MARINE ECOLOGY PROGRESS SERIES Vol. 84: 71-82,1992 Published July 23 Mar. Ecol. Prog. Ser. l l

Comparative ecophysiology of Baltic and Atlantic vesiculosus

'University of Helsinki, Department of Botany, Unioninkatu 44, SF-00170 Helsinki, Finland 'University of Liverpool, Department of Environmental & Evolutionary Biology, PO Box 147, Liverpool L69 3BX. United Kingdom

ABSTRACT: A sublittoral population of Baltic Fucus vesiculosus L. and an intertidal population from the Irish Sea have been compared in several aspects of ecophysiology under a wide range of short-term (24 h) salinity treatments (1.5 to 102 ppt). Both populations exhibited net photosynthesis over the range of salinity and in general Atlantic plants were more productive. Optimum salinity for photosynthesis was different for Baltic (6 ppt) and Atlantic (12 to 34 ppt) populations. There were no differences in total tissue water between the populations, but Atlantic plants contained a greater proportion of intracellular water. Salinity had an effect on the accumulation of osmotically active compounds and populations differed significantly in this respect; Atlantic contained higher ion and mannitol levels than Baltic. Both populations discriminated in favour of K in preference to Na and it seemed that KC1 was the main inorganic solute responsible for osmotic balance. Based on 42Ktracer experiments there was an un- exchangeable pool of K in both populations. After short-term salinity exposure mannitol showed no clear increase in Baltic plants, however mannitol levels were higher after long-term exposure (1 1 wk, 1.5 to 45 ppt). In long-term experiments tissue ion contents were lower and differences in ionic relations were not so evident between the populations as compared to the short-term exposure data. Intraspecific differences in physiology of F vesiculosus suggest that ecotypic differentiation has occurred to some extent between marine and brackish water populations.

INTRODUCTION Comparative studies on algae populations from varying salinity regimes have suggested that there One of major abiotic factors controlling growth of are intraspecific differences in osmoacclimation; for algae is salinity. Algae growing in estuaries evidence instance compared to marine populations, estuarine the salinity changes from freshwater to full seawater. ones appear to be more resistant to extreme hypo- Intertidal or rockpool algae face salinity stress during saline stress and less resistant to hypersaline condi- periods of emersion depending on weather conditions. tions (Reed & Barron 1983, Young et al. 1987b). Reed The most recent large-scale salinity change has hap- & Barron (1983) observed that there are differences in pened in the since the glacial period. The the relative importance of organic osmotica. Cellular late-glacial and the post-glacial history of the Baltic mannitol concentration was significantly lower in Sea is one of alternating saltwater and freshwater estuarine Pilayella littoralis plants throughout a salin- episodes (West 1968, Ignatius et al. 1981). The last ity range, as was the ion content; marine plants marine period occurred 7500 before present (BP) and had higher ionic contents. They also measured cell that would have been the most likely time for marine turgor, which was found to be higher in marine flora recruitment to the Baltic. The present Baltic Sea is plants. Young et al. (1987b) compared marine, rock- very young (3000 yr) and isolated from the Atlantic by pool and estuarine Enteromorpha intestinalis and its narrow exit, its non-tidal shores and its low, but found that the potassium levels within marine plants locally quite stable salinity. Plants with unusual os- were higher than in their estuarine and rockpool motic tolerances have been able to survive in the Baltic counterparts and proposed the existence of ecotypes Sea and the boundaries of the species coincide more or based on differences in the physiological responses in less with their salt tolerance limits (Hartog 1967). E. intestinalis.

O Inter-Research 1992 Mar. Ecol Prog. Ser. 84: 71-82, 1992

Despite numerous studies on the ionic relations and pieces of 0.1 to 0.2 g (fresh wt) were cut for analysis. In osmoadaptation of intertidal and estuarine algal popu- long-term experiments diluted or concentrated natural lation~there have been few studies on brackish water seawater was used. Diluted media were obtained by species such as those found in the Baltic. The most diluting seawater with distilled water; concentrated conspicuous alga of the sublittoral hard bottoms in seawater was obtained by evaporating natural sea- the Baltic Sea is Fucus vesiculosus L. On the intertidal water at 80 to 90 'C. Pieces of Fucus vesiculosus were Atlantic shores it grows in the eulittoral zone but in the cultivated for 12 wk in 250 cm3 media in beakers with Baltic it is always submerged, because of lack of tides, continuous aeration, media were changed and growth ice scouring and protracted periods of low water levels recorded once a week. Light conditions in long-term in spring. Due to its geographic isolation and the experiments were 100 pm01 m-' S-', (8 h light : 16 h unique hydrology of the Baltic Sea E vesiculosusmight dark) and the temperature was 10 'C. have developed special physiological characteristics, There are, however, problems which are encoun- which could be different from those of intertidal tered when studying the effects of low salinities on populations. photosynthesis. One such problem is that the amount The main aims of this comparative study were: to of dissolved carbon changes when media are diluted measure the photosynthesis and respiration as possible with distilled water and there is less HC03- available indicators of salinity stress; to quantify the main com- for photosynthesis (Jolliffe & Tregunna 1970). In ponents that restore turgor (Na, K, Cl, mannitol); to test general Baltic seawater has half the level of inorganic if populations are able to change their intracellular carbon compared to 35 ppt seawater (Raven & Samuei- solute content in response to salinity treatment; to son 1988). Also with decreasing salinity the concentra- determine whether these populations, from contrasting tions of all ions decrease by the same ratio. So the environments, show any of the previously observed question arises should dissolved carbon or certain ion patterns of differences in estuarine arid ll~dlir~ediyde; colicentraiioiis be kept coiistan: at norm;! se; -s;tcr and to study the long-term effects of osmotic stress on level during the experiment or should all components ion and organic solute accumulation. be diluted to obtain a certain salinity? In this study the approach was to mimic the changes in salinity as they might happen in nature by rainfall for instance. MATERIAL AND METHODS Thus it was decided to study the photosynthetic os- motic responses in natural Atlantic and Baltic sea- Plant material. Atlantic Fucus vesiculosus for short- waters without manipulating the pH, dissolved carbon term experiments was collected from the exposed sources or individual ion concentrations. eulittoral zone on the NW side of Hilbre Island, River Photosynthesis. Measurements of the net photo- Dee, Cheshire, UK. Material for long-term experi- synthetic rate and dark respiration after 24 h incuba- ments was obtained from the sheltered eulittoral zone tion in well-aerated media of a wide range of salinities of Derby Haven, Isle of Man, UK. Material was kept in in saturating light were carried out using a Clark-type running local-seawater tanks before being placed in oxygen electrode (Hansatech). The electrode was plastic bags and transported in 3 h to Liverpool. Baltic calibrated using aliquots of oxygen-saturated media. plants for short-term and long-term experiments were Experiments were conducted at 10 "C using a temper- collected from Brannskar, Tvarminne Archipelago, ature-controlled water jacket around the chamber. Gulf of Finland, SW Finland. After collection, material Samples of clean material were placed in the chamber was maintained in seawater-filled buckets overnight, together with 2 cm3 of medium. For each population, then packed into plastic bags and transferred to rates were measured using 3 separate plants. Produc- Liverpool in an insulated container. In the laboratory tion or consumption of 0, was displayed on a chart all plants were maintained in aerated filtered natural recorder and rates calculated from the slope of an 8 seawater (Atlantic, 34 ppt; Baltic, 6 ppt) at 10 "C under to 10 mln chart record and expressed as mg O2 h-' a light regime of 100 pm01 m-2 S-' PAR (8 h light 16 h (g dry wt)-l. Light was supplied from the halogen lamp dark). Experiments were carried out as soon as pos- of a slide projector. Light intensity was recorded by sible after collection, and always within 10 d. For all using a light meter placed behind the chamber unit. experiments material was cut from the apical 7 cm of Dark respiration was measured by covering the reac- the thallus. tion vessel completely with aluminium foil and black In short-term experiments plants were incubated plastic in a range of salinities (1.5, 6, 12, 34, 68, 102 ppt) of Water content. Tissue water content was deter- artificial seawater medium ASP12S without added mined by drying blotted preweighed fresh samples at vitamins (Reed et al. 1980a) for 24 h in continuous light 75 "C to constant weight. Tissue water content was (100 pm01 m-' S-') at 10 "C in 250 cm3 of media. Plant calculated as (fresh wt - dry wt)/fresh wt and ex- Back et al.: Ecophysiology of Fucusvesiculosus 73

pressed as a percentage. Intracellular water content The efflux of tracer can be calculated from the was calculated as (fresh wt) - (dry wt) - (extracellular equation water content). Extracellular water content was meas- U = kQ' ured using 14C-mannitol assuming that exogenously supplied 14C-mannitol is not respired and enters the where 0 = the flux rate, nmol S-' (kg fresh wt)-l; Q'= cell wall fraction only (Drew 1969, Diouris & Floc'h cellular tracer content, nmol (kg fresh wt)-l; k = rate 1984). Pieces of Fucusvesiculosus were incubated in constant, S-'. different salinities for 24 h. Pieces of 50 mg were The separate rate constants for both phases were cal- transferred to '4C-mannitol-labelled media for 20 min culated from slopes individually for every replicate followed by a 30 min efflux into 1 cm3 unlabelled dis- using simple linear regression analysis. The fast extra- tilled water. A toluene/Triton X-100 based scintilla- cellular and slow cellular tracer contents, Qf0andQ,' tion liquid was used for counting in a scintillation respectively, were calculated by extrapolating the counter (United Technologies Packard, Minaxi, 4000 phases to zero. Series). Mannitol concentration. Quantitative analysis of Chemical analyses. After incubation (as in photo- tissue mannitol content was carried out, after 24 h synthetic work) plants were blotted and 5 replicates incubation in a range of salinities, by gas-liquid were washed for 10 min in 50 cm3 ice-cold Ca(N03)2 chromatography. Amount of tissue used was 0.1 to solution isotonic with the treatment media. It was 0.2 g (fresh wt). Quantification of mannitol was per- assumed that this procedure would remove cations formed following extraction in 80 % v/v ethanol. bound to the cell wall. Plants were blotted dry and Boiling ethanol (30 cm3) was added and left for 20 h weighed. The dry weight was obtained after drying with continuous shaking at 40 "C. Another 20 cm3 for 2 d at 75 "C. For K and Na analysis material was boiling ethanol was then added and left for 2 h with digested in 1 cm3 concentrated nitric acid (Aristar, shaking. The procedure given by Holligan & Drew BDH) and the digest was diluted to 10 cm3. Ion con- (1971) was followed in preparing samples for analysis. centrations were measured using a flame-emission Samples were vacuum dried at 40 "C and dissolved in spectrophotometer (Perkin Elmer). For tissue C1 con- 0.7 cm3 pyridine (including arabitol as an internal centrations, plant material was extracted in hot water standard). For denvatization 0.2 cm3 hexamethyl- for approximately 1 h after washing and weighing. C1 disilane (HMDS) and 0.1 cm3 trichloromethylsilane was determined by electrometric titration against (TCMS) were used. Derivatized samples were shaken AgN03. well and left to stabilise for 20 h in a desiccator and 42~flux experiments. Plant material was incubated analysed the following day using a Philips series 304 at the same temperature and light conditions as de- gas-liquid chromatograph. Columns were packed with scribed previously. 42Kwas supplied by the University 2 % w/v SE 52 on Diatomite CQ 80-100. 2 to 3 p1 of Research Reactor, Risley, Cheshire, UK, in the form of sample was injected onto the column. The temperature irradiated K2CO3,All media used in flux experiments programme was as follows: column temperature 140 were artificial seawater with the required concen- to 280 "C, increasing at a rate of 20 "C min-l, holding tration of K supplied by the K2C03. The salinity range the initial and final temperatures for 1 min. Standards used in flux experiments was 1.5, 6, 12 and 34 ppt. containing known amounts of mannitol were prepared Samples were aerated throughout the experiment. and derivatized. From these a peak area vs concen- Samples were counted in a scintillation counter using tration standard curve was calculated. Peak areas Cherenkov radiation. Preweighed 50 mg Fucusvesi- were recorded and quantified using a Philips CDPl culosuspieces were loaded in high specific activity computing integrator. 42Kmedia of different salinities for 24 h in continuous light and 10 "C as described previously. After equili- RESULTS bration 5 replicates were blotted dry and transferred through unlabelled media (10 cm3) of the same Photosynthesis and respiration salinity at set time intervals. Wash samples and the F. vesiculosuspieces were counted at the end of the For salinity treatment experiments a light intensity of experiment. 400 pm01 m-' S-' was adopted. The effects of altered The procedure for calculating the effluxes of 42K is salinity on photosynthetic rates are shown in Fig. la. based on the determination of rate constants for The net photosynthetic rates varied significantly in exchange of tracer between tissue and solution. These response to salinity and the rates varied also between constants were used to calculate the potassium content the populations (p<0.001). In general Atlantic plants in extracellular and intracellular compartments based were more productive. Both populations exhibited on a biphasic model of efflux (Walker & Pitman 1976). differences in light-saturated photosynthetic responses Mar. Ecol. Prog. Ser. 84: 71-82. 1992

0 20 40 60 80 100 Salinityppt 0 20 40 60 80 100 Salinity ppt 45 -

40 -

-a- Atl. 35 - + Balt. 30 -

25 -

l of 0 20 40 60 80 I M) Sal~nityppt

0.0 0 20 40 60 80 100 Salinity ppt + Atl. + Balt. Fig. 1. Fucusvesiculosus. (a) Photosynthesis and (b) respira- 0.~1tion of Atlantic and Baltic plants (mean +SE) to salinity. The Baltic Fucusvesiculosus population had a clear peak of production in its normal seawater i salinity of 6 ppt, and showed a steady decrease in O2 00 production in hypersaline treatments. In Atlantic 0 20 40 M) 80 100 Salinityppt plants there was clearly suppression at hypo- and hypersaline conditions and net photosynthesis reached Fig. 2. Fucusvesiculosus. Variation of (a)tissue water, (b) ex- its peak at 12 and 34 ppt. tracellular water and (c)intracellular water content of Atlantic Respiration of Atlantic Fucusvesiculosus was greatest and Baltic plants in different salinities (mean +SE) at 1.5 ppt declining with increasing salinity until reaching its minimum in 68 ppt. Baltic plants showed surprisingly high respiration rates in their normal highly significant effect on water content (Fig. 2a) and seawater. However the highest respiration rates were there were no significant differences between these 2 obtained in 68 ppt (Fig. lb). Salinity had a highly populations. Results from partitioning tissue water into significant effect on respiration of Atlantic plants intracellular and extracellular water are shown in (p < 0.001) in contrast to Baltic plants (p < 0.05). Fig. 2b, c. Atlantic material showed a decrease in intracellular water content with increasing salinity, though the value in 1.5 ppt salinity was surprisingly Water content low. A very high percentage of water was present in the extracellular compartment (20 to 30 %). Similarly Tissue water content of Baltic and Atlantic plants Baltic material showed a decrease in intracellular was inversely related to external salinity which had a water content with increasing salinity. Compared to Back et al.: Ecophysiology of Fucus vesiculosus 75

Atlantic material the decrease was greater in Baltic plants particularly in salinities above 34 ppt. In Baltic plants a greater percentage of thallus water was in the extracellular compartment. Approximately 25 % of the tissue water was found as extracellular water in salini- ties below 12 ppt and 30 to 45 % was extracellular water in salinities above 34 ppt.

Q Na Atl. Ionic relations

o!.m.n.n.m.m. The K content of Atlantic plants was significantly 0 20 40 60 80 100 higher than in Baltic plants as were Na and C1 Salinity ppt contents. In both populations K was the major cation. The K and C1 contents showed a similar pattern in both populations. There was a highly significant difference between populations, and salinity had an effect on ion content. For K content there was no interaction, so populations reacted in a similar manner to increasing salinity, thus there was a clear increase in tissue K content in lower salinities and in higher salinities the response leveled off. In Atlantic plants C1 content leveled off in higher salinities, whereas Baltic plants showed a tendency to increase C1 content over the whole salinity range. 0 20 40 60 80 100 The total tissue contents calculated on the basis of Salinity ppt tissue fresh wt for Na, K, and C1 were determined as a function of salinity (Fig. 3a, b, c). Tissue Na content was highly dependent on salinity and also there was a highly significant difference between populations (Table 1). Increase of Na in Atlantic plants was nearly linear with increasing salinity, whereas in Baltic plants Na levels decreased in salinities above 34 ppt.

Table 1. Fucus vesiculosus. ANOVA-table. Effect of popu- lation and salinity on total (tissue) content of Na, K, Cl and mannitol 0 20 40 60 80 100 Salinity ppt Source of variation Significance level Na K C1 Mannitol Fig. 3. Fucus vesiculosus.Total tissue content (mean t SE)

a.. .m. of (a) Na, (b) K and (c) C1 of Atlantic and Baltic plants after Population ...... a.. a.. short-term (24 h) experiment in different salinities. Results Salinity ...... expressed in terms of tissue fresh wt Population X Salinity " NS "' NS: p>0.05; 'p<0.05, "p<0.01, "'p<0.001 increase in all ions with increasing salinity (Fig. 4a, b, c). The differences between populations except in C1 content were slight, though Atlantic plants still Ion concentrations, based on intracellular water con- tended to have higher concentrations than their Baltic tent, were estimated from the means of the measured counterparts. contents and the calculated intracellular water values. Because long-term salinity range was different (l.' The standard errors for these values were calculated to 45 ppt) from the short-term experiment and no using formulae adopted for cases involving sum or determination of intracellular water was made, com- quotient (Worthing & Geffner 1943). The general trend parisons were possible only in terms of tissue fresh wt of the ion concentrations for both populations was an (Fig. 5a, b, c). The Na content of Baltic plants followed 76 Mar. Ecol. Prog. Ser. 84: 71-82, 1992

+ Na Atl. + Na Balt.

0 20 40 60 80 100 30 40 Salinity ppt Salinity ppt

Y

E * K Atl. K Atl. 70 * + K Balt. * K Balt

" m 0 20 40 60 80 100 0 10 20 30 40 Salinlty ppt Salinityppt

+- Cl Atl. + C1 Atl. + Cl Balt. * C1 Balt.

0 20 40 60 80 100 0 10 20 30 40 Salinity ppt Salinity ppt

Fig. 4. Fucusvesiculosus. Total tissue content of (a) Na. (b) K Fig.5. Fucus vesiculosus. Total tissue content of (a) Na, (b) K and (c) C1 (mean f SE) of Atlantic and Baltic plants after and (c) C1 (mean f SE) of Atlantic and Baltic plants after short-term (24 h) experiment in different salinities. Results I1 wk growth experiment in different salinities. Results expressed in terms of intracellular water content expressed in terms of tissue fresh wt

the same pattern as in short-term experiments. In experiments. The pattern of C1 content was similar be- Atlantic plants Na content leveled off in salinities tween populations, though Atlantic plants contained above 12 ppt, whereas in the short-term Na increased less C1 than in short-term experiments. in the tissue with increasing salinity. Thus there was a trend for tissue Na content to be much lower in the long-term. 42Kflux experiments Both populations demonstrated clear differences in K content from short-term experiments. Atlantic plants Table 2 contains the results from radioisotope efflux still contained more K than their Baltic counterparts experiments in different salinities. Extracellular and but K contents were much lower than in short-term intracellular ion content calculated from 2 different Back et al.: Ecophysiology of Fucus vesjculosus 7 7

phases are presented together with total ion content Mannitol from chemical analysis. Cellular K content formed a major fraction of the total tissue K in all salinities, as Concentrations of mannitol increased linearly with there was very little adsorbed within cell walls. increasing salinity in Atlantic plants (Fig. 6a). There However it was obvious that between chemical and was a clear difference between populations, and radioisotopic experiments there was not very good salinity had an effect on mannitol accumulation agreement for total ion content. (Table 1). Baltic mannitol levels were only half those Table 3 shows data for efflux rates for both phases of the Atlantic plants. However, the Baltic Fucus calculated on the basis of tissue fresh wt and intra- vesiculosusdid not demonstrate any significant in- cellular water content. The fluxes from the fast phase crease in tissue mannitol content in the higher (O1.) increased with salinity in both Atlantic and salinities thus there is an interaction between popula- Baltic plants though the fluxes in Atlantic were tions and salinity. Calculated on the basis of intra- somewhat higher. The slow phase (0,) fluxes also cellular water content Atlantic plants still contained showed an increase with salinity, though this was higher concentrations of mannitol than Baltic plants nowhere near as clear as for the fast phase fluxes. (Fig. 6b). However, it is clear that Baltic plants do Again the Atlantic 0, tended to be greater than show an increase of mannitol concentration with for Baltic plants, though this was not so marked increasing salinity. as for Of. The observed pattern of higher efflux The long-term effects of salinity on mannitol accu- rates of Atlantic plants agreed with the observa- mulation differed from those previously described for tions that cellular K content was higher in Atlantic short-term treatments (Fig. 6c). In both populations plants. there was a linear increase in mannitol as salinity

Table 2. Fucus vesiculosus. Mean (SE) K content (Q,': extracellular; Q,': cellular) of Baltic and Atlantic plants in different salinities estimated by chemical and radioisotopic methods. All values are expressed in mm01 (kg fresh wt)-'

Salinity Qr ' Qs ' Q ' total Q ' total % K unexchangeable (PP~) radioisotopic chemical Atlantic Baltic Atlantic Baltic Atlantic Baltic Atlantic Baltic Atlantic Baltic

1.5 3.1 5.0 27.3 24.8 30.1 29.8 132.8 78.9 77 62 (0.2) (0.3) (1.3) (2.3) (1.2) (2.4) (6.6) (8.0) 6 8.2 6.9 75.3 59.2 83.8 66.1 113.3 97.2 26 31 (1.4) (1.2) (4.1) (4.1) (2.9) (4.5) (10.7) (6.1) 12 7.9 9.0 64.0 67.3 72.0 76.4 152.4 118.6 52 35 (1.2) (0.8) (4.1) (2.3) (3.6) (2.8) (7.9) (8.8) 34 22.7 20.6 165.6 96.1 188 3 116.9 209.8 171.0 10 3 1 (2.1) (0.5) (5.9) (5.9) (7.1) (6.5) (8.8) (3.2)

Table 3. Fucus vesiculosus. Mean (SE) efflux rates (0,: extracellular; 0,: cellular) of Baltic and Atlantic plants expressed in terms of both tissue fresh wt and intracellular water content

Salinity 01 0, 0, (PPt! nmol S-' (kg fresh wt)-' nmol S-' (kg fresh wt)-' nmol S-' (kg cellular H20)-' Atlantic Baltic Atlantic Baltic Atlantic Baltic

1.5 1683 1534 194 229 336 394 (180) (147) (20) (20) (38) (34) 6 4339 3214 1270 1098 2125 1945 (104) (648) (149) (128) (250) (225) 12 5706 3966 959 57 9 1780 1068 (237) (353) (77) (36) (140) (66) 34 15223 8410 4412 1087 8165 2277 (616) (425) (32) (53) (56) (111) Mar. Ecol. Prog. Ser. 84: 71-82, 1992

Table 4. Fucusvesiculosus. Total content of osmotically active compounds measured in Atlantic and Baltlc plants

Salinity Na + K + Cl + mannitol (ppt) (mOsmol kg-') (mOsmol kg-' intracellular H20) Atlantic Baltic

1.5 4 7 520 260 6 189 477 344 12 377 699 458 34 1070 1042 828 68 2140 1472 1225 102 3210 1504 1574 0 20 40 60 80 100 Salinityppt concentrations measured for Na, K, C1 and mannitol are added up they would account for 200 to 300 mm01 kg-' more than external osmotic pressure in salinities below 12 ppt. For salinities above 68 ppt for Atlantic plants and above 34 ppt for Baltic, measured internal solutes accounted for only 40 to 50 % of the externai osmolality. A positive turgor pressure is predicted in the Atlantic material in hyposaline treatment and up to 34 ppt, whereas for Baltic material turgor is posi- 1 I-- L--... .cP to 12 I IUACVCI, it must be rememberei? that this analysis takes no account of sugars, amino

0 20 40 60 80 100 acids and other ions. Salinityppt

DISCUSSION

As general responses of algae to sahnity, the growth rate and survival or photosynthetic and respiration rates have been studied most frequently ( Gordon et al. 1980, Reed et al. 1980b, Yarish & Edwards 1982, Smith & Berry 1986, Koch & Lawrence 1987, Russell 1987, - Atl. Back et al. 1992).The net photosynthetic responses of -C Balt. Atlantic and Baltic populations of Fucus vesiculosus to salinity showed that both populations have a wide 0 salinity range. Both exhibit net photosynthetic gain be- 0 10 20 40 Salinity ppt tween 1.5 and 102 ppt; the Atlantic population has an optimum between 12 to 34 ppt and the Baltic popula- Flg. 6.Fucus vesiculosus. Mannitol content (mean + SE) of tion at 6 ppt. The reduction of photosynthetic rate for Atlantic and Baltic plants in different salinities: (a)after short- term (24 h) experiment, results in terms of tissue fresh wt; Atlantic E vesiculosus plants in 1.5 and 102 ppt and (b) after short-term experiment, results in terms of intra- in higher salinities for Baltic plants demonstrates that cellular water, and (c) after long-term culture experiment, salinity does have an effect on photosynthesis. This is results in terms of tissue fresh tvt contrary to the observations by Reed et al. (1985) who found only a minor variation in the photosynthetic rate of F. serratus in the salinity range of 7 to 50 ppt. increased. The levels of mannitol, as before, were Many algal species exhibit the ability to photo- higher in Atlantic than Baltic plants. However, the synthesise over a broad salinity range, usually showing levels of mannitol were considerably higher after optimal photosynthesis in a certain range of salinity. the long-term experiments, especially in the Baltic Gracilaria verrucosa showed a positive photosynthetic material. response over a range of salinity, with a marked Normal Atlantic seawater has an osmolality of ca decrease in net photosynthesis below 10 and above 1070 mOsmol kg-' and the Baltic seawater of ca 40 ppt, and optimum being between 20 and 30 ppt 189 mOsmol kg-' (Table 4). If the total intracellular (Dawes et al. 1978). G. tlkvahiae showed broad toler- Back et al Ecophys~ology of Fucus ves~culosus 79

ance limits for net photosynthetic response between 5 than that found in our Atlantic (204 mm01 kg-') or and 40 ppt (Penniman & Mathieson 1985). An optimal Baltic (214 mm01 kg-') plants. range can be defined as those salinities causing mini- It is obvious that both the Baltic and Atlantic Fucus mal osnlotic stress and permitting the highest photo- vesiculosus discriminate in favour of K in preference to synthetic rates. Ogata & Schramm (1971) and Ohno Na and it seems that KC1 is the main inorganic solute (1976) reported depressed photosynthetic rates in responsible for osmotic balance. K is sufficient to bal- Porphyra un~bilicalisespecially in hypersaline condi- ance about 25 % in Baltic plants and about 30 % in tions and Thomas et al. (1988) showed that both fresh- Atlantic plants of the external solute concentration in and brackish water populations of Cladophora species 34 ppt seawater (calculated from radioisotopic experi- exhibited very little tolerance to higher salinities based ments). Both populations of F: vesiculosus fall into the on studies of net photosyntesis. category of algae having K as the main cation (Kirst The Fucus vesiculosus populations showed signifi- 1990). This is a common feature of many marine algae cantly different responses in dark respiration rates. (Raven 1976, K~rst& Bisson 1979); it is speculated that The responses of Atlantic plants followed the trends these species' turgor pressure is regulated by chang- shown by Ogata & Takada (1968) for some intertidal ing the activity of a K pump. Kirst & Bisson (1979) species in that the respiratory rate increases in hypo- found members of the Phaeophyta, e.g. Eclonia and saline media. Similarly Dawes et al. (1978) reported Dictyota, to have high intracellular K concentrations. high respiratory rates for intertidal and subtidal Similarly Young et al. (198713) reported that Entero- species after exposure to extreme hyposaline condi- morpha intestinalis has high K levels with very low tions. In general the observations in this study support intracellular Na values, though the Na levels in the cell the findings of Wilkinson (1980) that photosynthesis wall were high. Reed & Collins (1980) found that decreases and the rates of respiration increase at low Porphyra purpurea has most of its Na in the cell wall. In salinities, leading to reductions in net photosynthesis. the present study Na content was measured after a 10 Due to the conlplexity of the thallus structure of min wash in Ca(N03),which is assumed to remove ex- Fucus vesiculosus, partitioning the tissue water into tracellular ions, however such a simple procedure is intracellular and extracellular con~partmentscannot be unlikely to be totally efficient and probably gives a accepted without question. Normally extracellular large overestimate of intracellular Na and thus accu- water content has been measured after exposures of rate Na flux data are needed. If the intracellular Na about 2 min to 14C-labelled media (Reed 1980, Reed values here do epresent a serious overestimate, then et al. 1980a, 1985), but in our study the time used KC1 is even more important as an internal solute. was 20 min due to the complexity of tissue and to Certainly Na values of at least 200 mm01 kg-' cell allow external '4C-mannitol uptake to level off. Also water seem much too high (Flowers & Lauchli 1983); the possibility of intracellular accumulation, even of also K values seem to be quite high. seemingly negligible amounts of mannitol, could lead Another common phenomenon, an anion deficit, was to overestimation of the extracellular water. However, evident. The amount of C1 alone can not be responsible the calculated intracellular water content values for all negative charges in the cell but it has been appear to give reliable trends between populations. shown to be the main anion. In studies by Winter et al. Reed et al. (1985) gave values of water partitioning of (1987) and Young et al. (1987a) other anions such as Atlantic F vesiculosus which are in reasonable agree- SO,'-, HPOd2-have far lower concentrations than Cl. ment with the values from our study. Despite a similar Marine Fucus vesiculosus plants contaln significantly response between the populations it is worth noting higher amounts of C1 than their brackish water coun- that the changes in intracellular water vs salinity are terparts. In both populations C1 content is dependent greater in plants from the Baltic than from the Atlantic. on salinity. A similar pattern was observed by Reed & Barron Both Atlantic and Baltic Fucus vesiculosus showed (1983) where estuarine Pilayella littoralis had a greater biphasic K efflux. But there is great variation of the intracellular water content than marine material. initial fast phase duration which could be related to the All ion contents were calculated in the terms of both structure of the algae. The physical locations of the 2 tissue fresh wt and intracellular water content to make phases of K efflux are difficult to ascribe. It has been comparison of values easier with those existing in the assumed that the initial fast phase (tIl2= 30 min) comes literature. Davison & Reed (1985a) found a K concen- from extracellular films and intercellular spaces and tration of 499 mm01 kg-' in Laminaria digitata from the cell walls. The tIlzis long compared with values normal seawater. This is somewhat higher than the found by Ritchie & Larkum (1985) and Young et al. values found here: 388 and 358 rnrnol kg-' for Atlantic (1987a) (tIl2< 5 min) for Enteromorpha intestinalis. It is and Baltic Fucus vesiculosus respectively. However, also assumed that this is a reflection of the complexity their Na concentration of 59 mm01 kg-' was far lower of the multi-layered, leathery-like tissue of F vesicu- Mar. Ecol. Prog. Ser. 84: 71-82, 1992

losus as well as the copious mucus in the hyphae that pressure by means of organic solutes. These solutes is present. It is assumed that the slow second phase is usually exhibit some protective functions and they are intracellular in origin (Ritchie & Larkum 1985, Young generally less harmful to cellular metabolic function et al. 1987a). The location of phases is difficult, and in and enzyme activity than ions (Davison & Reed 1985b, the end may not be feasible. But certainly more experi- Edward et al. 1988). The short-term experiment might mentation is required in an effort to locate various ions have been too short to reach full mannitol adaptation, in the E vesiculosus thallus. Monophasic effluxes are though 24 h is surely a reasonable period allowing for found in Porphyra purpurea (Reed & Collins 1980, tidal cycles in nature. In the Baltic Sea Fucus vesicu- 1981) and E. intestinalis (Ritchie & Larkum 1985). How- losus does not experience tidal and short-term salinity ever, they found biphasic intracellular exchange in low changes and thus short-term mannitol production does salinlty grown E. intestinalis plants. The K efflux of not increase with increasing external salinity. Laminaria digitata (Davison & Reed 1985a) is mono- Broady et al. (1984) and Gibson et al. (1984) have phasic after an initial rapid phase. The efflux from the shown that ribosome stability in an in vitrotranslation intracellular slow phase is 2.9 pm01 (kg fresh wt)-' S-' of m-RNAs is salt sensitive for a wide range of plants. in normal seawater, this is only 65 % of the efflux Levels of K above 125 mM started to cause inhibition observed in Atlantic F. vesiculosus [4.4 ~mol(kg fresh and polysomes were also less stable in the presence wt)-' S-'] and nearly 3 times the value for Baltic plants of C1 than organic ions such as acelate. Similarly Na (1.0 firno1 (kg fresh wt)-' S-']. Reed (1984b) gives was more inhibitory than K. The intracellular levels vaiues of K efflux for Polysiphonia lanosa from marine reported here for all these ions (I<, Na and C!) seem and estuarine sites and when expressed with respect to markedly high. However, such seemingly high ionic plasmalemma surface area the K efflux is greater in levels have often been reported (Davison & Reed marine plants whereas flux rates, expressed in terms of 1985a, Young et a1 1987b, Thomas et al. 1989) for mar- cc!!u!ar water extent, are simi!sr. !E this I---nrosezt stndy ine --X--'alnao .4!so it is noteworthy th;rt long term ior! !m?- plasmalemma surfaces have not been calculated, but els are much lower than those found in the short term. the tendency of lower K flux rates was noticeable in There were clear differences in ion and mannitol Baltic plants for rates expressed in terms of cellular content between populations calculated on tissue fresh water. weight. Atlantic plants had consistently higher con- In the mannitol has been found to be the tents. This pattern was observed before when the same main organic compound involved in osmoacclimation, species from fully marine habitats and from estuarine varying as a direct function of salinity in all cases in the locations were compared (Reed & Barron 1983). They studied. It wo.uld seem that the role of observed that the cellular mannitol concentration n~annitolas an 'osmotic compatible solute' is clear (Reed was significantly lower in estuarine Pilayella littoralis et al. 1985, Davison & Reed 1985b). In both short-term plants throughout a salinity range, and that there were and long-term experiments mannitol levels increases higher ion levels in the marine plants as well. in Atlantic plants whereas in Baltic plants the increases It has been suggested that the wide salinity distribu- in mannitol levels were much more obvious only in tion of some species might be due to different 'osmotic long-term experiments. Similarly Reed (1983) found strategies' (Young et al. 198713) and osmoadaptation that organic solutes, e.g. di-methylsulphoniopropion- (Reed 1984a, b) has occurred. The presence of differ- ate (DMSP), formed a greater proportion of the internal ent strategies within a species might lead to the exis- osmotic potential of marine plants than of estuarine tence of ecotypes (Turesson 1922). The main question plants of Polysiphonia lanosa. A similar pattern was for this study was that are there differences between observed with mannitol content, where marine plants brackish and marine plants? The experimental data contained nearly 1.5 or double the level of mannitol show clearly that there are consistent differences that was found In brackish plants under the same between populations in photosynthetic responces, conditions. In our study mannitol counterbalances a intracellular water content, accumulation of ions and relatively small component of the external osmot~c the organic solute mannitol. All these intraspecific potential; it is relatively more important under hypo- differences in physiology suggest that ecotypic dif- saline conditions (20 % of external salinity) than ferentiation has occurred to some extent and has led hypersaline (10 %). Also mannitol seems to play a less to ecotypes of Fucus vesiculosus from marine and important role in Baltic plants, where ions play a cor- brackish water populations which live under com- respondingly greater role. pletely different saline environments and regimes. Both populations contained more mannitol after long-term exposure to salinity. Mannltol synthesis is Acknowledgements The authors thank the technical staff at Port Erin Marine Laboratory and Tvdrminne Zoological Sta- rapid (McLachlan & Bidwell 1978), and for cellular tion for help during the sampling. This work was supported functioning it is better to maintain a positive turgor by the Academy of Finland through a research grant to S.B. Back et al.: Ecophysiology of Fucusvesiculosus 8 1

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This article was submiftedto the editor Manuscript first received: November 1, 1991 Revised version accepted:April 28, 1992