Photosynthetic and cellular photoadaptive characteristics of three ecotypes of the marine diatom, Skeletonema costatum (Grev.) Cleve

233

J. Exp. Mar. Biol. Ecol., 1985, Vol. 94, pp. 233-250 Elsevier

JEM 594

PHOTOSYNTHETIC CHARACTERISTICS

AND CELLULAR PHOTOADAPTIVE

OF THREE ECOTYPES OF THE MARINE DIATOM,

SKELETONEMA

(Grev.) Cleve

COSTATUM

JANE C. GALLAGHER Biology Department

and Institute of Marine and Atmospheric Sciences, City College of the City University of New York, Convent Ave. at 138th St.. Mew York, NY 10031, U.S.A. and RANDALL

Department

S. ALBERTE

of Molecular Genetics and Cell Biology, Barnes Laboratory, 5630 S. Ingleside Ave., The University of Chicago, Chicago, IL 60637. U.S.A.

(Received

5 April 1985; revision

received

13 August

1985; accepted

5 September

1985)

Abstract: Physiological variation among genetically distinct clones of Skeletonema costatum (Grev.) Cleve, representative of the prevalent seasonal populations of this species in Narragansett Bay, were examined to determine the nature of their optimal survival strategies in response to changes in light intensity. The clones were found to exhibit significant differences in growth rates, cellular pigment content, PSU-RCI and PSU-RCII numbers per cell, ratios of RCI/RCII and photosynthetic capacities per cell. Respiration rates and I, values did not vary among clones. The variation among clones for many characteristics were similar in magnitude to those previously reported for differences among species. No single photosynthetic feature dominated or predicted the photoadaptive mechanism in any clone examined. Large clonal differences in photoadaptive features suggest that the evolution of the seasonal ecotypes of S. costatum was not a recent phenomenon. The integration of cell-dependent and cell-independent photosynthetic features in each clone and, by inference, the genetic groups which they represent, partially accounts for the seasonal pattern of abundance distinct ahochronic populations in the Narragansett Bay ecosystem. Key words: ecotypes;

photoadaptation;

photosynthesis;

respiration;

Skeletonema

costatum

INTRODUCTION

Skeletonemu costatum (Grev.) Cleve forms genetically distinct allochronic populations that dominate the flora in Narragansett Bay, R.I., during the summer and winter phytoplankton blooms (Gallagher, 1980). The regularity of population cycling (Gallagher, 1980), background data on the causes of bloom initiation (Hitchcock & Smayda, 1977; Deason & Smayda, 1982) and large phenotypic differences among strains representative of these seasonal populations indicate that the cycling between seasonal populations is probably due to cyclic natural selection (Gallagher, 1982). Intraspecific variation for photoadaptive characteristics is probably a particularly important factor influencing the changing patterns of local genetic diversity in this 0022-0981/85/$03.30

0

1985 Elsevier

Science

Publishers

B.V. (Biomedical

Division)

234

JANE C. GALLAGHER

AND RANDALL

S. ALBERTE

species because changes in light intensity appear to initiate the winter-spring bloom in Narragansett Bay (Riley, 1957; Hitchcock & Smayda, 1977). The goal of this investigation was to explore further the nature of variation of photosynthetic parameters among genetically different strains of the diatom, S. costaturn, in order to identify those characteristics that may be most important in determining differential growth and survival under light-limited and light-saturated regimes in the water column. The parameters examined in this investigation were those representing cellular features such as growth rate, cell size, numbers of reaction centers per cell and respiration, rather than the cell-independent features, such as pigment ratios and energy transfer efficiency, reported in a previous study (Gallagher et al., 1984). Although the responses of cell-independent features to growth light regimes have important implications for physiological studies, it is the integration of these features at the cell level that is ecologically important in determining the perpetuation of a genotype under differing conditions because the cell is the smallest unit of selection. The present investigation provides the most complete characterization to date of the variation in cellular photoadaptive strategies of a single phytoplankton species. MATERIALS

AND METHODS

The methods used in these experiments are reported in detail in Gallagher et al. (1984) and are summarized below. CULTURE

CONDITIONS

Three genetically distinct clones of Skeletonema costatum were used. Clones NY 17 and UP45 were used as representatives of the prevalent winter and summer bloom populations, respectively, in Narrgansett Bay, R.I., (Gallagher, 1980). Clone Skel, isolated from Long Island Sound in 1956, was obtained from the Culture Collection of Marine Algae, Bigelow Laboratory, and was included in order to serve as a point of reference between this investigation and prior studies of photoadaptation using this standard clone of Skeletonema. Details on the origins, genetics, and physiology of these clones are provided in Gallagher (1982). The Narragansett Bay clones have been continuously monitored since the time of isolation in 1977, and have not shown any evidence of change with time in culture (Gallagher, 1982, 1983). The cultures were grown at 20 “C in f/2 medium (Guillard & Ryther, 1962) (32%,) with sterile aeration in 2-8 1 batch cultures at 274 (high light) and 27 (low light) @.m-2.s-’ with a 14 : 10 L : D photoperiod using cool white fluorescent lights. This temperature and photoperiod are similar to those found in Narragansett Bay during the summer bloom. The high and low light intensities approximate 40-50 % and 5 y0 of the average incident radiation during the summer. The growth rates of Skeletonema were light-saturated under the high light regime and light-limited under the low light regime (Yoder, 1979). These growth conditions are similar to those used in previous studies of this organism (Gallagher, 1982).

PHOTOADAPTATION:

PHOTOSYNTHETIC

AND CELLULAR

FEATURES

235

For each experiment, log-phase, preadapted cells were inoculated into sterile media and were harvested after 2 days of growth. The final cell densities were 3-7 x lo4 cells * ml- i. These low cell densities were used to avoid self-shading and nutrient depletion. Replicate experiments were run sequentially and great care was taken to ensure that the culturing conditions at each light level were identical for all clones. Therefore, the observed physiological differences among clones with each light intensity were due to their genetic differences rather than to random environmental effects (Gallagher, 1982). Subsamples were taken from each of the cultures daily and were counted in a Sedgewick-Rafter cell. Growth rates were determined on a daily basis by changes in cell density expressed to the base 2 (div. +cell- ’ . day - ‘). Cell measurements on randomly selected chains (n = 16 per experiment) were determined using an ocular micrometer. PIGMENT

DETERMINATIONS

Cells were concentrated by centrifugation and were resuspended in f/2. The same cell concentrate was used for the determination of pigment concentrations, reaction center II (RCII) quantification, cell counts and for generation of photosynthesis versus h-radiance (P-I) curves. A minimum of four replicate subsamples (2-3 ml) from the cell concentrate for each experiment were collected on glass fiber filters. The filters were frozen and ground in 4-5 ml of 90% (v/v) acetone to extract pigments. The concentrations of chlorophyll (Chl) a and Chl c were determined spectrophotometrically in 90% (v/v) acetone, after the filter fragments and cell debris had been removed by centrifugation, using the equations of Jeffrey & Humphrey (1975). The concentration of fucoxanthin in each extract was determined after separation by high performance liquid chromatography (Waters Associates). Concentrations of all pigments were calculated as moles (mol). PHOTOSYNTHETIC

UNIT SIZES

AND NUMBERS

FOR PHOTOSYSTEMS

I AND II

The concentration of reaction centers I (RCI), P700, was determined by methods essentially identical to those of Perry et al. (1981) and Shiozawa et d. (1974). Photosynthetic unit (PSU) sizes for PS I (PSU-RCI) were calculated from Chl a to P700 ratios (mol/mol). The numbers of RCIs . cell - ’ were calculated by dividing the amount of Chl a per cell by the PSU-RCI size. The PSU-RCII sizes were estimated from determinations of the 0, yield per cell as described by Mishkind & Mauzerall (1980) as modified by Kursar & Alberte (1983). Oxygen produced per flash was used to calculate the Emerson & Arnold (1932) numbers which were then divided by four to obtain PSU,, sizes (Falkowski et al., 1981; Kursar & Alberte, 1983). The numbers of RCIIs . cell - ’ were calculated from the flash yield per cell.

236

JANE

C. GALLAGHER

PHOTOSYNTHESIS

MEASUREMENTS

AND RANDALL

S. ALBERTE

Rates of gross photosynthesis were determined using a Clark-type oxygen electrode (Rank Bros., England). All measurements were made at 20 “C, and illumination was provided by a 300-W tungsten-iodide slide projector lamp. The light intensities were varied by using neutral density filters. The initial slopes of the P-I curves were determined by regression of gross photosynthesis on light intensity for the first four points of each curve. The rate of maximum gross photosynthesis (P,,,) was calculated from the mean of the rates of 0, evolution at 400, 500, and 750 ZLLE * m - 2. s - 1 which were observed to be at or above the saturating light intensites in the P-I curves in all experiments. Respiration rates (R) were determined from the rates of 0, uptake in the dark. The ratio of net P,,, to dark respiration (P : R) ratio was also calculated. The compensation light intensities (Z,) were determined from the respiration rate and the rate of gross photosynthesis at the lowest light intensity tested in each experiment. Calculations of P-I curve parameters were based on cell number (Slope (cell), P,,, (cell), R (cell)), cell volume (Slope ( pm3), P,,, ( pm3), R ( pm3)), numbers of RCIs (Slope (RCI), P,,, (RCI)), and numbers of RCIIs (Slope (PSU-RCII), P,,, (PSU-RCII)). STATISTICAL

ANALYSES

The experimental matrix was designed as a two-way analysis of variance (ANOVA) with replicates (2 light regimes x 3 clones x 4 replicate cultures per clone per light intensity). Replicate measurements of some variables within each experiment were also taken. The following variables were measured: growth rate, mol of Chl a, Chl c and fucoxanthin per cell, cell diameter, cell volume, numbers of RCIs and RCIIs per cell, dark respiration rates, Z, values and slopes and P,,, values of the P-I curves. The total pigment per cell was calculated from the sum of the three pigments measured, and concentrations of each pigment were also standardized per ,um3 of cell volume. We have used the concentrations of pigment per pm3 as separate variables even though they were derived as ratios of two collected variables because this analysis yielded information not readily apparent from the inspection of each variable separately (Sokal & Rohlf, 198 1). Each variable was tested for homoscedasticity and independence of the variances and means (Gallagher, 1982). Where necessary, either log transformations or angular transformations of the data were used in order to satisfy the assumptions of the Analysis of Variance (Sokal & Rohlf, 1981). Each variable was tested separately in a two-way ANOVA and the means were compared by Welsch step-up, T- or GT-tests (Sokal & Rohlf, 198 1). Where replicate preparations within cultures were taken, the intermediate mean squares were tested by the methods of Bancroft (Sokal & Rohlf, 1981) and were pooled where possible. This means that some of the ANOVAs have more degrees of freedom than others. Since the replicate cultures were run sequentially, we also tested the variables as a randomized block to determine if the effect of time was significant. The use of single ANOVAs is the most common way to analyze laboratory data of this type (see Rockwell et al., 1975; Verity 8t Stoecker, 1982, as examples). However,

PHOTOADAPTATION:

PHOTOSYNTHETIC

AND CELLULAR

FEATURES

231

multiple analyses of variance and simultaneous analyses of variance are also appropriate (Morrison, 1976). These calculations were also performed and their results were similar to those of the univariate techniques. Therefore, only the results of the univariate analyses are reported. RESULTS

The results of the tests of significance are shown in Table I. Virtually all of the variables except R (cell) and 1, showed highly significant main effects and/or interaction terms. Analysis of the randomized blocks showed that time was not a significant factor for any variable. This indicates that running the replicates sequentially had no effect on the results. These findings are similar to those found for cell-independent features such as pigment ratios, PSU sizes for both photosystems, fluorescence excitation and emission characteristics and rates of photosynthesis per mol of pigment and per PSU (Gallagher et al., 1984). GROWTH

RATES,

PIGMENTS

AND

CELL VOLUME

Growth rates and pigment content per cell (Chl a, Chl c, fucoxanthin, and total pigments) are shown in Fig. 1. In high light, the summer bloom clone, UP45, had the highest growth rate and the standard clone (Skel) had the lowest. AU of the clones showed large decreases in growth in low light and the rank order of the clones was reversed compared to the high light regime. The relative changes in total pigments in response to growth light conditions are summarized in Table II. Although all of the clones showed increases in pigment content in low light, each clone showed a unique pattern of variation for each pigment. The winter clone had the least amount of pigment per cell but had the highest proportion of accessory pigments relative to Chl a. The summer clone, UP45, had the highest pigment content, but had intermediate proportions of accessory pigments relative to Chl a, and Skel had intermediate amounts of each pigment and had the lowest proportion of accessory pigments relative to Chl a. The responses of cell volume to light intensity are illustrated in Fig. 1. The clones differed from each other in volume within each light regime, and the two Narragansett Bay clones (NY17 and UP45) also showed significant (P < 0.05) decreases in volume in low light compared to high light. The analysis of randomized blocks indicates that these changes in size were not due to experimental artifacts created by running replicate experiments sequentially. Neither pigment content nor growth showed any correlation with cell volume among clones within each light regime (P > 0.05). This indicated that differences among clones with light regimes were not simple artifacts of differences in cell size. This confirms previous observations in this species (Gallagher, 1982). It is probable that size changes within clones of S. costutum are due to alteration in the size of the cell vacuole rather than to changes in the amount of living cytoplasm.

TABLE I

R.cell-’ MSb x 1O-2

3.10*** 0.02** 0.12*** 0.003

Cell volume MSb x lo2

O.SO** 1.29*** 5.92*** 0.09

Light Clones Interaction Error

Source

Light Clones Interaction Error 33.37*** 0.05* 0.18*** 0.01

Total.pm-3 MS” x 10 - i’

8.30*** 3.20*** 0.20 ns 0.20

Chlc.cell-’ MS? x 10-i’

1.13*** 1.31*** 0.02 ns 0.07

TABLE II

4.24*** 2.13** 0.55 ns 0.23

P,;pmA3 MSb x 10-i’

1.45* 5.39*** 1.15* 0.22

2.94 ns 4.00 ns 8.47** 1.35

4.01*** 5.4s*** 2.14*** 0.23

P/R ratio MS’ x lo2

0.23 ns 9.63*** 0.43 ns 0.55

PSUo, cell _ ’ MSb x lOWi*

R.prnm3 MSb x 10”

PSU,,,, cell - ’ MSb x 10-i*

1.18 ns 0.79 ns 0.94 ns 1.22

1, MSb x 10’

1.15** 1.79*** 0.33 ns 0.10

P,,, (cell) MSb x lo-l5

Skel

UP45

7.5 f 0.3

4.1 * 0.3 6.8 f 0.3

27

274

27

5.8 f 0.3

214

x IO-l6

3.0 * 0.2 3.8 f 0.1

274

NY17

Chla

27

Light intensity (pE.mm2.s-‘)

Clone

+66

i29

+ 21

% Change

+ 50

+ 58

1.2 * 0.1 1.8 f 0.1 1.2 r 0.1 1.9 + 0.1

% Change

+ 100

IO-l6

0.6 f 0.1 1.2 * 0.1

Chlcx

+82

+ 112

+ 225

1.7 f 0.2 3.6 + 0.3 0.4 * 0.0 1.3 * 0.1

% Change

2.0 f 0.0

1.1 f 0.1

Fuco x lo-l6

x IO- I6

10.1 + 0.2

5.6 + 0.3

12.9 k 0.7

8.7 k 0.5

7.0 f 0.2

4.7 f 0.3

Total pigments

+ 80

+48

+49

% Change

Pigment concentration per cell (mol): total pigments are Chl a + Chl c + fuco; percent changes were calculated as differences between values for high and low light grown cells expressed as a percent of the values for the high light grown cells; values are means + 1 SE.

1.02* 0.83* 0.31 ns 0.18

PSU,,,,~~m-3 MSb x 10-s’

PSUacrw -3 MSb x lo-”

6.90*** 4.17*** 0.36*** 0.05

Total . cell - ’ MS” x 10-i’

8.28*** 5.45*** 0.09*** 0.01

Fuco . cell - ’ MS” x lo-”

a Degrees of freedom: Light = 1, Clones = 2, Interaction = 2, Error = 186. b Degrees of freedom: Light = 1, Clones = 2, Interaction = 2, Error = 22.

0.04 ns 0.87 ns 1.72 ns 9.84

1.24*** 1.42*** 0.12*** 0.01

Growth MSb x 10”

Source

Chl a cell - ’ MS” x lo-l6

Results of univariate analyses of variables: results are presented as mean squares (MS) which were tested over the error term.

PHOTOADAPTATION:

PHOTOSYNTHETIC

AND CELLULAR

FEATURES

239

A. High

High

Fig. 1. Mean values ofgrowth rates, mol ofpigment per cell, and cell volume for Skeletonemu cost&m clones NY 17, UP45 and Skel: the top and bottom of each axis indicate the values obtained in high and low light, respectively; the boxes indicate the results of a posteriori tests for multiple comparisons of means (see text); if two or more clones are enclosed together in a box, their means were not significantly different at the 0.05 level; A, growth rate; B, Chl a per cell; C, Chl c per cell; D, fucoxanthin per cell; E, total pigment (Chl a + Chl c + fuco) per cell; F, volume per cell.

PHOTOSYNTHETIC

UNITS

PER CELL

AND

PER VOLUME

The numbers of PSUs for photosystems I and II per cell and per pm’ of cell volume are shown in Table III. Clone Skel had more RCIs per cell than the Narragansett Bay clones and also showed an increase (P < 0.05) in the numbers of RCIs per cell in low light. The Narragansett Bay clones did not show a change (P > 0.05) in this variable between the two light regimes. Growth light intensity did not alter the numbers of RCIIs per cell in any clone. In both light regimes, the winter clone, NY17, had significantly fewer RCIIs per cell than the other two clones which were similar to each other. When

240

JANE

C. GALLAGHER

AND RANDALL

S. ALBERTE

the numbers of RCIs and RCIIs were expressed per pm3 of cell volume, only clonal differences were apparent (Table III). TABLE III Numbers of photosynthetic means + 1 SE; ratios

units per cell and per pm3 of cell volume for RCI and-RCII: values of the two reaction centers (RCI and RCII) were determined by size. PSUs

Clone

Light intensity

. cell- ’

RCI x lOWI9

are

PSUs.pm-’

RCII x lo-l9

RCI x IO-*’

RCII x 10e2’

RCI:RCII

Sig.’

NY17

214 27

5.1 _+0.8 5.9 + 0.2

7.4 & 1.8 6.4 f 0.1

0.9 f 0.2 1.3 * 0.2

1.4 + 0.5 1.4 f 0.1

0.74 0.94

n.s. n.s.

UP45

274 27

6.0 k 1.0 5.6 f 0.6

11.6 _+ 1.8 12.6 + 0.3

0.4 & 0.7 0.7 f. 1.6

0.7 + 0.1 1.6 + 0.1

0.51 0.44

** *

Skel

274 27

8.0 f 1.1 12.3 + 0.2

12.5 k 1.1 14.4 + 0.8

1.1 + 0.1 1.6 it: 0.1

1.7 * 0.1 1.9 + 0.8

0.63 0.86

* ns.

’ Results

of test for comparisons

of the mean ratios of the reaction

centers

with 1.0.

Clone NY 17 maintained an RCI : RCII ratio that was not different (P > 0.05) from 1.0 in both light regimes. The summer clone, UP45, maintained an RCI : RCII ratio that was close to 0.5 in both high and low light. Clone Skel had ratios that were significantly different from 1.0 in high light but approximated 1.0 in low light. P-I RELATIONSHIPS

P-I curves standardized per ceil and per pm3 of cell volume are shown in Figs. 2 and 3, respectively. In high light, Skel had a steeper (P < 0.05) slope (cell) than the two Narragansett Bay clones, which were similar to each other. Both Skel and UP45 also showed increases (P < 0.05) in slope (cell) in low light compared to high light; NY17

rz$

A. High

$2

I

PSuP700cell-‘,

(x lo-‘g) *

4

8

II u-s.

Low 0.

P 2

High PSU

02

cell-’

(x Io-‘sl

3 CL? 307

c

2

Low

Fig. 2. Mean values of numbers

4

0

r5

10

12

1 9 3

141

P,

16

I I8

;;

of PSUs for PS I and PS II per cell: notation per cell; B, PSU-RCII per cell.

as in Fig. 1; A, PSU-RCI

PHOTOADAPTATION:

PHOTOSYNTHETIC

AND CELLULAR

FEATURES

241

maintained a constant slope in both light regimes. In low light all of the clones had different (P < 0.05) slopes. The patterns among clones for P,,, (cell) were similar to those for Slope (cell). NY 17 High Low

light light --•--

UP45

Skel

0

200

400

600

800

1000

Fig. 3. P vs. I curves standardized per cell for clones grown in high and low light: values for each point are means k 1 SE; A, NY17; B, UP45; C, Skel.

When the P-I curves were standardized to cell volume, NY 17 and Skel had similar slopes that were both greater than that for UP45. Only UP45 showed an increase (P < 0.05) in slope in low light compared to high light. The P,,, values per pm3 showed that all clones were different from each other in high light. Skel had the highest P,,, and UP45 had the lowest. The winter clone, NY 17, maintained a constant P,,, in both light regimes, but the other two clones both showed significant increases in P,,, ( pm3) in low light compared to high light.

8.65 i 2.50 4.93 + 0.47 15.7 2 4.95 35.8 + 1.16

3.45 5 0.45 2.20 f 0.20

0.26 fr 0.04 0.17 + 0.02

214 27

Skel

4.11 k 0.62 5.34 + 0.47 11.3 + 0.18 14.3 & 2.21

1.40 k 0.29 4.25 & 0.95

0.23 f 0.04 0.32 f 0.05

274 27

9.1 * 1.33 9.5 i: 1.98

UP45

214 27

P : R ratio

IrE.m~‘.s-~” -. 7.29 5 1.14 6.41 + 0.32 ~-

Respiration per pm3 mol 0,.flm-3.min-’ x lo-‘a ____3.80 4 0.73 4.85 k 0.73

Respiration per cell mol O,.cell-‘*min’ x IO-‘s -. 0.21 * 0.04 0.22 + 0.03

Clone -_____ NY17

Light intensity

Respiration rates per ceil and per pm' of cell volume, ratios of net photosynthesis versus respiration (P : R) and values of the compensation light intensity (I,): values are means + 1 SE.

TABLE IV

PHOTOADAPTATION:

PHOTOSYNTHETIC

AND CELLULARFEATURES

243

RESPIRATION

Dark resp~ation rates per cell and per pm3 are su~~zed in Table IV with P : R ratios and Z,. All of the clones had similar respiration rates per cell in both light regimes (P > 0.05). The clones had similar respiration rates per pm3 in high light, but clone Skel had a significantly lower (P < 0.05) rate than NY17 in low light. Only UP45 showed a significant decrease in respiration per pm3 in low light compared to high light. Cell size did not show a significant correlation with respiration rate per cell (r = 0.02, P > 0.05). Clonal differences in P : R ratios were similar to those shown for P,,, (cell) and were not correlated with growth rate (Z’> 0.05). In all cases, the P : R ratios approximated or exceeded the theoretical value of 10 reported for marine phytoplankton (Parsons et al., 1977). If daily carbon balances are determined assuming 24 h of respiration and 10 h of photos~thesis for the winter clone (winter photope~od) and 14 h for the summer clone (summer photoperiod), then the P: R ratios are about 3.5 for NY17 irrespective of light intensity and 11.2 for high light UP45 and 14 for low light. All of the clones showed similar Z, values in both light regimes (P > 0.05). The grand mean Z, for all of the data was 6.2 k 0.55 PIE*m-*0 s- ‘. Thus, all clones can reach compensation at very low light intensities. DISCUSSION Previous studies (Gallagher, 1982; Gallagher et al., 1984) indicate that the cycling between prevalent seasonal populations in N~ag~sett Bay is due to cyclic natural selection, rather than to random forces such as genetic drift (see Rockwell et al., 1985, for a discussion). Because the survival of individual cells is probably determined by their particular photosynthetic traits during at least part of the year (Hitchcock & Smayda, 1977), the patterns of variation in these traits among populations of S. costutum are probably largely determined by selective forces. This hypothesis implies that traits common to all S. co~tff~rn populations are probably not important in regulating seasonal cycling. Therefore, the present investigation sought to delineate those cellular photosynthetic features which differ among clones and which might contribute to the seasonal success of the distinct populations. The present study examined two clones of 5’. cosfatum from Narragansett Bay that are genetically and physiolo~~~ly representative of the prevafent winter (NY17) and summer (UP45) bloom populations (Gallagher, 1980, 1982). Although there is variance among clones within populations for photosynthetic features, this is much smaller than the variance between populations for the same features (Gallagher, 1982). Furthermore, there is no evidence that these clones have changed over time in culture (G~agher, 1983). Therefore, the broad patterns of differences shown by NY17 and UP45 are probably indicative of the major patterns of differences between the prevalent summer and winter bloom populations. This hypothesis may be revised as more clones

244

JANE

C. GALLAGHER

AND RANDALL

S. ALBERTE

are examined. Although it is unknown if the responses of the third clone, Skel, are generally representative of the population from which it was isolated, this clone was included in this investigation because it has been used in many previous studies of phytoplankton physiology (Davis, 1976; Falkowski & Owens, 1980; and many others), and therefore serves as an internal reference between the results of the present investigation and previous studies. 61

A

’ 18

NY 17

0-p F

70

q

ei:_ii-_l__l__~__:__l

B

6 5-

I

4-

,

!’ t

.cE 3-

P

n. ‘‘E 3_

7

I

’1

,

II

I.

------------I

Skel

0 0

200

600

400 -2

14 m

800

, 1000

s-1

Fig. 4. P vs. I curves standardized per pm3 of cell volume for clones grown in high and low light: values for each point are means f 1 SE; A, NY17; B, UP45; C, Skel.

PATTERNS

OF SIMILARITY

BETWEEN

CLONES

All of the clones responded to decreased light intensities by reducing growth rates and increasing the concentrations of pigments per cell. These observations are similar to those reported previously for this species (Yoder, 1979; Hitchcock, 1980). The P-I

PHOTOADAPTATION:

PHOTOSYNTHETIC AND CELLULAR FEATURES

245

curves showed that the slopes and P,,, values per cell of the low light grown cultures of each clone were either equal to, or were greater than their high light grown counterparts. This pattern of photoadaptation is similar to that found for another clone of Skeletonemu (Jorgensen, 1969). This type of response to low light has been attributed to the effects of nutrient limitation in batch culture (Beardall & Morris, 1976). Although nutrient effects may influence the photoadaptive response of S. costatum (Gallagher, unpubl.), the present data cannot be attributed to batch culture artifacts because all cultures were harvested at cell densities well below the carrying capacity of the medium. The changes in slope and P,, per cell for all clones were a function of cell size. Two out of three clones showed decreases in cell volume in low light, and the third remained constant. When the P-I curves were corrected for changes in cell size, all of the clones responded to low light by increasing their slopes and P,, values per pm3 of cell volume. Therefore, the cellular photoadaptive strategy of all of the clones was to alter the photosynthetic apparatus in order to increase the photosynthetic performance per pm3 of cell volume. These alterations involved changes in pigment content and PSU organization in all clones. The organizational differences involved both adjustment in PSU sizes and numbers per pm3. The clones examined either maximized their PSU sizes or number of PSUs per cell, but did not employ both adaptive strategies. This relationship is probably governed by limitations on maximum chloroplast size, packing of reaction centers, and, consequently, PSU packing along the chloroplast membranes (see Kursar et al., 1983). All of the clones showed similar values of respiration per cell and compensation light intensities. These data indicate that differences in dark respiration rate between clones are probably not an important selective factor at this growth temperature. Indeed, the P: R ratios all indicate very favorable daily carbon gains. The mean value of Z, (6.2 k 0.55 pE.m-2*s‘) for all clones is close to 1y0 of the incident light intensity during the summer in Narragansett Bay. As such, it is likely that all clones would maintain favorable carbon gains even at the bottom of the euphotic zone. PATTERNS OF VARIATION AMONG CLONES

Large differences among clones were found for cellular pigment content, P-I relationships, growth rates, changes in cell size and numbers of PSUs per cell. These differences complement those for cell-independent features such as pigment ratios and energy transfer efficiencies reported previously for the same clones (Gallagher et al., 1984). All of these characteristics are complex traits involving whole-cell and molecular changes. The lack of correlation of these traits with cell volume or cell diameter indicates that clonal variation was not due to differences in We cycle or developmental changes. This is consistent with previous findings (Paasche, 1973 ; Gallagher, 1982) and indicates that phenotypic variation among clones was a reflection of underlying genetic differences. The photosynthetic features identifed here are likely determined by a large number of structural and regulatory genes, therefore implying large genetic differences exist

246

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between clones. Such a large genetic divergence among clones indicates that they have evolved in the relatively distant past and have been maintained in nature. Variation in the organization of the photosynthetic apparatus among clones was particularly striking. The clones showed significant differences in the numbers of PSU-RCII and PSU-RCI per cell when grown under identical conditions. There was also significant variation in the numbers of RCIs per pm3 among clones in both light regimes. These differences in PSU numbers were inversely related to PSU size. Only clone Skel showed significant increases in the numbers of RCIs per cell in low light. Changes in numbers of RCIs per cell have been observed previously in other species and have been attributed to changes in cell volume (Perry et al., 1981; Alberte et al., 1984). Although differences in cell volume can account for the present observations in UP45 and NY 17, they cannot in Skel which maintained a constant volume in both light regimes. These and some previous observations (Kursar & Alberte, 1983; Perry, pers. comm.) indicated that PSU number per cell is not a static feature of marine diatoms, and that the expression of phenotypic variation for this trait may be clone-dependent. Consequently, a photoadaptation strategy for a species cannot be defined from examination of a single clone. The clones showed signiticant differences in the ratio of RCI : RCII when grown under identical light regimes. Many higher plants and clones of Synechoccocus spp. maintain a constant RCI : RCII of one regardless of growth light intensity (Haehnel, 1976; Barlow & Alberte, 1985); one of the clones examined here, NY17, showed this pattern of response. Clone UP45 maintained a constant RCI: RCII in both light regimes, but it was significantly less than one. In clone Skel, the RCI : RCII ratio was less than one in high light and approached one in low light. A previous report (Falkowski et al., 198 1) suggested that Skel shows changes in RCI : RCII ratios in response to light intensity, but.no statistical significance was placed on these changes. A possible explanation for RCI : RCII ratios of less than one in UP45 (high light) and perhaps Skel (high light) is that photosystem I is unstable under these growth light conditions. Photoinhibition in a diatom results in specific losses of RCI (P700) (Gerber & Burris, 1981). A similar finding has been made in clones of Synechococcus spp. (Barlow & Alberte, 1985 & in review). A similar response to high light could account for the apparent deficiency in RCI (RCI : RCII < 1) in UP45 grown under high light in as much as this clone showed photoinhibition of photosynthesis (Fig. 2). The instability and loss of RCI under low light in this clone is not readily explained at the physiological level, and may result from a genetic quality which distinguishes it from the other clones. An alternate explanation for RCI : RCII ratios less than one may lie in the fact the RCII content is estimated from an in vivo physiological measure of 0, yield under flashing light which is dependent upon PSI activity, whereas RCI (P700) content is estimated by an in vitro photochemical reaction independent of PSI1 activity. It is possible that the recovery of P700 activity from detergent solubilized thylakoids is not complete in UP45. However, this explanation is hard to reconcile with the good recovery of P700 in NY 17 under both light conditions and in Skel grown in high light since all clones were treated in an identical manner.

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The adaptive strategy shown by each clone was a function of the total integrated photosynthetic characteristics that were both dependent on and independent of cell size. Each clone showed a unique pattern of integration of all of these traits in response to growth light intensity. Skel showed an adaptive strategy that relied on high proportions of Chl a relative to the total pigments, small PSU sizes and flexible numbers of PSUs per cell. This clone had low growth rates in high light but high growth rates in low light. It also had the lowest rate of respiration per unit cell volume. Therefore, it appears that clone Skel has evolved to survive in low light environments, but is less successful under high light conditions. This adaptive strategy would be consistent with the fact that this clone was isolated from the turbid, highly polluted waters of Long Island Sound. This clone appears to compensate for its relatively small, less efficient PSUs by increasing the number per cell. It is reasonable, however, that this strategy which involves relatively large amounts of Chl a, may only be advantageous in white light regimes (for discussion, see Gallagher et al., 1984). It is also possible that the responses shown by this clone cannot be extrapolated to the populations from which it was isolated, and may be only a consequence of selection for growth in an incubator at the time of isolation. The cellular responses to light intensity suggest that two survival strategies may be employed for these clones. The first would be to maximize population size at the expense of the survival of single cells, and the second would be to maximize the survival of individual cells while sacrificing the probability of large population size (e.g. r or K strategy). The former strategy would probably be advantageous in areas of the water column where rate of loss of individuals is high due to wash-out or zooplankton grazing. The adaptive strategy shown by clone NY17 relied on very high proportions of accessory pigments in both high and low light, small PSU sizes and constant numbers of PSUs per cell. The density of PSUs per unit of cell volume was increased by decreasing cell size in low light. This clone had a relatively low growth rate in high light, but had a high growth rate in low light. The survival strategy of NY17 in low light appears to follow an r strategy, where population size is maximized, and is consistent with that expected for a clone representative of the prevalent winter bloom population whose growth rate may be regulated by light. Maximization of growth rate in low light in this clone might have been improved if there had not been a loss in energy transfer efficiency (Gallagher et al., 1984) at this temperature which is 18 “C higher than that present when populations of this type are prevalent in Narragansett Bay (Gallagher, 1980). Lower temperatures would probably also have reduced the respiration rate per pm3 in this clone. This is supported by the fact that the winter clones can grow at 0 ‘C, while the summer ecotypes cannot (Gallagher & DeVries, in prep.). The fact that this clone shows reasonably good growth and photosynthetic rates at high temperatures is consistent with the presence of winter types as rare forms during the summer (Gallagher, 1982).

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The adaptive strategy shown by UP45 relied on an intermediate amount of accessory pigments relative to Chl a, large PSU sizes and small numbers of PSUs per cell. It altered the number of PSUs by decreasing cell size in low light. In spite of its large PSU size and its apparent photoinhibition in high light, this clone appeared to utilize an r strategy in high light and a K strategy in low light. The latter strategy is similar to the long-term survival strategy shown by some marine dinoflagellates that live for long periods below the euphotic zone (Rivkin et al., 1982). This is probably advantageous in regions where the time scale of temporal variability in light is long. This would be an advantage to summer populations where storm-driven mixing is infrequent and waters are turbid, and to the possible over-wintering survival of these populations on the bottom during the winter in Narragansett Bay (see Gallagher et al., 1984). The success of this latter strategy would be dependent on a reduction in cell mortality due to reduced benthic grazing at low temperatures during the winter months (Nixon et al., 1975). In summary, the results of the present investigation showed that the photoadaptive strategy of Skeletonema costatum populations involves variation in the integration of many cellular photosynthetic features, and that no single factor dominates or predicts the mechanism of photosynthetic light adaptation in diatoms. Further, large clonal differences in photoadaptive features suggest significant genetic divergence in the distant past among ecotypes of this species. The patterns of variation among clones for the cellular photosynthetic parameters examined can, in part, account for changes in seasonal abundance of their respective genetic groups in natural ecosystems.

ACKNOWLEDGEMENTS

We wish to express our thanks to A. Friedman, M. DeVries, M. Abad, G. Fisher, and J. Downey. This research was supported by NSF Grant DEB80-21744, PSC-BHE Grant 664134 from the Research Foundation of the City University of New York (J.C.G.) and by NSF Grants PCM78-10535 and OCE82-14914 (R.S.A.).

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