'less' oceanic stations, the transition during this study must have been in the vicinity of P16, mostly evident in the N 0 3 \ S i (OH) 4 and H P 0 4 2 " distributions. P4 was the most different of all the stations, with the shallowest euphotic zone and lowest surface nutrient concentrations during spring and late summer. In contrast, P26 showed the deepest euphotic zone and persistently high macronutrient concentrations. Although Chi a concentrations were low along the entire region, the limiting factors during spring and summer were different along the transect. During the period of this study, a combination of iron limitation and high grazing pressure may have been responsible for the low phytoplankton biomass from P26 to P20/P16. In contrast, a combination of macronutrient (mainly N0 3 ") limitation and grazing pressure may have resulted in low phytoplankton stocks from P16/P12 to P4. 59 CHAPTER 2 S E A S O N A L V A R I A B I L I T Y IN N I T R O G E N O U S N U T R I T I O N O F N A T U R A L P H Y T O P L A N K T O N A S S E M B L A G E S IN T H E N E S U B A R C T I C P A C I F I C O C E A N I N T R O D U C T I O N The importance of regenerated nitrogen forms to the nitrogen nutrition by phytoplankton has been extensively documented (e.g. Carpenter et al., 1972; McCarthy, 1972a,b; McCarthy et al., 1977; Dortchera/., 1982; Cochlan and Harrison, 1991c; and see reviews by Paul, 1983; Dortch, 1990; Antia etal., 1991). Oligotrophic and H N L C regions rely on regenerative processes most of the year due to low availability of total nitrogen or some limitation on nitrate uptake. Other ecosystems, such as upwelling (e.g. Kudela etal, 1997), coastal (e.g. Kanda etal, 1990), offshore (e.g. Tamminen, 1995), and even marginal ice zones (e.g. Goeyens etal., 1995) seem to switch from nitrate-based systems at the beginning of the phytoplankton bloom to ammonium/urea-based systems as the bloom progresses and regenerated nitrogen becomes more abundant. Therefore, regenerated nitrogen forms are the basis of the nitrogenous nutrition of phytoplankton from many marine systems, regardless of the presence of new nitrogen. The nitrogenous nutrition of phytoplankton from the N E subarctic Pacific has been investigated by Wheeler etal. (1989), Wheeler and Kokkinakis (1990), Cochlan etal. (1991a), and Wheeler (1993); however these studies were limited to the oceanic region, and only covered the spring-summer periods. Thus, it is important to expand upon these investigations in space and time to more fully understand the nitrogen dynamics of H N L C regions and the role of these areas in biogeochemical cycles. 60 The present study is the first to report the uptake rates of inorganic and organic nitrogen by phytoplankton along a transect in the N E subarctic Pacific and to provide an estimate of new and regenerated primary production in a region extending eastward from station P26. It is also the first one to report winter values. The main goals of this chapter are to show: (1) the interannual, seasonal and spatial variability in the uptake rates of nitrate, urea and ammonium, (2) the relative proportions of new and regenerated primary production, and (3) the nitrogen preferences by natural assemblages of phytoplankton in the N E subarctic Pacific Ocean. M A T E R I A L S A N D M E T H O D S Nitrogen uptake rates were measured on water samples collected at the depths (100,55, 30, 10, 3.5 and 1% of I c), stations and cruises described in Chapter 1. Water samples were transferred to acid cleaned carboys, and subsamples obtained for chemical and biological measurements (presented in Chapter 1) as well as for nitrogen uptake experiments (presented here) at each depth. Nitrogen tracer experiments Nitrogen uptake rates were measured using the stable isotope 1 5 N as a tracer (Ness etal., 1962, Dugdale and Goering, 1967; Harrison, 1983a). A t each depth, three separate unfiltered water samples were collected from the Go-Flo sampler for nitrogen uptake experiments in 1 L polycarbonate Nalgene® bottles. Water samples were maintained under low light conditions during every manipulation preceding the start of the incubation period. Samples were inoculated with 1 S N labeled N a N 0 3 , urea or N H 4 C 1 (Cambridge Isotope Laboratories, 99 atom % 1 S N enriched) soon 61 after collection, and placed under neutral density screening to simulate the irradiance from which the samples were collected. The irradiance levels under the screens were precalibrated with a Biophysical Instruments Inc. light meter, model Q S L 100. The curves for incident surface irradiance during the incubations are presented in Appendix D for all Line P stations during March and May 1993, and M a y 1994 (Fig. D . 1). Incubations proceeded for 24 h in a temperature controlled on-deck incubator. Temperature was maintained at surface seawater values (see Table 1.2, Chapter 1) by continuous circulation of surface seawater. Incubations were terminated by gentle vacuum filtration (pressure < 125 mm Hg) through pre-combusted Whatman® G F / F filters. Filters were folded, placed in petri dishes and maintained at -20°C until analysed ashore. Isotopic additions were performed at low (generally trace) levels, at ca. 10% of ambient N0 3 " , urea or N H 4 + when ambient concentrations were above 0.5 pig-at L" 1 . A t most depths, however, ambient urea and N H 4 + concentrations were below 0.5 jAg-at L" 1 (see Figs. 1.4, 1.5 and 1.6 in Chapter 1). In those cases, 1 S N additions were made at the limit of detection (0.05 - 0.1 pig-at L" 1 ) ; thus, inoculations were not at trace levels. In most cases, nitrogen concentrations were determined before the start of the incubations, hence the isotopic additions were calculated based on these initial values. When the nitrogen concentrations were not yet available at the beginning of the incubations, tracer additions were estimated from previously collected data for the depth, station and cruise in question. Due to the length of the incubation period, nitrogen depletion was a concern. Two procedures were followed to check for nitrogen exhaustion. A t the end of the incubation period, N 0 3 , urea or N H 4 + concentrations were measured in the filtrate (through GF/F) of the 1 5 N 0 3 " , 1 5 N-urea and 1 S N H 4 + incubation bottles, respectively. Complete depletion of dissolved nitrogen at the end of the incubation was never observed. Another approach to check for nitrogen exhaustion is the calculation of the theoretical maximum atom % 1 S N that would be found in the particulates if all 1 S N added is taken up during the incubation. Based on the amount of tracer added, and the initial dissolved and particulate nitrogen concentrations, it was calculated that on average (± 1 S.E.) 10.1 ± 0.6% (n = 226), 19.0 ± 1.0% (n = 188) and 35.7 ± 1.0 % (n = 225) of dissolved 62 1 5 N 0 3 \ l s N - u r e a and 1 5 N H 4 + , respectively, were taken up during the incubation period. Even during periods of high rates of uptake (only at a few depths at P4 during September 1992, and May 1993 and 1994), never more than 85% was incorporated into the particulates. Thus, substrate exhaustion did not occur during these experiments. A t station P26 during September 1992, a one-time experiment was undertaken in order to compare N 0 3 " and N H 4 + uptake rates determined on unfiltered (entire community) and pre-filtered (116 pirn; without large grazers) seawater (see Appendix E). Measurement of atom % 1 5 N and calculation of nitrogen uptake rates The atom % 1 5 N in the particulates was measured by emission spectrometry (Fiedler and Proksch, 1975; Preston, 1993). Firstly, G F / F filters containing the sample were dried for 24-48 h at 60°C, and then the nitrogen in the particulates was converted into N 2 by the modified Dumas dry-combustion technique (Fiedler and Proksch, 1975; Preston, 1993). Finally, the 1 4 N / 1 S N ratio was determined with a J A S C O model N-150 emission spectrometer. The 1 4 N / 1 S N ratio for each sample was determined from the mean of duplicate combustion tubes prepared from the same filter sample. The 1 4 N / 1 S N ratio from each combustion tube was determined from the mean of at least three peak scans. Nitrogen uptake rates were calculated according to Dugdale and Wilkerson (1986) as summarized below. Absolute uptake rates (p N , N uptake in concentration units per unit time) were calculated from the constant transport model based on the particulate nitrogen concentration at the beginning of the incubation (equations (7) and (4) from Dugdale and Wilkerson (1986)): 0 = y * p \r where, V0 is the nitrogen-specific uptake rate calculated as: 63 V = -° C5N„-uiNl)*T 1 5 N X S is the atom % 1 S N excess in the sample calculated as: 1 5 = 1SNS - F 1 S N S is the atom % 1 S N in the sample F is the natural abundance of 1 5 N (0.365%) 1 5 N e n r is the atom % 1 5 N in the initially labeled fraction calculated as: [added 1 5TV* 0.99 + ambient N * 0.00365) added N + ambient N The '0.99' factor refers to the atom % 1 S N enrichment (99%) of the initially added isotope. T is the incubation time P N 0 is the particulate nitrogen concentration at the beginning of the incubation period. On a number of occasions, the P N was also measured at the end of the incubation, and showed no detectable changes from P N D . Chlorophyll a-specific uptake rates were calculated as p N /Chi a ( N taken up per unit Chi a per unit time). Chlorophyll a was also routinely measured at the beginning of the incubation period. However, a number of tests were run where Chi a was also measured at the end of the incubation period, and very little change, i f any, was observed. Nitrogen (N) -specific uptake rates ( V N , N taken up per unit particulate N per unit time) were calculated according to a constant specific uptake model (equation (6) from Dugdale and Wilkerson (1986)): where all symbols are as above. In the cases when ambient urea and N03" concentrations were undetectable (see Figs 1.4, 1.5 and 1.6 in Chapter 1), a value of zero was used as the 'ambient N ' for uptake calculations. * l n (15N -15 N ) V enr s J (Nenr-F) 64 Thus, the rate value obtained represents a conservative estimate of nitrogen uptake (Eppley etal., 1977). Nitrogen-15 uptake rates were not corrected for the simultaneous uptake of unlabelled nitrogen ( 1 4 N) sources, since no data were available for such a correction. Therefore, all rates are probably slightly underestimated (Dugdale and Wilkerson, 1986; Collos, 1987). Isotope dilution by remineralization of 1 4 N-urea or 1 4 N H 4 in the 1 5 N-urea or 1 S N H 4 + uptake experiments, respectively, was not estimated during this study, hence uptake rates were also not corrected for this effect. Isotope dilution could also contribute to underestimation of the rates (Harrison, 1983a; Dugdale and Wilkerson, 1986) Growth rate (//N) was calculated as V N / In 2 (div d"1). Due to the length of the incubation period (24 h), the term "uptake" used in this and the next chapters includes not only the transport of nitrogen through the membrane, but also the assimilation into organic metabolites and incorporation into macromolecules (i.e. growth; Collos and Slawyk, 1980; Goldman and Glibert, 1983; Wheeler, 1983). It is possible that part of the assimilated 1 S N was released back into the medium as dissolved organic 1 5 N ( D 0 1 S N ; see "Discussion"); thus, the calculated rates are considered net uptake rates. Calculation of indices of nitrogen nutrition Three methods were utilized to assess the relative importance of N 0 3 , urea and N H 4 + for phytoplankton nitrogen nutrition in the N E subarctic Pacific: (1) the / - rat io , (2) the relative preference index, and (3) the slope of the linear regression of the/-ratio vs. the nutrient concentration ratio. (1) The fraction of new production (due to N0 3~) to total production, / -ratio, was calculated according to Eppley and Peterson (1979) as: / - ratio = pNOj 65 where: p N 0 3 , purea, p N H 4 + are the absolute uptake rates of N0 3 ~, urea and N H 4 + , respectively, The/-ratio was also calculated by excluding purea from the denominator. (2) The relative preference indices (RPI) for N 0 3 " , urea and N H 4 + were calculated by comparing the rate of uptake (p N ) relative to availability by the method of McCarthy etal. (1977): pN03/ _ /(pNOj + purea + pNH4+ ) «riN03 - iNQ-y / ( [ M V ] + [urea] + [NH4+]) purea/ _ /pNQ3' + purea + pNH4 ) K™ure« - [ureay /([ NOf ] + [urea] + [NH4+ ]) PNH;/ npj / (pMV + purea + pNH4) /([NOf] + [urea] + [NH4 ]) where, pN0 3 " , purea, p N H 4 + are as above, and [ N 0 3 1 , [urea] and [NH 4 + ] are the concentrations (ambient + tracer additions). RPI values of unity indicate that uptake is proportional to availability; values above unity reflect preference and values below unity indicate discrimination (McCarthy etal., 1977). (3) Another indicator of the preference of phytoplankton for one nitrogen source or another is the slope of the linear regression of the/-ratio vs. the corresponding nutrient concentration ratio (Harrison etal., 1987). This method is equivalent to the RPI of McCarthy et al. (1977), but it is preferred by some investigators (e.g. Dortch, 1990; Tamminen, 1995). Slopes more than 1 indicate a preference for N 0 3 " , and slopes less than 1 indicate a preference for 66 regenerated nitrogen forms ( N H 4 + and/or urea). This relationship was studied for three different cases: (a) p N C V / (pN0 3 " + purea + p N H 4 + ) vs. [NO," / (NO," + urea + N H 4 + ) ] (b) p N 0 3 7 (pN03 + p N H 4 + ) vs. [N0 3 " / (N0 3 " + N H 4 + ) ] (c) p N 0 3 " / (pN0 3 " + purea) vs. [N0 3 " / ( N 0 3 " + urea)] where all symbols are as above. Statistical analyses Replication of water samples for 1SN uptake rate experiments Replication of complete vertical profiles was not routinely conducted because of time limitations in cruise schedules and the labor-intensive nature of the 1 S N technique. Routinely, a single water sample was collected from each light depth from which the three 1 S N incubations were done. However, during most cruises at P26, and occasionally at other stations, a number of replicate casts were taken from selected depths. Incubations were then carried out for 1 5 N 0 3 " , 1 S N -urea and 1 S N H 4 + from replicate water samples in order to examine the natural variability in uptake rates (Table 2.1). A complete set of replicates for the uptake rates of the three nitrogen sources for the entire vertical profile or at the surface is not available at P26 for every cruise (as was the case in Chapter 1 for chemical and biological measurements); thus statistical comparisons between cruises are not possible in this case. Therefore, the analysis of the data is based on the observation of trends. However, the variability in specific and absolute nitrogen uptake rates reported in Table 2.1 provides an estimate of the significance of the differences between the rates presented in this and the next Chapters. Precision of the 15N tracer technique Routinely, single samples were collected from the Go-Flo bottles for 1 5 N 0 3 " , 1 5 N-urea and 1 S N H 4 + incubations. During most cruises, however, subsamples (2 or 3) were taken from the 67 Table 2.1. Natural variability in absolute, and N- and Chi a -specific uptake rates for nitrate, urea and ammonium. Replicate tracer experiments were performed on samples taken from different bottle casts obtained in a 1 to 4 day period mainly at P26 during every cruise. Errors are expressed as the mean coefficient of variation (C.V., %) and mean standard deviation (S.D.) of groups (n) of replicates (2 to 7). Units for mean S.D. are: ng-at N L"1 d"1 for p N , d"1 for V N , and pig-al N (pig Chi a)1 d'1 for pN/Chl a Uptake rate Mean C.V. Mean S.D. (%) pN03" 26 10.33 13 VN0 3" 24 0.009 13 pN0 3 7Chla 30 0.047 13 purea 32 11.59 7 Vurea 31 0.010 7 purea/Chla 28 0.068 7 p N H 4 + 14 11.30 14 V N H 4 + 13 0.009 14 pNH 4 + /Chla 17 0.054 14 68 same Go-Flo bottles for 1 S N 0 3 ~ , 1 5 N-urea and 1 S N H 4 + incubations at selected stations and depths, in order to obtain an estimate of the precision of the 1 5 N technique. Appendix B presents a list of the analytical errors (as the mean coefficient of variation and mean standard deviation) associated with the measurement of absolute and specific uptake rates (Table B.2). RESULTS Depth profiles of nitrogen uptake rates and /-ratios The vertical profiles of absolute uptake rates of N0 3 ~, urea and N H 4 + (ng-at N L" 1 d"1) are shown for Line P stations for the winter (Fig. 2.1), spring (Fig. 2.2), and late summer (Fig. 2.3) cruises. For every station and cruise, uptake rates of the three nitrogen sources decreased with depth. Nitrogen uptake rates were lower at the 1% light level than at the surface, but always detectable. Ammonium uptake rates were always higher than N 0 3 " and, in most cases, higher than urea uptake rates throughout the euphotic zone. However, urea was taken up faster than N H 4 + at some depths, principally during the May 1993 cruise, e.g. at P16, P20 and P26 (Fig. 2.2 A ) , and the May 1994 cruise, e.g. P4 (Fig. 2.2 B) . Absolute uptake rates of NO a " , urea and N H 4 + are also presented in contour plots for March and M a y 1993 (Fig. 2.4), February and M a y 1994 (Fig. 2.5), and September 1992 (Fig. 2.6). During every cruise, the highest surface rates of N H 4 + uptake were measured at P4; this pattern was most clearly seen during May 1994 (Fig. 2.5 F). During May 1994, surface uptake rates of urea were also high at the coastal end of the transect, and were the highest urea uptake rates measured during this study (Fig. 2.5 D). However, surface urea uptake rates did not always exhibit consistently higher values at the eastern end of the transect, e.g. the highest values were observed towards P26 during May 1993 (Fig. 2.4 D), February 1994 (Fig. 2.5 C) and September 1992 (Fig. 2.6 B) . Surface N 0 3 " uptake rates did not exhibit a consistent longitudinal trend (Figs. 2.4 and 2.5, A & B , and 2.6 A ) . o o o o »/-> o o o o o o o in o >n o o o in < PQ • • . . a . g : : : : ^ : : : : : : : : : " : : : : : : : : : | : : : : • km...... # - ® <&••<] " - • ::^ -:::::5^ -r1 i 1 i 1 i 1 i 1 1 I 1 I I | I | I I C l — EC - a! • «k«::3V:V::a--.:-.::::::::----••e -%•!:':::::':':' - • » '•••«•••=:::::::#:::::! 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CD JD 3 S D- « CD CD 0 g> -° 5 O co S o 1 1 DHCU, CD C . —_i • i-H tn J <U ^  >< ^ o C CD 3 . 2 2 00 3 PU O o CN o NO o 00 o CN o NO o 00 (Ul) q jdQQ £ 6 d H S P6 dHS Mar 93 72 May 93 A . p N 0 3 ~ B . p N 0 3 ~ ()• 20 -g fl 4 0 -6 0 -8 0 -C . purea C L , ai a • • • • : • • • • <^40'-- • • • • '•••••• ••..20 * • ••••.'.'.'.20 • • • • <20 • • • • > : < 4 o f 80«J 9n • • • • • D . purea E . p N H 4 + F . p N H 4 + 130 145 140 Longi tude ( ° W ) 135 r o 1 i 1 1 1 i 1 120 240 1 I 1 360 480 130 p ^ n g - a t N U 1 d ' 1 ) F igure 2.4. Contour plots showing absolute uptake rates o f ( A , B ) nitrate, ( C , D ) urea, and ( E , F ) a m m o n i u m dur ing M a r c h and M a y 1993. S o l i d l ines indicate contour intervals o f 4 0 ng-at N L " 1 d" 1 ; dotted lines are intervals o f 20 ng-at N L " 1 d " . S o l i d c i rc les (•) indicate sampl ing depths. ( ) • 20-4 0 6 0 80-80 0 —i F e b 9 4 A. p N 0 3 Y • /••- - • • \40 *s * ' ,;'20 • <20 • • • • • • • • 1 C . 1 1 purea 1 t ....V.'.'.'.'.v*.-.-.20 • • • • • • • • • • <20 • • • • 1 E . l I p N H 4 + 1 • • * ^ • • • • M a y 9 4 B . p N Q 3 " 145 140 135 130 145 140 Longi tude ( ° W ) 135 130 I 1 1 1 I 1 0 120 240 360 1 1 I 480 P N (ng-at N U ' d " 1 ) F igure 2.5. Contour plots showing absolute uptake rates o f (A, B ) nitrate, ( C , D ) urea, and ( E , F) a m m o n i u m dur ing February and M a y 1994. S o l i d l ines indicate contour intervals o f 4 0 ng-at N L " 1 d" 1 ; dotted l ines are intervals o f 20 ng-at N L " 1 d" \ S o l i d c i rc les (•) indicate sampl ing depths. 74 Sep 92 A . p N 0 3 ~ 0 20 > ' 4 0 OH Q 6 0 80 0 20 4 0 60 80 145 140 135 130 Longitude (°W) "~I 1 120 240 ' 1 I 360 480 p N (ng-at N L - ' d " 1 ) F igure 2.6. Contour plots showing absolute uptake rates o f ( A ) nitrate, (B) urea, and (C) a m m o n i u m dur ing September 1992. S o l i d l ines indicate contour intervals o f 4 0 ng-at N L " 1 d" ; dotted lines are intervals o f 20 ng-at N L " 1 d" 1. S o l i d circles (•) indicate sampl ing depths. 75 The vertical profiles of/-ratios (including the three nitrogen sources) showed little change with depth, station or cruise (Fig. 2.7). The average (+ 1 S.E.) for the/-ratio was 0.23 + 0.01 (n = 156), ranging from 0.02 to 0.67, throughout the euphotic zone for the entire transect. The minimum value was calculated at the surface and the maximum at the 1% light depth; however, an increasing trend from surface to depth was not consistently observed at every station. Depth integrated nitrogen uptake rates and / -ratios Uptake rates ofN03, urea and NH/ Depth integrated uptake rates showed that N H 4 + was always taken up faster than N0 3 ~, and almost always faster than urea at every station and cruise (Tables 2.2 and 2.3, and Fig. 2.8). The only cases when urea integrated uptake rates were higher than N H 4 + were at P16 during March and May 1993, and P20 during May 1993 (Table 2.2 and Fig. 2.8). Seasonal changes were evident from winter to spring for the depth integrated uptake rates of all three nitrogen sources (Tables 2.2 and 2.3). Nitrate, urea and N H 4 + were taken up faster during the spring period compared to the winter. Depth integrated uptake rates for the three nitrogen sources were variable during September, with values ranging from lower than winter to higher than spring along the transect (Tables 2.2 and 2.3). Longitudinally, the highest depth integrated absolute N 0 3 " uptake rates occurred at P20-P26 during most cruises (Tables 2.2 and 2.3). Depth integrated urea uptake rates did not show a consistent longitudinal trend from cruise to cruise, while depth integrated absolute N H 4 + uptake rates showed higher values at P4 during most cruises (Table 2.2 and 2.3). f-ratios, new and regenerated primary production The depth integrated/-ratio did not show a consistent seasonal trend (Table 2.2). It ranged from 0.05 to 0.37 with an average (± 1 S.E.) of 0.21 ± 0.02 (n = 25) for all stations and cruises (Table 2.2). Thus, p N 0 3 " contributed between 5 to 37% with an average of 21 + 2% to the 76 ON ON ON 5 ON PJ ON PJ J3 -t—» OH CD Q 0-20-40-60-80-/ - ratio 0.0 0.5 0.0 0.5 . 0.0 0.5 0.0 0.5 0.0 0.5 I • • • • I • • I • • • • I • • I i i i i I • I • • • • I 0 -20-40-60-• • • 4 • - ! 4;' • * - •r • 4 • • • 80- • • • '» 0 -20 -1 , , , , 1 , , 1 i i i i 1 i i 1 . , . . 1 , . 1 . . . . 1 . . • • • * • • • 40 - • m • • 60 - • 4. 4 80 - P23A 4 B 20-40 • 60 • 80 • I i i i i I i i "I I I • . . . I I . . . . I I i . . . I • • 3? I i 0 - y 20- • 40- * 60 - • 80-0 - i • 4 20- 4 40 - • 60 -80 - 4 I • . . . I 4 I • . . . I I . . . . I D • 1 1 1 1 1 1 i . . . . i • 1 1 1 1 1 1 4 * P 2 0 P 1 6 P 1 2 P 4 P 2 6 F igure 2.7. Ve r t i c a l profiles o f / - r a t i o s for L i n e P stations dur ing ( A ) M a r c h 1993, (B) February 1994, (C) M a y 1993, (D) M a y 1994, (E) September 1992, and (F) September 1994. 77 Table 2.2. Depth integrated (100-1% I o) absolute uptake rates (mg-at N m 2 d"1) of nitrate, urea and ammonium, and depth integrated/-ratios for Line P stations during all cruises. Values shown for P26 in February 1994 corresponds to P23A. Seasonal means for winter and spring were obtained by averaging the two winter and the two spring cruises, respectively. Dashed line (-) indicates that data are not available. N uptake rate Station Cruise Seasonal Mean winter spring late summer winter spring Mar-93 Feb-94 May-93 May-94 Sep-92 Sep-94 pNCY P4 0.67 0.93 1.82 1.30 2.38 - 0.80 1.56 P12 0.73 0.79 1.39 1.94 0.27 - 0.76 1.66 P16 0.54 1.01 1.44 2.69 2.88 - 0.77 2.06 P20 1.04 - 1.59 4.95 1.59 - 1.04 3.27 P26 1.06 2.49 3.07 2.29 3.19 4.54 1.78 2.68 p urea p NH 4 + P4 1.30 0.31 3.03 9.17 0.29 - 0.81 6.10 P12 0.77 1.04 2.11 2.24 0.51 - 0.91 2.18 P16 1.74 1.52 4.59 1.25 2.98 - 1.63 2.92 P20 0.41 - 6.34 0.94 2.65 - 0.41 3.64 P26 0.34 1.57 4.57 2.13 3.88 3.28 0.96 3.35 P4 4.59 2.45 6.18 13.27 5.94 _ 3.52 9.73 P12 1.61 2.27 5.96 4.96 2.45 - 1.94 5.46 P16 1.53 2.28 4.02 5.79 7.30 - 1.90 4.90 P20 3.56 - 3.39 7.46 7.49 - 3.56 5.42 P26 2.37 4.08 5.11 5.76 5.85 5.75 3.23 5.43 f -ratio P4 0.10 0.25 0.16 0.05 0.28 - 0.18 0.11 P12 0.24 0.19 0.15 0.21 0.08 - 0.21 0.18 P16 0.14 0.21 0.14 0.28 0.22 - 0.18 0.21 P20 0.21 - 0.14 0.37 0.14 - 0.21 0.26 P26 0.28 0.31 0.24 0.22 0.25 0.33 0.29 0.23 78 Table 2.3. Dep th integrated (100-1% I o ) N-spec i f i c (d"1) and C h i a-specific uptake rates (^g-at (ptg C h i a)"1 d"1) for nitrate, urea and a m m o n i u m for L i n e P stations dur ing a l l cruises. Va lues shown for P 2 6 i n February 1994 correspond to P 2 3 A . Seasonal means for winter and spring were obtained by averaging the two winter and the two spring cruises, respectively. N uptake rate Station Cruise Seasonal Mean winter Mar-93 Feb-94 spring May-93 May-94 late Sep-92 summer Sep-94 winter spring . P4 0 014 0 020 0.036 0.015 0.029 0 017 0 025 P12 0 023 0 023 0.031 0.028 0.006 - 0 023 0 030 V N 0 3 P16 0 016 0 020 0.027 0.031 0.024 - 0 018 0 029 P20 0 026 - 0.029 0.056 0.014 - 0 026 0 042 P26 0 024 0 029 0.040 0.032 0.028 0.049 0 027 0 036 P4 0 026 0 005 0.060 0.088 0.004 0 016 0 074 P12 0 024 0 031 0.048 0.033 0.009 - 0 027 0 040 V urea P16 0 049 0 029 0.080 0.014 0.026 - 0 039 0 047 P20 0 009 - 0.099 0.011 0.023 - 0 009 0 055 P26 0 007 0 018 0.058 0.029 0.032 0.034 0 013 0 044 P4 0 084 0 045 0.118 0.135 0.119 _ 0 064 0 127 P12 0 049 0 067 0.126 0.071 0.041 - 0 058 0 098 V N H 4 + P16 0 042 0 044 0.073 0.072 0.063 - 0 043 0 072 P20 0 074 - 0.060 0.083 0.063 - 0 074 0 071 P26 0 051 0 047 0.067 0.078 0.050 0.060 0 049 0 072 p N0 3 7Chl a p urea/Chi a p NH 4 + /Chl a P4 0.029 0.113 0.087 0.103 0.102 - 0.071 0.095 P12 0.027 0.099 0.082 0.171 0.034 - 0.063 0.127 P16 0.038 0.111 0.120 0.127 0.073 - 0.075 0.124 P20 0.042 - 0.094 0.218 0.043 - 0.042 0.156 P26 0.072 0.197 0.123 0.163 0.091 0.202 0.134 0.143 P4 0.052 0.031 0.146 0.683 0.013 _ 0.042 0.414 P12 0.029 0.131 0.126 0.196 0.061 - 0.080 0.161 P16 0.116 0.161 0.392 0.059 0.080 - 0.138 0.225 P20 0.015 - 0.403 0.042 0.073 - 0.015 0.222 P26 0.021 0.123 0.252 0.157 0.099 0.140 0.072 0.205 P4 0.164 0.261 0.299 0.871 0.367 0.213 0.585 P12 0.059 0.283 0.359 0.435 0.236 - 0.171 0.397 P16 0.106 0.244 0.329 0.283 0.191 - 0.175 0.306 P20 0.135 - 0.206 0.310 0.205 - 0.135 0.258 P26 0.152 0.321 0.202 0.416 0.163 0.247 0.237 0.309 79 A. Mar 93 B . Feb 94 P26 P20 P16 P12 P4 P26 P20 P16 P12 P4 Station • N O 3 - 13 Urea • N H 4 + Figure 2.8. Contribution of the depth integrated absolute uptake rate of different nitrogen sources to the total nitrogen absolute uptake rate along Line P for (A) March 1993, (B) Feburary 1994, (C) May 1993, (D) May 1994, (E) September 1992 and (F) September 1994. 80 total depth integrated p N uptake (Fig. 2.8). The purea contribution was between 3 and 56% to the total integrated p N uptake, with an average (± 1 S.E.) of 24 ± 3%, and the p N H 4 + contribution ranged from 30 to 76% to the total integrated p N uptake, with an average (+ 1 S.E.) of 55 ± 2% (Fig. 2.8). Therefore, p N H 4 + had the highest contribution, followed by purea and pN0 3 ~. However, the contribution of purea and p N 0 3 " were not significantly different (r-test, P > 0.25). Longitudinally, the / - ra t io (and hence the % new production) showed slightly higher values at the western end of the transect for all cruises except for September 1992 (Table 2.2 and Fig . 2.8). The degree of overestimation of the / -ratio when purea was excluded from the calculation ranged from 4 to 130%, with an average of 36 ± 6% (± 1 S.E., n = 25; Fig . 2.9). The largest disagreement was found in May 1993, when urea was taken up at high rates compared with N 0 3 " and N H 4 + . New and regenerated production at P26 A t P26, the contribution of new production to total nitrogen production was slightly higher in winter (28 and 31% for March 1993 and February 1994, respectively) than in spring (24 and 22% for May 1993 and 1994, respectively; Table 2.2 and Fig. 2.8). The contribution of new production during the September cruises, however, fluctuated from 25 to 33% for 1992 and 1994, respectively (Table 2.2). Considering all cruises, the contributions to total integrated p N uptake were: 22 to 33% due to p N 0 3 " with an average (± 1 S.E.) of 27 ± 2%; 9 to 36% due to purea with an average (± 1 S.E.) of 23 ± 4%; and 40 to 63% due to p N H 4 + with an average (+ 1 S.E.) of 50 ± 4%. A t P26, p N H 4 + made the highest contribution, followed by p N 0 3 " and then purea. However, p N 0 3 " and purea contributions were not statistically different (Mest, P > 0.35). Thus, these results were very similar to those obtained when all stations were considered. Figure 2.9. Dep th integrated /-ratios calculated as p N 0 3 / ( p N 0 3 + pNFJ^ 4 ) and p N 0 3 ~ / ( p N 0 3 ~ + purea + p N F L / ) for a l l L i n e P stations and cruises plotted against each other. T h e 1:1 l ine is shown for reference. 82 Total nitrogen uptake rates Depth integrated uptake rates of total nitrogen are expressed as absolute (mg-at N rrf2 d"1), and averages for absolute (ng-at N L" 1 d"1) and N-specific (d"1) rates along Line P for each cruise (Fig. 2.10). Depth integrated averages were calculated by dividing the depth integrated rate by the depth of integration (i .e. depth of the euphotic zone). In every case, an increasing trend in depth integrated total nitrogen uptake was observed for most stations from winter to spring cruises. The seasonal trend was more evident for the absolute rates (Fig. 2.10 A & B) than for the N-specific rates (Fig. 2.10 C), suggesting that part of the spring and late summer increase in absolute uptake rates may have been due to a higher concentration of P N rather than an increase in the specific rates of uptake. Although depth integrated uptake rates of N 0 3 " , urea or N H 4 + for September were variable and did not show a seasonal trend relative to the winter or spring rates (Tables 2.2 and 2.3), depth integrated uptake rates of total nitrogen for September were intermediate between winter and spring levels for most stations (Fig. 2.10). When the integrated averages for absolute and N-specific uptake rates of total nitrogen were considered (Fig 2.10 B & C, respectively), the contribution of P4 was higher than when the integrated absolute uptake was taken into account (Fig 2.10 A ) . Because of the shallower euphotic zone generally measured at P4 (principally during May), integrated absolute uptake rates (expressed per m 2) were lower. West of P4, total nitrogen uptake rates did not show a consistent decrease or increase along the transect. Depth integrated growth rates (JAU; calculated from N-specific uptake rates of total nitrogen) for all stations and cruises varied from 0.08 to 0.34 div d"1. The seasonal averages for all stations along Line P were 0.14 ± 0.01 (± 1 S.E., n = 9) for the winter; 0.25 ± 0.02 (± 1 S.E., n = 10) for the spring; and 0.16 ± 0.02 (± 1 S.E., n = 6) for the late summer cruises. 83 Mar 93 Feb 94 M a y 93 M a y 94 Sep 92 Sep 94 Station • P26 • * P23A • P20 • P16 m P12 ED P4 Figure 2.10. Depth integrated (A) absolute uptake rates, and averages for (B) absolute and (C) N-specific uptake rates of total nitrogen for Line P stations during all cruises. Depth integrated averages (B and C) were calculated by dividing the depth integrated absolute and specific uptake rates by the depth of integration (i.e. depth of the euphotic zone). 84 Nitrogen uptake rates and ambient nitrogen concentrations Relative preference indices Relat ive preference indices were calculated for N 0 3 , urea and N H 4 + at every depth for every station and cruise, and a l l data were pooled together and plotted against total nitrogen concentration ( inc lud ing tracer addit ions; F i g . 2.11). T h e different number o f data points for RPI s o f N 0 3 \ urea and N H 4 + (147, 116 and 156, respectively) is due to the fact that i n a few instances ambient urea and N 0 3 concentrations were undetectable and, thus, R P I s cou ld not be calculated. T h e analysis o f R P I s was done w i t h data f rom a l l l ight depths since the separate analysis o f surface (100, 55 and 3 0 % ID) and deep (10, 3.5 and 1% I J samples y ie lded the same results. F igure 2.11 shows that for h igh total nitrogen concentrations (above ca. 2 /<g-at L " 1 ) the R P I s clustered i n three groups; one group be low 1 corresponding to R P I N 0 3 , and the other two groups above 1 corresponding to R P I ^ and R P I ^ . Hence, when total ni trogen was abundant, N H 4 + and urea were preferred over N 0 3 " relative to their availabil i t ies. R P I N H 4 values were the highest, indica t ing a clear preference for N H 4 + over urea. In contrast, at l o w total nitrogen concentrations, a l l R P I s fe l l around unity, indicat ing that phytoplankton d i d not have a single preferred source o f nitrogen. T h e var iabi l i ty i n total nitrogen concentration was most ly due to N 0 3 ~ , because the magnitude o f the concentration changes a long L i n e P was greater for N 0 3 " than for N H 4 + and urea (see Chapter 1). In summary, R P I s for the nitrogen sources considered i n this study showed a preference for N H 4 + , f o l l owed by urea, and no preference for N 0 3 " i n most cases. f-ratios vs. nutrient ratios Figure 2 .12 presents the analysis o f / -ratio vs. nutrient concentration ratio i n three cases: (a) when a l l three ni trogen sources are considered, (b) when urea is excluded, and (c) when N H 4 + is exc luded f rom the calculations. Note that s imple l inear regressions were appl ied to these data on ly for comparat ive purposes between the different relationships. T h e least squares regression fit to the data (n = 156 i n a l l cases) showed a preference for regenerated nitrogen forms (urea and 85 Figure 2 .11. Re la t ive preferences indices (RPIs) for nitrate, urea and a m m o n i u m calculated at a l l depths, stations and cruises. To ta l ni trogen concentrat ion includes tracer addit ions. 86 2 + 03 CD i-l + o 2 Q. 1.0-0.8-0.6 • y = 0.04 + 0.23 x r = 0.42 n= 156 N 0 3 ~ / ( N 0 3 ~ + urea + N H 4 ) X 2 a + cf 2 a O 2 1.0-0 .8-B y = 0.06 + 0.27 x r=0.39 n= 156 • 0.6-0 .4- 0 o o • * J i 0 .2 -0 .0 - CCD — o • ° • 0.0 0.2 0.4 0.6 0.8 1.0 N Q 3 " / ( N Q 3 " + N H 4 + ) 03 + cf 2 O 2 a. 1.0-0.8-0.6-0.4-0.2-0.0-y = -0.09 + 0.70 x • r=0.53 0 o: n= 156 O 100-30% I 0 • 10-1% L NC- 3 ~ / ( N Q 3 " + urea) F igure 2.12. / - ra t io vs nutrient ratio for a l l depths, stations and cruises. Nutr ient ratio includes tracer addit ions. ( A ) A m m o n i u m and urea, (B) o n l y a m m o n i u m , and (C) on ly urea are inc luded i n the calculat ion. Open symbols represent data from 100, 55 and 3 0 % I Q , and so l id symbol s represent data from 10 ,3 .5 and 1% I 0 . S o l i d l ine is the least squares fit to the data. 87 N H 4 + ) , with a slope (± 1 S.E.) of 0.23 + 0.003 (Fig. 2.12 A ) . When only N H 4 + was considered for the comparison, the slope (+ 1 S.E.) was 0.27 ± 0.004, indicating preference for N H 4 + over N 0 3 " (Fig. 2.12 B) . When only urea was considered in the analysis against N 0 3 " , then the slope (± 1 S.E.) was 0.70 ± 0.007, still indicating preference for urea (Fig. 2.14 C). However, in the latter case, the slope was closer to unity than in case (b) when only N H 4 + was used. Thus, judging from this analysis, the order of preference for nitrogen sources was N H 4 + , followed by urea and finally N0 3 ~. The results obtained from this approach agreed with the analysis of RPIs. The analysis of / - ra t io vs. nutrient ratio was done with data from all light depths since the separate analysis of surface (100, 55 and 30% I 0) and deep (10, 3.5 and 1% IG) samples yielded insignificant differences between the two slopes. N-specific uptake rates and f-ratios vs. nutrient concentrations The N-specific uptake rates of N 0 3 " and urea were plotted against ambient N H 4 + concentration for all depths, stations and cruises (Fig. 2.13). A t N H 4 + concentrations below 0.5-0.6 pig-at L" 1 , uptake rates varied widely indicating that other factors besides N H 4 + concentration are affecting these rates. A t N H 4 + > 0.5-0.6 ptg-at L" 1 , uptake rates of N 0 3 " and urea were lower. The threshold concentration of N H 4 + above which N-specific rates were depressed was about the same (0.5-0.6 pcg-at L" 1) for N0 3 "and urea. The low uptake rate values at high N H 4 + concentrations corresponded to greater depths (10, 3.5 and 1% IG) suggesting that the low rates may simply be the result of low irradiance, or a combination of low irradiance and an inhibitory effect o f N H 4 + . The relationship between the/ -ratio and N 0 3 " concentration for this complete data set can be observed in Figure 2.14. A n attempt was made to fit these data to an exponential model according to Piatt and Harrison (1985) and Harrison etal. (1987). The parameters of the model are the maximum/-ratio ( / j^) and the slope of the exponential curve (m). The estimates (± 1 S.E.) w e r e / m a x - O - 2 4 ± 0.001, and m = 1.02 ± 0.04 with n = 156. Although the exponential fit does 88 0.10 H 0.08-^ :o 0.06 -i 0.04-J 0.02-^ 0.00-^ o o o o o (§> o o o o o 100-30% Ic • 10-1% L o o o o o o o o 0 o o ° 8 • J g o 0 * S o o 0.20 -H 0.15 H 0.10-^ 0.05-J 0.00-J B o o o o o o o o o o o i 1 1 1 1 1 1 1 1 • r~ 0.0 0.2 0.4 0.6 0.8 1.0 Ambient N H 4 + concentration (pig-at L"1) Figure 2.13. N-specific nitrate and urea uptake rates vs. ambient ammonium concentration for all depths, stations and cruises. Open symbols represent data from 100, 55 and 3% IQ, and solid symbols represents data from 10, 3.5 and 1% I0. 89 Exponential model / - r a t i o = / m a x * ( i . e - ( m * N ° 3 / f m a x ) ) / m a x = 0.24 ± 0 . 0 0 1 m = 1.02 ± 0.04 n = 156 O 100-30% I c • 10-1% L o o o o o cm ..a.. V Q § o o o p. o • o Vd?....<i. o o o ~r 12 0 I 8 N 0 3 concentration (pig-at L"1) Figure 2.14. /-ratio vs. ambient nitrate concentration for all depths, stations and cruises. Nitrate concentration includes tracer additions. Open symbols represent data from 100, 55 and 30% IG, and solid symbols represent data from 10,3.5 and 1% IQ. The exponential model (Piatt and Harrison, 1985, and Harrison et al, 1987) applied to the data as well as the fitted parameters are included. 90 not seem to provide a significant description of this data set, it is included in Figure 2.14 for discussion purposes. Although the word "preference" is used in this section when referring to Figures 2.11 and 2.12, the potential inhibitory effects of ambient N H 4 + on N0 3 ~ and urea uptake rates cannot be disregarded. Therefore, these results mostly showed a discrimination against N 0 3 " due to preference and/or inhibition of N H 4 + . Urea followed in preference after N H 4 + , and its uptake also appeared to be affected by ambient N H 4 + at low irradiances. The effect of N H 4 + on N 0 3 " uptake rates by an ecologically relevant phytoplankton species from station P26 is investigated further in Chapter 4. D I S C U S S I O N New and regenerated production in the N E subarctic Pacific The results of this chapter show the importance of regenerated nitrogen forms in the nitrogen nutrition of phytoplankton from the N E subarctic Pacific during winter, spring and late summer. Of the two "recycled" nitrogen forms considered in this study, N H 4 + was almost always taken up faster than urea at every depth along the entire Line P transect. Nitrate uptake rates, which reflect the new production of the system, showed the lowest values and represented on average only 21% of the depth integrated total nitrogen uptake for every station and cruise (23% if considering all discrete depths), with no consistent seasonal variation. This is the first study to investigate the seasonal variability of the uptake rates of three nitrogen sources and to estimate new and regenerated production along a transect in the N E subarctic Pacific. It is also the first one to report winter uptake rates. Temporally consistent longitudinal trends were not evident for the integrated uptake rates of urea; however, slightly higher rates were measured for N H 4 + at P4, and for N 0 3 " at P20-P26 for 91 most cruises. Dissolved urea and N H 4 + concentrations did not show seasonal or longitudinal trends (see Chapter 1). This could explain the lack of a longitudinal pattern for urea uptake rates since they generally increased as a result of increases in dissolved urea concentrations. Probyn and Painting (1985) showed that uptake rates of urea in surface waters of the Antarctic were positively correlated with urea concentrations for some of their stations. The higher depth integrated N H 4 + uptake rates at P4 could be explained by the apparently higher biological activity at P4, and the preference for N H 4 + by the phytoplankton from this region. Ammonium uptake rates also increased with N H 4 + concentrations. Ambient N 0 3 " concentrations showed an increasing trend towards P26 during all cruises (see Chapter 1), and N 0 3 " uptake rates were also higher at P20-P26. There is not a simple explanation for those higher N 0 3 " rates, except that they were also a result of higher substrate availability. However, this assumption could not be confirmed since the plot of N 0 3 " uptake rate vs. ambient N 0 3 " for all discrete depths does not show any significant correlation (data not shown). A s discussed in Chapter 1, the high biological activity in the eastern end of the transect (P4 to P16) during May 1994 was reflected in a depletion of dissolved N 0 3 " and higher P N , but not Chi a concentrations. The higher rates of uptake of N H 4 + and urea measured at P4-P12 during May 1994 may have been the response to an increased supply of regenerated nutrients due to a higher abundance of heterotrophic nanoflagellates and mesozooplankton (Doherty, 1995; Wen, 1995) which were most likely responsible for the high P N signal (see "Discussion" in Chapter 1). This resembles post bloom situations, when N 0 3 " uptake is low, but uptake of regenerated nitrogen forms becomes dominant (Kanda etal, 1990). This dramatic pattern was not seen during the other M a y cruise in 1993. 92 Comparison with previous studies in the N E subarctic Pacific Previous studies in the N E subarctic Pacific have focused on the uptake and remineralization of nitrogen, but only at P26 during the May to October period (Wheeler etal., 1989; Wheeler and Kokkinakis, 1990; Cochlan etal, 1991a; Wheeler, 1993). It is important to note that Wheeler and Kokkinakis (1990) expressed their rates in moles rather than in gram-atoms (g-at) as reported here. In that case, the values for urea uptake rate are half of those expressed as g-at, and this has considerable consequences for the contribution of urea to total nitrogen uptake (lower when using moles), and/-ratios (higher when using moles). When molar units were used, the results obtained here gave a range of surface total nitrogen uptake from 65 to 489 n M d"1 for the May-September periods, which overlapped with the range reported by Wheeler and Kokkinakis (1990) of 84 to 374 n M d\ with a one-time high value of 732 n M d"1. The ranked proportional importance of the three nitrogen sources found during this study for surface waters of P26 considering both May-September periods is in close agreement with Wheeler and Kokkinakis (1990), although the values are different. When using molar units, N H 4 + represented 59 ± 13% (mean ± 1 S.D., n = 35) of the total nitrogen uptake, in comparison to 39 ± 9% reported by Wheeler and Kokkinakis (1990). Thus, as a result of the higher N H 4 + contribution to total nitrogen uptake found during this study, the relative contributions of N 0 3 " and urea were lower than those reported by Wheeler and Kokkinakis (1990). For the period May-October, Wheeler and Kokkinakis (1990) found no seasonal trend in the absolute uptake rates of total nitrogen for surface waters at P26. The present study showed that it was difficult to determine a seasonal trend between May and September (see Fig. 2.10 A ) ; however, a change was evident from winter to May/September. The integrated rate of N 0 3 " assimilation for P26 reported by Wheeler (1993) for the period May-September of 1987 and 1988 is remarkably close to the value found here for the same periods during 1992 and 1994. She reported an average (± 1 S.D.) of 45.0 ± 4.5 mg N m 2 d"1, while a similar calculation for this study gave a value of 45.8 + 13.1 mg N m 2 d"1. 93 Comparison of the N E subarctic Pacific with other H N L C regions The three H N L C regions are similar to each other with respect to phytoplankton nitrogen nutrition. In the equatorial Pacific, new production varied on average from 17% (Dugdale etal., 1992), to 23% (Murray etal., 1989) and to 26% (Pena etal., 1992) of total nitrogen assimilation, in comparison to 21% in the N E subarctic Pacific (this study). Absolute uptake rates of N03 and N H 4 + were also similar for both environments (this study, and Murray etal., 1989; Dugdale etal., 1992; Pena etal, 1992; Wilkerson and Dugdale, 1992). The pelagic realm of the Southern Ocean has also been identified as a H N L C region with low rates of nitrogen assimilation and variable /-ratios. During the winter-spring transition, Olson (1980) reported that the contribution of new to total production was 54% for the Scotia Sea and 40% for the Ross Sea. During the late summer, Glibert etal. (1982a) and Ko ike etal. (1986) found that, despite the abundance of N 0 3 " , there was a consistent preference for N H 4 + by phytoplankton in the Scotia Sea. On average, N 0 3 " accounted from 30% (Koike etal, 1986) to < 50% (Glibert etal, 1982a) of the total nitrogen utilized. These studies seem to show a trend of decreasing new production from winter-spring to late summer in the Scotia Sea, with a higher contribution of N H 4 + to total nitrogen uptake towards the end of the growing season. In contrast to the findings of Glibert etal. (1982a) and Koike etal. (1986), Collos and Slawyk (1986) reported that N 0 3 " was the major nitrogen source for phytoplankton from offshore areas south of the polar front in the Indian sector of the Antarctic Ocean during summer, with an average contribution of 70% to total nitrogen uptake. Although nitrogen uptake rates in the Antarctic were as low or lower than in the N E subarctic Pacific, the contribution of N 0 3 " to total nitrogen uptake appears to be more variable in Antarctic waters. The fraction of total production due to N 0 3 " may have been overestimated in the equatorial Pacific and in the Antarctic since urea was not considered in either of these studies. Probyn and Painting (1985) stressed the importance of urea in surface waters of the pelagic Southern Ocean, which supplied about 27% of the nitrogen requirements for phytoplankton. 94 Comparison of the N E subarctic Pacific with other North Pacific ecosystems The functioning of many other marine ecosystems also relies on regenerated nitrogen forms to support their primary production, but not always in such a consistent manner as in the N E subarctic Pacific. Studies of nitrogen utilization in the upwelling areas of Oregon and Washington revealed that N H 4 + and urea were the predominant nitrogen sources for phytoplankton nutrition when N 0 3 " was low (ca. < 5 /*g-at L" 1 ) , but N0 3 ~ became the dominant form when upwelling events occurred bringing higher concentrations of N 0 3 " to surface waters (Kokkinakis and Wheeler, 1987). In Monterey Bay (California), N 0 3 " was the predominant nitrogen source in freshly upwelled water, while N H 4 + utilization increased in older upwelled water, even in the presence of high concentrations of N 0 3 " (Kudela etal., 1997). Maximum new production in these ecosystems was on the order of 70% (Kudela etal, 1997) and 87% (Kokkinakis and Wheeler, 1987) of total primary production during upwelling events. The highest (surface and integrated) absolute uptake rates of N 0 3 " , urea and N H 4 + in the oceanic subarctic Pacific were 10 to 200 times lower than the highest rates during upwelling events (this study, and Kokkinakis and Wheeler, 1987; Kudela, 1995). This finding is in agreement with Dugdale and Wilkerson (1991), who postulated that N-specific N 0 3 " uptake rates in H N L C regions are an order of magnitude lower than in high iron upwelling areas. In coastal areas of the Gulf of Alaska, phytoplankton take up the nitrogen form which is the most available. In Auke Bay, Kanda etal. (1990) measured high N 0 3 " uptake rates when ambient N 0 3 " was abundant at the early stages of the spring bloom, but measured lower N 0 3 " uptake rates after the peak of the bloom. During the post bloom period, when N 0 3 " was depleted, N H 4 + showed the highest rates of uptake (Kanda etal., 1990). Urea was not measured during their study; however, it can be speculated that urea uptake rates may have followed the same pattern as those for N H 4 + since both forms are products of regenerative processes. Kanda etal. (1990) determined that through the bloom period, 62% of the total uptake was due to N 0 3 " utilization. Absolute nitrogen uptake rates in Auke Bay at the peak of the bloom were ca. 40 times 95 the highest rates measured in surface waters along Line P during May and September (Kanda et d., 1990 and this study). The use of regenerated nitrogen forms seems to be the basis of the functioning of oligotrophic oceans. In the central North Pacific Ocean, Eppley etal. (1977) determined that N 0 3 assimilation in surface waters was negligible. Even when potential rates (N03"-saturated) were estimated, the highest rates in surface waters of the North Pacific central gyre were ca. 20 times lower than the highest rates measured in the subarctic Pacific during the present study. The highest rates of N H 4 + and urea uptake were also 10-fold or more lower than in the subarctic Pacific (Eppley et al, 1977, and this study). Although Kanda etal. (1985) estimated that their 1 S N incorporation rates were 2-4 times higher than the ones determined by Eppley etal. (1977), values were still below the subarctic rates. In the northeastern Pacific subtropical gyre, Knauer et al. (1990) also reported rates of 1 S N 0 3 " incorporation at least 6 times lower than the values obtained during the present study. The mean annual/-ratio measured by Knauer etal. (1990) ranged from 0.11 to 0.14. Judging from these results, subarctic N 0 3 " uptake rates were at least 2 times higher than in the oligotrophic North Pacific gyre. In view of these comparisons, the N E subarctic Pacific seems to be more 'active' than the oligotrophic central North Pacific, but less so than the coastal region in Alaska, and much less than the Oregon-Washington-California upwelling systems. Although the subarctic Pacific could not be considered oligotrophic with respect to nitrogen levels, it may be considered functionally close to oligotrophic in terms of the phytoplankton-nitrogen interactions (Dugdale and Wilkerson, 1991). Preference of regenerated nitrogen over N03" In the N E subarctic Pacific, nitrogen forms were utilized by phytoplankton in the following order: N H 4 + > urea > N 0 3 . This implies that, on a yearly basis, N H 4 + was the dominant and preferred nitrogen source. Urea was generally the second most important nitrogen form for phytoplankton nutrition. A t times, however, N 0 3 " utilization became more dominant than urea, 96 although urea was always preferred over N0 3 ~. Similar results were reported for a wide range of environments by Harvey and Caperon (1976) in Kaneohe Bay (Hawaii), McCarthy etal. (1977) in the Chesapeake Bay (eastern U S A ) , Metzler etal. (1997) in inshore and offshore sites in the South Atlantic off Brazi l , Harrison etal. (1985) in the eastern Canadian Arctic, and Mitamura and Saijo (1986) for Lake B i w a (Japan), among many others. It is uncertain i f the proportionally higher rates of N H 4 + uptake in this study resulted from the inhibitory effects of ambient N H 4 + concentration on the rates of uptake of N 0 3 " and urea. A t high ambient N H 4 + , the rates of uptake of N 0 3 " and urea were low; however, these rates corresponded to low light conditions (< 10% I 0). Although low irradiance may account for the low values, compounding inhibitory effects can not be disregarded. Further evidence that N H 4 + may be affecting N 0 3 " uptake rates was given by the analysis of the relationship between the/-ratio vs. the N 0 3 concentration. Piatt and Harrison (1985) and Harrison etal. (1987) used an exponential model to fit data sets from different environments. In regions with low N H 4 + concentrations, the shape of the relationship between /-ratio vs. N 0 3 " showed an asymptotic increase with high slopes a n d / m a x values, while for regions with high N H 4 + concentrations (up to 1 /•<g-at L" 1 ) , the model yielded low slopes a n d / ^ values (Harrison et al., 1987). The data set presented in Figure 2.14 has a lot of scatter, but it weakly resembles the relationships shown by Harrison etal. (1987) for environments with high ambient N H 4 + . In those cases, Harrison etal. (1987) claimed that N H 4 + concentrations were affecting the/-ratio by inhibiting N 0 3 " uptake, and hence, the shape of the curve was not a result of inherent differences to the relationship between / and N0 3 " . Inhibitory effects of N H 4 + on uptake rates of N 0 3 " (see review by Dortch, 1990; and e.g. Tamminen, 1995; Chapter 4 of this thesis) and urea (e.g. Kristiansen, 1983; Kokkinakis and Wheeler, 1988; Tamminen and Irmisch, 1996; this chapter) have been inferred from field as well as laboratory studies. In Chapter 4, the effect of N H 4 + on N 0 3 " uptake rate is investigated for an ecologically relevant phytoplankter from the oceanic subarctic Pacific. In view of these results, an inhibitory effect of ambient N H 4 + on N 0 3 " (and urea) uptake rates is strongly suspected for the subarctic Pacific. 97 Although the importance of N H 4 + as well as urea for phytoplankton nitrogen nutrition has been extensively documented, many studies still ignore the measurement of urea uptake rates. The data presented here showed that the overestimation of the/-ratios (and, hence, new production) was on average ca. 36%, when urea was excluded from the calculation. Wafar etal. (1995) showed that the omission of urea overestimated / to a degree which depended on the ecosystem type, with a maximal overestimation of 55% in estuarine waters. Their review only included one reference from an H N L C region (Antarctic). Metzler etal. (1997) also calculated overestimates of 3-50% in South Atlantic waters. Therefore, new production wi l l be more accurately estimated if urea (and even other D O N sources) are not neglected in the studies of nitrogen uptake rates in the oceans. Factors affecting nitrogen uptake in the NE subarctic Pacific Rates of new (pN0 3~) and regenerated production (purea and p N H 4 + ) are calculated from the product of the N-specific rates of N0 3 ~, urea and N H 4 + uptake, and P N . Hence, those rates wi l l be dependent on the magnitude of the N-specific uptake rates, the amount of phytoplankton biomass, or both. Factors such as light, temperature, nutrient limitation, and grazing wi l l affect the N-specific rates, which in turn wi l l yield low biomass accumulation. Although this study did not investigate the environmental factors affecting N-specific uptake rates, it can be suggested that the seasonal patterns in nitrogen uptake were mainly a product of light and temperature effects on phytoplankton metabolism. A s seen in Chapter 1, light levels were lower in winter than in spring. During every cruise, the effect of irradiance on nitrogen uptake rates was reflected in the decrease of these rates from the surface to the bottom of the euphotic zone. However, the vertical decrease of uptake rates may also be due to other variables (e.g. nutrient concentrations, phytoplankton assemblage composition). The single effect of irradiance on rates of nitrogen uptake can be investigated by performing uptake vs. irradiance experiments which can often be described with a Michaelis-Menten type relationship (Maclsaac 98 and Dugdale, 1972). Appendix F presents the results of V N vs. I experiments performed at P16 during February 1994 and at P26 during May 1994. These experiments should be interpreted cautiously since too few data points have been obtained for low irradiances and no dark uptake experiments were conducted. Dark uptake, however, occurs for all three nitrogen sources (e.g. Maclsaac and Dugdale; 1972; Kanda etal., 1989; Wheeler etal., 1989; Wheeler and Kokkinakis, 1990; Cochlan etal., 1991a,b; Kudela etal., 1997). Moreover, the experiments presented in Appendix F were done only at two stations during two cruises. Nevertheless, these experiments showed that the uptake rates of the three nitrogen sources decreased at low irradiances (3.5 and 1% I G; Fig. F. 1). Judging from the estimated parameters ( V ^ and K L T ) from the model applied to V N vs. I, N H 4 + uptake rates were the least light dependent during both cruises, with V N 0 3 " being the most light dependent in February, and Vurea most light dependent in May (Fig. F. 1 and Table F. 1). Less light dependency for N H 4 + uptake rates was observed by Dodds and Priscu (1989), Muggli and Smith (1993), and Kudela etal. (1997), among others. The half-saturation constants for irradiance for N 0 3 , urea and N H 4 + ranged from 0.7 to 9.4% of ID, within the range found in other studies (e.g. Maclsaac and Dugdale; 1972; Cochlan etal., 1991b). Thus, the dependency of N-specific uptake rates on irradiance suggests that light was, at least in part, responsible for the seasonal variations. It has been claimed that deep mixing can also induce light limitation in phytoplankton from the subarctic Pacific and the Antarctic (Dugdale and Wilkerson, 1991). During the May and September cruises, the mixed layer depth was generally shallower than the depth of the euphotic zone along the entire transect (see Fig. 1.3 in Chapter 1). Thus, low rates during those times are not the result of light limitation due to deep mixing. During the winter cruises, however, wind mixing caused the mixed layer depth to exceed the 1% light depth, implying that light limitation was possible i f phytoplankton were carried below their critical depth (Parsons et al., 1966; Parsons and LeBrasseur, 1968). Temperature can also affect physiological rates (Eppley, 1972); however, no measurements were made during this study to investigate the temperature effect on N-specific uptake rates. 99 Surface uptake rates of N 0 3 \ urea and N H 4 + vs. mixed layer temperature from all stations during the May and September cruises did not show a consistent trend (data not shown). This is not unexpected due to the complexity resulting from the compounding effects of different environmental factors on nitrogen uptake rates. Other studies showed that rates of N 0 3 " , urea and N H 4 + uptake covaried with ambient water temperature (Kanda et al., 1985; L e Bouteiller, 1986). Nitrogen uptake rates for the three nitrogen sources have been found to increase 1.7 to 2.9 times with a 10°C rise in temperature (the Q 1 0 factor; Paasche and Kristiansen, 1982; Mitamura, 1986). It has been stated, however, that the Southern Ocean may be the only H N L C region where temperature is a limiting factor on N-specific rates (Dugdale and Wilkerson, 1991 and references therein). Although light and possibly temperature were depressing the rates at least during the winter, N-specific uptake rates still did not increase during the spring-summer to values as high as in other regions with similar nitrogen concentrations. In the subarctic Pacific, N 0 3 " uptake rates seem to be impaired by iron limitation (Boyd etal., 1996). Iron is essential for the synthesis of the two enzymes ( N 0 3 " and N 0 2 " reductases) responsible for N 0 3 " assimilation in phytoplankton (e.g. Sunda, 1988/1989), which partly explains why regions with low dissolved iron have persistently high levels of N 0 3 " . In the Gulf of Alaska, Martin and Fitzwater (1988) showed that N 0 3 " was depleted from experimental vessels only when iron was added. Although Banse (1991b) challenged their interpretation of the results for two of their three stations, he still agreed that at P26 the depletion of N 0 3 " was significant when iron was added in comparison to a control with no iron additions. Boyd etal. (1996) went a step further by directly investigating the influence of iron supply on the uptake and partitioning of inorganic nitrogen by two size-classes of phytoplankton at P26. They showed that N-specific uptake rates of 1 S N 0 3 " increased after 4 days of enrichment with iron, and this was only the case for the cells > 5 /-mi. The N-specific uptake rates of 1 S N 0 3 " for the smaller cells were unaffected by the trace metal enrichment. B y contrast, the N-specific uptake rates of 1 S N H 4 + were unaffected by iron additions to either size class (Boyd etal., 1996). Price etal. (1991, 1994) also observed that iron enrichment stimulated the rates of N O s " uptake by 100 entire assemblages of phytoplankton from the equatorial Pacific, while N H 4 + uptake rates did not increase as a result of iron additions. The effect of iron on urea uptake rates has not been investigated to date. Thus, low availability of dissolved iron may have been responsible for low N-specific uptake rates of N 0 3 " at least for the western part of Line P (e.g. P16, P20 and P26; see L a Roche etal., 1996). East of P16, dissolved iron concentrations are higher (La Roche etal., 1996), and therefore low N 0 3 " uptake rates may have been due to N 0 3 " limitation, at least for May 1994 and September 1992 (see Chapter 1). The activity of the dominant microzooplankton grazers could have also reduced the uptake rates of the three nitrogen forms in the N E subarctic Pacific at all times of the year. Intensive grazing can rapidly recycle the N 0 3 " , N H 4 + and urea incorporated into phytoplankton biomass which wi l l result in low net rates of N-specific nitrogen uptake. Growth rates derived from N -specific nitrogen uptake yielded very low values (range: 0.08 to 0.34 div d"1), comparable to the net growth rates measured previously from chlorophyll changes (-0.6 to 1 div d"1; Landry etal., 1993; 0.23 to 0.32 div d"1; Boyd etal., 1996). Although gross N-specific rates of nitrogen uptake may have been higher, the measured (net) rates were low due to high grazing pressure. Another consequence of grazing is the production of N H 4 + (see "Discussion" in Chapter 1) which was preferentially utilized and/or may have inhibited N-specific rates of N 0 3 " (and urea) uptake (see "Results" from this Chapter and Chapter 4). A l l of these limiting factors wi l l not only reduce the N-specific nitrogen uptake rates, but also the build-up of biomass, which in turn wi l l yield low rates of new and regenerated primary production. 101 Limitations of the 1 5 N tracer technique Problems associated with sample confinement The estimation of physiological rates in phytoplankton from long term experiments is subject to potential errors (e.g. Venrick etal., 1977); however, the calculation of rates from short incubations is also subject to ambiguities. For example, the conversion of hourly rates of nitrogen uptake derived from short daytime incubations to daily rates can be significantly over- or underestimated i f the effects of diel periodicity are not considered (see Chapter 4, and e.g. Price et al, 1985; Wheeler etal, 1989; Wheeler and Kokkinakis, 1990; Cochlan etal, 1991a,b). The rates of nighttime uptake vary for different nitrogen sources in different environments and conditions, and this variability is not always known. Another problem that occurs during all incubations, but has a substantially greater effect on the estimation of nitrogen uptake rates from short experiments, is the relatively more rapid uptake at the beginning of the incubation period (e.g. Conway etal, 1976; Conway and Harrison, 1977; Glibert and Goldman, 1981; Harrison, 1983b; Harrison etal, 1989), mainly for 1 S N H 4 + and l s N-urea at high 1 5 N enrichments. Rapid initial uptake, however, seems to be less problematic in eutrophic waters (e.g. Harrison and Davis, 1977). Thus, longer incubations would minimize these potential errors. Other problems such as nutrient exhaustion, uptake rate of unlabelled nitrogen sources, release of D 0 1 5 N and isotope dilution could be potentially more important in long term incubations. A s mentioned in the "Materials and Methods" section of this Chapter, nutrient exhaustion did not occur during the nitrogen uptake experiments performed during this study. The uptake of unlabelled nitrogen sources was not assessed, and therefore the calculated rates may underestimate the true value. The processes involved in the release of D O N and isotope dilution are complex, and it is not the intention of this discussion to do a comprehensive review of these mechanisms; however, brief descriptions are given below. The release of D 0 1 S N by phytoplankton has been recognized as an ubiquitous process (Laws, 1984; Hansell and Goering, 1989; Bronk and Glibert, 1991, 1993; Collos, 1992; Collos et 102 al., 1992; Bronk and Glibert, 1994; Bronk et al, 1994). Losses of 1 S N through D 0 1 5 N release by phytoplankton during the incubation period result in an underestimation of the gross uptake rates (Bronk etal, 1994). Bronk etal. (1994) indicated that in a variety of marine systems an average of 25 to 41% of the N 0 3 " and N H 4 + taken up by phytoplankton could be released as D O N . Hansell and Goering (1989) also reported high losses (37 to 58%) of assimilated 1 5 N-urea into the D 0 1 5 N pool in the northeastern Bering Sea. However, other studies showed that D O N release was only a small fraction of the assimilated 1 S N ; on the order of 13-24% in oligotrophic waters (Raimbault and Slawyk, 1997) and < 5% in areas of high substrate concentrations (Raimbault and Slawyk, 1997; Bronk and Glibert; 1994). The amount of D 0 1 5 N released also depends on the nitrogen species being incorporated (generally higher for 1 S N H 4 + ) , and the amount released increases with the length of the incubation (Bronk and Glibert, 1994). Although the process of dissolved organic matter release by 'healthy' phytoplankton is well known, some researchers attribute this excessive loss of D O N to 'unhealthy' phytoplankton exposed to unfavourable conditions, or to methodological problems (e.g. cell breakage during filtration) (e.g. Hellebust, 1974; Sharp, 1977). D 0 1 S N release by phytoplankton during the experiments presented here was not assessed, and it is difficult to speculate on the magnitude of the problem. Thus, values obtained are considered conservative estimates or net uptake rates. Isotope dilution results from regenerative processes that return unlabelled nitrogen (generally as 1 4 N H 4 + and/or 1 4N-urea) into the dissolved fraction and dilute the initial 1 S N H 4 + or I S N-urea atom % enrichment (e.g. Harrison, 1983a, Dugdale and Wilkerson, 1986; Slawyk etal., 1990). This process w i l l ultimately result in an underestimation of the rates of uptake. The magnitude of the underestimation varies depending on incubation time, culture conditions, community structure, and other factors. Glibert etal. (1982b) indicated that traditional 1 5 N uptake rates could be underestimated by a factor of ca. 2 i f isotope dilution is not considered, and suggested the determination of the atom % 1 S N enrichment of the N H 4 + pool at different times throughout the incubation. These measurements are extremely labor intensive, and are rarely performed on every sample when the objective of the study is the estimation of nitrogen uptake 103 rather than remineralization rates. In the subarctic Pacific, Wheeler etal. (1989) showed that the atom % 1 S N H 4 + enrichment decreased from 96 to 71% during the first 24 h of the incubation, and the regeneration was occurring exclusively during the night period. Although isotope dilution occurred during their experiments, the bias associated with this process may not be as great as in temperate and tropical waters (Harrison, 1993b). Harrison (1993b) showed linearity of uptake rates of N 0 3 " , N H 4 + and urea for at least 30 h of incubation despite low initial nutrient concentrations in the Canadian Arctic. They suggested that low temperatures kept phytoplankton growth at low levels which lengthened turnover times and decreased the possibility of isotope dilution (and substrate exhaustion). It is not clear if their results can be applied to the subarctic Pacific; however, they suggest that the problem is more serious in temperate and tropical waters characterized by high turnover rates. Furthermore, an underestimation of N H 4 + and urea uptake rates should have little impact on the conclusions of this chapter (i.e. nitrogen forms taken up in the order : N H 4 + > urea > N0 3 ~). Contribution of heterotrophic bacteria to nitrogen uptake It is well documented that marine heterotrophic bacteria can utilize D I N , mainly N H 4 + , as a nitrogen source (see review by Kirchman, 1994). The contribution of bacteria to total N H 4 + uptake rate ranges between 8-25% (Tupas etal., 1994), 33% (Fuhrman etal., 1988), > 50% (Wheeler and Kirchman, 1986) to 50-75% (Laws etal., 1985) in different regions. Although this list of references is not a comprehensive coverage of all the studies on bacterial assimilation of N H 4 + , it illustrates that the relative importance of bacteria in total N H 4 + uptake is extremely variable. A more limited number of studies indicated that bacteria are also capable of assimilating urea (e.g. Jahns, 1992), and N 0 3 " (e.g. Kirchman etal., 1992; Kirchman and Wheeler, in press); however rates of uptake are lower than those of phytoplankton (see Kirchman, 1994, e.g. Tamminen and Irmisch, 1996). Although heterotrophic bacteria are smaller (mostly < 0.8 pirn) than most phytoplankton, part of the bacterial biomass from a water sample can be retained by G F / F filters (commonly used 104 in studies o f 1 N incorporation by phytoplankton), especially when plankton biomass is high. The percent of bacteria caught on a G F / F filter may be as high as 30% (Fuhrman et al., 1988) or 40% (Kirchman etal, 1989). Thus, i f bacteria are taking up N H 4 + (and possibly N 0 3 " and urea), the 'phytoplankton' uptake rates measured from P 0 1 S N retained on G F / F filters would be overestimated. The degree of overestimation, however, wi l l depend on the number of bacteria retained on the filters and on how actively they are taking up N H 4 + . No estimates of bacterial numbers caught by the G F / F filters are available for this study. However, even i f high numbers were retained, their contribution to phytoplankton nitrogen uptake rates may have been low since it has been observed that heterotrophic bacteria in the subarctic Pacific are limited by the supply of organic carbon (i.e. glucose; Kirchman etal, 1990). Moreover, bacterial specific growth rates are low (< 0.1 d"1; Kirchman etal, 1993) in comparison to phytoplankton specific growth rates (up to 1 d"1; Booth etal, 1988; Kirchman etal, 1993; Boyd etal, 1996) in subarctic Pacific waters. Although the contribution of bacteria to the uptake of 1 S N may not have been large, the rates presented here are somewhere in between net phytoplankton rates and net community rates. To date, methodological limitations complicate the simultaneous estimation of the contribution of heterotrophic bacteria and phytoplankton to nitrogen uptake by means of the 1 S N technique. Better estimates of nitrogen uptake rates are undoubtedly achieved when corrections for the aforementioned problems are obtained. However, in practical terms, due to the labor-intensive nature of these analyses, it is generally not feasible to measure all those processes while performing a comprehensive temporal and spatial study of 1 S N uptake rates. Therefore, during the present study, the decision on the experimental design (24 h incubations) was made under the assumption that in a region like the N E subarctic Pacific, where phytoplankton biomass is low and relatively constant over the incubation period, most of the errors associated with long incubations may be minimized. Although the contribution of heterotrophic bacteria to total nitrogen uptake still remains to be assessed in the N E subarctic Pacific, it is not expected to be high due to the low specific growth rates of bacteria in this region. 105 S U M M A R Y The nitrogenous nutrition of phytoplankton from the subarctic Pacific was based on regenerated nitrogen forms at every depth, station and season. The ranked proportional importance of nitrogen was N H 4 + > urea > N0 3 " . Ammonium was the dominant and preferred nitrogen source. Urea was generally the second most important nitrogen form for phytoplankton nitrogen nutrition. When urea was excluded from the calculation of the/-ratio, the overestimation of the/-ratio ranged from 4 to 130%, with an average of 36%. Annually, new production represented 21% of the depth integrated total nitrogen production along the transect. Longitudinally, ammonium uptake rates were generally higher at the coastal end of Line P, while nitrate uptake rates showed higher values at the oceanic sites. In contrast, urea uptake rates did not show a consistent longitudinal trend. It is suggested that ammonium may have inhibited the uptake rates of nitrate and urea. 106 CHAPTER 3 N I T R O G E N U P T A K E B Y S I Z E - F R A C T I O N A T E D P H Y T O P L A N K T O N A S S E M B L A G E S F R O M T H E O C E A N I C N E P A C I F I C O C E A N I N T R O D U C T I O N Oligotrophic oceans are dominated by small phytoplankton cells (pico- and/or nanoplankton) on a yearly basis, while regions with relatively higher nitrate fluxes are dominated by larger cells (Parsons and Takahashi, 1973; Malone 1971, 1980). It has been suggested from these observations that regenerated forms of nitrogen (ammonium and urea) support primary productivity in oligotrophic waters, and new nitrogen (nitrate) sustains productivity in coastal and upwelling systems. The dominance of small or large cells in these environments could also be the result of physical forcing and not necessarily a sole response to nutrient preferences (Malone, 1971, 1975). For example, Taylor and Joint (1990) showed that intense mixing resulted in increases of large phytoplankton relative to the smaller cells, whereas low mixing had the opposite effect. Therefore, the physical characteristics of the environment could partly explain the dominance of small or large cells. However, the different size fractions may also be responding to nutrient enrichment, and, hence, their dominance in one environment or another could also be dependent on the main nitrogen forms available for their nutrition. This hypothesis has been tested by investigating the nitrogen uptake dynamics of natural phytoplankton assemblages of different sizes. Many studies supported the initial nutrient hypothesis stated by Malone (1971), but exceptions to that trend have also been observed (Furnas, 1983; Chisholm, 1992; Dauchez etal., 1996). The structure and activity of food webs in the surface of the oceans may influence in several ways the export fluxes of biogenic carbon towards the deep ocean (see "General 107 Introduction"; Legendre and Le Fevre, 1989, 1991, 1995). The oceanic N E subarctic Pacific is dominated by phytoplankton cells < 5 pim (mainly represented by Micromonaspusilla, < 2 pim; Chapter 1), and primary production by the entire phytoplankton assemblage is mainly based on regenerated nitrogen forms (Chapter 2), similar to oligotrophic systems. However, up to now the role that the numerically dominant small phytoplankton have on the nitrogen dynamics of this region has not been considered. Thus, it is useful to understand how new and regenerated nitrogen forms flow through the different compartments of the subarctic ecosystem to better estimate the role of this and other H N L C regions in the global nitrogen and carbon cycles. The purpose of the study presented in this chapter was to: (1) determine the rates of uptake of ambient nitrogen (nitrate, ammonium and urea) by small phytoplankton cells (< 2 pan), and to compare them to the rates of uptake by the rest of the assemblage, and (2) evaluate the nitrogen preferences by the < 2 and > 2 pim phytoplankton in the oceanic region of the N E subarctic Pacific. M A T E R I A L S A N D M E T H O D S Studies were conducted at station P26 during two cruises: September 1992 and May 1994. Water samples for chemical and biological parameters, and for nitrogen uptake rate experiments were collected from 6 light depths and processed as described in Chapters 1 and 2. Chemical and biological measurements A t each depth during both cruises, samples were obtained for the measurement of dissolved and particulate nitrogen, and chlorophyll a concentrations. Samples for dissolved nitrogen were collected as described in Chapter 1. A t each depth, two separate subsamples for P N were filtered, one through a pre-combusted Whatman® G F / F filter, and the other through a 108 Poretics® 2 pim porosity polycarbonate filter, using a pressure of < 125 mm Hg. The 2 pim filter was discarded and the filtrate re-filtered through a pre-combusted G F / F filter. Thus, two filter samples for P N were obtained per depth: one containing the P N from the entire assemblage (> 0.7 pim) and the other containing the P N for the fraction 0.7-2 pim. The P N for the > 2 pim size fraction was obtained by subtraction of the 0.7-2 pim from the > 0.7 pim fraction. Samples for Chi a were size fractionated only in May 1994 following the same procedure described for P N , except that the > 0.7 pim and the 0.7-2 pim fractions were obtained from different casts done on consecutive days. Analytical procedures for the measurement of dissolved and particulate nitrogen, and chlorophyll a were described in Chapter 1. Nitrogen uptake experiments During September 1992, nitrogen uptake rate experiments were conducted by filling four 1 L polycarbonate bottles per depth, and spiking two of them with 1 S N 0 3 " , and the other two with 1 5 N H 4 + . During M a y 1994, three 2 L bottles were filled per depth and each one spiked with 1 S N 0 3 ~ , I S N H 4 + , or 1 5N-urea. Water samples were inoculated with 1 S N at trace amounts (ca. 10% of ambient N 0 3 " , N H 4 + , or urea concentration), and incubated for ca. 24 h. A t the end of the incubation period, 1 L was filtered using a pressure of < 125 mm H g through a pre-combusted Whatman® G F / F filter, while the other litre was filtered through a Poretics® 2 pim polycarbonate filter for each isotope and depth. The 2 pim polycarbonate filter was discarded and the filtrate re-filtered through a pre-combusted G F / F filter. Hence, as in the case for P N and Chi a, two G F / F filter samples were obtained: one with P 1 S N from the entire assemblage (> 0.7 pim), and the other with P 1 S N from the 0.7-2 pim size fraction. A l l 1 5 N experimental and analytical procedures, and final calculations of N-specific and absolute uptake rates were done as described in Chapter 2. The rates of uptake for the > 2 pim size fraction were obtained by subtraction of the uptake rates for the 0.7-2 pim fraction from the uptake rates for the > 0.7 pim size fraction. The incident surface 109 irradiance during the incubation period on 21 May 1994 is shown in Figure D.3 (Appendix D). Incident irradiance data were not available for September 1992. The/-ratios and relative preference indices (RPIs) were calculated as described in Chapter 2. During September 1994,/-ratios and RPIs were calculated only with N 0 3 " and N H 4 + , because data for urea uptake rates were not available. In contrast, during May 1994,/-ratios and RPIs were calculated including urea since data were available for this cruise. However,/-ratios and RPIs for May 1994 were also calculated excluding urea so that they could be compared with the RPIs from September 1992. R E S U L T S Initial environmental conditions The water column properties at P26 during September 1992 and May 1994 are shown in Table 3.1. Particulate nitrogen for the 0.7-2 pim size fraction ranged between 41 to 94%, and 64 to 80% of the total P N throughout the euphotic zone during September 1992 and May 1994, respectively (Table 3.1). Depth integrated P N for the 0.7-2 pim fraction represented 74-75% of the integrated > 0.7 pim P N during both cruises. Chlorophyll a for the small fraction ranged from 44 to 81% of the total phytoplankton biomass throughout the euphotic zone (Table 3.1), and was 58% of the total depth integrated Chi a during May 1994. Contribution of each size class to nitrogen uptake by the entire assemblage The uptake rates of N 0 3 " , N H 4 + and urea by both fractions and by the entire assemblage decreased from the 100% to the 1% I 0 depth (Figs. 3.1 and 3.2). N-specific uptake rates for the 0.7-2 pim size fraction were higher than the rates for the > 2 pim fraction for all nitrogen sources at every depth during both cruises (Figs. 3.1 A & C, and 3.2 A , C & E). When uptake of N 0 3 " , T3 s § CO 3, 3 A § § 00 CO 2 £ •a s T 3 CO co co ' c CO co-ed a o co •— cfa co co & NO cd * s 1 8 O CO 1§ 8 3 ' E 2 cd ,u» 1 § CO 'co 0 g co X, CO '3 A J '•a CO cd " cd S-l > fa £ O > o c 0 1 CO T3 2 oo co •S o 'S-fa S ° ^ Q co « . p s o x: b S /P CO Q CO co CO co N •*—> — C co s S •>> * ^ co O co s =5, £ as. A S ZJ as. a oo CN u 3, t\ O 15 as. i \ cS A B as. CN A S as. 00 CN t\ CJ B at. O A ed Ure ca 12 00 Cf z K CD Q Cd Q 5 h N M ^ W ^ d d d d d d ^ o\ oo t o r~ o m --i \o M d d d d d d CO ON ON O NO —< *0 CO ON ON CO o —; o —i d d h CO 00 ON - i CN tf CO CN V) V-) ON O ON 00 CN O CN CO CN CN CO d d d d d d H IS Ob £ o «o Q >/"> —< ^ NO 8 OH CN 00 NO 00 CO O NO O O O — i — i d d d d d d NO h t ' t M d d d d d d CO N H h IT) 00 CN CN CO ^ - i CN CN d d d d d d o co NO NO CO CO CN CN CN CN d d d d d d CO U") co CN o x ON ON oo r-~ >o d d d d d d o co 3 NO CN — I l> NO o ON — 1 ~ —' O O CN CN CN d d oo CN d t~- 00 CN ^ CN O O CN CO NO d d d d d ON NO 00 00 00 O >-« —i ^ - J CN m ft o <5 ^ CO "^1" NO at cd CN I l l V N (cf1) PN (ng-at N L - V 1 ) 0.00 _L 0.04 0.08 0 4 0 80 120 X3 PH Q 0 - Q ®. A °-. fP '® B 2 0 - # 6 ® # ®' 4 0 -6 0 - id.<k c * 4 8 0 - • ® N 0 3 " N 0 3 -I . I , i 1 , 1 , i , i , 0 - 1 C ? D •;;0E1 2 0 -4 0 - i d Kf' 6 0 -irj . i 8 0 - • m'' N H 4+ N H 4 + - ® - N 0 3 " , - H - - N H 4 + > 0.7 pim •o-- N 0 3 \ • • • • • N H 4 + 0.7-2 pim N 0 3 \ • • • • • N H 4 + > 2 pim Figure 3 .1 . D e p t h profiles o f N- spec i f i c uptake rates o f ( A ) nitrate and (C) a m m o n i u m , and absolute uptake rates o f (B) nitrate and (D) a m m o n i u m for the entire assemblage o f phytoplankton (> 0.7 pim) and for the 0.7-2 pim and > 2 pim s ize fractions at P 2 6 dur ing September 1992. 112 VN (d 1) 0.00 0.04 0.08 20 H 4 0 H 60 H O H • o. _L _L P ® •O" '" ®" • 6® NO, 0 - 1 TTxl • (m) 2 0 - jc Depth i 4 0 - 1 1 1  P'W 6 0 - 1 i dm N H 4 + (ng-at N L" •d"1) 0 4 0 80 I . I . I . 120 I . * ^ ® - ~ • ;0 '® <#' ®"' B •» •® N 0 3 -i . i . i . i . f •? ) D i P W p'" BT"' •1 IE!'' N H 4 + 0 - \ E — > A : A F - A' A > T " 2 0 - -i fa A A > 4 0 -k A*" ' ' 6 0 - M Urea i Urea NO3", N H 4 + , •urea > 0.7 pim -o-- N 0 3 ~ , N H 4 + , -A- urea 0.7-2 pim N 0 3 \ • • • • • N H 4 + , urea > 2 pim Figure 3.2. Dep th profiles o f N- spec i f i c uptake rates o f ( A ) nitrate, (C) a m m o n i u m , and (E) urea, and absolute uptake rates o f (B) nitrate, (D) a m m o n i u m , and (F) urea for the entire assemblage o f phytoplankton (> 0.7 pim) and for the 0.7-2 pim and > 2 pim s ize fractions at P26 dur ing M a y 1994. 113 N H 4 + and urea were expressed as absolute rates ( V N * PN) , the > 2 pim size fraction exhibited higher contributions to the total (> 0.7 ^m) rate of uptake (Figs. 3.1 B & D , and 3.2 B , D & F), since the proportional contributions of > 2 pim P N to > 0.7 pim P N were higher than the proportional contributions of V N by > 2 pim to V N by > 0.7 pim. Nevertheless, absolute uptake rates of N H 4 + and urea by the 0.7-2 pim fraction were still higher than those by the > 2 pim fraction (Figs. 3.1 D , and 3.2 D & F), but those of N 0 3 showed no detectable differences between the two fractions (Figs. 3.1 B and 3.2 B). The contribution of the small fraction to the integrated N0 3 ~, N H 4 + , urea and total nitrogen uptake rate by the total assemblage also showed higher values for V N than for p N (Table 3.2). The V N by the 0.7-2 pim size fraction ranged between 69 to 74% of the V N 0 3 " , 81 to 90% of the V N H 4 + , and was 90% of the Vurea by the > 0.7 pim size fraction. The p N by the 0.7-2 pim size fraction varied between 50 to 55% of the p N 0 3 , 60 to 68% of the p N H 4 + , and was 68% of the purea by the > 0.7 pim size fraction. Thus, most of the uptake of the three nitrogen sources was by the fraction smaller than 2 pim. Hence, the small cells were responsible for 77 to 87% of the Vtotal nitrogen uptake, and 56 to 66% of the ptotal nitrogen uptake by the > 0.7 pim size fraction. Partitioning of the nitrogen forms used by each size class Both phytoplankton size fractions were taking up N H 4 + at higher rates than N 0 3 " and urea (Fig. 3.3). However, in every case N 0 3 " represented a larger proportion of the nitrogen requirements for the > 2 pim cells than for the 0.7-2 pim cells. The 0.7-2 pim cells took up urea after N H 4 + , while the > 2 pim cells seemed to take urea at lower or equal rates to those of N 0 3 " (Fig. 3.3 B & D). The/-ratios showed slightly higher values for the > 2 pim than for the 0.7-2 pim size fraction throughout the euphotic zone for both cruises (Fig. 3.4). However, the profiles corresponding to the > 0.7, 0.7-2 and > 2 pim were not significantly different from one another in Table 3.2. Contribution of the 0.7-2 pim size fraction to the depth integrated N-specific and absolute uptake rates of nitrate, ammonium, urea and total nitrogen by the entire assemblage (> pim). Sep 92 May 94 N-source vN P N P N (%) (%) (%) ( % ) N 0 3 " 69 50 74 55 N H 4 + 81 60 90 68 Urea - - 90 68 Total N 77 56 87 66 115 0.05 0.04 0.03 H Sep 92 May 94 ^ 0.02 0.01 H 0.00 a i oo a 2 0.7-2 pim > 2 pim 0.08 0.06-^ 0.04 0.02 H 0.00 0.7-2 pim >2pim ED N0 3" • N H 4 Q Urea Figure 3.3. Depth integrated (A) N-specific and (C) absolute uptake rates of nitrate and ammonium for September 1992, and (B) N-specific and (D) absolute uptake rates of nitrate, ammonium and urea for May 1994 for the 0.7-2 pim and > 2 pim size fractions. The numbers on top of the bars indicate the percent contribution of that nitrogen source to the total nitrogen requirements for each size fraction. 116 Sep 92 May 94 / - ratio 0.0 0.4 0.8 OH CD Q 0 ^ 2 0 - ^ 4 0 H 6 0 - ^ a so-i 0.0 0.4 J i L 0.8 q <a».... . 0 -9 4 • 2 0 -4 0 -Cjb'' 6 0 -" t o 0 - cm „ q » , C 6 4 > 0.7 2 0 -•o - 0 . 7 - 2 / m i > 2 /<m 4 0 -• | 6 0 -c * F igure 3.4. Dep th profiles o f /-ratios for ( A ) September 1992 (no urea), and (B) M a y 1994 exc lud ing urea and (C) M a y 1994 inc lud ing urea for the entire assemblage o f phytoplankton (> 0.7 pim), and for the 0.7-2 pim and > 2 pim s ize fractions. 117 either case (single factor A N O V A s calculated considering all six depths of each profile as replicates, P > 0.1). Depth integrated/-ratios for the small fraction varied between 0.22 to 0.31 when urea was excluded, and was 0.16 when urea was included (Table 3.3). The integrated/-ratios for the > 2 pim fraction ranged between 0.33 to 0.41 when urea was excluded, and was 0.25 when urea was included (Table 3.3). Hence, the > 2 pim cells were slightly greater contributors to the euphotic zone new production. Preference of nitrogen sources by each size class The RPIs for N0 3 ~, N H 4 + and urea for the 0.7-2 pim and > 2 pim fractions are shown in Figure 3.5. For both size fractions and cruises, R P I ^ and R P I ^ values were above unity, indicating preference (utilization proportional to their concentrations), while R P I N 0 3 values were below unity, suggesting discrimination against N0 3 " . R P I N Q 3 values were slightly higher, and R P I N H 4 values were slightly lower for the > 2 pim size fraction (Fig. 3.5 C & D) than for the 0.7-2 pim size fraction (Fig. 3.5 A & B) . However, R P I N 0 3 as well as R P I ^ were not significantly different between size fractions for either cruise (Mest, P > 0.1). R P I ^ values, however, were significantly higher for the > 2 pim size fraction (Fig. 3.5 D) than for the small fraction (Fig. 3.5 B ; Mest, P < 0.01). Therefore, the order of preference as defined from the RPIs was N H 4 + > urea > N 0 3 " for the 0.7-2 pim size fraction, and N H 4 + = urea > N 0 3 " for the > 2 pim fraction. In summary, RPIs showed a preference for regenerated forms of nitrogen for both size fractions and cruises. 1 Table 3.3. Depth integrated/-ratios for the entire assemblage (> 0.7 pim), and the 0.7-2 pim and 2 pim size fractions for both experiments. During May 1994,/-ratios were calculated including and excluding urea. Cruise /-ratio by size fraction Notes > 0.7 pim 0.7-2 pim > 2 pim Sep 92 0.36 0.31 0.41 no urea May 94 0.26 0.22 0.19 0.16 0.33 0.25 no urea with urea 119 Sep 92 May 94 A a • • o 8 o 0.7-2 pim 4 2-10-=] 6-4-6-A-m u fl B • A A > A 8 • $ 0.7-2 / a n c • -D • • A o o o o > 2 pim o 0 • >^  O > 2 /^m 8 9 1 10 1 r • 2 • i • i • i 13.2 13.6 14.0 Total nitrogen (//g-at L"1) • N 0 3 " ( a l l ) o N 0 3 (no urea) • N H 4 + ( a l l ) A U r e a (all) N H 4 + (no urea) F igure 3.5. Nitrate, a m m o n i u m and urea relative preference indices (RPIs) for the ( A ) 0.7-2 pim and (C) > 2 pim s ize fractions for September 1992, and (B) 0.7-2 pim and (D) > 2 pim s ize fractions for M a y 1994. R P I s were calculated wi thout urea ('no urea') for September 1992, and w i t h ('all') and wi thout urea ('no urea') dur ing M a y 1994. See text for details on R P I calcula t ion. 120 D I S C U S S I O N Absolute and N-specific uptake rates of all nitrogen sources were higher for phytoplankton < 2 pim than for the > 2 pim cells. This implies that picoplankton accounted for most of the regenerated as well as new production in the oceanic subarctic Pacific during September 1992 and May 1994. For both size classes, N H 4 + was taken up at higher rates than urea and N 0 3 " , and N H 4 + and urea were utilized preferentially over N 0 3 \ Although these results only apply to station P26, due to the similarities in the longitudinal patterns of phytoplankton species composition (Chapter 1) and nitrogen uptake rates for the entire assemblage (Chapter 2), it is very likely that these conclusions can be extended to most of Line P. The contribution of the small cells to the uptake of nitrogen by whole assemblages of phytoplankton seems to be higher in oceanic than in coastal regions. For example, in the Benguela upwelling system, nitrogen uptake by cells < 1 pim increased from 10 to 25% of the nitrogen uptake by the whole assemblage from coastal to oceanic sites (Probyn, 1985). A l so Harrison and Wood (1988) indicated that the relative importance of < 1 pim cells to total nitrogen uptake increased from highly productive coastal waters of Georges Bank to the oligotrophic open N W Atlantic. The results of the present study did not allow a coastal to oceanic comparison, but agreed with previous results in that picoplankton cells were responsible for most of the (recycled and new) nitrogen uptake in the oceanic environment. It has been suggested that small cells (< 10 pim) satisfy their nitrogen requirements with regenerated nitrogen while primary productivity of larger cells is based on new nitrogen (Parsons and Takahashi, 1974; Malone, 1980). These suggestions were based on the observation that small cells dominated stable oligotrophic environments where the only source of nitrogen came from recycling, while large cells (diatoms and occasionally dinoflagellates) dominated the plankton and were able to bloom when environmental conditions enhanced the upward flux of N 0 3 " from deep waters into the euphotic zone (Malone, 1971). A physiological basis for this hypothesis was presented by Eppley etal. (1969b) who compared the half-saturation constants ( K s , the 121 concentration supporting an uptake rate of half the m a x i m u m rate) for 16 species o f phytoplankton of various sizes and environments. T h e y showed that smal l -ce l led oceanic species had low K s values for N H 4 + and N 0 3 ~ , and that K s var ied i n proport ion to ce l l size. M o r e recently, H e i n etal. (1995) reported s imi la r results by rev iewing the Michae l i s -Men ten kinet ic parameters from the literature for 76 algal species, f rom mic ro - to macroalgae. K i n e t i c parameters were closely related to changes i n the surface area to vo lume ratio, and, thus, H e i n etal. (1995) concluded that smal l algae were more efficient at harvesting nitrogen from the environment than larger algae. It w o u l d be expected then that h igh N 0 3 " regions such as the N E subarctic Pac i f i c should have a phytoplankton assemblage wi th a higher representation o f large cells . A l t h o u g h sporadic diatom ' b l o o m s ' have occurred i n the station P26 region (see " D i s c u s s i o n " i n Chapter 1), the predominant species are smal l (see Chapter 1), and those < 2 pim cel ls satisfied most o f their nitrogen requirements w i t h N H 4 + and urea. However , the larger cells also satisfied more than half o f their nitrogen requirements w i t h regenerated nitrogen. M o s t f ie ld studies agree that reduced nitrogen is the most important source for the nitrogen nutrit ion o f smal l cel ls . Howeve r , there is no agreement on the part i t ioning o f nitrogen uptake by larger cel ls w h i c h seems to vary depending on environmental condit ions. Despite the dominance of N 0 3 " i n the d issolved nitrogen pool (96%) i n Anta rc t ic surface waters, N H 4 + and urea accounted for, on average, up to 7 5 % o f the N-requirements for cel ls <1 pim, and 6 2 % for cel ls < 15 pim (Probyn and Pa in t ing , 1985). In another An ta rc t i c study, N H 4 + was found to account for over 7 5 % o f the total inorganic nitrogen uptake by cells < 20 pim, w h i l e cells > 20 pim took up most ly N 0 3 " ( K o i k e etal., 1986). L e Boute i l l e r (1986) also reported that phytoplankton < 3 pim ma in ly assimilated N H 4 + , and large cells assimilated N 0 3 " i n the equatorial At l an t i c . Na lewajko and Gars ide (1983), and Har r i son and W o o d (1988) found that smal l cel ls (< 3 and < 1 pim, respectively) used more N H 4 + than N 0 3 " , wh i l e larger cells seemed to take up new and regenerated nitrogen i n equal amounts. In the Benguela upwe l l ing system, N H 4 + accounted for most o f the nitrogen required by cel ls < Ipim at coastal and offshore stations (Probyn , 1985). In contrast, N 0 3 " contributed more to the nitrogen requirements o f larger cells i n higher N 0 3 waters at coastal 122 stations where the active upwelling sites were located; however, larger cells took up mostly reduced nitrogen at oceanic sites where N0 3 ~ concentrations were low (Probyn, 1985). In a more recent study of the Benguela system, Probyn etal. (1990) reported that recycled nitrogen contributed 94% of the nitrogen used by cells < 10 pim, and 63% (range: 0-100%) by larger cells not only for offshore, but also for inshore stations with aged upwelled waters. These two studies on the Benguela system seem to be contradictory in the forms of nitrogen mostly used by large cells for inshore areas. The explanation may lie on the different water conditions to which the cells were exposed. The predominance of N 0 3 " uptake in freshly upwelled waters and an increased assimilation of reduced nitrogen in aged upwelled waters was observed by Kudela etal. (1997) for intact assemblages. The activity of large cells may have produced those differences in the study by Kudela etal. (1997), since small cells seem to be always more inclined to assimilate reduced nitrogen forms. Thus, large cells take up regenerated, or new, or both nitrogen forms depending on the environment. Results from the present chapter agreed with all these studies regarding the partitioning of nitrogen by small cells. However, although phytoplankton > 2 pim showed proportionally higher N 0 3 " uptake than the smaller cells, N H 4 + and urea were still the main nitrogen sources. Thus, the subarctic Pacific phytoplankton > 2 pim did not respond to high N 0 3 " concentrations as they did in other environments, and this is probably due to iron (Boyd etal., 1996) and grazing limitation of N 0 3 " uptake (as discussed in Chapter 2). In a laboratory study, Muggli etal. (1996) showed that the diatom Actinocyclus sp. isolated from station P26 appeared to have a physiological advantage when grown with N H 4 + (as opposed to N0 3 ") under iron-stressed conditions. The results from this chapter complemented the study by Muggl i etal. (1996) by proving that in situ, under ambient iron conditions, larger cells (in this case, > 2 pim) were taking up N H 4 + at a higher rate than N0 3 " . The N H 4 + > urea > N 0 3 " ranked preference for all size fractions observed here has also been reported for other marine systems (e.g. Probyn, 1985; Probyn and Painting, 1985, Tremblay, 1995). Kokkinakis and Wheeler (1988) also observed that N H 4 + was more important than urea as a regenerated nitrogen source for the 1-10 and 10-200 pim size fractions at low and 123 high N 0 3 " stations in the coastal upwelling region off Oregon and Washington. Further support to the findings presented in this chapter are given by laboratory experiments with Micromonaspusilla (the one species that dominated the < 2 pim assemblages during this study). It has been shown that N 0 3 " was not taken up as readily as the reduced nitrogen forms by M. pusilla under N-starved (Cochlan and Harrison, 1991c) and N-replete (Cochlan and Harrison, 1991a) conditions, indicating a preference for N H 4 + over urea and N 0 3 by M. pusilla. S U M M A R Y In the subarctic Pacific, most of the phytoplankton biomass was in the < 2 pim size fraction, and these cells were responsible for most of the new and regenerated primary production. For the < 2 and > 2 pim size classes, N H 4 + was taken up at higher rates than urea and N0 3 ~, and N H 4 + and urea were utilized preferentially over N 0 3 \ The order of preference as defined from the RPIs was N H 4 + > urea > N 0 3 " for the 0.7-2 pim size fraction, and N H 4 + = urea > N 0 3 " for the > 2 pim fraction Depth integrated/-ratios for the < 2 pim size fraction varied between 0.22 to 0.31 when urea was excluded, and was 0.16 when urea was included. The integrated/-ratios for the > 2 pim fraction ranged between 0.33 to 0.41 when urea was excluded, and was 0.25 when urea was included. Hence, the > 2 pim cells were slightly greater contributors to the euphotic zone new production. 124 CHAPTER 4 E F F E C T O F A M M O N I U M O N N I T R A T E U T I L I Z A T I O N B Y EMILIANIA HUXLEYI, A C O C C O L I T H O P H O R E F R O M T H E O C E A N I C N E PACIFIC, G R O W N O N A L I G H T : D A R K C Y C L E I N T R O D U C T I O N Numerous studies have shown that ammonium exerts an effect on the nitrate metabolism of marine algae. Ammonium has been found to be the preferred nitrogen source for most species of marine phytoplankton as well as to inhibit the utilization of nitrate in a more direct manner (see review by Dortch, 1990). Concentrations of ammonium lower than 1 ptM may readily inhibit nitrate uptake (e.g. Eppley etal., 1969a, McCarthy etal., 1977, Haines and Wheeler, 1978, Harrison etal., 1996). However, in some cases ammonium had little or no effect on nitrate uptake (e.g. Haines and Wheeler, 1978; Kokkinakis and Wheeler, 1987; Kristiansen and Lund, 1989) and in other cases ammonium enhanced nitrate uptake rates (e.g. Dortch etal., 1991). Therefore, although there is undoubtedly an interaction between ammonium and nitrate uptake, it appears that the extent and threshold concentrations involved depend on the species under study, its physiological status, and the environmental conditions to which this particular species or the natural assemblage of phytoplankton has been exposed (e.g. Bates, 1976; Dortch and Conway, 1984; Dortch, 1990; Dortch etal., 1991, Harrison etal., 1996). Under controlled laboratory conditions, it is possible to differentiate between the nitrogen uptake interactions of preference and inhibition. The preference that phytoplankton have for one nitrogen source over another can be investigated by comparing the parameters derived from Michaelis-Menten kinetics. These can be obtained by measuring the uptake rate for each nitrogen source at increasing concentrations (e.g. see Dortch, 1990; Cochlan and Harrison, 1991a). 125 Inhibition can be quantified by comparing the uptake rate for one nitrogen source alone with its uptake in the presence of increasing concentrations of the inhibiting nitrogen source (e.g. see Dortch, 1990; Cochlan and Harrison, 1991b). These experiments are rarely possible in the field because of the near universal presence of more than one nitrogen source at any time; therefore, controls become problematical. In addition, the coexistence of different groups of microorganisms in natural samples complicates even further the study of the nitrogen uptake dynamics of phytoplankton. Nitrogen can be excreted by zooplankton and remineralized by heterotrophic bacteria (Harrison, 1980, 1992), but bacteria can also compete with phytoplankton for the uptake of the same nitrogen sources (Kirchman, 1994). Moreover, uptake of nitrogen by phytoplankton wi l l be affected differently by environmental variables. Therefore, separating and quantifying the phytoplankton uptake interactions between different nitrogen sources in the field is an extremely complicated task. The study of the interaction between uptake of nitrate and ammonium in the natural environment, however, is still useful because it allows the determination of their overall relationship and effects on natural assemblages of phytoplankton under combined environmental conditions. In the oceanic N E subarctic Pacific, concentrations of nitrate are high while ammonium concentrations are low and fairly constant annually (see Chapter 1). Despite the high nitrate concentrations, phytoplankton biomass remains low (see Chapter 1) even during times of the year when other environmental variables are favorable for rapid growth. A number of factors have been cited as responsible for this phenomenon, such as iron limitation and grazing pressure (see "Discussions" Chapters 1 and 2; and e.g. Martin and Fitzwater, 1988; Martin etal., 1989; Coale, 1991; Mil ler etal., 1991a, b; Frost, 1991, 1993; Landry etal., 1993a, b; Mi l le r , 1993; Strom et al., 1993; Boyd etal., 1996). Wheeler and Kokkinakis (1990) added anew candidate: ammonium inhibition of nitrate uptake. From their field study, they suggested that the constant recycling of ammonium in subarctic waters can also limit the utilization of nitrate by phytoplankton, and thus prevent its depletion. However, their conclusions were based on the field observation that nitrate uptake rates decreased with ambient ammonium concentrations. This observation deserves further 126 testing both in the field and in the laboratory with unialgal cultures of ecologically relevant species isolated from subarctic waters. It is important that the laboratory studies are carried out under conditions that best simulate the natural environment in terms of light intensity and periodicity, nutrient concentrations, and temperature, so that the results can be extrapolated to the field. Numerous physiological studies have focused their attention on the coccolithophore Emiliania huxleyi (Class Prymnesiophyceae), formerly called Coccolithus huxleyi. This cosmopolitan species can form massive blooms which may play an important role in the biogeochemical cycle of carbon (Holligan et al.; 1983; Fernandez et al, 1993; Holligan et al, 1993; Brand, 1994; Townsend etal., 1994). It is not yet clear whether E. huxleyi contributes significantly to the sink of atmospheric carbon or acts as a net source of C 0 2 (Flynn, 1990; Holligan, 1992; Paasche, 1992; Sikes and Fabry, 1994; Crawford and Purdie, 1997). Emiliania huxleyi blooms can also be relevant in climate change, by releasing dimethyl sulfide that can enhance the process of cloud formation (Keller, 1988/1989; Keller et al., 1989; Holligan, 1992; Holligan etal., 1993). Thus, this coccolithophore is of great importance in global nutrient cycles. Emiliania huxleyi has been found to be the most abundant coccolithophore in surface waters of the oceanic N E subarctic Pacific (Honjo and Okada, 1974) and is one of the representatives of the dominant < 5 /*m autotrophic size class (see Chapter 1; Booth et al., 1982; Taylor and Waters, 1982; Booth, 1988; Booth etal., 1993). The majority of studies onEmiliania huxleyi, however, have been conducted on isolates obtained from the North Atlantic (e.g. Brand, 1982), because up to now isolates from the oceanic N E Pacific or any other H N L C region have not been available. However, Emiliania huxleyi was successfully isolated in 1991 from water samples from station P26 (or Papa) in the oceanic N E Pacific (see Muggl i , 1995). Since then, experiments on this isolate have focused mainly on metal nutrition (Muggli and Harrison, 1996a,b) and sinking rates (Lecourt etal., 1996). Studies are needed to understand other physiological aspects, such as the nitrogenous nutrition, of this coccolithophore from an H N L C region. The primary purpose of the present study was to investigate the effect of changing ammonium concentrations on nitrate uptake rates in Emiliania huxleyi isolated from the oceanic N E 127 subarctic Pacific. However, additional laboratory and field data are also provided here. Thus, this chapter presents: (1) the physiological characteristics for E. huxleyi grown under environmental conditions typical of the oceanic N E Pacific; (2) the diel periodicity of nitrate uptake rate by E. huxleyi and the response of other physiological characteristics to irradiance; (3) the effect of increasing ammonium concentration on nitrate uptake rate in E. huxleyi; (4) in situ nitrate uptake rate by natural assemblages of phytoplankton from the oceanic N E Pacific over a range of ambient ammonium concentrations, and the effect of ammonium additions. M A T E R I A L S A N D M E T H O D S Laboratory experiments Culture conditions Maintenance cultures: Emiliania huxleyi was isolated from water samples collected at station P26 in November, 1991 (Muggli , 1995). Recently isolated cells had multiple layers of coccoliths, and in order to preserve the original morphology, E. huxleyi was maintained in nutrient-enriched microwave-sterilized station P26 water (Muggli , 1995). It has been observed that with the use of artificial or nutrient-enriched coastal seawater media, E. huxleyi looses its coccoliths, becoming naked and motile after some time (D. Muggl i , E . Simons, pers. comm.). It is not clear why E. huxleyi does not experience those changes when using nutrient-enriched station P26 water as the maintenance medium. The maintenance medium for the cells utilized in the present study was enriched with macronutrients (30pM N 0 3 " and 5 ptM HP0 4 2 " ) , metals (23 n M M n , 8 n M Z n , 1 n M Cu, 2.5 n M Co, 100 n M M o , 10 n M Se and 100 n M Fe), E D T A (10 yM) and vitamins (1 x 10"4 g L" 1 thiamine and 5 x 10"7 g L" 1 biotin) (Price etal, 1988/1989; Muggl i , 1995). Cultures were maintained at 128 16°C under low irradiance (ca. 30 pimol photons m"2 s"1 provided by Vita-lite™ fluorescent tubes and measured with a Biophysical Instruments Inc. light meter, model Q S L 100) on a 14:10 h light: dark cycle. Experimental cultures: Experimental cultures were grown in conditions that closely simulated E. huxleyi's natural oceanic environment for the summer period. Cells were grown in semi-continuous batch cultures with filter-sterilized (0.22 yim) nutrient-enriched artificial seawater medium ( E S A W ; Harrison et ah, 1980) in 1 or 2 L acid washed glass flasks. The original E S A W was modified by reducing 50 times the concentrations of Fe, M n , Zn , Co and E D T A , adding 0.02 n M N i and M o , adding 1 n M Se, decreasing the concentrations of N 0 3 " to 30 yM and H P 0 4 2 " (as N a ^ P O J to 3 piM, and not adding Si . Cultures were exposed to saturating irradiance (ca. 180 frniol photons m"2 s"1; Muggli and Harrison, 1996b) with a 14:10 h light:dark cycle while immersed in a temperature-regulated water bath maintained at 10.5 ± 0.5°C. Cultures were manually agitated once or twice daily because the coccoliths were knocked off i f a stir bar was used. Cultures were not exposed to bubbling in order to avoid any possible contamination by N H 4 + from the air (since very low ammonium concentrations were used for the experiments). Since cultures were not bubbled with air, care was taken to allow a constant air space in the culture flasks to avoid carbon limitation. Moreover, the concentration of total dissolved inorganic carbon (DIC) was measured on two occasions with an A D C type 225 M k 3 infra-red gas analyser during the logarithmic phase of growth to check for carbon availability. D I C values were found to be 1.92 and 2.05 m M , close to the typical DIC concentration in the oceans (i.e. around 2 mM) . The p H of the medium was measured frequently and found to range between 7.9 and 8.1. Cells were acclimated to the described conditions for > 10 generations. During the acclimation period, cell growth was monitored daily (at the same time every day) using cell counts and in vivo fluorescence. A l l transfers and sampling were conducted in mid logarithmic growth phase. Careful microscopic examination of the cultures was done at the time of 129 the transfers to ensure that the morphology of the cells remained unchanged, mainly in terms of coccolith abundance, for the entire experiment. Cultures were discarded if the number of layers of coccoliths on the cells was decreasing in any of the flasks. Cultures were unialgal and sterile techniques were employed in order to reduce bacterial contamination. Sterile techniques included sterilization by autoclaving of all glassware (flasks for media and cultures), filter-sterilization of media, and use of laminar flow hood for all transfers and manipulation of cultures. Although bacterial counts were not performed, due to the precautions taken, bacteria were most likely maintained at low enough levels so that their effect would have been negligible relative to the response of E. huxleyi. After the acclimation period, two separate experiments were performed: (1) Diel periodicity ofN03 uptake rate and other physiological characteristics in a 14:10 L:D cycle in the absence of NH/: Every 3 h during a 24 h period, samples were drawn from triplicate cultures for the determination of cell number, cell volume, in vivo fluorescence, Chi a, P N , P C , and dissolved N 0 3 " concentrations. Nitrate uptake rates (fmoles cell" 1 h"1) were calculated for each 3 h period from the disappearance of N 0 3 " from the medium and then normalized to the average cell number over the same period. Ammonium concentration was also measured, even if no additions of N H 4 + were made. (2) N03 uptake rate with NH4+ additions: These experiments were carried out by adding N H 4 + to the N0 3 "-grown cultures of E. huxleyi and monitoring the concentrations of N0 3 "and N H 4 + in the medium. Ammonium additions to multiple cultures ranged from 0 (control) to ca. 3 piM. Immediately after the N H 4 + addition, samples were drawn for the determination of cell counts, cell volume, in vivo fluorescence, Chi a, P N , P C , and dissolved N0 3 "and N H 4 + concentrations. Twenty-four hours after the N H 4 + addition, cultures were re-sampled for the same measurements. This procedure was repeated every 24 h until N H 4 + was undetectable (< 0.05 piM) in the medium, and then samples were taken again 24 h after N H 4 + became undetectable. Nitrate uptake rates (fmoles cell" 1 d"1) were estimated from the disappearance of N 0 3 " from the medium by calculating the decrease in concentration between the beginning and the end of 130 every 24 h period, and then normalizing them to the average cell number over that period. The concentration of N H 4 + for the same 24 h period was calculated as the average between the beginning and the final concentrations. Replicate cultures were run simultaneously as well as staggered in time. Analytical methods Growth rate was calculated as the slope of the natural log of cell number vs. time, and of the natural log of in vivo fluorescence vs. time. Cell counts and average cell volumes were determined with a Coulter Counter® Model T A I I particle counter equipped with a population accessory using a 70 pim aperture. Before making measurements with the Coulter Counter®, the p H of the samples was decreased to ca. 5 with 5% HC1 to dissolve the coccoliths, which could interfere with cell counts in some of the channels. In vivo fluorescence was measured with a Turner Designs™ Model 1 0 - A U fluorometer (Lorenzen, 1966). Samples for Chi a were filtered through Whatman® G F / F filters and stored at -20°C in a desiccator until analysis. Extraction of Chi a was done as explained in Chapter 1. Chlorophyll a concentrations were measured using in vitro fluorometry (Yentsch and Menzel, 1963) with a Turner Designs ™ Model 10 -AU fluorometer as outlined by Parsons et al. (1984). Samples for P N and PC(t) (total, including coccoliths) were filtered through pre-combusted Whatman® G F / F filters and stored at -20°C in a desiccator. After drying at ca. 60°C for 24 h, samples were prepared for analysis with a Carlo Erba Model NA-1500 Element Analyzer as described by Verardo etal. (1990). Samples for dissolved N H 4 + and N 0 3 " were filtered through pre-combusted Whatman® G F / F filters into acid cleaned polypropylene bottles. Nutrient samples were processed immediately or kept at 4°C until analysis within a few hours. Dissolved N H 4 + and N0 3 ~ concentrations were measured colorimetrically with aTechnicon® Autoanalyzer® II following the procedures of Slawyk and Maclsaac (1972) and Wood etal. (1967), respectively. 131 Nitrate uptake rates were estimated from the disappearance of N 0 3 " from the medium, as explained above. Due to the length of the period over which the rates were calculated (3 h in experiment (1) and 24 h in experiment (2)), the term "uptake" includes transport of nitrogen through the membrane and also assimilation into amino acids and proteins (i.e. growth; Collos and Slawyk, 1980; Goldman and Glibert, 1983; Wheeler, 1983). Efforts were made to carry out short-term measurements in experiment (2), but due to the low biomass of the cultures, the calculation of nutrient disappearance was very imprecise. Precision was greatly enhanced by performing 24-h measurements, which were also more appropriate for comparison with field data. Models used The N 0 3 " uptake rate vs. N H 4 + concentration data were modeled using three non-linear least-squares fits: (1) a reversed Michaelis-Menten equation of the form: where, V N 0 3 " is the N 0 3 " uptake rate (fmoles cell" 1 d"1, or d"1); is the maximum rate of N 0 3 " uptake at undetectable N H 4 + concentrations (fmoles cell" 1 d"1, or K ; is the N H 4 + concentration at which N 0 3 " uptake rate is reduced to half of its maximum rate (i.e. N H 4 + is the N H 4 + concentration (piM). 132 (2) a modi f i ed M i c h a e l i s - M e n t e n equation (Harr ison et al., 1996) o f the form: pNO~(rel) = 1 -I * NH + •'max  l y n 4 {KT + NH; ) where, p N 0 3 " (rel) is the N 0 3 " uptake rate normal ized to the m a x i m u m observed uptake rate at undetectable N H 4 + levels (values f rom 0 to 1); 1 ^ is the m a x i m u m realized inhib i t ion (values from 0 to 1); K ; and N H 4 + are as above. (3) a s imple exponential model o f the form: where , A + B is the m a x i m u m rate o f N 0 3 " uptake (i.e. V ^ J at undetectable N H 4 + concentrations; C is the exponential decay constant; p N 0 3 (rel) and N H 4 + are as above. Field experiments Data f rom two f ie ld experiments conducted at station P 2 6 are also u t i l ized i n this chapter. 1) N03 uptake rate with NH4+ additions: O n M a y 20, 1994, a water sample f rom 5 m depth was col lected at P 2 6 f o l l o w i n g the same sampl ing procedures as specified i n Chapters 1 and 2, and transferred to eight, 2 L ac id cleaned polycarbonate Nalgene® bottles. Increasing additions o f N H 4 + (0, 0.2, 0.6 and 0.8 /<M) were made to duplicate samples. T h e ambient N H 4 + concentration was 0.17 ptM, thus mak ing the final concentration i n the duplicate samples 0.17, V7V0 3 " or pNQ3~(rel) = A+ B* e (-NH4+/C) 133 0.37, 0.77 and 0.97 JAM. The same water samples also received a 1 S N 0 3 " enrichment at a concentration of 10% of ambient (11 pM) N0 3 ~. Inoculated samples were incubated for 24 h in on-deck incubators immersed in flowing surface seawater. A t the end of the incubation period, samples were filtered through pre-combusted Whatman® G F / F filters and stored at -20°C until analysis in the laboratory. The 1 S N / 1 4 N ratio in the particulates was analysed and N-specific N 0 3 " uptake rates (d 1 ) calculated as explained in Chapter 2. Samples for P N (2 L ) , and dissolved N 0 3 " and N H 4 + concentrations were collected at the beginning of the experiment, and handled and analysed as described in Chapter 1. Particulate nitrogen and dissolved nutrients were also collected from the incubation bottles at the end of the 24 h incubation period to check for changes in P N concentration and nutrient exhaustion. The concentration of P N at the end of the incubation period showed no detectable differences from the P N concentration at the beginning; thus, initial P N values were used for uptake rate calculations in order to be consistent with all other calculations used in this thesis. Measurement of dissolved N 0 3 " and N H 4 + at the end of the incubation period showed no nutrient exhaustion during the 24 h incubation period. 2) N03 uptake rate vs. ambient NH/ concentrations: Nitrate uptake rate data corresponding to the depths of 100, 55 and 30% of ID for P26 from all 6 cruises (Chapter 2) were utilized in this section. N-specific N 0 3 " uptake rates were plotted against the corresponding ambient N H 4 + concentrations (Chapter 1) and pooled with the data obtained in the field experiment with N H 4 + additions described above. The field data were fit to the (1) reversed Michaelis-Menten and (3) simple exponential models explained above. Because of the length of the incubation period utilized for the experiments, "uptake" calculations included not only transport, but also assimilation and incorporation of N 0 3 " (i.e. growth; Collos and Slawyk, 1980; Goldman and Glibert, 1983; Wheeler, 1983). 134 R E S U L T S Laboratory experiments Physiological characteristics for Emiliania huxleyi Table 4.1 summarizes some physiological parameters for the experimental cultures of E. huxleyi grown under the conditions specified in the "Materials and Methods" section of this Chapter. The growth rate (0.52 + 0.01 d"1; mean + 1 S.E., n = 52) shown in the table was calculated from cell numbers and was not significantly different from the growth rate (0.49 ± 0.01 d"1; mean + 1 S.E., n = 55) calculated from in vivo fluorescence (Rest, P > 0.01). Doubling time for these cultures was 32 h (0.75 div d"1). Diel periodicity ofN03 uptake rate and other physiological parameters for Emiliania huxleyi Nitrate concentrations in the culture medium were followed every 3 h for a 24 h cycle and showed a more rapid decrease during the 14 h light period than during the 10 h dark period (Fig. 4.1 A ) . Although the objective of this experiment was to observe the diel variation of the rate of N 0 3 " uptake in the absence of N H 4 + , N H 4 + concentrations of 0.1 yM were measured during the first 6 h of the experiment. However, for the rest of the incubation period, N H 4 + was undetectable (< 0.05 pM). The slopes of N 0 3 " disappearance for the period 0-3 and 3-6 h were lower than the slopes from 6-15 h. The presence of 0.1 pM of N H 4 + could have been responsible for the slower rate of N 0 3 " disappearance (see next section and Fig 4.4). Due to the presence of N H 4 + during the first 6 h of the experiment, only N 0 3 " uptake rates from 6 to 24 h are shown in Fig 4.1 B . Cell specific N 0 3 " uptake rates were significantly higher during the light period (2.53 ± 0.04 fmoles of NCVce l l " 1 h"1; mean + S.E.) than during the dark period (0.69 ± 0.02 fmoles of N 0 3 " c e l l _ 1 h"1, mean ± S.E.) (f-test, P « 0.001). Thus, in a 24 h diel cycle of 14 L : 10 D , 84% of the total daily N 0 3 " was taken up during the light period, and the remaining 16% during the dark period. 135 Table 4 .1 . Phys io log i ca l parameters for Emiliania huxleyi cultures g rown i n mod i f i ed E S A W wi th 30 ptM nitrate under saturating irradiance on a 14:10 L : D cyc le at 1 0 . 5 ° C . Parameter M e a n ± 1 S . E . (n) G r o w t h rate (d' 1) 0.52 ± 0.01 (52) G r o w t h rate (div d"1) 0.75 + 0.01 (52) C e l l vo lume * ( /mi 3 ) 42 .7 + 0.8 (52) C h i a quota (pg ce l l " 1 ) 0.138 ± 0.003 (29) Ni t rogen quota (pg cell" 1) 1.7 + 0.1 (27) Ca rbon (t) quota * (pg cell" 1) 14.6 ± 0.7 (28) * C e l l v o l u m e = without coccoli ths. * Ca rbon (t) = total carbon, inc lud ing coccoli ths. 136 S a O a o o CD o c/j J D "o I -4—» CD OH dP 2 0 -1 8 -1 6 - "°--„ 1 4 -^ 0 r\ 1 2 -A i i i i i i i , , | , , 3 . 0 -2 . 5 -2 . 0 -1 . 5 -1 . 0 -0 . 5 -B • • • ^ • • 1 0 I 1 ' , 9 12 15 18 I n c u b a t i o n t i m e (h ) 21 24 Figure 4 .1 . Changes i n ( A ) nitrate concentration and (B) nitrate uptake rate (calculated for each 3 h per iod, starting at 6 h ; see text for details), for Emiliania huxleyi cultures dur ing the 14:10 L : D experiment. T h e gray area (14 to 24 h) indicates the dark per iod. E a c h s y m b o l represents the mean o f triplicate cultures ± 1 S . E . If no error bars are v is ib le , they are smal ler than the symbo l . 137 During the L : D cycle, other physiological parameters were also measured (Figs. 4.2 and 4.3). Cel l numbers showed a rapid increase during the last part of the dark period (Fig 4.2 A ) . During most of the light period and the first 4 h of darkness, cell numbers remained constant. In contrast, in vivo fluorescence increased during the light period but changed little during the dark (Fig. 4.2 B) . A s a result of these diel patterns of cell numbers and in vivo fluorescence, fluorescence per cell increased during the light period, but decreased during the dark (Fig 4.2 C). The rate of accumulation of P N and PC(t) in the cultures was higher during the day than during the night, while Chi a showed a constant increase during the 24 h cycle (data not shown). When these parameters were expressed as cell quotas (Fig. 4.3 A to C) , they all showed a similar pattern of increase during the light period and decrease during the dark due to the higher cell number during the dark period. Cell volume also increased during the light hours, but decreased at night (Fig. 4.3 D). Effect of increasing NH/ concentrations on N03 uptake rate in Emiliania huxleyi The presence of increasing N H 4 + concentrations resulted in a decrease in N 0 3 " uptake rates (Fig. 4.4). When N H 4 + concentrations were undetectable in the culture medium, N 0 3 " was taken up at maximal rates; however rates decreased sharply when N H 4 + was present even at low concentrations (0.1-0.5 pM), and was almost completely inhibited at 2.2 pM N H 4 + . Figures 4.4 A and B present the same data set (n = 56). In Figure 4.4 A , data are shown as fmoles of N 0 3 " taken up cell" 1 d"1 vs. N H 4 + concentration, and in Figure 4.4 B , data have been transformed to relative N 0 3 " uptake rate (0 to 1 scale) vs. N H 4 + concentration in order to apply the modified Michaelis-Menten model (Harrison etal., 1996; see "Materials and Methods" of this Chapter). Figure 4.4 A shows that the reversed Michaelis-Menten and exponential models fit the data well. The values (+ S.E.) calculated by both models are in good agreement, with an estimate of 53.24 ± 0.24 fmoles of N 0 3 " cell"1 h"1 from the reversed Michaelis-Menten fit and 51.34 ± 0.61 fmoles of N Q j ' c e i r 1 d"1 from the exponential fit. The reversed Michaelis-Menten model also estimates a K ; (± S.E.) of 0.24 ± 0.004 pM of N H 4 + . 138 CO e £ -a ^2 B ° 3 2 U w CO GO = 2 o oo CD B > FT P _ 1/1 — s O • s 3 CO S-H § 2 240 -I 200 -4 160 H A / . . , 5 l - ^ § ^ 1—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—r 7.0 H 6.0-1 5.0 -A 4.0 -4 B Q * Cr"' ..a .o-o o--" I 1 1 I 1 1 I 1 1 I - i — i — i — i — r I 1 1 I 1 1 I 1 1 I 36 - c 5 32 -...Ci"' ' I^BllBUlll 28 -5 Q 2 4 -2 0 -T 9 12 15 Incubation time (h) 18 21 1 1 I 24 Figure 4.2. Changes in (A) culture density, (B) culture fluorescence, and (C) fluorescence per cell for Emiliania huxleyi during the 14:10 L:D experiment. The gray area (14 to 24 h) indicates the dark period. Each symbol represents the average of triplicate cultures + 1 S.E. If no error bars are visible, they are smaller than the symbol. 139 CD O 00 w o 3 1.6 H 14 H 1.2 H 1.0 4 o ^ } D T — i — i — T - T — r - r <D o 00 OH o 3 ? Q CD O oo a . o 3 <3 u <D CJ m s CD a 3 > U 14-12 • 10-0.16 H 0.14 Hi 0.12 H 48-44-40-36-32 • l — | — i — i — | — i — i — | — i — i — i — i — r B A f §|ll^i§||fli 0 o c T • [ 1 • • • ( • I B I 1 1 I D p l l l l l l l l l ..-0-"'" •)••-••" J § l l l l i l l B l l l l IHIH111 c7 T 9 12 15 18 Incubation t ime (h) 21 24 Figure 4.3. Changes i n ( A ) nitrogen quota, (B) total carbon quota ( inc lud ing coccol i ths) , (C) ch lo rophy l l a quota, and (D) ce l l v o l u m e for Emiliania huxleyi dur ing a 14:10 L : D cyc le . T h e gray area (14 to 24 h) indicates the dark period. E a c h s y m b o l represents the average o f triplicate cultures ± 1 S . E . If no error bars are v i s ib l e , they are smaller than the symbo l . 140 0.0 0.4 0.8 1.2 1.6 2.0 NH 4 + concentration (j/M) Figure 4.4. (A) Nitrate uptake rate and (B) relative nitrate uptake rate at increasing ammonium concentrations for cultures of Emiliania huxleyi grown in a 14:10 L:D cycle. Each symbol represents a determination from a single culture (n = 56). The models applied to the data as well as the fitted parameters (± S.E.) are included. 141 When applying the modified Michaelis-Menten model (Fig. 4.4 B) , the estimation of K ; (± S.E.) is 0.38 + 0.01 pM of N H 4 + . However, judging from the graphical representation of the modified Michaelis-Menten fit and by calculating the concentration of N H 4 + at using the equation derived from this model, K ; is ca. 0.28 pM (which is also in agreement with the estimate from the reversed Michaelis-Menten model). Even i f the modified Michaelis-Menten model seems to fit the data well , the estimated K ; does not agree with the value of N H 4 + concentration at which the of N 0 3 " is reduced to half. It is suggested here that the problem with the modified Michaelis-Menten model is that 1^ is not constrained, when it should only vary between 0 and 1 (0 to 100% inhibition), and in this particular case, it yields an 1^ value of 1.17 ± 0.01 (> 100%; shown as « 1 0 0 % in Figure 4.4 B) . If the modified Michaelis-Menten model is forced to run with i m a x = 1, then the fit overlaps with the reversed Michaelis-Menten curve. Therefore, the reversed Michaelis-Menten model seems to be the most appropriate fit for this data set. A l l models agree, however, in estimating almost complete inhibition at the highest concentrations of N H 4 + utilized. The exponential model is included in both Figure 4.4 A & B for comparative purposes. Field experiments Effect of increasing NH/ concentrations on N03 uptake rate by natural assemblages of phytoplankton from station P26 Figure 4.5 shows N-specific N 0 3 " uptake rates from surface waters of station P26 from 6 cruises, measured with the 1 5 N technique, plotted against N H 4 + concentration. Data from the N H 4 + addition experiment conducted during May 1994 are also included in Figure 4.5. One major drawback of these field data is the limited range of ambient N H 4 + concentrations over which the N 0 3 " - N H 4 + relationship could be studied. No rate values for concentrations of N H 4 + < 0.17 pM were available (0.17 pM was the lowest N H 4 + concentration in surface waters measured during the field component of this thesis), and hence no controls (N0 3 " uptake rates at zero N H 4 + ) were possible. The highest end of the range of ambient N H 4 + concentrations was only 0.54 pM; thus, 142 o.io —r 0.08-^  0.06-^  0.OM O O V V V T • 0 % Reversed Michaelis-Menten model V m a x = 0.066 ± 0.003 d"1 K; = 0.61 + 0.07 yM Exponential model V m a x = 0.061 + 0.020 d"1 I m a x - 100% o i j p v XI N o Sep 92 N Mar 93 • May 93 A Feb 94 V May 94 T May 94, + N H 4 + o Sep 94 0.02-^  0.0 I 1 I 1 I 1 I 0.2 0.4 0.6 0.8 NH4+ concentration (//M) 1.0 Figure 4.5. N-specific nitrate uptake rate vs. ambient ammonium concentration for the natural assemblage of phytoplankton in surface waters at station P26 in the NE Pacific. Each symbol represents a single determination (n = 40). Filled symbols ( T ) are for the N H 4 + addition experiment (+ NH 4 + ) done at P26 during May 1994. The models applied to the data as well as the fitted parameters (± S.E.) are included. 143 the N H 4 + addition experiment was performed to investigate the N 0 3 " uptake response at higher N H 4 + values. A t 0.77 and 0.97 piM N H 4 + , N 0 3 " uptake rates were low; however, between 0.17 and 0.54 piM N H 4 + , N 0 3 " uptake rates ranged widely between 0.02 and 0.095 d: 1 for any time of the year. A seasonal trend is not evident from these data. The reversed Michaelis-Menten and exponential models fit the data poorly; i.e. and K ; are estimated with considerable error (C. V . > 29% and = 73%, respectively), and due to the lack of N 0 3 " uptake rates at undetectable N H 4 + concentrations, the modified Michaelis-Menten model could not be applied. D I S C U S S I O N Physiological characteristics for Emiliania huxleyi Over the four year period since E. huxleyi was isolated, much care has been taken to maintain this isolate under conditions typical of the oceanic N E Pacific in order to avoid adaptation to unrealistic culture conditions. Except for total carbon quotas, every other physiological parameter of E. huxleyi measured during the present study was in close agreement with the measurements made soon after its isolation (Muggli and Harrison, 1996b). However, total carbon per cell was lower in the present study, 14.6 ± 0.7 compared to 27.6 + 1.1 pg cell" 1 in Muggli and Harrison (1996b), probably because of the presence of more layers of coccoliths on the cells of the original cultures. Most of the other physiological characteristics measured here can be regarded as representative of the original isolate. In comparing physiological parameters for this subarctic isolate, it should be noted that different experimental conditions have been utilized in previous studies. In the present study, cultures were grown at 10.5°C and on a 14:10 L : D cycle. Muggli and Harrison (1996b) exposed the cultures to 16°C and an identical light regime, and Lecourt et al. (1996) utilized 17°C and continuous illumination. In the present study, growth rates were 0.75 div d"1 and cell volumes were 42.7 pim3, compared with 0.63 div d"1 and 48.9 pim3, respectively, in Muggli and Harrison 144 (1996b). Lecourt etal. (1996) reported the lowest growth rates (ca. 0.5 div d"1) and cell volumes (ca. 27 pim3). The greater differences observed between Lecourt etal. (1996) and the other two studies may be attributed to the different photoperiods utilized. Brand and Guillard (1981) indicated that continuous illumination was slightly inhibitory for E. huxleyi growth. Price etal. (in press) also showed that although the responses of nine coccolithophores to different photoperiods were variable, lower growth rates were measured under continuous light than on a 14:10 L : D cycle for some species. A greater degree of variability is observed when comparing physiological parameters of this isolate with other isolates from different regions. A coccolith-forming coastal clone of E. huxleyi from the Oslo Fjord was found to have growth rates of 1.85 div d"1 (Paasche, 1967), and 1.68 div d"1 (Paasche and Klaveness, 1970), compared to 0.75 div d"1 for the oceanic isolate in the present study. However, the growth rates measured by Paasche (1967) and Paasche and Klaveness (1970) were obtained at 21°C, which is close to the optimum temperature for growth at saturating irradiance (Paasche, 1967), and therefore the observed higher rates are expected. In fact, Paasche (1967) followed the growth rate of E. huxleyi over a range of temperatures for different photoperiods. Thus, growth rates at 10°C for a 16:8 L : D were ca. 0.6 div d"1, similar to the rates found for the subarctic isolate (this study, Muggli and Harrison, 1996b, and Lecourt et al., 1996). In other studies, however, growth rates were found to be higher, even at temperatures and photoperiods similar to the ones used here and in Muggli and Harrison (1996b). Brand (1982) measured the growth rates of 73 clones of E. huxleyi from the N W Atlantic at 16°C on a 14:10 L : D cycle and found values ranging from 1.5 to 1.72 div d"1. Another isolate from the North Sea showed rates of growth averaging 1.23 div d"1 when grown at 10°C and on a 16:8 L : D cycle (Van Bleijswijk etal., 1994). In these cases, the cause for the difference in growth rates is likely due to genetic differentiation in isolates from different locations (Brand, 1982; Brand, 1988/1989), and not to the growth conditions. 145 Diel periodicity of N03" uptake rate and other physiological parameters in Emiliania huxleyi A l l the physiological characteristics measured in E. huxleyi showed diel changes during a 14:10 L : D cycle. The stimulation of N 0 3 " utilization by light is thought to be primarily at the assimilation rather than at the transport level (see Vincent, 1992). The higher rate of N 0 3 " uptake observed here for E. huxleyi during the light period is probably the result of the dependency of the N0 3 -assimilat ing enzymes on photosynthetic energy. Data from Eppley etal. (1971) confirm this suggestion; they found that the activity of N 0 3 " reductase (NR) and N 0 2 " reductase measured in cell-free extracts of E. huxleyi was higher during the day than during the night. These results have also been observed for other algal groups (see Syrett, 1981). Berges etal. (1995) reported that diel patterns of N R activity closely followed changes in nitrogen uptake rate under high light in Thalassiosirapseudonana. The diel pattern of N 0 3 " uptake rate observed by Berges etal. (1995) closely resembles the pattern shown in this study for E. huxleyi. The diel periodicity of nitrate uptake rate observed in this and other laboratory studies is supported by observations from field experiments from various locations (e.g. Goering etal., 1964; Maclsaac, 1978; Koike etal., 1986; Eppley etal., 1990; Cochlan etal, 1991a,b). In a few cases, diel periodicity of N R activity was also measured for natural populations and showed patterns similar to the ones observed in laboratory cultures (Eppley etal., 1970; Berges etal., 1995). In the oceanic subarctic Pacific, phytoplankton N 0 3 " uptake rates showed a clear diel pattern, with daytime rates about twice nighttime values (Cochlan etal., 1991a). A similar pattern was also reported by Wheeler et al. (1989) and Wheeler and Kokkinakis (1990). Although N R activity has not been measured in the N E subarctic Pacific, diel variations would probably show patterns similar to those found by Cochlan etal. (1991a) for N 0 3 " uptake rates. The synchronization of cell division observed during the present study, with very little i f any increase in cell number during the light period but a sudden increase during the dark, has been reported previously for other isolates of E. huxleyi (Paasche, 1967; Eppley etal., 1971; Nelson 146 and Brand, 1979; Linschooten et al, 1991; Van Bleijswijk etal., 1994). The division rate for E. huxleyi responded to diel periodicity in irradiance in the same manner as other algal species (e.g. Nelson and Brand, 1979; Berdalet etal., 1992). Of all the algal groups, the most complex and varied patterns of diel periodicity in cell number have been exhibited by diatoms. Diatom division rates have been shown to increase in the light (Nelson & Brand, 1979; Chisholm and Costello, 1980), in the dark (Eppley and Renger, 1974), in both light and dark periods (Chisholm and Costello, 1980) or to remain constant over the light:dark cycle (Nelson and Brand, 1979; Berges et al., 1995). Thus, diel periodicity in cell division seems to be species as well as clone specific (Nelson and Brand, 1979; see reviews by Chisholm, 1981 and Prezelin, 1992). The diel pattern of in vivo fluorescence and fluorescence per cell observed during this study coincided with the results of Van Bleijswijk etal. (1994) for a North Sea isolate of the same species and with Berges (1993) for the diatom Thalassiosirapseudonana. In the present study, specific growth rates calculated from in vivo fluorescence were not significantly different from those calculated from cell counts over 24 h (see "Results" of this Chapter). However, considering the diel pattern of cell number and in vivo fluorescence, it is evident that: (1) growth rate calculations for less than 24 h wi l l differ depending on the parameter used, and (2) cell numbers and in vivo fluorescence should be measured every day at the same time to obtain meaningful growth rate estimates. Rates of accumulation of total P C and P N , being light-dependent processes, were enhanced during the light period, while cell division occurred in the dark. A s a result, carbon and nitrogen quotas decreased during the dark. In terms of carbon, this coccolith-forming clone of E. huxleyi incorporated carbon not only for photosynthesis, but also for calcification. In spite of two processes being involved in carbon accumulation, the total carbon content per cell in E. huxleyi showed a similar diel variation to the one observed for the organic carbon content of the diatom Thalassiosirapseudonana (Berges etal, 1995). In contrast, the nitrogen content of T. pseudonana exhibited no variation during the diel cycle (Berges etal., 1995). 147 Chlorophyll a synthesis did not seem to decrease at night, but Chi a cell quotas were lower during the night due to the sudden increase in cell number. The same pattern of Chi a accumulation found here was reported by Paasche (1967) for the same species, and also for other phytoplankton (Latasaetal., 1992). However, for Thalassiosirapseudonana, Berges (1993) reported no variation in Chi a content during the L : D cycle. The diel pattern of cell volume observed in the present study is the result of the synthesis of cellular components during the light, and cell division during the dark. The diel pattern in mean cell size of E. huxleyi reported by Van Bleijswijk etal. (1994) showed a similar trend during the L : D cycle to the one found here. Latasa et al. (1992) reported similar trends for Heterocapsa sp. and Olisthodiscus luteus. When comparing cell volume patterns of E. huxleyi with diatom species, more complex patterns are observed for diatoms, with two peaks found during the 24 h diel period (Chisholm, 1981; Berges etal, 1995) as opposed to one for E. huxleyi. The observation of a strong diel periodicity in N 0 3 " uptake rates as well as in other physiological parameters for E. huxleyi helped in choosing appropriate sampling intervals for the calculation of N 0 3 " uptake rates at increasing N H 4 + concentrations. Measurements every 24 h were preferred so that all diel changes were accounted for. Effect of increasing N H 4 + concentrations on N 0 3 uptake rate The culture experiments reported here showed the inhibitory effect that N H 4 + has on N 0 3 " uptake in the subarctic isolate of E. huxleyi. Maximal N 0 3 " uptake rates observed at undetectable concentrations of N H 4 + were rapidly reduced when N H 4 + was present. This is the first study to investigate this phenomenon for any phytoplankton species isolated from a H N L C region. Inhibition of N 0 3 " uptake by N H 4 + has been seen in a many laboratory studies on unialgal cultures of phytoplankton and also in field studies of natural assemblages of phytoplankton (see review by Dortch, 1990). Most of the phytoplankton utilized in the laboratory have been species easily grown in culture and therefore only a handful of studies have been dedicated to ecologically 148 significant species. The culture conditions adopted in the present study for E. huxleyi were very similar to those found in the surface waters of the oceanic N E subarctic Pacific during the summer period, in terms of temperature, photoperiod and composition of the medium. The resulting biomass exceeded the natural values, but it was not unrealistically high, and thus, extrapolation to a field scenario is reasonable. Cochlan and Harrison (1991b) also simulated natural conditions as close as possible when studying nitrogen uptake interactions with the picoplankter Micromonas pusilla, and found that N 0 3 " uptake rate was completely inhibited at 1 pM N H 4 + . The original uptake rate of N 0 3 " was only resumed when N H 4 + was exhausted from the medium. Similar findings were reported for more commonly used species such as Chlorellavulgaris (Syrett and Morris , 1963), Skeletonema costatum (Bates, 1976; Conway, 1977; Dortch & Conway, 1984; Lund, 1987) and Thalassiosirapseudonana (Yin , 1988; Dortch etal. 1991; Berges etal., 1995). In all of the above studies, significant or complete inhibition of N 0 3 uptake was observed between 0.5 and 1 pM N H 4 + . In the isolate of E. huxleyi studied here, however, inhibition at 0.5 and 1 pM N H 4 + was ca. 67 and 84%, respectively; and complete inhibition was not achieved below 2.2 pM. These differences are not unexpected because inhibition is species specific and is also affected by the preconditioning of the cultures, light levels and other environmental factors, such as temperature. Nitrate uptake rate in nitrogen-sufficient cultures of Skeletonema costatum was completely inhibited by N H 4 + when the cells were preconditioned with N H 4 + as the sole nitrogen source (Dortch and Conway, 1984). In contrast, the same study showed that when nitrogen-sufficient cultures of S. costatum were preconditioned with N 0 3 " , the extent of the inhibition was very small (ca. 6%). In nitrogen-deficient cultures of S. costatum, the degree of inhibition was not only dependent on the preconditioning nitrogen source, but also on the growth rate. Inhibition was higher when cultures were preconditioned with N H 4 + , and was inversely related to growth rate (Dortch and Conway, 1984). Nitrogen^starved S. costatum showed complete inhibition of N 0 3 " uptake by N H 4 + (Dortch and Conway, 1984). Dortch and Conway (1984) also found that nitrogen-deficient cultures of Chaetoceros debilis showed much greater inhibition than S. costatum with similar preconditioning, whereas there were no significant 149 differences when g r o w n i n N 0 3 ~ or N H 4 + . Dor t ch etal. (1991) d i d s imi la r experiments wi th Thalassiosirapseudonana. Contrary to the f indings o f D o r t c h and C o n w a y (1984), they observed that inhib i t ion o f N 0 3 " uptake i n nitrogen-deficient cultures o f T. pseudonana g r o w n on N H 4 + was less than i n the cultures g rown on N 0 3 " . Furthermore, the degree o f inh ib i t ion is also affected by l ight intensity. Bates (1976) showed that N 0 3 " uptake rates by Skeletonema costatum and a chlorophyte were depressed by N H 4 + at a l l l ight intensities, but more i n l igh t - l imi ted cultures of both species, and more i n S. costatum. In contrast, Y i n (1988) found that Thalassiosira pseudonana d i d not show inh ib i t ion o f N 0 3 uptake by N H 4 + at l o w l ight intensities. It is not yet clear how temperature w o u l d affect the N 0 3 " - N H 4 + interactions (Dor tch , 1990). These results illustrate the complex i ty o f the nitrogen uptake interactions when species are exposed to different environmental condi t ions, and serve to emphasize the mul t ip le responses that can be found i n different laboratory studies and i n the f ie ld . In spite o f the var iabi l i ty of condit ions found i n the natural environment, f ie ld studies o f nitrogen uptake interactions seem to suggest that N H 4 + inh ib i t ion o f N 0 3 " uptake c o m m o n l y occurs i n the oceans. Such conclusions based upon f ie ld data generally come from two sources: (1) the observation o f the relationship between the measured N 0 3 " uptake rate and the ambient N H 4 + concentration (e.g. M a c l s s a c and Dugdale , 1972; M c C a r t h y etal, 1975; Gars ide , 1981; Paasche and Kr i s t i ansen , 1982; Co c h l a n , 1986; D o r t c h and Postel , 1989; Whee le r and K o k k i n a k i s , 1990), and (2) the results f rom control led experiments on N 0 3 " uptake rates where N H 4 + is added incremental ly to water samples (e.g. B l a s c o and C o n w a y , 1982; M u g g l i and S m i t h , 1993; Har r i son etal., 1996). In the first case, it is diff icul t to separate the effect o f inh ib i t ion from other interactions (e.g. preference), so that the results show the response o f the rate o f N 0 3 " uptake to N H 4 + concentrations under a range o f condit ions. In the second case, the direct effect o f N H 4 + on the N 0 3 " uptake rate is better observed and the results are closer to the response expected f rom an exc lus ive ly inhib i tory effect. In the present study, f ie ld results for station P 2 6 obtained from both approaches are plotted together (F ig . 4.5). A t N H 4 + concentrations < 0.6 pM, a w i d e range o f uptake rate values was observed. H o w e v e r , at concentrations > 0.6 pM N H 4 + , N 0 3 " uptake rates 150 remained low, implying a negative non-linear effect of N H 4 + on N 0 3 " uptake. Those low N 0 3 " uptake values at high N H 4 + additions may also be due to low irradiance conditions on the day of the experiment (20 May 1994, see Fig. 1.2 C in Chapter 1). Similar results were shown in Figure 2.13 (Chapter 2) for a complete data set of the entire Line P transect. Among other studies, McCarthy etal. (1975), Cochlan (1986), and Dortch and Postel (1989) showed a similar relationship to the one found here between the fraction of N 0 3 " to total nitrogen utilized and the ambient concentration of N H 4 + for phytoplankton assemblages. The scatter of data observed here is not unusual for field studies due to the diversity of natural assemblages during different times of the year and the array of conditions that can affect the N 0 3 " - N H 4 + relationship. Field data that entirely corresponds to the observational approach mentioned above were presented by Wheeler and Kokkinakis (1990) in the first study of nitrogen uptake interactions in the oceanic N E subarctic Pacific. They indicated that N 0 3 " assimilation was completely inhibited at 0.1-0.3 yM N H 4 + , and the shape of the relationship was linear. Although the field data from the present study does not confirm their findings with certainty, an effect of ambient N H 4 + on N 0 3 " uptake rate is strongly suspected. Moreover, the results obtained for E. huxleyi in this laboratory experiment agree in that a negative relationship exits. However, inhibition of N 0 3 " uptake was never complete for E. huxleyi at concentrations as low as 0.1-0.3 yM N H 4 + and the relationship was non-linear. It was observed, however, that at ca. 0.24 yM N H 4 \ N 0 3 " uptake rate by E. huxleyi was reduced to 50% of its maximal rate and that the rate only reached undetectable values at 2.2 piM N H 4 + . Although the results of the present study and those of Wheeler and Kokkinakis (1990) did not entirely agree, both studies demonstrated that a suppression of N 0 3 " uptake occurs both in subarctic waters and in a phytoplankton species isolated from the same region. Non-linear exponential relationships between N 0 3 " uptake rate and N H 4 + concentration were obtained for field data when controlled experiments were performed (Blasco and Conway, 1982; Muggl i and Smith, 1993; Harrison etal., 1996). Those experiments are more comparable to the laboratory study presented here, as semi-controlled conditions were imposed during the field manipulations. In all cases, there was agreement in the shape of the relationship; however, there is 151 disagreement in the extent of the inhibition. Those field experiments showed a reduction of the rates of N 0 3 " uptake when N H 4 + increased, but did not show complete inhibition at any particular concentration of N H 4 + , in contrast to the findings for E. huxleyi in this study. Although N H 4 + inhibition of N 0 3 " uptake has been documented in many studies, not only in microalgae (see Dortch, 1990), but also in higher plants (e.g., see Glass and Siddiqi, 1995), the mechanism of this effect is still unclear. If inhibition is expressed on a short time scale, it is probably taking place at the transport level; however, i f inhibition is apparent over longer time scales, the transport as well as the assimilatory systems must also be affected. Lee and Drew (1989) noted that inhibition of N 0 3 " uptake in barley appeared within 3 minutes after the addition of N H 4 + . They suggested that N H 4 + inhibition was occurring at the membrane level. However, Blasco and Conway (1982) noted that inhibition of N 0 3 " uptake was maximal 6 h after the addition of N H 4 + , and that N R activity was significantly reduced after 24 h for marine phytoplankton assemblages from eutrophic areas. Their results showed that inhibition occurred at both levels, first on transport and then on assimilation. Effects of N H 4 + on N R activity were also noted by Eppley etal. (1969a) in cultures of Ditylum brightwellii, Cachoninaniei, Gonyaulaxpolyedra and Phaeocystis sp. For all these species, N R activity was suppressed during N H 4 + assimilation and started to increase when N H 4 + concentrations were lower than 1 pM for Cachoninaniei and 0.5 pM for the other species. In Thalassiosirapseudonana, Berges etal. (1995) noted that N R activity was completely absent when N H 4 + was present in the culture medium. Therefore, inhibition of N 0 3 " utilization by N H 4 + is reflected in a reduced transport capacity as well as a decrease in N R activity. In the present study, the time period between measurements was 24 h. Therefore, it was not possible to determine if inhibition was taking place at the transport level, assimilatory level, or both levels. It was also not possible to determine the time scales of this effect on these two physiological processes. The results reported here showed an overall effect on transport as well as assimilation and incorporation of N0 3 " . The biochemical basis for such an inhibitory effect is not clear. Syrett and Morris (1963) stated that inhibition was not due to N H 4 + per se, but to a product of its assimilation. Conway 152 (1977) proposed that the level of total amino acids in the cell suppressed N0 3" uptake rate through feedback control on the membrane permease system and/or on NR. It has also been suggested that glutamine, a product of N H 4 + assimilation, may cause a reversible (and partly irreversible) inactivation of NR, thus suppressing the assimilation of N03" (Syrett, 1981; Flynn, 1991). Ammonium is also considered energetically advantageous for assimilation over N03" due to its already reduced state. Although this would just be a 'preference' at the biochemical level, it could result in the production of a signal that would also give an inhibitory effect (due to the production of glutamine). Although a great deal of research is presently being done on the biochemical processes involved in N H 4 + inhibition of N03" uptake, an explanation is not yet available. Ecological implications On the basis of the findings of this laboratory study on Emiliania huxleyi, inhibition of N03" uptake by N H 4 + may be one of the factors contributing to the low rates of N0 3" uptake in surface waters of the oceanic NE subarctic Pacific during those times of the year when irradiance is saturating for growth. Nitrate-grown E. huxleyi showed higher growth rates than NH4 +-grown cells under low iron-conditions (Muggli and Harrison, 1996b). These findings suggest that the small cells in the subarctic Pacific could grow faster if N H 4 + was not depressing N0 3" uptake rates. The impact of this NH 4 + -N0 3 " uptake interaction on E. huxleyi on the subarctic ecosystem cannot be neglected since this species forms part of the dominant size class (< 5 ym) of this region. The degree to which the utilization of N03" is prevented in the small phytoplankton size class will depend on the N H 4 + concentration present in the water. The surface N H 4 + concentrations measured at station P26 for all cruises during this study varied from 0.17 to 0.54 JAM. This laboratory study showed that N03" uptake rates decreased most rapidly over this range (see Fig. 4.4). The laboratory data imply inhibition of N0 3" uptake rate by N H 4 + of between 37 to 69%, respectively, at these N H 4 + levels (Fig. 4.6). This may partly contribute to the low/-ratios found at station P26 throughout the water column at various times of the year (Chapter 2). 153 Figure 4.6. Percent inhibition of nitrate uptake rate at increasing ammonium concentrations for cultures of Emiliania huxleyi grown in a 14:10 light:dark cycle. Same data as used in Figure 4.4 but transformed to percentages. Each symbol represents a determination from a single culture (n = 56). The three models used to fit the data are also included. Arrows point to minimum and maximum surface ammonium concentrations measured at station P26 in the N E Pacific during all cruises. 154 S U M M A R Y Nitrate uptake rate as well as other physiological characteristics of E. huxleyi exhibited a strong diel periodicity during the 14:10 L:D cycle. About 84% of the total daily N0 3" was taken up during the light period. The presence of N H 4 + inhibited N03~ uptake in E. huxleyi. Nitrate uptake rates were reduced to half the maximum value at 0.24 piM. N H 4 \ and maximum inhibition was « 100% at 2.2 piM N H 4 + . If this laboratory result is extrapolated to field conditions, the inhibition of nitrate uptake rates for the small size class of phytoplankton would be predicted to be 37 to 69% for the range of ambient ammonium concentrations found in the NE subarctic Pacific. 155 GENERAL CONCLUSIONS This thesis has described the nitrogenous nutrition of natural assemblages of phytoplankton from the N E subarctic Pacific. This was the first study to investigate: (a) interannual and seasonal rates of inorganic and organic nitrogen uptake by phytoplankton along a longitudinal gradient in the N E subarctic Pacific, and the first one to report winter values; (b) the contribution of small cells (picoplankton) to the nitrogen uptake rates by intact assemblages in the NE Pacific; and (c) the effect of ammonium concentration on nitrate uptake rate by Emiliania huxleyi, a coccolithophore isolated from the NE Pacific, under laboratory conditions that closely simulated E. huxleyVs natural oceanic environment. The general conclusions of this thesis are best summarized by highlighting the main results from each chapter. Chapter 1 offered a description of the physical, chemical and biological characteristics of the euphotic zone along Line P from winter to late summer for the period 1992-1994. The results of this chapter confirmed previous studies in that nitrate, silicic acid and phosphate increased towards the oceanic end of the transect during every season. Lowest nutrient values were observed during late summer and spring, and highest during winter throughout the euphotic zone. For spring and late summer, surface depletion of nitrate and very low silicic acid and phosphate were observed at the coastal end of the transect, while concentrations at the oceanic sites were never limiting for phytoplankton growth. Ammonium and urea concentrations were measured for the first time along Line P, and exhibited a patchy distribution along the transect with no seasonal variation. Chlorophyll a, and particulate nitrogen and carbon did not show a consistent longitudinal pattern from year to year. In general, the highest concentrations of chlorophyll a, and particulate nitrogen and carbon were observed during the late summer cruises, with lower values in spring and lowest in winter. The highest chlorophyll a concentration ever measured during this study was 0.66 pig L"1. Interannual variability was evident for nitrate, silicic acid, phosphate, chlorophyll a, and particulate nitrogen and carbon concentrations. Phytoplankton assemblages 156 were numerically dominated by flagellates < 5 pim, vertically and longitudinally during every cruise. Diatoms remained at very low levels, with only sporadically higher numbers due to increases in pennates. The euphotic zone of the entire Line P transect showed similar chemical and biological characteristics. However, based on the distribution of nitrate, silicic acid and phosphate concentrations, a distinction between 'more' and 'less' oceanic stations could be made in the vicinity of P16. Although chlorophyll a concentrations were low along the entire region, the limiting factors during spring and summer were different from one end of the transect to the other. During the period of this study, a combination of iron limitation and high grazing pressure may have been responsible for the low phytoplankton biomass from P26 to P20/P16. In contrast, a combination of macronutrient (mainly nitrate) limitation and grazing pressure may have resulted in low phytoplankton stocks from P16/P12 to P4. Chapter 2 showed that nitrogen forms were utilized by phytoplankton from the N E subarctic Pacific in the following order: N H 4 + > urea > N 0 3 \ Ammonium was almost always taken up at higher rates and was used preferentially over urea and nitrate at every depth along Line P throughout the year. Urea was taken up at lower rates than ammonium; however, higher values were occasionally observed, particularly during the spring of 1993. New production represented on average 21% of the depth integrated total nitrogen production along Line P for every season. When urea was excluded from the calculation of the/-ratio, the overestimation of the /-ratio ranged from 4 to 130%, with an average of 36%. Longitudinally, ammonium uptake rates were generally higher at the coastal end of the transect, while nitrate uptake rates showed higher values at the oceanic sites. In contrast, urea uptake rates did not exhibit a consistent longitudinal trend. It is suggested that ammonium may have inhibited the uptake rates of nitrate and urea. This chapter showed that this system was functioning on regenerated nitrogen forms year round, despite the availability of dissolved nitrate in most of the region. Chapter 3 investigated the nitrogen uptake rates by size-fractionated phytoplankton from the oceanic N E subarctic Pacific. Small (< 2 pim) and large (> 2 pim) phytoplankton cells took up ammonium at higher rates than urea and nitrate. The order of preference for the nitrogen sources 157 by the small cells was N H 4 + > urea > N0 3 ~, and for the larger cells it was N H 4 + = urea > N 0 3 . Depth integrated/-ratios were 0.16 for the small fraction, and 0.25 for the larger fraction (when urea was included), indicating that > 2 pim cells were slightly greater contributors to the euphotic zone new production. However, picoplankton cells (< 2 pim) were responsible for most of the new and regenerated primary production in the oceanic region of the N E subarctic Pacific. Chapter 4 demonstrated that nitrate uptake was affected by diel cycles and increasing concentrations of ammonium in the ecologically relevant coccolithophore Emiliania huxleyi. Nitrate uptake rate as well as other physiological characteristics of E. huxleyi exhibited a strong diel periodicity during the L : D cycle; about 84% of the total daily nitrate was taken up during the light period. The presence of ammonium inhibited nitrate uptake in E. huxleyi. Nitrate uptake rates were reduced to half the maximum value at 0.24 piM. ammonium, and maximum inhibition was « 100% at ammonium concentrations of 2.2 piM. If this laboratory result is extrapolated to field conditions, the inhibition of nitrate uptake rates for the small size class of phytoplankton would be predicted to be 37 to 69% for the range of ambient ammonium concentrations found in the N E subarctic Pacific. In summary, on a yearly basis primary productivity in the N E subarctic Pacific was based on regenerated nitrogen forms, which were mostly utilized by the small phytoplankton cells (< 2 pim). New production in this region was low, despite the persistently high N 0 3 " concentrations. Ammonium inhibition of nitrate uptake could be claimed as one of the factors responsible for the low rates of new production, at least for the small size class of phytoplankton. In conclusion, the N E subarctic Pacific is one of those regions of the world's oceans with low levels of new primary production. L o w rates of nitrate uptake, the lack of a sizable phytoplankton bloom and the dominance of phytoplankton cells < 5 pim may result in low export fluxes of particles towards deep waters. Although occasional high export fluxes have been inferred from material collected with sediment traps (e.g. Takahashi, 1986; Takahashi etal., 1990), on a yearly basis the export of particles may be too low to make a significant contribution to 158 the global sequestration of carbon and other nutrients in deep waters and to the support of high densities of deep water heterotrophs in the open N E subarctic Pacific. A potentially low vertical export of nutrients still has important implications for the biogeochemical cycles since most particles would be retained and nutrients recycled within the surface of the ocean during most of the year. The dominance of a microbial system in surface waters of the N E subarctic Pacific has interesting implications for the ecology of the region. Efficient recycling of organic matter in surface waters provides the required nitrogen for the nutrition of the dominant phytoplankton cells. Moreover, a constant supply of regenerated nitrogen affects the nitrogen metabolism of phytoplankton. The preference for ammonium and urea over nitrate, as well as the ammonium inhibition of nitrate uptake by the dominant small phytoplankton may contribute to the low rates of nitrate uptake in the N E subarctic Pacific. Rates of nitrate uptake (and, hence, new production) would be expected to be higher at undetectable concentrations of ammonium. However, important increases in new production in the subarctic Pacific may also depend on the abundance of larger cells (i.e. diatoms). Growth and nitrate uptake rates by the larger cells, however, seem to be limited by iron availability at least in the oceanic regions (Boyd etal., 1996). Hence, neither large nor small cells can make full use of the persistently high nitrate concentrations in surface waters of most of the N E subarctic Pacific. 159 FUTURE STUDIES This thesis leads to a number of additional studies that would complement the results obtained here. Interesting future research may include: a) Further studies on size-fractionation of nitrogen uptake rates along Line P. These experiments should also include the bacterial fraction, in order to determine the contribution of heterotrophic bacteria to total, new and regenerated primary production. b) Determination of regeneration rates along Line P. These measurements will show if autochthonous sources of regenerated nitrogen are in balance with the nitrogen requirements by phytoplankton. Regeneration rates would also provide a correction for the underestimation of nitrogen uptake rates due to isotope dilution. c) Comparison of the rates of new production obtained during this study with sediment trap data. Sediment traps were deployed in the N E subarctic Pacific during the 1992-1994 period, hence, the magnitude of the fluxes of particulates to deep waters can be compared to the rates of N03" uptake. Then, rates of export and new production could be compared. d) Ammonium inhibition of urea uptake rates by a representative of the small (e.g. Emiliania huxleyi) and the large (e.g. Actinocyclus sp) phytoplankton size fraction. Because the field data presented here suggest a possible inhibition of urea uptake at increasing ammonium concentrations, it would be of interest to determine if the decrease in urea uptake rates was due to a direct effect of ammonium or to other environmental factors. 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Prentice Hal l , New Jersey. 661 pp. 182 APPENDIX A SAMPLING STATIONS Table A . 1. L o c a t i o n and water depth o f sampl ing stations i n the N E subarctic Pac i f ic . Station Lati tude d e g ° m i n ' sec" N Longi tude d e g ° m i n ' sec" W Depth (m) P4 4 8 ° 3 9 ' 0 0 " 126° 4 0 ' 0 0 " 1342 P12 4 8 ° 5 8 ' 12" 130° 4 0 ' 0 0 " 3 2 6 0 P16 49° 17' 0 0 " 134° 4 0 ' 0 0 " 3656 P20 4 9 ° 3 4 ' 0 0 " 138° 4 0 ' 0 0 " 3 9 8 4 P 2 3 A 4 9 ° 4 8 ' 3 0 " 142° 2 6 ' 2 4 " 4 0 3 4 P26 , Papa or P 5 0 ° 0 0 ' 0 0 " 145° 0 0 ' 0 0 " 4 2 6 5 183 APPENDIX B PRECISION O F A N A L Y T I C A L T E C H N I Q U E S The precision of the analytical techniques used during the field study of this thesis are reported in Tables B . l and B.2 as the mean coefficient of variation ( C . V . (%) = [s.D. / x j * 100) and mean standard deviation (S.D.) of multiple groups (n) of subsamples (2 to 3). Subsamples were collected from the same water sample (i.e. same Go-Flo bottle) at selected stations and depths during every cruise and, therefore, the errors reported here represent "within bottle" variability. The estimated errors include all manipulation errors, from collection to final measurements. For a few parameters, high mean C . V . resulted from ambient values near the detection limit of the analytical techniques. Water samples were collected and processed as outlined in Chapters 1 and 2. 184 Table B . 1. Precision of the analytical techniques for dissolved nutrient, chlorophyll a, and particulate nitrogen and carbon concentrations employed during the field component of this thesis. Units for mean S.D. are: pig L" 1 for Chi a and pig-at L" 1 for all other parameters. Parameter Mean C . V . (%) Mean S.D. n N 0 3 - 1.4 0.10 16 N 0 2 3.3 0.01 6 N H 4 + 12.3 0.04 36 Urea 27.0 0.08 26 S i (OH) 4 1.1 0.14 16 H P 0 4 2 " 1.6 0.01 15 C h l a 3.0 0.01 16 P N 3.4 0.03 6 P C 4.2 0.30 6 185 Table B.2. Precision of the 1 5 N tracer technique employed during the field component of this thesis. Errors are shown for absolute, and N - and Chi a -specific uptake rates. Units for mean S.D. are: ng-at N L" 1 d 1 for p N , d ' 1 for V N , and ^g-at N (jtg Chi a)'1 d 1 for p ^ C h l a. Uptake rate Mean C . V . Mean S.D. (%) p N 0 3 " 8.8 4.18 17 V N 0 3 - 8.6 0.002 17 p N 0 3 / C h l a 8.8 0.010 17 purea 15.2 8.74 8 Vurea 14.9 0.006 8 purea/Chla 15.2 0.019 8 p N H 4 + 7.5 10.85 11 V N H 4 + 7.2 0.008 11 p N H 4 + / C h l a 7.5 0.038 11 186 APPENDIX C V E R T I C A L P R O F I L E S O F T E M P E R A T U R E , SALINITY A N D <Jt Vertical profiles of temperature, salinity and Ot for the upper 200 m at the Line P stations are presented for the winter (Fig. C l ) , spring (Fig. C.2), and late summer (Fig. C.3) cruises. The dates when those profiles were measured are specified in Table 1.1. Note from Tables 1.1 and 1.2 that during September 1992 and May 1994, two profiles were obtained at P26. Since both profiles were very similar, only the first one is depicted in each case (see Figs. C.2 B and C.3 A). (ui) mdarj e6 a w i 6^ and 188 £6 AWi (in) indaa P6 AVW (ui) indarj Z6 d3S P6 dHS 190 APPENDIX D INCIDENT S U R F A C E I R R A D I A N C E D U R I N G 1 5 N E X P E R I M E N T S Figure D . 1 shows the incident surface solar irradiance (i.e. incident surface photosynthetic active radiation) for a l l L i n e P stations dur ing the 1 S N incubations presented i n Chapters 2 and 3. Data are o n l y avai lable for M a r c h 1993, M a y 1993, and M a y 1994. 191 CQ U o CN u q o CN \- P o CN U P s s o 03 O Xi CN S O c L- <=>. o CN U P o CN ( s u i suojoqd pratf) aouEipmji sorbins j u a p p u j co 03 ON 5 2 1 — 1 *> x = > 2 03 03 3^. < 3 CO 00 CD s -fi o s co u §.2P co CD CL h co 3 03 XI O s Z co . J5 op 5 •C « § ^ 12 ^ C ON s w co -a T3 3 'o rt 3 CO ON . ON i—• i—i Q ^ § 2 3 00 co in CD ~-, J3 O 03 CD .„ co 3 O <U o 3 03 CD 3 u O o *-> CO J3 e s CD 03 •I ^ rvi U O W P-. +-> ^ f> CO 03 3 "S.s 3 CO O ~ CO 3 ^ I -I—J r \ >7 C ^ § l 'C 7 3 co « ^ ^ 3 2 x co ™ <U t» 3 -3 cx o CO co £ 3 £ V") co X _ t CD CD 3 CO • J3 Ui CL 3 co E CO ON a o .2 0 10 es U. O 03 00 0 3 3 03 S fi .S 3 2 CO T3 a 3 5 o CO OS CL o CD CQ J3 3 . .2? * CD 73 3 ~ ^ ^ 1 ^ 3 o 5 i3 u 6 s 3 3 *?<u 3 -C 3 O 3 03 S -a H3 T3 ^ -co co SI "o S CO CO 3 CO €6 HOHVTY £6AVW v6 AVTM 192 APPENDIX E 1 5 N U P T A K E E X P E R I M E N T S W I T H P R E - F I L T E R E D A N D U N F I L T E R E D W A T E R Objective: Compare 1 S N 0 3 ~ and 1 S N H 4 + uptake rates of pre-filtered (116pim; without large grazers) with unfiltered seawater (intact community). Introduction: The rationale for pre-filtering seawater before inoculation and incubation with 1 S N isotopes is to exclude large grazers which may produce an underestimation of the gross rates through their feeding activity. However, the filtration step is not ideal since it can contribute to nutrient contamination (principally ammonium and trace metals), and to cellular damage of phytoplankton. Moreover, only a fraction of the grazers is removed, generally the larger mesozooplankton. In the subarctic Pacific, nano- and microzooplankton are the main grazers of the dominant small phytoplankton cells (see "Discussion" in Chapter 1), and therefore little may be gained by screening out the less abundant mesozooplankton. During September 1992 at P26, a test was done to check for differences in N 0 3 " and N H 4 + uptake rates between pre-filtered (< 116 pim) and unfiltered samples. Methods: A t each one of the 6 light depths, two samples were drawn for N 0 3 " , and two samples for N H 4 + uptake experiments. One sample for each nitrogen source was pre-filtered through a net of 116 pan pore size. Thus, mesozooplankton and a portion of microzooplankton would have been removed in the pre-filtered samples. A t each depth, one unfiltered and one pre-filtered sample were inoculated with 1 S N 0 3 \ and another set with 1 5 N H 4 + at trace levels. A l l procedures, from sampling to calculation of nitrogen uptake rates, were done as specified in the "Material and Methods" section in Chapter 2. 193 Results: F igu re E . 1 shows the vert ical profiles o f N03" and N H 4 + uptake rates for pre-filtered (< 116 pim) and unfiltered (entire communi ty) water. T h e profiles for fi l tered and unfiltered samples for each nitrogen form were very s imi la r to each other, suggesting that there was not a detectable difference between both treatments. A statistical compar ison was done between both treatments for each nitrogen form by using a l l six samples i n each profi le as replicates. A r-test showed no statistical difference at P > 0.80 between filtered and unfiltered samples for 15N03" or 1 S N H 4 + . D u r i n g this thesis, the nitrogen uptake experiments were performed on unfiltered water samples; thus, the response of an intact communi ty was measured. p (ng-at N L / 1 d"1) Figure E . l . Ve r t i ca l profiles o f absolute uptake rates o f N-nitrate and 1 5 N - a m m o n i u m measured on pre-filtered (116 pim) and unfil tered water at station P 2 6 dur ing September 1992. 195 APPENDIX F E F F E C T O F I R R A D I A N C E O N N I T R O G E N U P T A K E R A T E Objective: Investigate the effect of irradiance on the uptake rate of N 0 3 " , urea and N H 4 + . Introduction: The response of nitrogen uptake rate to irradiance can be described by a Michaelis-Menten expression (Maclssac and Dugdale, 1972): V * I 1/ max N = K a + I where V N is the rate of nitrogen uptake, is the maximum rate of nitrogen uptake, I is irradiance, and K L T is the irradiance at which V N = V^Jl. In some cases, a term for dark uptake is introduced in the equation to better describe the V N vs. I relationship (e.g. Maclssac and Dugdale, 1972; Cochlan etal., 1991b, Kudela etal., 1997). During the present study, few data points were obtained at low irradiances, and dark uptake was not measured. Although dark uptake is known to occur for nitrogen (see references cited above), the equation with no dark uptake component was a better fit for these data. Thus, the estimates from these fits should be interpreted cautiously since the introduction of the dark uptake term could produce considerable changes. Although limited, the analysis presented here provides an approximate representation of the effects of light on nitrogen uptake rates by phytoplankton from the N E subarctic Pacific. Methods: On February 10, 1994 at P16, water samples were collected from a clean seawater line that pumped water from 3-5 m depth to the ship's laboratory. On M a y 21, 1994 at P26, water samples were obtained from a bottle cast at 5 m depth. During each cruise, water was transferred to 18 ,1-L polycarbonate Nalgene® bottles. Six bottles were inoculated with 1 S N 0 3 " , 196 another 6 wi th 1 5 N - u r e a and the f inal 6 wi th 1 5 N H 4 + . F o r each isotope, bottles were exposed to 6 levels o f i rradiance by p lac ing them i n neutral density screen bags s imulat ing: 100, 55, 30 , 10 ,3 .5 and 1% of incident solar irradiance. Detai ls of water sampl ing, incubat ion procedures, analytical techniques and nitrogen uptake calculations are described i n Chapters 1 and 2. Results: T h e uptake rates o f the three nitrogen sources decreased at l o w irradiances (3.5 and 1% ID) du r ing both cruises (F ig . F . 1). A t saturating irradiance, N H 4 + exhibi ted the highest rates o f uptake dur ing February and M a y 1994. A t l o w irradiance, N H 4 + showed the steepest slopes dur ing February and M a y 1994. Judging f rom and K L T (Table F . 1), N H 4 + was the least l ight dependent o f the three nitrogen sources dur ing winter and spring. Nitrate was the most l ight dependent i n winter, and urea i n spring. A . Feb94-P16 CD -i—> 2 & OH o o CD O h C/2 I 0.12 H 0.08 4 0.04 4 0.00-1 0.12 4 0.08 4 0.04 4 0.00-1 1 1 1 1 1 1 1 1 1 1 B. May94-P26 • * • @ • ©" A*"" 20 40 60 80 Relative irradiance (%) too © N 0 3 " A U r e a • N H 4 + | F igure F . l . N - spec i f i c uptake rate o f nitrate, urea and a m m o n i u m as a function o f % I 0 f o r ( A ) P 1 6 dur ing February 1994 and (B) P 2 6 dur ing M a y 1994. T h e least squares M i c h a e l i s - M e n t e n fit to the data is shown i n dotted l ines . 198 Tab le F . 1. Est imated parameters ( V ^ and K L T ) f rom the least squares M i c h a e l i s - M e n t e n fit to the uptake vs. i rradiance data for P 1 6 dur ing February 1994 and P 2 6 dur ing M a y 1994. Errors for the parameters are g iven as ± 1 S . E . (n = 6). K L T is g iven i n % of surface incident irradiance. Cruise and station Ni t rogen source V max K L T ( d 1 ) ( % U N 0 3 " 0 .038 ± 0.002 9.4 ± 1.8 Feb 94 - P 1 6 U r e a 0.060 ± 0.002 0.7 ± 0.2 N H 4 + 0.095 ± 0.003 0.9 ± 0.2 N 0 3 " 0 .075 ± 0.002 4 .9 ± 0.6 M a y 94 - P 2 6 U r e a 0.020 ± 0 .0004 3.6 ± 0.3 N H 4 + 0.121 ± 0.003 1.3 ± 0.2 

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