Freezing tolerance in the intertidal red algaeChondrus crispus andMastocarpus stellatus: Relative importance of acclimation and adaptation

Marine Biology 106, 427-436 (1990)

M a r i n e ................ B io logy

@ Springer-Verlag 1990

Freezing tolerance and Mastocarpus and adaptation

in the intertidal red algae Chondrus crispus stellatus: relative importance of acclimation

S.R. Dudgeon, I.R. Davison and R.L. Vadas

Department of Botany and Plant Pathology and Center for Marine Studies, University of Maine, Orono, Maine 04469, USA

Date of final manuscript acceptance: May 18, 1990. Communicated by J. Grassle, Woods Hole

Abstract. The effect of repeated daily freezing on photosynthesis, growth and phenotypic acclimation to freezing was studied in the red algae Chondrus crispus Stackhouse and Mastocarpus stellatus (Stackhouse in With.) Guiry. Algae used for experiments were collected from Cham- berlain, Maine, between March and August 1987, and field observations and experiments were carried out at Chamberlain and Kresge Point, Maine between March 1987 and March 1989. After ca 30 d of daily freezing for 3 h at -5 °C photosynthesis of C. crispus was reduced to 55% of control values. Growth rates of C. crispus were also reduced in fronds frozen daily compared to unfrozen controls, and eventually fronds became bleached and fragmented resulting in biomass losses. Fronds of C. crispus, frozen daily, had higher photosynthetic rates following freezing events than unfrozen controls indicating that this species can acclimate to freezing conditions. Accli- mation to freezing involves the light-harvesting reactions of photosynthesis. In contrast, photosynthesis and growth in M. stellatus were unaffected by repeated daily freezing for 3 h at -5 °C for 36 d. No differences in photosynthesis following freezing were observed between frozen and control fronds suggesting that M. stellatus does not phenotypically acclimate to freezing. The greater freezing tolerance of M. stellatus relative to C. crispus results, in part, from genetic adaptations associated with plasma membranes and the light-harvesting reactions of photosynthesis.

Introduction

Freezing during low tide exposure is likely to be an important stress affecting organisms inhabiting sub-arctic and boreal shores. Freezing temperatures prevail during most winter low-tides in the northern Gulf of Maine and intertidal macroalgae may be frozen three times within 24 h. Macroalgae may also be exposed for several hours to extremely cold temperatures (e.g. -20 °C) either occasionally or repeatedly during the winter months (Kan-

wisher 1957, Parker 1960, Bird and McLachlan 1974). A single freezing exposure can have profound impacts on metabolism and survival of macroalgae (Parker 1960, Bird and McLachlan 1974, Green 1983, Frazer etal. 1988, Davison et al. 1989, Dudgeon et al. 1989). The fre- quency and extreme range of sub-zero temperatures that occur during much of the year suggest freezing may be a strong selective force on the physiology and ecology of perennial intertidal macroalgae inhabiting high latitude shores.

Our previous studies on two co-occurring intertidal algae indicate that photosynthesis in Chondrus crispus was much less tolerant of a single freezing event than photosynthesis in gametophytes ofMastocarpus stellatus, which occurs slightly higher on the shore (Dudgeon et al. 1989). The reduction of photosynthesis by freezing was associated with damage to the plasma membrane and reduced efficiency of energy transfer in the photosynthetic light harvesting reactions. This is consistent with other studies which have also indicated that the degree of freezing tolerance is positively correlated with position on the shore with high intertidal species being most tolerant, low intertidal species having low tolerance and sub-littoral and rock pool species being intolerant of freezing (Biebl 1972, Frazer et al. 1988, Davison et al. 1989). To date, most studies on freezing have examined the effect of a single exposure to sub-zero temperatures (Kanwisher 1957, Biebl 1972, Frazer etal. 1988, Dudgeon etal. 1989). Less is known regarding the effect of repeated exposures to mild freezing conditions (e.g. - 5 °C) on metabolism and growth of macroalgae, although Davi- son et al. (1989) reported that repeated exposures for 7 d to -20 °C affected photosynthesis in lower, but not upper eulittoral species from the Gulf of Maine.

The interspecific differences in freezing tolerance related to vertical zonation reported previously (Biebl 1972, Frazer et al. 1988, Davison et al. 1989, Dudgeon et al. 1989) may have resulted from either phenotypic acclimation or genetic differences since the more tolerant species grow higher on the shore and are, therefore, exposed to more severe and frequent freezing prior to use in

428

experiments. Seasonality in freezing tolerance within fucoid algae suggests phenotypic acclimation is likely to occur (Parker 1960, Bird and McLachlan 1974). Howev- er, this does not preclude the existence of genetic differences in freezing tolerance. Genetic adaptation and phenotypic acclimation are both important in macroalgae. Ecotypes of kelps (Laminaria sp.) have been reported for nitrogen assimilation and low light levels indicating the importance of genetic adaptation to determining their physiological stress tolerance (Espinoza and Chapman 1983, Gerard 1988). Phenotypic acclimation to alleviate stress occurs in intertidal fucoids in response to desiccation and in L. saccharina in response to temperature (Schonbeck and Norton 1979 a, b, Davison 1987). Expo- sure to chilling temperatures (5 °C) has been shown to induce greater freezing tolerance in some terrestrial crop species (Guy and ttaskell 1987, Singh and Johnson- Flanagan 1987).

This study examined the effects of repeated freezing exposures on growth and metabolism of Chondrus crispus and Mastocarpus stellatus. We addressed the following questions: (1) What effect does repeated freezing have on photosynthesis, growth and survival? (2) Is the loss of pigments (hereafter referred to as bleaching) and loss of biomass in late winter in field populations of C. crispus caused by freezing? (3) Is there phenotypic acclimation in the freezing tolerance of either species, or is tolerance genetically determined? These questions were addressed by measuring responses of photosynthesis, growth, pigment concentrations, light harvesting and membrane characteristics under different air and seawater temperatures in the laboratory and survival following winter in the field. To determine whether the difference in freezing tolerance between species resulted from genetic adaptation or phenotypic acclimation, experiments were performed on algae collected in March and August 1987. In the August samples neither species had been frozen for ca 5 mo and presumably were not acclimated to freezing.

Materials and methods

S.R. Dudgeon et al.: Freezing in seaweeds

arbitrarily defined; no damage, I to 20, 21 to 40, 41 to 60, 61 to 80 and 81 to 100% of the thallus bleached.

To determine the range and duration of sub-zero temperatures in the intertidal zone a Tempmentor thermograph (Ryan Instru- ments, Redmond, Washington), set immediately above the intertidal zone at Chamberlain recorded air temperatures every 30 min from December 7, 1988 through March 13, 1989. Temperature data were compared to tide tables to determine the approximate air temperature within the intertidal zone during low tide exposure on minus tide days. Bi-weekly observations throughout the winter of 1988-1989 were made to establish the approximate time that bleaching of algae might occur.

Algal material

Chondrus crispus and Mastocarpus stellatus were hand collected from the shore at Long Cove Point, Chamberlain, Maine (43°56'N; 69°54'W) between March and August 1987. Fronds were transport- ed to the laboratory in seawater, cleaned of visible epibionts and maintained in plexiglass aquaria containing 61 of aerated GF/C (Whatman) filtered enriched seawater (PES, Provasoli 1968) that was changed every 3 to 4 d. There were two types of freezing exposures: chronic daily exposures to relatively mild freezing temperatures (e.g. -5°C), and single acute exposures to extremely cold temperatures (e.g. < -20 °C). Fronds used for chronic freezing experiments were collected in March 1987 and maintained at 5°C. Fronds used in chronic freezing acclimation experiments were collected in August 1987 and maintained at 15 °C for 7 to 10 d prior to use in experiments. Cultures (5 ° and 15°C) were maintained at a photon flux density (PFD) of 70 to 80 pmol photons m - z s- ~ in a 16 h light :8 h dark cycle.

Photosynthesis

Photosynthesis in all experiments was measured by incubating excised apices of fronds for 30 min at 15 °C at a saturating photon flux density of 500 #tool photons m - z s- 1 (Mathieson and Burns 1971) in 200 ml of (Millipore, 0.45 #m) filtered seawater (pH 8.0) containing 10 #Ci H~CO3 in a total HCO~ concentration of 2 raM. Car- bon-14 incorporation by fronds was measured as described by Davison and Davison (1987). Apices were cut following the exposure of fronds to their respective treatments the day before measuring photosynthesis. Thus, apices were given approximately a 24 h recovery period from freezing before use in experiments.

Winter bleaching of fronds Effect of chronic freezing

To quantify the bleaching of fronds observed during late winter (late February/March; R. L. Vardas unpublished observations) intertidal samples were harvested in mid-March 1987. Transects parallel to the shoreline were laid out in the Chondrus crispus Stackhouse/Masto- carpus stellatus (Stackhouse in With.) Guiry mixed assemblage [ca 0.5 m above mean low water (MLW) - hereafter the mid zone] and in the C. crispus dominated low zone (ca 0.0 m MLW) at two sites, Chamberlain and Kresge Point (adjacent to Pemaquid Point), Maine. Seven to ten 20 x 20 cm quadrats were harvested from the substra- tum with paint scrapers at randomly selected points along each transect in the two zones and returned to the laboratory. The mussel shell/algal crust/sediment matrix in which fronds were embedded usually remained intact after scraping the plot, which facilitated discrimination between canopy and understory fronds of each species. In addition, canopy and understory fronds were distinguished by size. Thus, samples were sorted by species and as canopy or understory fronds. Twenty fronds of each species randomly selected from each group were assessed for bleaching damage, expressed as percent of thallus bleached. Six categories of bleaching damage were

Large, whole fronds collected in March 1987 and maintained in seawater at 5 °C were emersed daily for 3 h either at 5 °C (controls) or -5 °C (experimental) for 36 d. Plants were emersed in the dark in closed plexiglass boxes. Photosynthesis was measured every 5 to 7 d.

Freezing acclimation

To determine whether either species acclimates to freezing, fronds collected in August 1987, when seawater temperatures were approximately 15 °C, were maintained in laboratory culture for 7 to 10 d at 15 °C. Fronds of each species were randomly assigned to one of four treatments: seawater at 20 °C; seawater at 5 °C; seawater at 5 °C with daily freezing for 3 h at -5 °C; and seawater at 5 °C with daily freezing for I h at -20°C. Experimental treatments lasted 26 d. Treated and control fronds were assayed for photosynthesis, phycoerythrin fluorescence, plasma membrane permeability, phycobiliprotein pigment concentrations and growth.

S.R. Dudgeon et al. : Freezing in seaweeds

Photosynthesis of excised apices of both species (from the above treatments) was measured following the initial 10 d period in 15 °C seawater and every 5 to 7 d thereafter for 26 d. Photosynthesis was measured in two conditions; as controls and following 3 h at -20 °C. The acclimation characteristics of the light harvesting reactions were assessed by measuring fluorescence ofphycoerythrin after 16 d exposure to the conditions listed above. Phycoerythrin fluorescence was measured in vivo in controls maintained at 5 °C and following 3 h at -20°C as previously described (Dudgeon etal. 1989). Changes in plasma membrane permeability, after 21 d exposure to the above conditions, were assessed indirectly using the amino acid release technique of Dudgeon et al. (1989) in control (5°C) and experimental apices (3 h a t -20 °C), In both experiments only one freezing treatment, seawater at 5 °C and 1 h d-1 at -20 °C, was utilized.

The effect of repeated freezing events in causing bleaching of fronds was determined by comparing phycobiliprotein pigment concentrations after 26 d between the two "non-frozen" and two "frozen" culture treatments using the method of Rosenberg (1981). To distinguish whether bleaching results from chronic or acute freezing, fronds of both species were exposed once to -20 °C for 12 h (controls were maintained in seawater at 5 °C) following which they were returned to culture conditions at 5 °C for 26 d prior to analysis of phycobiliprotein pigment concentrations.

Two methods were used to determine the effect of freezing on growth of Chondrus crispus and Mastocarpus stellatus fronds. The linear apical extension from a marked position (a pin hole) on tagged fronds following 25 d exposure to the acclimation experiment treatments above was used to quantify linear growth. Increases (or decreases) in fresh weight (fwt) biomass of tagged fronds under each treatment were measured and converted to growth (gfwt g- fwt mo-1) as described by Evans (1972).

Win te r survival of Chondrus crispus holdfasts

Our own observations suggested that Chondrus crispus fronds bleached during winter were dislodged by wave-action in early spring. However, the holdfasts were capable of regenerating erect stipes in the spring. To determine whether holdfasts of C. crispus were protected from lethal damage through insulation by the canopy a field removal experiment was established in the low intertidal zone at Chamberlain during the period November 20 to De- cember 4, 1987. A randomized complete block design was utilized consisting of 3 blocks (spatially separated along the shoreline) with six 50 x 50 cm plots in each block. Blocks were established in essen- tially homogeneous C. crispus stands within the low intertidal zone (ca 0.25 m above MLW). Initial percent cover of each plot was visually estimated following which one of three treatments was randomly assigned; (1) controls - no manipulation; (2) thallus removal all erect fronds scraped from the substrate; and (3) complete removal - erect fronds scraped away and algal crusts killed by burning for 5 to 10 min with a propane torch. Two replicates of each treatment were established per block. Final percent cover of C. crispus in each plot was visually censused in August 1988.

Statistical analyses

Comparisons between species, and between canopy and understory fronds within species, of bleaching damage of fronds was made using the Z 2 test on raw data. Photosynthetic measurements over time in the chronic freezing and acclimation experiments were analyzed with repeated measures ANOVA (analysis of variance). All other experimental data were analyzed with one- or two-way ANOVA. Non-normal data were either log or arc-sine transformed prior to analysis. Multiple comparisons were performed using the Student-Newman-Kuels procedure.

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Fig. 1. Chondrus crispus and Mastocarpus stellatus. Proportion of fronds at each tidal height exhibiting a given degree of bleaching damage at Chamberlain in March 1987: (a) C. crispus in the low zone, (b) C. crispus in the mid zone and (c) M. stellatus in the mid zone. Filled bars: data for canopy fronds; hatched bars: data for understory fronds; mean proportion__+ SE (n = 10 quadrats)

Results

Winte r b leaching of f ronds

The shoreline at Chamber l a in faces east and at Kresge Po in t faces southwest , bu t the extent of b leaching of bo th species at the two sites was similar. Thus, only data for C ha mbe r l a i n are shown in Fig. 1.

Mos t canopy fronds of Chondrus crispus in the inter- t idal zone at C ha mbe r l a i n were bleached by m i d - M a r c h 1987. The p ropor t ion of bleached fronds and the extent of b leaching per f rond was less in C. crispus f rom the low zone than in f ronds f rom the mid zone (Fig. 1 a, b; Z2=60.24, p < 0 . 0 1 , d f = 5;). In the mid zone, where the species co-occur, the p ropo r t i on of f ronds bleached and the extent of b leaching per f rond was greater in C. crispus t han Mastocarpus stellatus (Fig. l b , c; Z2=187.01, p < 0.01, d f = 5). In bo th species the degree of b leaching

430

Table 1. Summary of air temperature data recorded at 30 mininter- vals during minus low tides at Chamberlain, Maine from December 1988 to March 1989

Data Dec. Jan. Feb. Mar.

Total days 20 15 11 8 of minus tides

Days frozen 10 9 10 7

Proportion of exposure 0.48 0.60 0.90 0.76 time spent frozen

Most consecutive 5 5 (2) 9 (6) 6 (6) days frozen a, b

Mean time period 45.5 16.6 10.3 13 between freezing exposures (h)a

Mean temperature - 1.59 - 1.21 -- 5.31 - 5.16 during exposure (°C)

a Only data for days within a minus tide series are considered b Number in parenthesis indicates the number of consecutive days freezing conditions prevailed during two minus tides within 24 h

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Fig. 2. Chondrus crispus and Mastocarpus stellatus. Effect of daily freezing for 3 h at - 5 °C on photosynthesis. Fronds were not frozen on day of measurement. Open circles: data for C. crispus; filled circles: data for M. stellatus; mean 4- SE (n = 7).

damage was less in understory than canopy fronds (C. c?'ispbls" )~2 = 56.42, p < 0.01, d f = 4; M. stellatus: Z 2 = 15.92, p < 0.01, d f = 2). Thus, there appeared to be a microclimate effect whereby understory fronds were protected from freezing through insulation provided by canopy fronds.

Air temperatures (recorded every 30 rain) within the intertidal zone at Chamberlain for the 3 h period (1.5 h before and 1.5 h after) around low tide for each minus tide from December 7, 1988 to March 13, 1989 indicated that temperatures _<-10°C occurred during ca 20% of low tide exposures. Although the minimum temperatures recorded in each month were similar (-13.6 ° to -21.1 °C), freezing temperatures occurred only sporadically in De- cember and January, whereas freezing temperatures prevailed during most low tide exposures in February and March (Table 1). Freezing rarely occurred twice during any 24 h period in December and January, making the time period between freezing relatively long (46 and 17 h, respectively). In contrast, in February and March freezing frequently occurred on consecutive days, and often, three times within 24 h, reducing the recovery time between exposures (10 and 13 h, respectively). During winter 1988/1989 bleached Chondrus crispus were first observed on March 9, 1989 indicating that bleaching of fronds occurred between February 20 (the previous observation) and March 9.

Effect of chronic freezing on photosynthetic rates

Repeated daily freezing to -5°C for 3 h progressively inhibited photosynthesis in Chondrus crispus (Fig. 2). Al- though no effect occurred prior to 12 d, beyond 30 d photosynthesis in C. crispus had declined significantly to 55% or less of controls (F=4.95, p<0.05) .

Daily freezing had no significant effect on photosynthesis of Mastocarpus stellatus up to 36 d (F= 1.07,

p = 0.32; Fig. 2). Photosynthesis in treated fronds typically ranged from 90 to 140% of control fronds on any given day. A significant difference in photosynthetic tolerance to chronic freezing existed between the two species (Fsp. × trz = 5.97, p < 0.03).

Photosynthetic acclimation to freezing

There was no significant difference in rates of photosynthesis between control fronds in the various acclimation treatments in either species (Chondrus crispus: F= 1.55, p = 0.22; Mastocarpus stelIatus: F= 1.91, p = 0.15). Thus, rates of photosynthesis for fronds in each acclimation treatment following 3 h at -20 °C are expressed as a per- centage of the mean of all controls for each species (Fig. 3).

Photosynthetic rates of Chondrus crispus fronds measured immediately following 3 h at -20 °C were strongly dependent upon the acclimation regime to which they had been exposed and the duration of acclimation (F = 87.34, p < 0.01; Fig. 3 a). Initially, exposure t o -20 °C for 3 h reduced photosynthesis to ca 30% of controls. A similar degree of inhibition persisted in 5 °C grown fronds for the duration of the experiment, whereas photosynthesis in 20 °C grown fronds became progressively more inhibited by -20 °C exposure, reaching 10 % of control rates after 12 d. In contrast, fronds subjected to daily freezing (either 1 h at -20 °C or 3 h at -5 °C) exhibited increased freezing tolerance that was maximum following 12 d acclimation, with photosynthesis being reduced to approximately 80% of controls after 3 h at -20 °C.

Photosynthesis of Mastocarpus stellatus fronds following 3 h at 20 °C showed no clear trend between acclimation treatments (Fig. 3 b). Three hours at -20 °C had no significant effect on photosynthesis of M. stellatus fronds neither at time zero, nor at any time in the acclimation treatments. This suggests that M. stelIatus does

S.R. Dudgeon et al. : Freezing in seaweeds 431

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Fig. 3. Chondrus crispus and Mastocarpus stellatus. Photosynthesis in (a) C. crispus and (b) M. stellatus fronds following 3 h at - 20 °C exposed to different culture conditions over time. Symbols are: data for fronds continuously in seawater at 20 °C (open circles), seawater at 5 °C (filled circles), seawater at 5 °C and frozen daily in air for 3 h to - 5 °C (open triangles), and seawater at 5 °C and frozen daily in air for 1 h to -20 °C (filled triangles); mean _+ SE (n = 4)

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Fig. 4. Chondrus crispus and Mastocarpus stellatus. Fluorescence of phycoerythrin (expressed as a ratio of initial values) in (a) C. erispus and (b) M. stellatus following 16 d exposure either to seawater at 20 °C, seawater at 5 °C, or seawater at 5 °C and frozen 1 h d-x at -20 °C. Filled bars: data for controls, hatched bars: data following 3 h at -20°C; mean+ SE (n=3)

not acclimate, but that it is genetically adapted to tolerate such freezing exposures.

Photosynthetic rates in control fronds (all acclimation regimes combined) of both species varied consider- ably. However, Chondrus crispus control fronds had slightly higher photosynthetic rates than those of Masto- carpus stellatus (data not shown). In contrast, photosynthetic rates in C. crispus fronds (all acclimation regimes combined) were affected more than M. stellatus fronds following 3 h at -20 °C, although C. crispus fronds acclimated to freezing exhibited rates similar to M. stellatus. Thus, overall C. crispus fronds were less tolerant of freezing than M. stellatus fronds (Fsp" × te,,p = 60.10, p < 0.01).

Mechanisms of photosynthetic acclimation

Phycoerythrin fluorescence of Chondrus crispus fronds cultured in 20 ° and 5 °C seawater increased approximately 1.5-fold over that of controls following 3 h a t - 2 0 °C (Fig. 4a). In contrast, fluorescence values of C. crispus fronds cultured at 5 °C and frozen daily for 1 h d-~ for 16 d were similar to unfrozen controls following 3 h at -20 °C, indicating less disruption of energy transfer rela-

tive to "non-frozen" cultured fronds following 3 h at -20°C ( F = 15.31, p<0 .01) .

There was no change in phycoerythrin fluorescence of Mastocarpus stellatus fronds following 3 h at -20 °C from any culture t reatment after 16d (F=0.11, p=0 .90 ; Fig. 4b). Over all culture treatments, there was a significant difference between species in degree of phycoerythrin fluorescence in response to freezing (Fsv. × temp = 6.06, p < 0.02).

Exposure to the different acclimation regimes for 21 d had no effect on plasma membrane permeabili ty following freezing in either species ( F = 1.00, p = 0.39). Chondrus crispus fronds f rom each culture regime typically released 55 to 65% of their total amino acid content following 3 h at -20 °C (Fig. 5 a). Amino acid release in Mastocarpus stellatus fronds following 3 h at -20°C in all culture regimes was significantly less than in C. crispus ( F = 38.57,p < 0.01). Release of amino acids following 3 h a t - 2 0 °C in M. stellatus ranged f rom 22% in 20 °C seawater acclimated fronds to 5% in freezing acclimated fronds, however, there was no significant difference between acclimation regimes due to large variation among fronds cultured at 20 °C (Fig. 5 b).

432

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Fig. 5. Chondrus crispus and Mastocarpus stellatus. Release of amino acids in (a) C. crispus and (b) M. stellatus following 21 d exposure either to seawater at 20 °C, seawater at 5 °C or seawater at 5 °C and frozen 1 h d -1 at -20°C. Filled bars: data for controls; hatched bars: data following 3 h at -20°C; mean +_SE (n=3)

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Fig. 6. Chondrus crispus and Mastocarpus stellatus. Phyco- biliprotein pigment concentrations in (a) C. crispus and (b) M. stellatus following 26 d exposure to either seawater at 20°C (filled bars), seawater at 5 °C (hatched bars), seawater at 5 °C and frozen daily for 3 h to - 5 °C (open bars ), and seawater at 5 °C and frozen daily for 1 h to -20 °C (cross hatched); mean_+ SE (n = 3)

Effect of freezing on bleaching of fronds

Labora tory experiments to determine whether freezing could bleach fronds indicated that a single acute exposure (12 h at -20 °C) followed by a 26 d recovery period had little effect on phycobiliprotein pigment concentrations in either species (data not shown). However, chronic freezing exposures did affect phycobiliprotein pigment concentrations (Fig. 6). Da ta for each pigment, pooled into frozen and non-frozen treatments, indicated that freezing typically reduced pigment concentrations in Chondrus crispus by 50% but had little effect on Masto- carpus stellatus (Phycoerythrin: F,v" × t , t = 6.48, p < 0.02; Phycocyanin: F,p. × trt = 6.62, p < 0.02; Allophycocyanin: Fsv. × t r t = 4.61, p < 0.05). Following approximately 15 d of repeated freezing C. crispus fronds began showing signs of bleaching and following 26 d much of the thallus of most fronds was bleached (S. R. Dudgeon personal observation). No significant reductions in phycobiliprotein pigment concentrations were observed in control fronds which were immersed continuously in seawater or those emersed in air (5 °C) but not frozen (data not shown). Thus, freeze-induced bleaching of C. crispus, but not M. stellatus, in the laboratory after approximately 2 wk sup-

ports field observations that winter bleaching of C. crispus coincides with frequent low tide freezing exposures.

Effect of freezing on growth

Chronic freezing significantly reduced apical growth rates in Chondrus crispus (F=7,71, p<0 .01) . Apical growth of fronds maintained continuously in seawater at 20 ° and 5 °C were 3.00 and 2.28 m m m o - 1, respectively. In contrast, apical growth in treated fronds was reduced to 0.36 m m mo -1 in fronds exposed 3 h daily to - 5 ° C and 0.24 m m m o - 1 in fronds exposed daily to -20 °C for 1 h (Fig. 7 a).

Chronic freezing tended to slow apical growth in Mastocarpus stellatus, however, there was no significant difference between frozen and non-frozen treatments (F = 2.33, p = 0.11). Fronds of M . stellatus in non-frozen treatments grew more slowly than C. crispus, but because M. steIlatus was less affected by freezing (Fig. 7 b), it was able to grow faster than C. crispus under chronic freezing stress (F~p . . . . . ~ = 2.92, p < 0.05).

Growth measured as biomass changes also showed that Chondrus crispus was more susceptible to freezing


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Fig. 7. Chondrus crispus and Mastocarpus stellatus. Apical growth in (a) C. crispus and (b) M. stellatus exposed either to seawater at 20 °C, seawater at 5 °C, seawater at 5 °C and frozen daily for 3 h at -5°C, or seawater at 5°C and frozen daily for 1 h at -20°C; mean-I- SE (n = 5)

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Fig. 8. Chondrus crispus and Mastocarpus stellatus. Changes in frond biomass in (a) C. crispus and (b) M. stellatus exposed either to seawater at 20 °C, seawater at 5 °C, seawater at 5 °C and frozen daily for 3 h to - 5 °C and seawater at 5 °C and frozen daily for 1 h to -20°C; mean _+SE (n=5)

than Mastocarpus stellatus (Fig. 8). Repeated freezing caused C. crispus fronds to fragment and lose biomass (F= 9.55, p < 0.01), whereas non-frozen C. crispus grew by approximately 0.40 gfwt g - 1 fwt m o - 1. There was no significant difference between growth of frozen and non- frozen fronds of M. stellatus ( F = 1.96, p =0.16). Frozen fronds of M. stellatus did not lose biomass and, thus, were more tolerant of freezing than C. crispus (F = 5.28, p<0.01) .

Winter survival of Chondrus crispus holdfasts

Since understory fronds exhibited less bleaching damage than canopy fronds, removals of erect Chondrus crispus fronds were performed to determine whether C. crispus holdfasts exposed to freezing during winter would be killed. The initial percent cover of C. crispus prior to manipulation was ca 95%. This value remained constant in control plots sampled the following summer at which time the percent cover of C. crispus in thallus removal plots was 83%, with 6% in thallus and holdfast removal plots. This indicates that the presence of C. crispus in manipulated plots resulted primarily from holdfast re-

generation and not recruitment, and that holdfasts were not killed during winter (Fig. 9).

D i s c u s s i o n

Chronic freezing effects on bleaching, photosynthesis, and growth

These results indicate that Chondrus crispus is less tolerant of chronic freezing exposures than Mastocarpus stellatus. Daily freezing exposure in the laboratory resulted in bleaching and fragmentation of fronds, and reduced photosynthesis (following 20 to 25 d exposure) and growth in C. crispus, but not in M. stellatus. These find- ings are consistent with results of a previous investigation examining the effect of an acute (single) freezing event at -20 °C on photosynthetic metabolism of these two species (Dudgeon et al. 1989). In addition, results of the laboratory experiments presented here support the hypothesis, based on field observations, that freezing is involved in bleaching and fragmentation of C. erispus fronds, but has little effect on M. stellatus.

Although sub-zero temperatures occurred during tidal emersion from December to March, bleaching of

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100

80

0 6O

40 11.

20

controls thallus removal total removal

Treatment

Fig. 9. Chondrus crispus. Percent cover o f C. crispus in plots at Chamber l a in exposed to one o f the following t r ea tmen t s : cont ro ls = u n m a n i p u l a t e d ; thal lus r emova l and total r emova l = thal lus and holdfast. Filled bars: pre-manipulation cover; hatched bars: percent cover the following summer in each treatment; mean percent cover +_SE (n=6)

field populations of Chondrus crispus did not occur until the period February 20 to March 9 1989. Bleaching of C. crispus in the late, but not early, winter probably occurs every year (R. L. Vadas personal observations). The de- lay in the onset of bleaching until late winter could be caused by several factors. First, bleaching may only occur after repeated freezing for several months. However, this is contradicted by our laboratory studies that indicate damage occurs 15 to 20 d after the onset of daily freezing. A second explanation is that bleaching resulted from the more frequent freezing and shorter recovery periods that prevailed in February and March. Finally, photoperiod and light intensity also increase in February and March and it is possible that bleaching is caused by the combined effects of low temperature stress and high irradiance. Photoinhibition and subsequent photooxidation (bleaching) of photosynthetic pigments in higher plants exposed to low temperatures is well established (Van Hasselt 1972, Martin et al. 1978, Martin and Oquist 1979, Spearing and Karlander 1979, 0quist et al. 1980, Powles 1984). How- ever, it should be noted that bleaching of C. crispus occurred in laboratory experiments in plants grown in mod- erate light intensities and frozen in darkness, suggesting that high irradiance is not required for bleaching of photosynthetic pigments at sub-zero temperatures. The greater severity of bleaching observed in the field (complete loss of all pigments) may result from greater light intensities encountered during, and following, freezing, and the shorter recovery periods between exposures in the field compared to the laboratory.

Breakdown of plasma membrane integrity following freezing has been shown to be one factor responsible for the freezing susceptibility of Chondrus crispus in particu- lar (Dudgeon et al. 1989), and in plants in general (Lyons et al. 1979, Berry and Bj6rkmann 1980, Gordon-Kamm and Steponkus 1984, Singh and Johnson-Flanagan 1987). Similarly, freezing adversely affects chloroplast and thylakoid membranes and is aggravated further in


high light (Garber 1977, Martin and Oquist 1979, 0quist et al. 1980). Although membrane integrity may be quick- ly restored (Davison et al. 1989) we suggest cumulative damage to membranes and, perhaps, cell walls may result from frequent repeated freezing events. This cumulative damage may contribute to bleaching and fragmenting of C. crispus fronds observed in February and March. In contrast, plasma membranes of Mastocarpus stellatus, which rarely exhibits bleaching or fragmenting, are affected little by freezing (Davison et al. 1989, Dudgeon et al. 1989).

Ultra-violet radiation (UV) and desiccation are also potential causes of bleaching of Chondrus crispus since bleached fronds are occasionally observed at other times of the year, particularly in summer. UV radiation is known to cause bleaching in Ecklonia radiata (Phaeophyceae: Laminariales; Wood 1987) and in phyto- plankton and corals (Worrest 1982). However, UV radiation is probably not at damaging levels during winter (Wood 1987) and significantly less bleaching was observed in fronds growing only 0.5 m lower on the shore, which probably experience similar UV levels. Similarly, it is unlikely that the damage is due to desiccation, since this stress is probably more severe in summer than winter (Schonbeck and Norton 1978).

Genetic adaptation vs phenotypic acclimation in freezing tolerance

The Chondrus crispus and Mastocarpus stellatus fronds collected and used for experiments in late August 1987 had not been frozen for ca 5 mo and presumably were not acclimated to freezing conditions. This enabled us to determine whether different chronic and acute freezing tolerances of C. crispus and M. stellatus resulted from genetic differences between species or from phenotypic acclimation in M. stellatus. This experiment was also designed to determine if either species can phenotypically acclimate to freezing. Photosynthesis in C. crispus was significantly less tolerant of freezing than that of M. stellatus regardless of season (winter and late summer; Dudgeon et al. 1989, this study). Large, persistent differences observed in freezing tolerance between the two species cultured under identical conditions in different seasons of the year indicate that genetic differences are responsible for the greater tolerance of M. steIlatus. This is further supported by our observation that photosynthetic metabolism in M. stelIatus did not acclimate to daily freezing.

An alternative hypothesis is that Mastocarpus stellatus may acclimate following freezing exposures at an early stage by switching on "cold-hardiness" genes and re- main acclimated for the life of the frond. This is the developmental conversion type of phenotypic adjustment (Mayr 1963, Levins 1968, Etter 1988). However, the adaptive value of such a mechanism would seem small, particularly in summer, if the mechanism is energetically expensive and serves no additional function other than conferring freezing tolerance. It is also possible, given the similarity between desiccation and freezing tolerance in intertidal algae (e.g. Dring and Brown 1982, Dudgeon

S.R. Dudgeon et al.: Freezing in seaweeds

et al. 1989), that the same mechanism confers tolerance to both types of stress. If this were so, then, the acclimation of M. stellatus to desiccation during summer would explain the apparent lack of freezing acclimation observed in this study.

Although Chondrus crispus is freezing intolerant relative to Mastocarpus stellatus, the adverse effects of chronic freezing (at -5 °C) were manifest only following ca 20 d of continual exposure. The greater photosynthetic rates exhibited by freezing-exposed fronds in comparison to non-frozen cultured fronds following 3 h at -20 °C indicate that C. crispus combats freezing stress by phenotypic acclimation. Peak acclimation occurred between Days 12 and 23 of the experiment. The period up to 12 d probably represents the onset of acclimation and beyond 23 d the cumulative damage incurred by a frond may overwhelm the benefit of acclimation. We suggest that acclimation to freezing in C. erispus maintains photosynthesis (and other metabolic processes) at near normal levels to tolerate mild freezing stress (e.g. exposure to -5 °C) until irre- versible damage accumulates (after ca 25 exposures). Ac- climation to freezing has been observed in Fucus vesiculosus (Phaeophyta; Parker 1960, Bird and McLachlan 1974) and in several higher plants (Martin et al. 1978, Von Swaaij et al. 1985, Guy and Haskell 1987, Singh and Johnson-Flanagan 1987).

One aspect of photosynthetic metabolism in Chon- drus crispus that acclimated to freezing was the light-harvesting reactions. Fronds acclimated to freezing for 16 d exhibited less phycoerythrin fluorescence following 3 h at -20 °C than non-acclimated fronds. This suggests that the degree of freezing induced disruption of energy transfer in the light reactions was less in freezing-acclimated than non-acclimated fronds, possibly allowing the former to maintain higher photosynthetic rates. However, the specific component(s) of the light-harvesting complex un- dergoing acclimation cannot be determined from these data. Acclimation may occur within the thylakoid membrane, in the phycobilisomes or chlorophyll reaction center themselves, or may reflect changes within the photosynthetic electron transport chain. Reduced capacity of the photosynthetic electron transport chain is responsible for decreases in photosynthesis of higher plants during periods of freezing (()quist et al. 1980). Conversely, the maintenance of photosynthesis in acclimated fronds of C. crispus following freezing may result from acclimation of the photosynthetic electron transport chain.

No acclimation was observed in the plasma membranes of Chondrus crispus exposed daily to freezing for 21 d. Experimental fronds released more of their total amino acid content following 3 h at -20 °C, in contrast to controls upon re-immersion in seawater. Large losses of amino acids following freezing were also observed in fronds tested during winter (Dudgeon et al. 1989) further indicating that plasma membranes of C. crispus do not acclimate to freezing.

Freezing tolerance of Mastocarpus stellatus appears to be determined primarily by genetic adaptation. Previ- ous studies indicate that only extreme exposures (> 8 h at -20 °C) reduced photosynthesis in M. stellatus, and that full recovery was achieved rapidly (Davison et al. 1989,

435

Dudgeon et al. 1989). In addition, photosynthesis and growth were unaffected by chronic exposures up to 36 d regardless of the season tested. No differences were observed in photosynthesis, fluorescence of phycoerythrin and membrane permeability between M. stellatus fronds from the different acclimation treatments in response to 3 h at -20 °C. Photosynthetic rates of fronds were no different from controls following 3 h at -20 °C whether tested in winter or summer. Frozen fronds released between 5 and 18% of their total amino acids following 3 h at -20 °C in each treatment and similar releases were observed in winter following freezing (Dudgeon et al. 1989). Similarly, exposures of ca 3 h at-20 °C had little effect on phycoerythrin fluorescence in either winter or summer.

Freezing as a stress and disturbance

Grime (1979) defines stress as external constraints that limit dry matter production in organisms, and disturbance as external factors that contribute to the removal of biomass of an organism. This study indicates that freezing acts as a stress, limiting the productivity and growth of Chondrus crispus in winter and early spring, and as a disturbance, with the consequence that physio- logically inactive tissue (bleached) is removed by water motion (S. R. Dudgeon personal observation). This reduction in productivity and biomass may be important in limiting space occupation by C. crispus, a strong competi- tor in the New England rocky intertidal zone (Lubchenco 1980). Freezing has also been shown to adversely affect productivity and survival in several other perennial seaweeds from the Gulf of Maine, implicating freezing as a selective agent on sub-arctic intertidal shores (Kanwisher 1957, Bird and McLachlan 1974. Davison et al. 1989). It should be noted that although C. crispus fronds (ramets; sensu Harper 1977) are very susceptible to freezing damage (reduced metabolism, bleaching and fragmentation) freezing is usually not lethal to the genet (sensu Harper 1977), because holdfasts exposed all winter survived and regenerated erect fronds in the spring.

In spite of morphological and taxonomic similarity (both are in the order Gigartinales) between Chondrus crispus and Mastocarpus stellatus they exhibit different strategies to tolerate freezing stress. Freezing appears to be of little consequence to M. stellatus which exhibits a more stress-tolerant strategy (Grime 1979) than C. crispus, a more competitive species. At least two adaptations are linked to stress tolerance in M. stelIatus; the resistance of the light-harvesting reactions of photosynthesis and plasma membranes to freezing damage. In higher plants (Division Coniferophyta), reduced capacity of electron transport in the chloroplast thylakoids, (i.e. the activity of plastoquinone) is responsible for reduced photosynthesis during winter (Oquist etal. 1980). The maintenance of photosynthesis following freezing in M. stellatus may result from tolerance of the photosynthetic electron transport chain reflected in the lack of increased fluorescence of phycoerythrin. In addition to M. stellatus, thermally stable plasma membranes are also observed in other freezing-tolerant macroalgae, Fucus spiralis and E vesiculosus (Davison et al. 1989), suggesting

436

m e m b r a n e s tabi l i ty is o f w idesp read i m p o r t a n c e in m a c r o a l g a l freezing to lerance.

Acknowledgements. We would like to express sincere thanks to Mr and Mrs Murray of Chamberlain for allowing us to place a thermograph on their property. Thanks are also due to Dr L. Mayer, L. Schick and the staff of the Ira Darling Center for use of the fluorescence spectrophotometer, hospitality, and for providing other facil- ities. E. J. Podolak and J. E. Kuebler provided valuable laboratory assistance and M. Lesser, S. Sawyer and W. Stochaj offered insights and discussion. To all we are grateful. This research was supported by a University of Maine Center for Marine Studies research assis- tantship to Steve Dudgeon, and in part by a National Science Foundation Grant (OCE-8 700 763) and University of Maine equi)ment fund awards to I. R. Davison, and Maine Agricultural Experiment Station funds to R. L. Vadas.

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Freezing tolerance in the intertidal red algaeChondrus crispus andMastocarpus stellatus: Relative importance of acclimation and adaptation