Physiological response of fucoid algae to environmental stress: comparing range centre and southern populations

Physiological response of fucoid algae to environmental stress:comparing range centre and southern populations

Jo~ao G. Ferreira1, Francisco Arenas2, Brezo Mart�ınez3, Stephen J. Hawkins1,4 and Stuart R. Jenkins1

1School of Ocean Sciences, Bangor University, Menai Bridge, Anglesey, LL59 5AB, UK; 2Laboratory of Coastal Biodiversity, Interdisciplinary Centre of Marine and Environmental Research

(CIIMAR), University of Porto, Rua dos Bragas 289, 4050-123 Porto, Portugal; 3�Area de Biodiversidad y Conservaci�on, Rey Juan Carlos University, 28933 M�ostoles, Madrid, Spain; 4Ocean

and Earth Science, National Oceanography Centre Southampton, University of Southampton, European Way, Southampton, SO14 3ZH, UK

Author for correspondence:Jo~ao G. Ferreira

Tel: +351 918300561Email: [email protected]

Received: 30 July 2013

Accepted: 28 January 2014

New Phytologist (2014)doi: 10.1111/nph.12749

Key words: abiotic, air temperature, Fucusspiralis, Fucus vesiculosus, photosynthesis,physiological limit, range edge, solar radia-tion.

Summary

� Climate change has led to alterations in assemblage composition. Species of temperate

macroalgae at their southern limits in the Iberian Peninsula have shown shifts in geographical

range and a decline in abundance ultimately related to climate, but with the proximate factors

largely unknown.� We performed manipulative experiments to compare physiological responses of Fucus

vesiculosus and Fucus spiralis from Portugal and Wales (UK), representing, respectively,

southern and central areas of their distribution, to different intensities of solar radiation and

different air temperatures.� Following exposure to stressful emerged conditions, Portuguese and Welsh individuals of

both fucoid species showed increased frond temperature, high desiccation levels and reduced

photophysiological performance that was evident even after a 16 h recovery period, with

light and temperature acting in an additive, not an interactive, manner. The level of physio-

logical decline was influenced by geographical origin of populations and species identity, with

algae from the south and those living higher on the shore coping better with stressful condi-

tions.� The negative effect of summer conditions on photophysiology may contribute to changes

in fucoid abundance and distribution in southern Europe. Our results emphasise how physio-

logical performance of geographically distinct populations can differ, which is particularly rele-

vant when predicting responses to climate change.

Introduction

Fucoid algae are important primary producers on intertidal rockyshores worldwide (Connell, 1972; Littler & Murray, 1974;Chapman, 1995; Connell & Irving, 2008; Jenkins et al., 2008;Konar et al., 2010). As ecosystem engineers (Jones et al., 1994),their canopies modify habitat conditions, facilitating the exis-tence and survival of other intertidal species and thereforestrongly influencing the structure and functioning of coastal eco-systems (Jenkins et al., 1999a, 2008). Their importance has ledto their use as model species for ecological and ecophysiologicalstudies of causes of distribution patterns (Baker, 1909, 1910) anddevelopment (Berger et al., 1994; Brownlee, 1994).

In the intertidal zone of the northeast Atlantic, abundance ofmacroalgae declines with latitude (Ballantine, 1961; Hawkins &Hartnoll, 1983; Jenkins et al., 2008), with fucoid cover and bio-mass decreasing rapidly near its southern limit of distribution inPortugal (Ballantine, 1961; Ferreira, 2012). Portugal is known asa biogeographical transition zone, a region where species

composition changes markedly (L€uning, 1990; Southward et al.,1995; Lima et al., 2007; Tuya et al., 2012) and some fucoid spe-cies reach (e.g. Fucus vesiculosus (Nicastro et al., 2013) orapproach (e.g. Fucus spiralis (Ribera et al., 1992) their southernlimit of distribution. Intertidal species are subjected to stressesduring low tides, which can take them close to their physiologicaltolerance threshold (Helmuth et al., 2006); such species can thusbe especially susceptible to climatic stress, particularly towardstheir southern limits of distribution. For example, in Europe, anumber of time series have been used to demonstrate significantchanges in the abundance and range limits of intertidal species inresponse to climatic fluctuations (Southward et al., 1995; Mies-zkowska et al., 2006; Hawkins et al., 2008; Poloczanska et al.,2008). Changes in assemblage composition and distribution ofmacroalgae have occurred in Portugal with a clear northwardexpansion of warm-water species (Lima et al., 2007). Range con-tractions of temperate macroalgal species, although not evidentin Portugal (Lima et al., 2007), have occurred in areas adjacent toPortugal: the southern range edge of F. vesiculosus in Morocco

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(Dangeard, 1949; Benhissoune et al., 2002; Canovas et al., 2011)has recently retracted to Portugal and a decline was also recordedin the Bay of Biscay (Nicastro et al., 2013). These recent findingsconfirm results from previous studies, which showed a markeddecline in the abundance of F. vesiculosus in northern Spain (Fer-nandez & Anadon, 2008; Lamela-Silvarrey et al., 2012) and sug-gest likely northward contraction of range edge populations inPortugal if the warming trend continues.

The distribution of fucoid algae in rocky intertidal areas isdetermined by a combination of physical and biological factors(Schonbeck & Norton, 1978; Lubchenco, 1980; Hawkins &Hartnoll, 1985; Menge, 2000; Jenkins et al., 2008). Variations inthe magnitude, duration and frequency of wave forces create dif-ferent environmental conditions, leading to the development ofhorizontal gradients of macroalgae abundance (Jones & Deme-tropoulos, 1968; Blanchette, 1997; Jenkins et al., 2008). Physicalfactors can also lead to vertical zonation; factors associated withtidal emersion have long been known to influence the verticalabundance of organisms in the rocky intertidal zone (Evans,1948; Dring & Brown, 1982; Chapman, 1995). The pervasiveinfluence of adverse physical conditions on algal distribution hasbeen demonstrated numerous times; brown algae species such asF. spiralis, Pelvetia canaliculata, Ascophyllum nodosum andLaminaria digitata have all been shown to have their upper limiton the shore directly set by physical factors (Schonbeck &Norton, 1978; Todd & Lewis, 1984; Hawkins & Hartnoll,1985; Skene, 2004).

In temperate regions, the peak of environmental stress occursin the summer, when mid-afternoon low-tide periods occur onclear, calm days (Helmuth et al., 2002). These conditions lead toharsh thermal and desiccation stresses, particularly at the upperlevels of the shore (Doty, 1946; Davison & Pearson, 1996;Denny & Wethey, 2001). Solar radiance, when composed ofhigh irradiances of photosynthetically active and ultraviolet radia-tion, can lead to oxidative stress and consequent photoinhibitionin intertidal macroalgae (Hader & Figueroa, 1997; Flores-Moyaet al., 1998; Figueroa & Vi~negla, 2001). Photoinhibition occursbecause oxidative stress prevents the synthesis of plastid encodedproteins, essential for the repair of photosystem II (PSII) machin-ery, leading to a consequent decrease in photosynthetic activity(Nishiyama et al., 2011; Takahashi & Badger, 2011). Such effectsof light on the physiological performance of macroalgae may beaggravated, at local and regional scales, if climatic conditions pro-mote harsher stressful regimes of temperature (Dromgoole,1980; Altamirano et al., 2000; Helmuth et al., 2006). For exam-ple, Martinez et al. (2012) showed that warmer seawater tempera-tures can have a negative effect on growth rate of F. serratus.Overheating of tissues can have an impact on protein and mem-brane stability, as well as on enzymatic reaction rates, and cantherefore affect physiological performance and growth rates oforganisms (Lobban & Harrison, 1997; Chen et al., 2012; Marti-nez et al., 2012). In addition to effects of light and temperatureon a local scale, such stressors influence the geographical distribu-tion limits of fucoid taxa over large scales. Physical stressors mayact additively, synergistically or antagonistically (Darling & Cote,2008). If physical stressors act synergistically, they may have

larger impacts and cause unexpected negative responses, as theresponse will be superior to that predicted from the effect of eachindividual stress (reviewed in Darling & Cote, 2008). Therefore,the exact contribution of each factor to the combined effect cre-ated by multiple interacting stressors needs to be further studiedand understood (Darling & Cote, 2008; Martinez et al., 2012).The determination of stress levels that cause direct physical limi-tation leading to death and elimination of populations is certainlyimportant, but nonlethal amounts of stress that can influence fit-ness and hence recruitment at range edges are probably equallyimportant, especially for understanding the possible conse-quences of climate change (Davison & Pearson, 1996; Harleyet al., 2012).

A useful way to understand and measure the complex effects ofmultiple factors is to perform experiments where multiple stres-sors may be tested simultaneously and thus allow for the detec-tion of interactive effects among them. One of the most efficientmeans to measure the effect of stress in plants is the use of thenonintrusive pulse amplitude modulated (PAM) fluorometer. Inparticular, the maximum photosynthetic quantum yield (Fv/Fm)is used to estimate photoinhibition, as the balance of photodam-age and repair (Krause & Weis, 1991; Hader & Figueroa, 1997;Figueroa et al., 2003). Fv/Fm has frequently been used and is welldocumented in fucoid experimental procedures, showing thatdifferent fucoid species or populations may have distinct toler-ance thresholds to wave action, extreme temperatures, solar radia-tion or ambient humidity among other factors (Coelho et al.,2001; Malm & Kautsky, 2003; Gylle et al., 2009; Martinez et al.,2012).

The effects of stress have been extensively documented inFucus species (see Wahl et al., 2011 for review). Zardi et al.(2011) examined photophysiological resilience to emersion stress,under different air temperature regimes (33–40°C), of three sym-patric Fucus species from a single rocky shore in northern Portu-gal. Physiological resilience was consistent with the verticaldistribution of fucoids on the shore (F. spiralis > F. guiryi > F. ves-iculosus). The photophysiological resilience of central and south-ern-edge fucoid populations has also been compared. Noregional differences in the physiological performance amongF. vesiculosus populations were detected (Pearson et al., 2009;Zardi et al., 2013); however, a degree of maladaptation of south-ern F. serratus populations to desiccation and to high seawatertemperatures (32 and 36°C) was shown (Pearson et al., 2009).

Given the clear decline in fucoid biomass from northern tosouthern European areas (Ballantine, 1961; Ferreira, 2012) andthe presence of southern distributional limits of F. vesiculosus andF. spiralis in Portugal (Nicastro et al., 2013) and Morocco (Riberaet al., 1992; Southward et al., 1995), respectively, we aimed to testthe effects of abiotic summer stress on fucoid physiologicalresponse capacity. Previous experiments were carried out underlaboratory conditions with artificial light sources of low intensity.However, stressful conditions during emersion periods on theshore in the summer are likely to occur under the influence ofhigh solar radiation, high air temperatures and low humidity(Martinez et al., 2012). Taking that into consideration, we per-formed manipulative experiments with a robust hierarchical

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design that allowed us to compare the physiological responses oftwo populations of F. vesiculosus or F. spiralis from two geograph-ical origins to different combinations of light intensity and airtemperature. The main aims of the experiments were to deter-mine if stress from high temperature and high light intensities,experienced during low water periods, would lead to elevatedamounts of stress in fucoids, independently of their origin, andto determine the nature of the interactions among abiotic stresses– are they additive or synergistic? By using populations from dif-ferent origins and through the application of combined stressconditions, we evaluated the extent to which southern and centralfucoid populations are different in terms of their physiologicaltolerance and performance when exposed to complex abioticstresses. We hypothesize the existence of some local populationadjustment to regional environmental pressures and thereforepossible greater resistance to strong light intensity and high airtemperatures in Portuguese populations, resulting from theirexposure to longer and harsher summer conditions. We testedwhether such regional variation is consistent among algae sub-jected to different stress conditions on the shore through the useof both high-shore (F. spiralis) and mid-shore (F. vesiculosus) spe-cies. The effects of abiotic stresses on the species used areexpected to be stronger on the mid-shore species, as high-shorespecies tend to be more resilient to harsh atmospheric conditions(Viejo, 2009).

Materials and Methods

During September 2010, experiments in Porto, Portugal,assessed the effects of temperature and light stress on algae popu-lations from Portugal and Wales. Two experiments were per-formed, one for Fucus vesiculosus (Linnaeus, 1753) and one forFucus spiralis (Linnaeus, 1753) (both Ochrophyta, Fucales). Foreach experiment, two trials were conducted on consecutive daysto assess the generality and repeatability of results. Stress manipu-lations were performed to assess the effects on overheating, desic-cation resilience and, through the use of PAM fluorometry,photophysiological performance of fronds.

Collection

Vegetative fronds of F. vesiculosus and F. spiralis were collectedfrom central and southern regions of their geographical distribu-tion. The central populations were from northern shores locatedin Anglesey, north Wales, Porth Cwyfan (53.182821°N,4.489829°W) and Cemlyn Bay (53.407460°N, 4.533636°W). Inthe south, the Portuguese populations came from Viana do Castel-o (41.690403°N, 8.849988°W) and Carrec�o (41.716555°N,8.866798°W). At each of the four shores, 48 F. vesiculosus andF. spiralis fronds from healthy individuals, c. 10–15 cm in length,were collected. F. vesiculosus plants were easily identifiable becauseof their vesicles, while F. spiralis fronds were collected very high upon the shore to avoid areas where the presence of fucoid hybridswas more probable. The collection of specimens was undertakenthroughout August 2010 during low tide, on shores moderatelyexposed to wave action and easily accessible. This stratified

approach allowed us to formally remove the influence of exposurefrom subsequent analyses. Within each geographical region, theshores were at least 3 km apart. They all had a typical mosaicpatchy community, composed of fucoids, barnacles, bare rock andlimpets, were gently sloping and exposed to full salinity.

Transport, acclimatization and maintenance

After collection, the algae were transported in dark, cold andhumid conditions to the laboratory. The algae from Welsh shoreswere wrapped in blotting paper, to keep them in a hydrated condi-tion, and transported to Portugal by plane in dark, cold andhumid cool boxes. The transport took no more than 1 h from theshore to the laboratory and the air transport of Welsh fucoids toPortugal took less than 20 h. After the transport to Portugal, algaehad a 15 d period of acclimatization in laboratory conditions toallow a steady growth response to be attained. There were no dele-terious effects of transportation with respect to photophysiologicalperformance, as Fv/Fm values measured at the start of the experi-ment remained high in Portuguese and Welsh algae (see theResults section). All the individuals were cultured in 300 l tanksunder ambient day-length conditions, reaching a maximum of1400 lmol photons m�2 s�1, in aerated and circulating seawatercontrolled at 16°C by water refrigerators. Following recommen-dations described in Martinez et al. (2012), seawater was enrichedtwice a week to avoid nutrient limitation by adding inorganicnitrogen (NaNO3) and phosphorus (NaH3PO4) to final concen-trations of at least 50 and 5 µM, respectively.

Experimental design and setup

For each of the four trials (two for F. vesiculosus and two forF. spiralis) the same design was used; algal fronds from two shoresnested within each of two geographical regions (Wales and Portu-gal) were exposed to a factorial combination of two light intensi-ties and two temperatures. Within each of these region/shore/temperature/light combinations, a total of six fronds were used(divided spatially between two containers; see later). All the trials,performed on 4 d during September 2010, were made inPortugal in warm cloudless summer conditions around noon ona rooftop location to allow the use of naturally high solar radia-tion (TrialFves1, 2038.41� 545.60; TrialFves2, 2185.50� 44.82;TrialFspi1, 2213.73� 184.15; TrialFspi2, 2141.06� 8.34µmol photons m�2 s�1 (mean� SD); n = 24010; measured witha spherical quantum scalar sensor (QSL-2100, BiosphericalInstruments Inc., San Diego, CA, USA)) and exposure to realisticambient temperatures (TrialFves1, 21.50� 0.40; TrialFves2,22.43� 0.72; TrialFspi1, 20.70� 0.60; TrialFspi2, 20.10� 2.08;°C; n = 3; measured with temperature-humidity data loggers(MicroLog EC650, Fourier Systems Ltd, Barrington, RI, USA)).There were no differences between days in air temperature(ANOVA, F3,8 = 2.3, P = 0.156), but there were significant dif-ferences in solar radiation (ANOVA, F3,1596 = 28.7, P < 0.001).The difference between maximal and minimal means of solarradiation among days was still small (175.38 µmol pho-tons m�2 s�1), indicating that conditions were similar.


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The experimental design allowed simultaneous testing oforthogonal combinations of light intensity (natural Portuguesesummer radiance values and a 65% reduction provided by shadingmaterial; see Martinez et al. (2012) for further details) and air tem-perature regimes (warm and cold), which was possible because thealgae were kept in plastic containers (479 309 13 cm) sealedwith UV-transparent methacrylate covers (Plexiglas GS2458; Evo-nik Rohm GmbH, Darmstadt, Germany). The range of physicalconditions, air temperatures and light intensities, measured insidethe experimental units was in accordance with values observedunder field conditions at Welsh and Portuguese rocky intertidalareas during the summer season (verified through the parametersof earth surface skin temperature and earth photosyntheticallyavailable radiation data available from NASA Aqua (http://disc.sci.gsfc.nasa.gov/giovanni/overview/index.html) – AIRS standardand MODIS-Aqua missions, respectively). Half of the containerswere exposed to full light and half were overlaid with two layers ofneutral fibreglass mesh to reduce photosynthetic active radiation.By using an air conditioning unit, significant differences of mean airtemperature between warm and cold treatments were achieved(ANOVA of test day, F1,12 = 84.9, P < 0.001) (cold containers,23.79� 2.70°C; warm containers, 32.09� 1.07°C; n = 8) (Sup-porting Information, Table S1). All containers were connected tothe air conditioning unit and air flow was maintained by an electricfan inside each container that createdmean wind speeds of 2.8 m s�2

(as recommended by Martinez et al., 2012). Humidity inside thecontainers was also considered to remain at natural values. This issupported by results from a preliminary 3 d experiment showing thathumidity conditions in containers linked to air conditioning werenot significantly different from those in containers subjected toambient conditions (ANOVA, F1,8 = 1.77, P = 0.22).

Measurement of response variables

For each trial, 96 randomly selected vegetative fronds were blot-ted dry, cleaned with seawater and weighed the day before experi-mentally induced stress. Distal algal vegetative fronds of similarsize were used to restrict the effects of biomass variation (F. vesi-culosus, 1.79� 0.16 g; F. spiralis, 1.84� 0.16 g; n = 192). Thefronds were then left in seawater over night (12 h) in a 300 l tankwith aerated and circulating seawater at 16°C. Just before appli-cation of experimental treatments, the fronds were subjected to aminimum of 25 min in dark conditions, whilst in seawater, toguarantee an equilibrium state of the photosynthetic electrontransport chain before assessing for the first time the photosyn-thetic performance of the algae (pre-stress measurements).

The photosynthetic performance was assessed, on apices of thealgae, as the ratio of variable to maximal Chl fluorescence, Fv/Fm,where Fv = Fm – F0, Fm is the maximal fluorescence and F0 is theinitial fluorescence in dark-adapted algae (Krause & Weis, 1991).Measurements were made with a WATER-PAM ChlorophyllFluorometer (Heinz Walz GmbH, Effeltrich, Germany) using sat-urating pulse intensities (800 ms, 4350 lmol photons m�2 s�1)for both fucoid species. The Fv/Fm ratio is an indicator of the max-imal quantum yield of PSII photochemistry (Maxwell & Johnson,2000; Baker, 2008), which responds to the alteration of optimum

conditions. Variability of this ratio indicates that photosyntheticperformance of algae is affected (Butler, 1978; Long et al., 1994;Baker &Oxborough, 2004).

The pre-stress measurement of Fv/Fm indicated the maximumquantum yield of the algae in optimum physiological conditionsand was used directly as a response variable to test for theamounts of initial stress presented by fucoid algae from differentpopulations. Samples were then left for an extra 30 min in thetanks exposed to natural light conditions, allowing the adaptationof the PSII reaction centres to natural sunlight in a hydrated envi-ronment. Algae were then transferred from the seawater tanksinto aerial conditions in experimental units at the assigned tem-perature and light intensities for an exposure period of 75 min.Periods longer than 90 min resulted in the death of fronds (per-sonal observation), probably because the single fronds used donot have the protection offered by self-covering. After 35 min,the surface temperature of each frond was directly measured withthermocouple thermometers (Easyview 15, Extech InstrumentsCorp., Nashua, NH, USA). At the end of the stress period, algaewere blotted dry and weighed before being resubmerged in the300 l tank with aerated and circulating seawater at 16°C for arehydration period of 25 min. During this period, algae werekept in the dark to allow the relaxation and oxidation of reactioncentres in the PSII (Gylle et al., 2009) before PAM measurementswere carried out again. Algae were then left submerged for 16 hovernight to assess recovery in optimum conditions. They werethen exposed to 25 min of dark conditions before the final PAMmeasurements were completed. These PAM measurements wereused to evaluate the percentage of pre-stress Fv/Fm lost as a resultof stress and exhibited after 16 h, through the following formulas:

% of pre-stress Fv=Fm lost as a result of stress

¼ Pre-stress Fv=Fm � Fv=Fm after stress

Pre-stress Fv=Fm� 100 Eqn 1

% of pre-stress Fv=Fmexhibited after16 h

¼ Fv=Fm after16 h

Pre-stress Fv=Fm� 100 Eqn 2

Finally, samples were removed from the tank and dried at60°C for 48 h in order to determine their DW. The quantifica-tion of hydrated, post-stress and dry weights allowed the watercontent to be determined at the different stages of each trial. Thepercentage of water in fucoid tissue before the application ofstress was determined by measuring the hydrated and dry weightsof specimens, while the percentage of initial water lost as a resultof the stress period was estimated by incorporating the weight ofthe specimens after the stress period.

Data analysis

In order to analyse the results, each of the four trials was consid-ered individually and the results across trials for each of the twospecies were qualitatively compared. The effects of treatments on



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response variables were analysed using mixed-model ANOVAwith five factors allowing formal comparisons among geographi-cal regions (fixed, two levels), shores (random nested in geo-graphical regions, two levels), light intensities (fixed, two levels),air temperatures (fixed, two levels) and container (random nestedin the interaction between geographical regions, shores, lightintensities and air temperatures, two levels). Significant resultswere explored further with Student–Newman–Keuls (SNK)multiple comparisons (Underwood, 1997), with results fromnested factors presented but not described in detail. An ANOVAwith only two factors, geographical region and shore, was usedfor response variables measured before stress was applied (pre-stress Fv/Fm values and initial water content). Cochran’s test wasused to test the data for heterogeneity of variance (Cochran,1951) and transformations were made where appropriate. All theanalyses were performed using the statistical package WinG-MAV5 (EICC, University of Sydney).

Results

Before application of stress

Water content of fronds All algae used in experiments were wellhydrated before applying stress, with at least 63.8% of their weightcomposed of water. No significant differences in water contentwere detected between algae from different geographical regions,although there was some variation detected between shores. ForF. vesiculosus, differences among shores occurred only in Portugal(ANOVAs, TrialFves1, F2,92 = 5.2, P = 0.008; TrialFves2, F2,92 =2.6, P = 0.084), while in F. spiralis there were differences in bothPortugal and Wales (TrialFspi1, F2,92 = 12.1, P < 0.001; TrialFspi2,F2,92 = 0.4, P = 0.651). However, the amplitude of the differencesfound was small, never more than 3.2% (Fig. 1).

Pre-stress Fv/Fm values The mean pre-stress values of Fv/Fm forF. vesiculosus and F. spiralis populations were naturally high(Fig. 2). No differences were detected between regions, althoughthere were some significant differences, of small magnitude, inboth fucoid species between Portuguese shores (ANOVAs,TrialFves1, F2,92 = 16.3, P < 0.001; TrialFves2, F2,92 = 8.2,P < 0.001; TrialFspi1, F2,92 = 2.1, P = 0.132; TrialFspi2, F2,92 = 4.0,P = 0.021).Despite these differences, all pre-stress values of Fv/Fmmeasured are equivalent to those for healthy unstressed specimensobserved in other studies (Magnusson, 1997; Pearson et al., 2000;Skene, 2004; Gylle et al., 2009) and indicate that the photosys-tems of specimens used were in a good state.

During and after stress application

Frond surface temperature during stress Frond surface temper-atures were elevated, from 16°C in hydrated conditions to meanvalues as high as 35°C, as a result of both increasing temperatureand light in both species after 35 min of stress application (Fig. 3).There was no interaction between the two stressors, but the effectof treatments was large (Table 1, Fig. 3). The effects sizes of thelight treatment (other factors pooled) were 6.3°C (TrialFves1) and8.1°C (TrialFves2) in F. vesiculosus and 8.1°C (TrialFspi1) and10.2°C (TrialFspi2) in F. spiralis, while the effects sizes of tempera-ture treatment (other factors pooled) were 7.1°C (TrialFves1) and7.7°C (TrialFves2) in F. vesiculosus and 7.8°C (TrialFspi1) and7.9°C (TrialFspi2) in F. spiralis (Fig. 3). For both species, there wasa significant container effect (Table 1), although the SNK test ofthis factor showed there was no consistent pattern.

Water lost as a result of stress (indicative of desiccationstate) Light intensity was a consistent factor determining theamounts of water loss for both fucoid species; plants exposed to

(a)

(c)

(b)

(d)

Fig. 1 Percentage of water, before stressapplication, in tissues of Fucus vesiculosus –TrialFves1 (a) and TrialFves2 (b) – and Fucus

spiralis – TrialFspi1 (c) and TrialFspi2 (d) – fromPortuguese and Welsh, UK, shores. Errorbars, � 1SE; significant difference:*, P < 0.05; **, P < 0.01. Via, Viana doCastelo; Car, Carrec�o; Cwy, Porth Cwyfan;Cem, Cemlyn Bay.





full light showed consistently higher water loss than shaded plantsacross all trials (Table 2, Fig. 4). The differences in mean waterloss between high and low light treatments (other factors pooled)were 8.4% (TrialFves1) and 16.8% (TrialFves2) in F. vesiculosus,and 11.1% (TrialFspi1) and 7.2% (TrialFspi2) in F. spiralis (Fig. 4).There was an effect of air temperature on water loss in one out of

the two trials for each of the fucoid species (Table 2: Trials Fves2,

Fspi1, Fig. 4), although the effect size was much lower than forlight intensity. None of the interactions between these physicalstressors were significant, suggesting additive effects. Comparisonbetween regions generally did not show a difference in theamount of water lost (Table 2; TrialFves2, TrialFspi1 and

(a)

(c)

(b)

(d)

Fig. 2 Pre-stress Fv/Fm values in Fucus

vesiculosus – TrialFves1 (a) and TrialFves2 (b) –and Fucus spiralis – TrialFspi1 (c) andTrialFspi2 (d) – from Portuguese and Welsh,UK, shores. Error bars, �1SE; significantdifference: **, P < 0.01. Via, Viana doCastelo; Car, Carrec�o; Cwy, Porth Cwyfan;Cem, Cemlyn Bay.

(a) (a′) (a′′) (b′) (b′′)

(c′) (c′′) (d′) (d′′)

(b)

(c) (d)

Fig. 3 Frond surface temperature of Fucusvesiculosus – TrialFves1 (a) and TrialFves2 (b) –and Fucus spiralis – TrialFspi1 (c) andTrialFspi2 (d) – from Portuguese and Welsh,UK, populations after being exposed for35min to stress. Smaller graphs representsignificant effects of fix factors on each trial:TrialFves1, effect of light intensity (a’) and airtemperature (a”); TrialFves2, effect of lightintensity (b’) and air temperature (b”);TrialFspi1, effect of light intensity (c’) and airtemperature (c”); TrialFspi2, effect of lightintensity (d’) and air temperature (d”). Errorbars, �1SE; significant difference: *, P < 0.05;**, P < 0.01. COL, cold; WAR, warm; UNS,unshaded (white bars where not labelled);SHA, shaded (shaded bars where notlabelled).



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TrialFspi2). However, in Trial Fves1, F. vesiculosus plants fromWales retained significantly more water than those from Portu-gal, although the magnitude of the differences was small (Table 2,Fig. 4).

Percentage of pre-stress Fv/Fm lost as a result of stress Acrossall trials carried out, Fv/Fm values declined, as expected, whenmeasured 25 min after the application of stress (Fig. 5). Fucoidspecimens of both species, subjected to higher light intensities,lost a significantly greater percentage of pre-stress Fv/Fm thanthose confined to shaded containers (Table 3). The differences inthe mean percentage of pre-stress Fv/Fm lost as a result of stressbetween specimens subjected to high and low light treatments(other factors pooled) were 51.8% (TrialFves1) and 41.0%(TrialFves2) in F. vesiculosus and 38.5% (TrialFspi1) and 47.8%(TrialFspi2) in F. spiralis, respectively (Fig. 5). In Trial Fspi2, aneffect of geographical region dependent on the interaction with

air temperature and light controlled during the experimental pro-cedure was also detected (Table 3; SNK of Re9 Te9 Li).

After a 16 h period of recovery

Percentage of pre-stress Fv/Fm exhibited 16 h afterstress Although Fv/Fm values improved from the values observedafter stress, only algae exposed to low light intensities achievedcomplete or high degrees of recovery (Fig. 6). In high-light treat-ments, mean Fv/Fm values (mean� SE; n = 48) after 16 h onlycorresponded to 71.8� 8.7% (TrialFves1) and 66.7� 9.4%(TrialFves2) in F. vesiculosus and 78.7� 6.7% (TrialFspi1) and74.5� 7.5% (TrialFspi2) in F. spiralis of observed pre-stress levels(other factors pooled), respectively. In all trials there was a signifi-cant effect of light (Table 4, Fig. 6). This effect was consistentacross regions and shores for F. spiralis, but varied by region forF. vesiculosus (Fig. 6). Specimens of F. vesiculosus collected in

Table 1 Mixed-model ANOVA of frond surface temperature from Fucus vesiculosus (TrialFves1 and TrialFves2) and Fucus spiralis (TrialFspi1 and TrialFspi2)after being exposed for 35min to stress

Source df

F. vesiculosus

F ratio vs

TrialFves1 TrialFves2

MS F P MS F P

Re 1 2.84E+08 0.1 0.825 0.14 0.0 0.944 Sh (Re)Sh (Re) 2 4.50E+09 1.3 0.289 22.31 0.5 0.634 Co (Re9 Sh(Re) 9 Te9 Li)Te 1 5.53E+10 48.5 0.020 1419.11 293.5 0.003 Te9 Sh (Re)Li 1 4.53E+10 21.7 0.043 1564.13 72.8 0.014 Li9 Sh (Re)Co (Re9 Sh(Re)9 Te9 Li) 16 3.35E+09 34.2 < 0.001 47.63 15.5 < 0.001 ResRe9 Te 1 1.46E+08 0.1 0.755 2.19 0.5 0.570 Te9 Sh (Re)Re9 Li 1 1.13E+08 0.1 0.838 13.13 0.6 0.516 Li9 Sh (Re)Te9 Sh (Re) 2 1.14E+09 0.3 0.716 4.84 0.1 0.904 Co (Re9 Sh(Re)9 Te9 Li)Li9 Sh (Re) 2 2.09E+09 0.6 0.549 21.50 0.5 0.645 Co (RexSh(Re) 9 Te9 Li)Te9 Li 1 4.19E+08 1.0 0.418 17.94 0.2 0.698 Li9 Te9 Sh (Re)Re9 Te9 Li 1 5.61E+07 0.1 0.747 35.89 0.4 0.591 Li9 Te9 Sh (Re)Li9 Te9 Sh (Re) 2 4.09E+08 0.1 0.886 89.20 1.9 0.186 Co (Re9 Sh(Re) 9 Te9 Li)Res 64 9.79E+07 3.07Cochran’s test C = 0.1974, P > 0.05 C = 0.1373, P > 0.05

F. spiralis

F ratio vs

TrialFspi1 TrialFspi2

MS F P MS F P

Re 1 149.24 0.1 0.743 3.05 0.3 0.640 Sh (Re)Sh (Re) 2 1056.70 1.2 0.317 10.24 0.1 0.868 Co (Re9 Sh(Re) 9 Te9 Li)Te 1 38 944.60 491.1 0.002 1487.59 58.2 0.017 Te9 Sh (Re)Li 1 41 846.22 66.4 0.015 2485.75 158.4 0.006 Li9 Sh (Re)Co (Re x Sh(Re)9 Te9 Li) 16 856.02 10.2 < 0.001 71.41 37.1 < 0.001 ResRe9 Te 1 257.75 3.3 0.213 0.11 0.0 0.953 Te9 Sh (Re)Re9 Li 1 981.32 1.6 0.338 0.01 0.0 0.980 Li9 Sh (Re)Te9 Sh (Re) 2 79.31 0.1 0.912 25.57 0.4 0.705 Co (Re9 Sh(Re) 9 Te9 Li)Li9 Sh (Re) 2 630.36 0.7 0.494 15.70 0.2 0.805 Co (Re9 Sh(Re) 9 Te9 Li)Te9 Li 1 630.76 0.4 0.581 2.31 0.1 0.850 Li9 Te9 Sh (Re)Re9 Te9 Li 1 64.71 0.0 0.854 5.27 0.1 0.776 Li9 Te9 Sh (Re)Li9 Te9 Sh (Re) 2 1476.61 1.7 0.210 50.06 0.7 0.511 Co (Re9 Sh(Re) 9 Te9 Li)Res 64 84.21 1.93Cochran’s test C = 0.1918, P > 0.05 C = 0.1329, P > 0.05

Re, geographical region; Sh, shore; Te, air temperature; Li, light intensity; Co, container; Res, residual. Bold indicates P < 0.05.





Portugal, which had previously been stressed in high-light condi-tions, achieved significantly greater degrees of recovery than indi-viduals from Wales. The mean differences between F. vesiculosusspecimens previously subjected to high-light conditions from Por-tugal and Wales were 20.2% and 11.5% in TrialFves1 andTrialFves2, respectively (Table 4; SNK of Re9 Li, Fig. 6). Simi-larly, F. vesiculosus specimens collected in Portugal also achievedsignificantly greater degrees of recovery than individuals fromWales when subjected to low-light conditions (other factorspooled), although the effect sizes were smaller (TrialFves1, 1.7%;and TrialFves2, 2.6%) (Fig. 6).

Variation in air temperatures during the stress period alsoaffected photophysiological state after recovery in F. spiralis but

not in F. vesiculosus (Table 4). Fv/Fm values of F. spiralis weregreater in cold than in warm treatments, but only under certainconditions of light and/or shores from which specimens were col-lected (Table 4: TrialFspi1, SNK of Te9 Li; TrialFspi2, SNK ofLi9 Te9 Sh (Re)).

Discussion

The warming of the Earth’s climate system is unequivocal,with increases over the last 40 yr in global air and ocean tem-perature (from the surface to a depth of 700 m) averaging 0.5and 0.1°C, respectively (IPCC, 2007). Can the observeddecline in the abundance of fucoid species in southern

Table 2 Mixed-model ANOVA of the percentage of water content lost as a result of the stress period (indicative of desiccation state) from Fucusvesiculosus tissues (TrialFves1 and TrialFves2) and from Fucus spiralis tissues (TrialFspi1 and TrialFspi2)

Source df

F. vesiculosus

F ratio vs


MS F P MS F P

Re 1 2.37 39.8 0.024 78.10 0.1 0.851 Sh (Re)Sh (Re) 2 0.06 0.5 0.598 1708.81 18.9 < 0.001 Co (Re9 Sh(Re)9 Te9 Li)Te 1 1.32 5.2 0.149 1044.32 193.9 0.005 Te9 Sh (Re)Li 1 5.09 41.7 0.023 6741.38 106.2 0.009 Li9 Sh (Re)Co (Re9 Sh (Re)9 Te9 Li) 16 0.11 1.4 0.177 90.30 1.8 0.058 ResRe9 Te 1 0.01 0.0 0.896 225.00 41.8 0.023 Te9 Sh (Re)Re9 Li 1 0.01 0.1 0.813 150.48 2.4 0.264 Li9 Sh (Re)Te9 Sh (Re) 2 0.25 2.2 0.139 5.39 0.1 0.942 Co (Re9 Sh (Re)9 Te9 Li)Li9 Sh (Re) 2 0.12 1.1 0.361 63.50 0.7 0.510 Co (Re9 Sh (Re) 9 Te9 Li)Te9 Li 1 0.26 1.9 0.305 204.37 3.7 0.196 Li9 Te9 Sh (Re)Re9 Te9 Li 1 0.27 1.9 0.303 16.91 0.3 0.638 Li9 Te9 Sh (Re)Li9 Te9 Sh (Re) 2 0.14 1.3 0.314 55.87 0.6 0.551 Co (Re9 Sh (Re)9 Te9 Li)Res 64 0.08 51.37Cochran’s test C = 0.1889, P > 0.05 C = 0.1687, P > 0.05SNK Re9 Te

War – Por >WalAll Re –War >Col

Source df

F. spiralis

F ratio vs


MS F P MS F P

Re 1 8.99E+10 0.1 0.817 31 341.87 4.8 0.161 Sh (Re)Sh (Re) 2 1.30E+12 2.3 0.137 6587.28 0.2 0.790 Co (Re9 Sh (Re) 9 Te9 Li)Te 1 6.49E+12 45.4 0.021 57 681.54 2.9 0.232 Te9 Sh (Re)Li 1 5.54E+13 916.7 0.001 6.54E+05 18.9 0.049 Li9 Sh (Re)Co (Re9 Sh (Re) 9 Te9 Li) 16 5.76E+11 1.4 0.184 27 586.18 4.4 < 0.001 ResRe9 Te 1 4.48E+08 0.0 0.960 3757.42 0.2 0.707 Te9 Sh (Re)Re9 Li 1 2.53E+11 4.2 0.177 501.12 0.0 0.915 Li9 Sh (Re)Te9 Sh (Re) 2 1.43E+11 0.3 0.783 20 049.88 0.7 0.499 Co (Re9 Sh (Re) 9 Te9 Li)Li9 Sh (Re) 2 6.04E+10 0.1 0.901 34 608.20 1.3 0.312 Co (Re9 Sh (Re) 9 Te9 Li)Te9 Li 1 3.07E+12 5.3 0.148 36 051.55 3.5 0.201 Li9 Te9 Sh (Re)Re9 Te9 Li 1 5.36E+11 0.9 0.438 36 365.24 3.6 0.200 Li9 Te9 Sh (Re)Li9 Te9 Sh (Re) 2 5.81E+11 1.0 0.386 10 226.16 0.4 0.696 Co (Re9 Sh (Re) 9 Te9 Li)Res 64 4.19E+11 6267.12Cochran’s test C = 0.1967, P > 0.05 C = 0.1961, P > 0.05

Complex post hoc SNK tests of significant differences are presented. Bold indicates P < 0.05.Re, geographical region (Por, Portugal; Wal, Wales); Sh, shore; Te, air temperature (Col, cold; War, warm); Li, light intensity; Co, container; Res, residual.



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NewPhytologist8

Europe and contractions in adjacent areas be linked with anincreasing difficulty in dealing with the physical environment?Are the harsher summer conditions limiting the fitness of

species such as F. vesiculosus and F. spiralis in Portugal or arethe local populations adapted to deal with those amounts ofstress?

(a) (a′) (a′′) (b′)

(c′) (c′′) (d′)

(b)

(c) (d)

Fig. 4 Percentage of water content lost as aresult of stress (indicative of desiccationstate) in Fucus vesiculosus tissues – TrialFves1(a) and TrialFves2 (b) – and in Fucus spiralis

tissues – TrialsFspi1 (c) and TrialFspi2 (d) –from Portuguese (POR) and Welsh (WAL),UK, populations. Smaller graphs representsignificant effects or significant interactionsof fix factors on each trial: TrialFves1, effect oflight intensity (a’) and geographical region(a”); TrialFves2, interaction of geographicalregion with air temperature (b’); TrialFspi1,effect of light intensity (c’) and airtemperature (c”); TrialFspi2, effect of lightintensity (d’). Error bars, �1SE; lowercaseletters indicate comparison of responses offucoid specimens from different geographicalregions when exposed to equal airtemperature independently of solar radiationintensity; significant difference: *, P < 0.05;**, P < 0.01. COL, Cold; WAR, Warm; UNS,unshaded (white bars where not labelled);SHA, shaded (shaded bars where notlabelled).

(a) (b)

(c) (d)

(a′) (b′)

(c′)

Fig. 5 Percentage of pre-stress Fv/Fm lost asa result of stress in Fucus vesiculosus tissues –TrialFves1 (a) and TrialFves2 (b) – and in Fucusspiralis tissues – TrialFspi1 (c) and TrialFspi2 (d)– from Portuguese and Welsh, UK,populations. Smaller graphs representsignificant interactions of fix factors on eachtrial: TrialFves1, effect of light intensity (a’);TrialFves2, effect of light intensity (b’);TrialFspi1, effect of light intensity (c’). Errorbars, �1SE; lowercase letters indicatecomparison of responses of fucoid specimensfrom different geographical regions whenexposed to equal air temperature and solarradiation intensity, while uppercase lettersindicate comparison of responses of fucoidspecimens from the same geographicalregions when exposed to equal solarradiation intensity but also to different airtemperatures. Significant differences:*, P < 0.05; **, P < 0.01. UNS, unshaded(white bars where not labelled); SHA, shaded(shaded bars where not labelled).





Solar irradiance is considered a major influence on distributionof plants at local and regional scales (Austin & Van Niel, 2011).An increase in solar radiance has been noted in the Iberian Penin-sula over recent decades, owing to a decrease in cloud cover and anincrease in sunshine duration (Calbo & Sanchez-Lorenzo, 2009;Sanchez-Lorenzo et al., 2009, 2013). The importance of solarradiance for algae has previously been shown by studies describingpatterns of photosynthesis in macroalgae (Gomez et al., 2004;Rohde et al., 2008), where light intensity is described as a major

physical factor driving the shore height occupied by photosyn-thetic algae species. In our study, the photophysiological responseof fucoid algae to stressful conditions indicates that harsh summerconditions can influence the physiological capacity of F. vesiculosusand F. spiralis. Specimens of both species exposed to high lightintensities during emersion in our trials lost a greater photosyn-thetic capacity and showed increased frond temperature and desic-cation when compared with specimens subjected to shadedtreatments, following the results also observed by Martinez et al.

Table 3 Mixed-model ANOVA of the percentage of pre-stress Fv/Fm lost as a result of stress in Fucus vesiculosus tissues (TrialFves1 and TrialFves2) and inFucus spiralis tissues (TrialFspi1 and TrialFspi2)

Source df

F. vesiculosus

F ratio vs


MS F P MS F P

Re 1 1165.45 14.2 0.064 4108.82 6.8 0.121 Sh (Re)Sh(Re) 2 82.12 0.3 0.721 606.70 1.0 0.381 Co (Re9 Sh (Re) 9 Te9 Li)Te 1 3142.00 11.0 0.080 779.36 0.8 0.456 Te9 Sh (Re)Li 1 64 386.88 178.3 0.006 40 423.99 105.6 0.009 Li9 Sh (Re)Co(Re9 Sh (Re) 9 Te9 Li) 16 245.61 0.8 0.662 591.64 3.3 < 0.001 ResRe9 Te 1 215.61 0.8 0.476 126.48 0.1 0.747 Te9 Sh (Re)Re9 Li 1 1312.98 3.6 0.197 6813.64 17.8 0.052 Li9 Sh (Re)Te9 Sh (Re) 2 285.22 1.2 0.338 925.05 1.6 0.240 Co (Re9 Sh (Re) 9 Te9 Li)Li9 Sh (Re) 2 361.07 1.5 0.259 382.66 0.7 0.537 Co (Re9 Sh (Re) 9 Te9 Li)Te9 Li 1 627.25 4.0 0.182 2013.83 12.4 0.072 Li9 Te9 Sh (Re)Re9 Te9 Li 1 544.97 3.5 0.202 80.03 0.5 0.556 Li9 Te9 Sh (Re)Li9 Te9 Sh (Re) 2 155.29 0.6 0.544 163.05 0.3 0.763 Co (Re9 Sh (Re)9 Te9 Li)Res 64 300.75 177.34Cochran’s test C = 0.1270, P > 0.05 C = 0.1360, P > 0.05

Source df

F. spiralis

F ratio vs

Trial Fspi1 Trial Fspi2

MS F P MS F P

Re 1 1974.27 6.4 0.127 301.01 0.4 0.589 Sh (Re)Sh (Re) 2 308.78 1.1 0.344 739.22 2.6 0.109 Co (Re9 Sh (Re) 9 Te9 Li)Te 1 1808.13 2.6 0.250 1712.56 5.0 0.156 Te9 Sh (Re)Li 1 35 552.06 94.9 0.010 54 811.79 14 872.1 < 0.001 Li9 Sh (Re)Co (Re9 Sh (Re) 9 Te9 Li) 16 270.60 1.4 0.152 289.68 2.4 0.007 ResRe9 Te 1 208.24 0.3 0.641 390.71 1.1 0.399 Te9 Sh (Re)Re9 Li 1 331.79 0.9 0.446 30.23 8.2 0.103 Li9 Sh (Re)Te9 Sh (Re) 2 702.79 2.6 0.106 345.87 1.2 0.329 Co (Re9 Sh (Re) 9 Te9 Li)Li9 Sh (Re) 2 374.53 1.4 0.279 3.69 0.0 0.987 Co (Re9 Sh (Re) 9 Te9 Li)Te9 Li 1 1590.15 7.6 0.111 393.94 57.4 0.017 Li9 Te9 Sh (Re)Re9 Te9 Li 1 1.27 0.0 0.945 1418.27 206.5 0.005 Li9 Te9 Sh (Re)Li9 Te9 Sh (Re) 2 210.04 0.8 0.477 6.868 0.0 0.977 Co (Re9 Sh (Re) 9 Te9 Li)Res 64 187.76 120.75Cochran’s test C = 0.1110, P > 0.05 C = 0.1200, P > 0.05SNK Re9 Te9 Li

Col9Uns –Wal > PorWar9Uns – Por >WalWar9 Sha –Wal > PorPor9Uns –War >ColWal9 Sha –War >ColAll Re at all Te – Uns > Sha

Complex post hoc SNK tests of significant differences are presented. Bold indicates P < 0.05.Re, geographical region (Por, Portugal; Wal, Wales); Sh, shore; Te, air temperature (Col, cold; War, warm); Li, light intensity (Uns, unshaded; Sha, shaded);Co, container; Res, residual.



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NewPhytologist10

(2012) for F. serratus. This is especially evident when high intensi-ties of solar radiation reach the fronds, as specimens subjected tosuch treatments were not able to physiologically recover 100% oftheir photosynthetic capacities even after 16 h in hydrated condi-tions (a period approximately twice that experienced in the field).When subjected to low light intensities, both fucoid species werecapable, after the recovery period in hydrated conditions, ofregaining a photosynthetic performance very similar to that shownbefore the application of stress. These results demonstrate theimportance of ameliorated conditions and the impact that stron-ger solar radiation can have on the photosystem of fucoid algae.The fact that the Fv/Fm values of specimens subjected to high lightintensity were not completely re-established suggests damage ofthe electron transport chain by oxidation or denaturalization ofpigments and proteins at the PSII, probably as a result of the insuf-ficient capacity of photoprotection mechanisms (Nishiyama et al.,2011; Takahashi & Badger, 2011). Excessive radiation can affectconcentrations of reactive oxygen species that prevent repair ofPSII machinery, leading to decreased photosynthetic activity (Col-len & Davison, 2001; Nishiyama et al., 2011), which ultimatelycould limit the rates of resource acquisition, growth, reproductivecapacity and consequently population survival (Aguilera et al.,1999; Dethier et al., 2005; Wernberg et al., 2010; Takahashi &Badger, 2011;Martinez et al., 2012).

The effects of temperature variation on the performance offucoid species under emerged conditions were also clear in ourstudy, similar to previous work in southern Europe (Zardi et al.,2011; Martinez et al., 2012). Results from both species indicatethat warmer air conditions, such as those observed during hotsummer days throughout southern Europe, promote desiccation

and lead to the decline of Fv/Fm values observed after stress, limit-ing the photosynthetic recovery success over time. The influenceof air temperature variation was most pronounced on the controlof frond temperature, contributing to tissue overheating duringthe experiments. This is critical, as increased tissue temperaturecan lead to a reduction in growth rates and to changes in thechemical composition of algae, owing to its effects on proteindenaturation, kinetics of cellular enzymes, membrane stabilityand active influence on membrane transport (Lobban & Harri-son, 1997; Chen et al., 2012; Martinez et al., 2012).

Our experimental design allowed us to establish that solar radi-ation and air temperature act additively for the values tested. Itshould be pointed out that maximal air temperature and solarradiation, naturally reached in Portugal, can be higher than thoseused in our experiment and therefore impacts on physiologicalperformance may be greater than those observed. Interactions insome conditions still appeared, but not as a generalized phenom-enon, and where present were associated with a response of lowmagnitude. Therefore, we propose that solar radiation and airtemperature can be seen as important additive, rather than inter-active, summer emersion stresses acting on F. vesiculosus andF. spiralis populations. This idea is supported by Martinez et al.(2012), who also showed no synergistic effects acting onF. serratus populations at southern limits. This is a particularlyrelevant point, as additivity of abiotic stresses is normallyassumed in the development of species distribution models(SDMs) (Darling & Cote, 2008), which attempt to predict spe-cies biogeographic shifts in response to climatic change.

Our results further indicate that the ability to tolerate amuntsof summer emersion stress is dependent on the species concerned,

(a)

(c)

(b)

(d)

(a′) (b′)

(c′)

Fig. 6 Percentage of pre-stress Fv/Fmexhibited 16 h after stress in Fucus

vesiculosus tissues – TrialFves1 (a) andTrialFves2 (b) – and in Fucus spiralis tissues –TrialFspi1 (c) and TrialFspi2 (d) – fromPortuguese (POR) and Welsh (WAL), UK,populations. Smaller graphs representsignificant effects or significant interactionsof fix factors on each trial: Trial Fves1,interaction of geographical region with lightintensity (a’); TrialFves2, interaction ofgeographical region with light intensity (b’);TrialFspi1, interaction of air temperature withlight intensity (c’). Error bars, �1SE;lowercase letters indicate comparison ofresponses of fucoid specimens from differentgeographical regions (TrialFves1 andTrialFves2) or different air temperatures(TrialFspi1) when exposed to equal solarradiation intensity. Significant differences:**, P < 0.01. COL, cold; WAR, warm; uns,unshaded (white bars where not labelled);sha, shaded (shaded bars where notlabelled).





with results reflecting, as we hypothesised, their position in theintertidal. As proposed by Ji et al. (2005), it seems that it is theability to withstand desiccation stress that determines the distri-bution of intertidal seaweeds on the shore. Previous work hasshown that tolerance to climatic variability and the capacity totolerate desiccation is greater in F. spiralis than in fucoid speciesliving on lower areas of the shore (Schonbeck & Norton, 1978;

Dring & Brown, 1982). Dring & Brown (1982) showed thatF. spiralis specimens were capable of a complete recovery of pho-tosynthetic levels, even when subjected to tissue water loss of 80–90%, a result similar to those presented here for specimens ofboth species subjected to shaded conditions. Comparison of theresults of our experiments, run separately on different fucoid spe-cies, seems to support our initial expectation, as F. spiralis

Table 4 Mixed-model ANOVA of the percentage of pre-stress Fv/Fm exhibited 16 h after stress in Fucus vesiculosus tissues (TrialFves1 and TrialFves2) and inFucus spiralis tissues (TrialFspi3 and TrialFspi4)

Source df

F. vesiculosus

F ratio vs


MS F P MS F P

Re 1 3.32E+06 117.2 0.008 1.45E+06 25.1 0.038 Sh (Re)Sh (Re) 2 28295.97 0.2 0.810 57801.30 0.1 0.869 Co (Re9 Sh (Re) 9 Te9 Li)Te 1 1.31E+06 9.6 0.090 7.29E+05 4.3 0.175 Te9 Sh (Re)Li 1 2.33E+07 88754.4 < 0.001 2.02E+07 19928.2 < 0.001 Li9 Sh (Re)Co (Re9 Sh (Re) 9 Te9 Li) 16 1.33E+05 1.1 0.409 4.07E+05 3.4 < 0.001 ResRe9 Te 1 28166.21 0.2 0.694 3.69E+05 2.2 0.279 Te9 Sh (Re)Re9 Li 1 2.17E+06 8265.2 < 0.001 4.86E+05 479.0 0.002 Li9 Sh (Re)Te9 Sh (Re) 2 1.37E+05 1.0 0.380 1.71E+05 0.4 0.664 Co (Re9 Sh (Re) 9 Te9 Li)Li9 Sh (Re) 2 262.29 0.0 0.998 1015.47 0.0 0.998 Co (Re9 Sh (Re) 9 Te9 Li)Te9 Li 1 2.98E+05 4.1 0.180 1806.35 0.0 0.910 Li9 Te9 Sh (Re)Re9 Te9 Li 1 22 609.67 0.3 0.633 43 770.77 0.4 0.594 Li9 Te9 Sh (Re)Li9 Te9 Sh (Re) 2 72 467.41 0.6 0.589 1.11E+05 0.3 0.765 Co (Re9 Sh (Re)xTe9 Li)Res 64 1.25E+05 1.19E+05Cochran’s test C = 0.1944, P > 0.05 C = 0.1975, P > 0.05SNK Re9 Li Re9 Li

All Li – Por >Wal All Li – Por >WalAll Re – Sha >Uns All Re – Sha >Uns

Source df

F. spiralis

F ratio vs


MS F P MS F P

Re 1 2.31E+08 4.4 0.171 3.50E+06 19.3 0.048 Sh (Re)Sh (Re) 2 5.24E+07 0.4 0.656 1.81E+05 1.0 0.401 Co (Re9 Sh (Re) 9 Te9 Li)Te 1 1.14E+09 17.4 0.053 2.41E+06 3.9 0.187 Te9 Sh (Re)Li 1 1.52E+10 1259.3 < 0.001 4.65E+07 278.7 0.004 Li9 Sh (Re)Co (Re9 Sh (Re) 9 Te9 Li) 16 1.21E+08 1.4 0.153 1.87E+05 0.7 0.785 ResRe9 Te 1 3.67E+07 0.6 0.532 33565.23 0.1 0.837 Te9 Sh (Re)Re9 Li 1 1.58E+07 1.3 0.372 1.55E+06 9.3 0.093 Li9 Sh (Re)Te9 Sh (Re) 2 6.54E+07 0.5 0.592 6.18E+05 3.3 0.063 Co (Re9 Sh (Re) 9 Te9 Li)Li9 Sh (Re) 2 1.21E+07 0.1 0.906 1.67E+05 0.9 0.429 Co (Re9 Sh (Re) 9 Te9 Li)Te9 Li 1 9.25E+08 89.2 0.011 1.26E+06 1.4 0.363 Li9 Te9 Sh (Re)Re9 Te9 Li 1 51692.21 0.0 0.950 6.30E+05 0.7 0.496 Li9 Te9 Sh (Re)Li9 Te9 Sh (Re) 2 1.04E+07 0.1 0.918 9.23E+05 4.9 0.021 Co (Re9 Sh (Re) 9 Te9 Li)Res 64 8.41E+07 2.68E+05Cochran’s test C = 0.1937, P > 0.05 C = 0.1951, P > 0.05SNK Te9 Li Li9 Te9 Sh(Re)

Uns – Col >War Via9Uns – Col >WarAll Te – Sha >Uns Cwy9Uns – Col >War

Uns9Col – Via >CarUns9War – Cem >CwyAll Sh (Re)9 all TE (exceptVia9 Col) – Sha >Uns

Complex post hoc SNK tests of significant differences are presented. Bold indicates P < 0.05.Re, geographical region (Por, Portugal; Wal, Wales); Sh, shore (Via, Viana do Castelo; Car, Carrec�o; Cwy,Porth Cwyfan; Cem, Cemlyn Bay); Te, airtemperature (Col, cold; War, warm); Li, light intensity (Uns, unshaded; Sha, shaded); Co, container; Res, residual.



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NewPhytologist12

specimens, when subjected to an environment with high air tem-perature and strong solar irradiance, were able to recover photo-synthetic performance more efficiently than specimens ofF. vesiculosus, despite displaying similar levels of desiccation andfrond overheating after stress application. These results, compa-rable to those of Zardi et al. (2011), are in accordance with thevertical distribution of these two fucoid species on the shore andcould form part of the explanation as to why F. vesiculosus hasbeen reported to be retracting in areas adjacent to Portugal, suchas Morocco and northern Spain, while F. spiralis has not.

Our comparative experimental approach shows that, withinthe same fucoid species, specimens from southern and centralpopulations can have distinct physiological tolerances and perfor-mances when exposed to similar abiotic stresses. Harsh condi-tions of high air temperature and solar radiation vary betweenregions and occur more frequently in southern than in northernEuropean regions (validated by mean photosynthetically availableradiation and mean air temperature data from NASA Aqua(http://disc.sci.gsfc.nasa.gov/giovanni) – AIRS standard & MO-DIS-Aqua missions, respectively). Our results suggest that thephotosynthetic systems of specimens from northern populationsof F. vesiculosus were significantly more affected than the onesfrom southern populations, a result that contrasts with previousphysiological studies that showed no regional differentiation inthe physiological response of F. vesiculosus (Pearson et al., 2009;Zardi et al., 2013). These contrasting results may be explained bythe nature of the stress provided in our experiments, whichallowed for investigation of the additive effects of temperatureand solar radiance. When strong light intensities were applied inour experiments, the effect of these stressful summer emersionconditions was felt on all specimens, but the effects were moremarked in Welsh than in Portuguese populations. This was par-ticularly evident for values of Fv/Fm sustained by F. vesiculosusspecimens after 16 h of recovery from stress. For F. spiralis, differ-ences between central and southern populations, observed inFv/Fm values immediately after stress, are not so obvious and con-sistent, reflecting the greater physiological resilience of this spe-cies (Schonbeck & Norton, 1978; Dring & Brown, 1982;Chapman, 1995). Differences in physiological capacity amongnorthern and southern populations may reflect phenotypicplasticity of species or the strong effect of natural selection byenvironmental factors on genetically distinct fixed traits. Thesealternatives cannot be distinguished by our study and requireadditional work using a true common garden approach that elim-inates long-term carryover effects, supplemented by molecularapproaches, to determine if this is a case of phenotypic plasticityor genotype9 environment interactions, which are the essence oflocal adaption (Scheiner, 1993; Rutter & Fenster, 2007; Berg-mann et al., 2010). Despite greater resistance to stress of southernalgae, the fact that F. vesiculosus and F. spiralis specimens fromPortuguese populations are clearly physiologically stressed and donot recover for a considerable time period may have importantconsequences. The conditions of solar radiation and air tempera-ture used in our experiments are experienced regularly and oftenon consecutive tides in Portugal. Environmental stress may there-fore have a significant nonlethal effect on Portuguese algae, with

consequent negative effects on fitness. Such nonlethal effects havebeen observed in F. vesiculosus through a reduction in reproduc-tive capacity in Iberian populations (Viejo et al., 2011; Ferreira,2012) and no doubt contribute to the generally low level ofrecruitment and adult abundance in southern Europe.

In summary, the present study further clarifies which proxi-mate ecological processes may cause the observed decline of somefucoid species near their southern limits of distribution (Ballan-tine, 1961; Hawkins & Hartnoll, 1983; Jenkins et al., 2008;Ferreira, 2012). Biological processes, such as grazing pressure,competition and facilitation, have been shown to influence fucoidabundance (Jenkins et al., 1999b, 2005; Arrontes et al., 2004;Coleman et al., 2006). However, the experiments performed hereclearly demonstrate that physical stress, mainly exposure to theadditive effects of high air temperatures and high intensities ofsolar radiation caused by emersion on warm cloudless summerdays, has an important nonlethal effect on photophysiology,frond temperature and desiccation of specimens, affecting theirphysiological performance and potentially leading to reductionsin abundance of F. vesiculosus and F. spiralis. Our results alsodemonstrate that the ability to tolerate summer emersion stressvaries with both species identity and geographical origin of popu-lations. Greater resilience to emersion stress at the studied area isapparent in F. spiralis, the species living higher on the shore. Fur-thermore, we show how fucoid populations inhabiting geograph-ical regions with distinct climatic backgrounds can havedifferential photosynthetic capacities and consequently show dif-ferent degrees of resistance when exposed to equal amounts ofphysical stress. This observation, combined with the fact thatgenetic differentiation has been shown in populations exposed todifferent physical conditions (Billard et al., 2005), highlights theimportance of considering differences between populations, espe-cially when analysing effects of climate change or local anthropo-genic stresses.

The capacity to identify the multiple drivers and comprehendtheir possible effects on fucoid species abundance in southernEuropean regions is essential for understanding and predictingthe consequences of future climatic variations on the biogeo-graphical ranges and intertidal primary productivity. Given theimportance of these fucoid species as intertidal ecosystem engi-neers and primary producers (Hawkins et al., 2009), the acquisi-tion of such information is essential for successful projections offuture broad ecological changes on the intertidal ecosystem (Dar-ling & Cote, 2008). Therefore, we propose future studies tofocus on the clarification of the processes controlling the distribu-tion of ecologically important species, as such knowledge willallow advanced predictions of the consequences of eventual cli-matic alterations on the intertidal ecosystem balance.

Acknowledgements

Our research was supported by a PhD grant from Fundac�~ao paraa Ciencia e Tecnologia (FCT) (SFRH/BD/41541/2007) awardedto J.G.F. and by the FCT project PHYSIOGRAPHY (PTDC/MAR/105147/2008 cofunded by FEDER through the programCOMPETE-QREN). Support to B.M. was provided by the





project CGL2010-19301 funded by the Spanish Ministry of Sci-ence. We would also like to thank Dr Bruno Jesus and Dr MartinSkov for their expert advice; all the members of the LCB fromCIIMAR, Porto, especially Alba Trilla, for their valuable helpduring the experimental period; and finally Ian Nicolas for hisindispensable technical support. This manuscript was improvedby editorial comments from anonymous reviewers.

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Physiological response of fucoid algae to environmental stress: comparing range centre and southern populations