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

Authors


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 physiological 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 conditions.
  • The negative effect of summer conditions on photophysiology may contribute to changes in fucoid abundance and distribution in southern Europe. Our results emphasise how physiological performance of geographically distinct populations can differ, which is particularly relevant when predicting responses to climate change.

Introduction

Fucoid algae are important primary producers on intertidal rocky shores 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 existence and survival of other intertidal species and therefore strongly influencing the structure and functioning of coastal ecosystems (Jenkins et al., 1999a, 2008). Their importance has led to their use as model species for ecological and ecophysiological studies of causes of distribution patterns (Baker, 1909, 1910) and development (Berger et al., 1994; Brownlee, 1994).

In the intertidal zone of the northeast Atlantic, abundance of macroalgae declines with latitude (Ballantine, 1961; Hawkins & Hartnoll, 1983; Jenkins et al., 2008), with fucoid cover and biomass decreasing rapidly near its southern limit of distribution in Portugal (Ballantine, 1961; Ferreira, 2012). Portugal is known as a biogeographical transition zone, a region where species composition changes markedly (Lüning, 1990; Southward et al., 1995; Lima et al., 2007; Tuya et al., 2012) and some fucoid species reach (e.g. Fucus vesiculosus (Nicastro et al., 2013) or approach (e.g. Fucus spiralis (Ribera et al., 1992) their southern limit of distribution. Intertidal species are subjected to stresses during low tides, which can take them close to their physiological tolerance threshold (Helmuth et al., 2006); such species can thus be especially susceptible to climatic stress, particularly towards their southern limits of distribution. For example, in Europe, a number of time series have been used to demonstrate significant changes in the abundance and range limits of intertidal species in response to climatic fluctuations (Southward et al., 1995; Mieszkowska et al., 2006; Hawkins et al., 2008; Poloczanska et al., 2008). Changes in assemblage composition and distribution of macroalgae have occurred in Portugal with a clear northward expansion of warm-water species (Lima et al., 2007). Range contractions of temperate macroalgal species, although not evident in Portugal (Lima et al., 2007), have occurred in areas adjacent to Portugal: the southern range edge of F. vesiculosus in Morocco (Dangeard, 1949; Benhissoune et al., 2002; Canovas et al., 2011) has recently retracted to Portugal and a decline was also recorded in the Bay of Biscay (Nicastro et al., 2013). These recent findings confirm results from previous studies, which showed a marked decline in the abundance of F. vesiculosus in northern Spain (Fernandez & Anadon, 2008; Lamela-Silvarrey et al., 2012) and suggest likely northward contraction of range edge populations in Portugal if the warming trend continues.

The distribution of fucoid algae in rocky intertidal areas is determined by a combination of physical and biological factors (Schonbeck & Norton, 1978; Lubchenco, 1980; Hawkins & Hartnoll, 1985; Menge, 2000; Jenkins et al., 2008). Variations in the magnitude, duration and frequency of wave forces create different environmental conditions, leading to the development of horizontal gradients of macroalgae abundance (Jones & Demetropoulos, 1968; Blanchette, 1997; Jenkins et al., 2008). Physical factors can also lead to vertical zonation; factors associated with tidal emersion have long been known to influence the vertical abundance of organisms in the rocky intertidal zone (Evans, 1948; Dring & Brown, 1982; Chapman, 1995). The pervasive influence of adverse physical conditions on algal distribution has been demonstrated numerous times; brown algae species such as F. spiralis, Pelvetia canaliculata, Ascophyllum nodosum and Laminaria digitata have all been shown to have their upper limit on 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 occurs in the summer, when mid-afternoon low-tide periods occur on clear, calm days (Helmuth et al., 2002). These conditions lead to harsh thermal and desiccation stresses, particularly at the upper levels of the shore (Doty, 1946; Davison & Pearson, 1996; Denny & Wethey, 2001). Solar radiance, when composed of high irradiances of photosynthetically active and ultraviolet radiation, can lead to oxidative stress and consequent photoinhibition in intertidal macroalgae (Hader & Figueroa, 1997; Flores-Moya et al., 1998; Figueroa & Viñegla, 2001). Photoinhibition occurs because oxidative stress prevents the synthesis of plastid encoded proteins, essential for the repair of photosystem II (PSII) machinery, leading to a consequent decrease in photosynthetic activity (Nishiyama et al., 2011; Takahashi & Badger, 2011). Such effects of light on the physiological performance of macroalgae may be aggravated, at local and regional scales, if climatic conditions promote harsher stressful regimes of temperature (Dromgoole, 1980; Altamirano et al., 2000; Helmuth et al., 2006). For example, Martinez et al. (2012) showed that warmer seawater temperatures can have a negative effect on growth rate of F. serratus. Overheating of tissues can have an impact on protein and membrane stability, as well as on enzymatic reaction rates, and can therefore affect physiological performance and growth rates of organisms (Lobban & Harrison, 1997; Chen et al., 2012; Martinez et al., 2012). In addition to effects of light and temperature on a local scale, such stressors influence the geographical distribution limits of fucoid taxa over large scales. Physical stressors may act additively, synergistically or antagonistically (Darling & Cote, 2008). If physical stressors act synergistically, they may have larger impacts and cause unexpected negative responses, as the response will be superior to that predicted from the effect of each individual stress (reviewed in Darling & Cote, 2008). Therefore, the exact contribution of each factor to the combined effect created by multiple interacting stressors needs to be further studied and understood (Darling & Cote, 2008; Martinez et al., 2012). The determination of stress levels that cause direct physical limitation leading to death and elimination of populations is certainly important, but nonlethal amounts of stress that can influence fitness and hence recruitment at range edges are probably equally important, especially for understanding the possible consequences of climate change (Davison & Pearson, 1996; Harley et al., 2012).

A useful way to understand and measure the complex effects of multiple factors is to perform experiments where multiple stressors may be tested simultaneously and thus allow for the detection of interactive effects among them. One of the most efficient means to measure the effect of stress in plants is the use of the nonintrusive pulse amplitude modulated (PAM) fluorometer. In particular, the maximum photosynthetic quantum yield (Fv/Fm) is used to estimate photoinhibition, as the balance of photodamage and repair (Krause & Weis, 1991; Hader & Figueroa, 1997; Figueroa et al., 2003). Fv/Fm has frequently been used and is well documented in fucoid experimental procedures, showing that different fucoid species or populations may have distinct tolerance thresholds to wave action, extreme temperatures, solar radiation 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 in Fucus 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 sympatric Fucus species from a single rocky shore in northern Portugal. Physiological resilience was consistent with the vertical distribution of fucoids on the shore (F. spiralis > F. guiryi > F. vesiculosus). The photophysiological resilience of central and southern-edge fucoid populations has also been compared. No regional differences in the physiological performance among F. vesiculosus populations were detected (Pearson et al., 2009; Zardi et al., 2013); however, a degree of maladaptation of southern F. serratus populations to desiccation and to high seawater temperatures (32 and 36°C) was shown (Pearson et al., 2009).

Given the clear decline in fucoid biomass from northern to southern European areas (Ballantine, 1961; Ferreira, 2012) and the presence of southern distributional limits of F. vesiculosus and F. spiralis in Portugal (Nicastro et al., 2013) and Morocco (Ribera et al., 1992; Southward et al., 1995), respectively, we aimed to test the effects of abiotic summer stress on fucoid physiological response capacity. Previous experiments were carried out under laboratory conditions with artificial light sources of low intensity. However, stressful conditions during emersion periods on the shore in the summer are likely to occur under the influence of high solar radiation, high air temperatures and low humidity (Martinez et al., 2012). Taking that into consideration, we performed manipulative experiments with a robust hierarchical design that allowed us to compare the physiological responses of two populations of F. vesiculosus or F. spiralis from two geographical origins to different combinations of light intensity and air temperature. The main aims of the experiments were to determine if stress from high temperature and high light intensities, experienced during low water periods, would lead to elevated amounts of stress in fucoids, independently of their origin, and to determine the nature of the interactions among abiotic stresses – are they additive or synergistic? By using populations from different origins and through the application of combined stress conditions, we evaluated the extent to which southern and central fucoid populations are different in terms of their physiological tolerance and performance when exposed to complex abiotic stresses. We hypothesize the existence of some local population adjustment to regional environmental pressures and therefore possible greater resistance to strong light intensity and high air temperatures in Portuguese populations, resulting from their exposure to longer and harsher summer conditions. We tested whether such regional variation is consistent among algae subjected to different stress conditions on the shore through the use of both high-shore (F. spiralis) and mid-shore (F. vesiculosus) species. The effects of abiotic stresses on the species used are expected to be stronger on the mid-shore species, as high-shore species 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 populations from Portugal and Wales. Two experiments were performed, one for Fucus vesiculosus (Linnaeus, 1753) and one for Fucus spiralis (Linnaeus, 1753) (both Ochrophyta, Fucales). For each experiment, two trials were conducted on consecutive days to assess the generality and repeatability of results. Stress manipulations were performed to assess the effects on overheating, desiccation resilience and, through the use of PAM fluorometry, photophysiological performance of fronds.

Collection

Vegetative fronds of F. vesiculosus and F. spiralis were collected from central and southern regions of their geographical distribution. The central populations were from northern shores located in Anglesey, north Wales, Porth Cwyfan (53.182821°N, 4.489829°W) and Cemlyn Bay (53.407460°N, 4.533636°W). In the south, the Portuguese populations came from Viana do Castelo (41.690403°N, 8.849988°W) and Carreço (41.716555°N, 8.866798°W). At each of the four shores, 48 F. vesiculosus and F. spiralis fronds from healthy individuals, c. 10–15 cm in length, were collected. F. vesiculosus plants were easily identifiable because of their vesicles, while F. spiralis fronds were collected very high up on the shore to avoid areas where the presence of fucoid hybrids was more probable. The collection of specimens was undertaken throughout August 2010 during low tide, on shores moderately exposed to wave action and easily accessible. This stratified approach allowed us to formally remove the influence of exposure from subsequent analyses. Within each geographical region, the shores were at least 3 km apart. They all had a typical mosaic patchy community, composed of fucoids, barnacles, bare rock and limpets, were gently sloping and exposed to full salinity.

Transport, acclimatization and maintenance

After collection, the algae were transported in dark, cold and humid conditions to the laboratory. The algae from Welsh shores were wrapped in blotting paper, to keep them in a hydrated condition, and transported to Portugal by plane in dark, cold and humid cool boxes. The transport took no more than 1 h from the shore to the laboratory and the air transport of Welsh fucoids to Portugal took less than 20 h. After the transport to Portugal, algae had a 15 d period of acclimatization in laboratory conditions to allow a steady growth response to be attained. There were no deleterious effects of transportation with respect to photophysiological performance, as Fv/Fm values measured at the start of the experiment remained high in Portuguese and Welsh algae (see the 'Results' section). All the individuals were cultured in 300 l tanks under ambient day-length conditions, reaching a maximum of 1400 μmol photons m−2 s−1, in aerated and circulating seawater controlled at 16°C by water refrigerators. Following recommendations described in Martinez et al. (2012), seawater was enriched twice a week to avoid nutrient limitation by adding inorganic nitrogen (NaNO3) and phosphorus (NaH3PO4) to final concentrations of at least 50 and 5 µM, respectively.

Experimental design and setup

For each of the four trials (two for F. vesiculosus and two for F. spiralis) the same design was used; algal fronds from two shores nested within each of two geographical regions (Wales and Portugal) were exposed to a factorial combination of two light intensities 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 in Portugal in warm cloudless summer conditions around noon on a rooftop location to allow the use of naturally high solar radiation (TrialFves1, 2038.41 ± 545.60; TrialFves2, 2185.50 ± 44.82; TrialFspi1, 2213.73 ± 184.15; TrialFspi2, 2141.06 ± 8.34 µmol photons m−2 s−1 (mean ± SD); = 24010; measured with a spherical quantum scalar sensor (QSL-2100, Biospherical Instruments Inc., San Diego, CA, USA)) and exposure to realistic ambient temperatures (TrialFves1, 21.50 ± 0.40; TrialFves2, 22.43 ± 0.72; TrialFspi1, 20.70 ± 0.60; TrialFspi2, 20.10 ± 2.08; °C; = 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, = 0.156), but there were significant differences in solar radiation (ANOVA, F3,1596 = 28.7, < 0.001). The difference between maximal and minimal means of solar radiation among days was still small (175.38 µmol photons m−2 s−1), indicating that conditions were similar.

The experimental design allowed simultaneous testing of orthogonal combinations of light intensity (natural Portuguese summer radiance values and a 65% reduction provided by shading material; see Martinez et al. (2012) for further details) and air temperature regimes (warm and cold), which was possible because the algae were kept in plastic containers (47 × 30 × 13 cm) sealed with UV-transparent methacrylate covers (Plexiglas GS2458; Evonik Rohm GmbH, Darmstadt, Germany). The range of physical conditions, air temperatures and light intensities, measured inside the experimental units was in accordance with values observed under field conditions at Welsh and Portuguese rocky intertidal areas during the summer season (verified through the parameters of earth surface skin temperature and earth photosynthetically available radiation data available from NASA Aqua (http://disc.sci.gsfc.nasa.gov/giovanni/overview/index.html) – AIRS standard and MODIS-Aqua missions, respectively). Half of the containers were exposed to full light and half were overlaid with two layers of neutral fibreglass mesh to reduce photosynthetic active radiation. By using an air conditioning unit, significant differences of mean air temperature between warm and cold treatments were achieved (ANOVA of test day, F1,12 = 84.9, < 0.001) (cold containers, 23.79 ± 2.70°C; warm containers, 32.09 ± 1.07°C; = 8) (Supporting Information, Table S1). All containers were connected to the air conditioning unit and air flow was maintained by an electric fan inside each container that created mean wind speeds of 2.8 m s−2 (as recommended by Martinez et al., 2012). Humidity inside the containers was also considered to remain at natural values. This is supported by results from a preliminary 3 d experiment showing that humidity conditions in containers linked to air conditioning were not significantly different from those in containers subjected to ambient conditions (ANOVA, F1,8 = 1.77, = 0.22).

Measurement of response variables

For each trial, 96 randomly selected vegetative fronds were blotted dry, cleaned with seawater and weighed the day before experimentally induced stress. Distal algal vegetative fronds of similar size were used to restrict the effects of biomass variation (F. vesiculosus, 1.79 ± 0.16 g; F. spiralis, 1.84 ± 0.16 g; = 192). The fronds were then left in seawater over night (12 h) in a 300 l tank with aerated and circulating seawater at 16°C. Just before application of experimental treatments, the fronds were subjected to a minimum of 25 min in dark conditions, whilst in seawater, to guarantee an equilibrium state of the photosynthetic electron transport chain before assessing for the first time the photosynthetic performance of the algae (pre-stress measurements).

The photosynthetic performance was assessed, on apices of the algae, as the ratio of variable to maximal Chl fluorescence, Fv/Fm, where Fv = Fm – F0, Fm is the maximal fluorescence and F0 is the initial fluorescence in dark-adapted algae (Krause & Weis, 1991). Measurements were made with a WATER-PAM Chlorophyll Fluorometer (Heinz Walz GmbH, Effeltrich, Germany) using saturating pulse intensities (800 ms, 4350 μmol photons m−2 s−1) for both fucoid species. The Fv/Fm ratio is an indicator of the maximal quantum yield of PSII photochemistry (Maxwell & Johnson, 2000; Baker, 2008), which responds to the alteration of optimum conditions. Variability of this ratio indicates that photosynthetic performance of algae is affected (Butler, 1978; Long et al., 1994; Baker & Oxborough, 2004).

The pre-stress measurement of Fv/Fm indicated the maximum quantum yield of the algae in optimum physiological conditions and was used directly as a response variable to test for the amounts of initial stress presented by fucoid algae from different populations. Samples were then left for an extra 30 min in the tanks exposed to natural light conditions, allowing the adaptation of the PSII reaction centres to natural sunlight in a hydrated environment. Algae were then transferred from the seawater tanks into aerial conditions in experimental units at the assigned temperature and light intensities for an exposure period of 75 min. Periods longer than 90 min resulted in the death of fronds (personal observation), probably because the single fronds used do not have the protection offered by self-covering. After 35 min, the surface temperature of each frond was directly measured with thermocouple thermometers (Easyview 15, Extech Instruments Corp., Nashua, NH, USA). At the end of the stress period, algae were blotted dry and weighed before being resubmerged in the 300 l tank with aerated and circulating seawater at 16°C for a rehydration period of 25 min. During this period, algae were kept in the dark to allow the relaxation and oxidation of reaction centres in the PSII (Gylle et al., 2009) before PAM measurements were carried out again. Algae were then left submerged for 16 h overnight to assess recovery in optimum conditions. They were then exposed to 25 min of dark conditions before the final PAM measurements were completed. These PAM measurements were used to evaluate the percentage of pre-stress Fv/Fm lost as a result of stress and exhibited after 16 h, through the following formulas:

display math(Eqn 1)
display math(Eqn 2)

Finally, samples were removed from the tank and dried at 60°C for 48 h in order to determine their DW. The quantification of hydrated, post-stress and dry weights allowed the water content to be determined at the different stages of each trial. The percentage of water in fucoid tissue before the application of stress was determined by measuring the hydrated and dry weights of specimens, while the percentage of initial water lost as a result of the stress period was estimated by incorporating the weight of the specimens after the stress period.

Data analysis

In order to analyse the results, each of the four trials was considered individually and the results across trials for each of the two species were qualitatively compared. The effects of treatments on response variables were analysed using mixed-model ANOVA with five factors allowing formal comparisons among geographical regions (fixed, two levels), shores (random nested in geographical regions, two levels), light intensities (fixed, two levels), air temperatures (fixed, two levels) and container (random nested in the interaction between geographical regions, shores, light intensities and air temperatures, two levels). Significant results were explored further with Student–Newman–Keuls (SNK) multiple comparisons (Underwood, 1997), with results from nested factors presented but not described in detail. An ANOVA with only two factors, geographical region and shore, was used for response variables measured before stress was applied (prestress Fv/Fm values and initial water content). Cochran's test was used to test the data for heterogeneity of variance (Cochran, 1951) and transformations were made where appropriate. All the analyses were performed using the statistical package WinGMAV5 (EICC, University of Sydney).

Results

Before application of stress

Water content of fronds

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

Figure 1.

Percentage of water, before stress application, in tissues of Fucus vesiculosus – TrialFves1 (a) and TrialFves2 (b) – and Fucus spiralis – TrialFspi1 (c) and TrialFspi2 (d) – from Portuguese and Welsh, UK, shores. Error bars, ± 1SE; significant difference: *, < 0.05; **, < 0.01. Via, Viana do Castelo; Car, Carreço; Cwy, Porth Cwyfan; Cem, Cemlyn Bay.

Pre-stress Fv/Fm values

The mean pre-stress values of Fv/Fm for F. vesiculosus and F. spiralis populations were naturally high (Fig. 2). No differences were detected between regions, although there were some significant differences, of small magnitude, in both fucoid species between Portuguese shores (ANOVAs, TrialFves1, F2,92 = 16.3, < 0.001; TrialFves2, F2,92 = 8.2, < 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/Fm measured are equivalent to those for healthy unstressed specimens observed in other studies (Magnusson, 1997; Pearson et al., 2000; Skene, 2004; Gylle et al., 2009) and indicate that the photosystems of specimens used were in a good state.

Figure 2.

Pre-stress Fv/Fm values in Fucus vesiculosus – TrialFves1 (a) and TrialFves2 (b) – and Fucus spiralis – TrialFspi1 (c) and TrialFspi2 (d) – from Portuguese and Welsh, UK, shores. Error bars, ±1SE; significant difference: **, < 0.01. Via, Viana do Castelo; Car, Carreço; Cwy, Porth Cwyfan; Cem, Cemlyn Bay.

During and after stress application

Frond surface temperature during stress

Frond surface temperatures were elevated, from 16°C in hydrated conditions to mean values as high as 35°C, as a result of both increasing temperature and light in both species after 35 min of stress application (Fig. 3). There was no interaction between the two stressors, but the effect of treatments was large (Table 1, Fig. 3). The effects sizes of the light treatment (other factors pooled) were 6.3°C (TrialFves1) and 8.1°C (TrialFves2) in F. vesiculosus and 8.1°C (TrialFspi1) and 10.2°C (TrialFspi2) in F. spiralis, while the effects sizes of temperature treatment (other factors pooled) were 7.1°C (TrialFves1) and 7.7°C (TrialFves2) in F. vesiculosus and 7.8°C (TrialFspi1) and 7.9°C (TrialFspi2) in F. spiralis (Fig. 3). For both species, there was a significant container effect (Table 1), although the SNK test of this factor showed there was no consistent pattern.

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 35 min to stress
SourcedfF. vesiculosusF ratio vs
TrialFves1TrialFves2
MSFPMSFP
Re12.84E+080.10.8250.140.00.944Sh (Re)
Sh (Re)24.50E+091.30.28922.310.50.634Co (Re × Sh(Re)  × Te × Li)
Te15.53E+1048.5 0.0201419.11293.5 0.003Te × Sh (Re)
Li14.53E+1021.7 0.0431564.1372.8 0.014Li × Sh (Re)
Co (Re × Sh(Re) × Te × Li)163.35E+0934.2 < 0.00147.6315.5 < 0.001Res
Re × Te11.46E+080.10.7552.190.50.570Te × Sh (Re)
Re × Li11.13E+080.10.83813.130.60.516Li × Sh (Re)
Te × Sh (Re)21.14E+090.30.7164.840.10.904Co (Re × Sh(Re) × Te × Li)
Li × Sh (Re)22.09E+090.60.54921.500.50.645Co (RexSh(Re)  × Te × Li)
Te × Li14.19E+081.00.41817.940.20.698Li × Te × Sh (Re)
Re × Te × Li15.61E+070.10.74735.890.40.591Li × Te × Sh (Re)
Li × Te × Sh (Re)24.09E+080.10.88689.201.90.186Co (Re × Sh(Re)  × Te × Li)
Res649.79E+07  3.07   
Cochran's test C = 0.1974, P > 0.05C = 0.1373, P > 0.05 
  F. spiralisF ratio vs
TrialFspi1TrialFspi2
MSFPMSFP
  1. Re, geographical region; Sh, shore; Te, air temperature; Li, light intensity; Co, container; Res, residual. Bold indicates P < 0.05.

Re1149.240.10.7433.050.30.640Sh (Re)
Sh (Re)21056.701.20.31710.240.10.868Co (Re × Sh(Re)  × Te × Li)
Te138 944.60491.1 0.0021487.5958.2 0.017Te × Sh (Re)
Li141 846.2266.4 0.0152485.75158.4 0.006Li × Sh (Re)
Co (Re x Sh(Re) × Te × Li)16856.0210.2 < 0.00171.4137.1 < 0.001Res
Re × Te1257.753.30.2130.110.00.953Te × Sh (Re)
Re × Li1981.321.60.3380.010.00.980Li × Sh (Re)
Te × Sh (Re)279.310.10.91225.570.40.705Co (Re × Sh(Re)  × Te × Li)
Li × Sh (Re)2630.360.70.49415.700.20.805Co (Re × Sh(Re)  × Te × Li)
Te × Li1630.760.40.5812.310.10.850Li × Te × Sh (Re)
Re × Te × Li164.710.00.8545.270.10.776Li × Te × Sh (Re)
Li × Te × Sh (Re)21476.611.70.21050.060.70.511Co (Re × Sh(Re)  × Te × Li)
Res6484.21  1.93   
Cochran's test C = 0.1918, P > 0.05C = 0.1329, P > 0.05 
Figure 3.

Frond surface temperature of Fucus vesiculosus – TrialFves1 (a) and TrialFves2 (b) – and Fucus spiralis – TrialFspi1 (c) and TrialFspi2 (d) – from Portuguese and Welsh, UK, populations after being exposed for 35 min to stress. Smaller graphs represent significant effects of fix factors on each trial: TrialFves1, effect of light intensity (a') and air temperature (a”); TrialFves2, effect of light intensity (b') and air temperature (b”); TrialFspi1, effect of light intensity (c') and air temperature (c”); TrialFspi2, effect of light intensity (d') and air temperature (d”). Error bars, ±1SE; significant difference: *, < 0.05; **, < 0.01. COL, cold; WAR, warm; UNS, unshaded (white bars where not labelled); SHA, shaded (shaded bars where not labelled).

Water lost as a result of stress (indicative of desiccation state)

Light intensity was a consistent factor determining the amounts of water loss for both fucoid species; plants exposed to full light showed consistently higher water loss than shaded plants across all trials (Table 2, Fig. 4). The differences in mean water loss 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 for light intensity. None of the interactions between these physical stressors were significant, suggesting additive effects. Comparison between regions generally did not show a difference in the amount of water lost (Table 2; TrialFves2, TrialFspi1 and TrialFspi2). However, in Trial Fves1, F. vesiculosus plants from Wales retained significantly more water than those from Portugal, although the magnitude of the differences was small (Table 2, Fig. 4).

Table 2. Mixed-model ANOVA of the percentage of water content lost as a result of the stress period (indicative of desiccation state) from Fucus vesiculosus tissues (TrialFves1 and TrialFves2) and from Fucus spiralis tissues (TrialFspi1 and TrialFspi2)
Sourcedf F. vesiculosus F ratio vs
TrialFves1TrialFves2
MS F P MS F P
Re12.3739.80.02478.100.10.851Sh (Re)
Sh (Re)20.060.50.5981708.8118.9 < 0.001 Co (Re × Sh(Re) × Te × Li)
Te11.325.20.1491044.32193.9 0.005 Te × Sh (Re)
Li15.0941.7 0.023 6741.38106.2 0.009 Li × Sh (Re)
Co (Re × Sh (Re) × Te × Li)160.111.40.17790.301.80.058Res
Re × Te10.010.00.896225.0041.8 0.023 Te × Sh (Re)
Re × Li10.010.10.813150.482.40.264Li × Sh (Re)
Te × Sh (Re)20.252.20.1395.390.10.942Co (Re × Sh (Re) × Te × Li)
Li × Sh (Re)20.121.10.36163.500.70.510Co (Re × Sh (Re)  × Te × Li)
Te × Li10.261.90.305204.373.70.196Li × Te × Sh (Re)
Re × Te × Li10.271.90.30316.910.30.638Li × Te × Sh (Re)
Li × Te × Sh (Re)20.141.30.31455.870.60.551Co (Re × Sh (Re) × Te × Li)
Res640.08  51.37   
Cochran's test C = 0.1889, P > 0.05C = 0.1687, P > 0.05 
SNK  Re × Te 
   War – Por > Wal 
   All Re – War > Col 
Sourcedf F. spiralis F ratio vs
TrialFspi1TrialFspi2
MS F P MS F P
  1. Complex post hoc SNK tests of significant differences are presented. Bold indicates P < 0.05.

  2. Re, geographical region (Por, Portugal; Wal, Wales); Sh, shore; Te, air temperature (Col, cold; War, warm); Li, light intensity; Co, container; Res, residual.

Re18.99E+100.10.81731 341.874.80.161Sh (Re)
Sh (Re)21.30E+122.30.1376587.280.20.790Co (Re × Sh (Re)  × Te × Li)
Te16.49E+1245.4 0.021 57 681.542.90.232Te × Sh (Re)
Li15.54E+13916.7 0.001 6.54E+0518.9 0.049 Li × Sh (Re)
Co (Re × Sh (Re)  × Te × Li)165.76E+111.40.18427 586.184.4 < 0.001 Res
Re × Te14.48E+080.00.9603757.420.20.707Te × Sh (Re)
Re × Li12.53E+114.20.177501.120.00.915Li × Sh (Re)
Te × Sh (Re)21.43E+110.30.78320 049.880.70.499Co (Re × Sh (Re)  × Te × Li)
Li × Sh (Re)26.04E+100.10.90134 608.201.30.312Co (Re × Sh (Re)  × Te × Li)
Te × Li13.07E+125.30.14836 051.553.50.201Li × Te × Sh (Re)
Re × Te × Li15.36E+110.90.43836 365.243.60.200Li × Te × Sh (Re)
Li × Te × Sh (Re)25.81E+111.00.38610 226.160.40.696Co (Re × Sh (Re)  × Te × Li)
Res644.19E+11  6267.12   
Cochran's test  C = 0.1967, P > 0.05C = 0.1961, P > 0.05 
Figure 4.

Percentage of water content lost as a result of stress (indicative of desiccation state) 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 represent significant effects or significant interactions of fix factors on each trial: TrialFves1, effect of light intensity (a') and geographical region (a”); TrialFves2, interaction of geographical region with air temperature (b'); TrialFspi1, effect of light intensity (c') and air temperature (c”); TrialFspi2, effect of light intensity (d'). Error bars, ±1SE; lowercase letters indicate comparison of responses of fucoid specimens from different geographical regions when exposed to equal air temperature independently of solar radiation intensity; significant difference: *, < 0.05; **, < 0.01. COL, Cold; WAR, Warm; UNS, unshaded (white bars where not labelled); SHA, shaded (shaded bars where not labelled).

Percentage of pre-stress Fv/Fm lost as a result of stress

Across all trials carried out, Fv/Fm values declined, as expected, when measured 25 min after the application of stress (Fig. 5). Fucoid specimens of both species, subjected to higher light intensities, lost a significantly greater percentage of pre-stress Fv/Fm than those confined to shaded containers (Table 3). The differences in the mean percentage of pre-stress Fv/Fm lost as a result of stress between 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, an effect of geographical region dependent on the interaction with air temperature and light controlled during the experimental procedure was also detected (Table 3; SNK of Re × Te × Li).

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 in Fucus spiralis tissues (TrialFspi1 and TrialFspi2)
Sourcedf F. vesiculosus F ratio vs
TrialFves1TrialFves2
MS F P MS F P
Re11165.4514.20.0644108.826.80.121Sh (Re)
Sh(Re)282.120.30.721606.701.00.381Co (Re × Sh (Re)  × Te × Li)
Te13142.0011.00.080779.360.80.456Te × Sh (Re)
Li164 386.88178.3 0.006 40 423.99105.6 0.009 Li × Sh (Re)
Co(Re × Sh (Re)  × Te × Li)16245.610.80.662591.643.3 < 0.001 Res
Re × Te1215.610.80.476126.480.10.747Te × Sh (Re)
Re × Li11312.983.60.1976813.6417.80.052Li × Sh (Re)
Te × Sh (Re)2285.221.20.338925.051.60.240Co (Re × Sh (Re)  × Te × Li)
Li × Sh (Re)2361.071.50.259382.660.70.537Co (Re × Sh (Re)  × Te × Li)
Te × Li1627.254.00.1822013.8312.40.072Li × Te × Sh (Re)
Re × Te × Li1544.973.50.20280.030.50.556Li × Te × Sh (Re)
Li × Te × Sh (Re)2155.290.60.544163.050.30.763Co (Re × Sh (Re) × Te × Li)
Res64300.75  177.34   
Cochran's test C = 0.1270, P > 0.05C = 0.1360, P > 0.05 
Sourcedf F. spiralis F ratio vs
Trial Fspi1Trial Fspi2
MS F P MS F P
  1. Complex post hoc SNK tests of significant differences are presented. Bold indicates P < 0.05.

  2. 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.

Re11974.276.40.127301.010.40.589Sh (Re)
Sh (Re)2308.781.10.344739.222.60.109Co (Re × Sh (Re)  × Te × Li)
Te11808.132.60.2501712.565.00.156Te × Sh (Re)
Li135 552.0694.9 0.010 54 811.7914 872.1 < 0.001 Li × Sh (Re)
Co (Re × Sh (Re)  × Te × Li)16270.601.40.152289.682.4 0.007 Res
Re × Te1208.240.30.641390.711.10.399Te × Sh (Re)
Re × Li1331.790.90.44630.238.20.103Li × Sh (Re)
Te × Sh (Re)2702.792.60.106345.871.20.329Co (Re × Sh (Re)  × Te × Li)
Li × Sh (Re)2374.531.40.2793.690.00.987Co (Re × Sh (Re)  × Te × Li)
Te × Li11590.157.60.111393.9457.40.017Li × Te × Sh (Re)
Re × Te × Li11.270.00.9451418.27206.5 0.005 Li × Te × Sh (Re)
Li × Te × Sh (Re)2210.040.80.4776.8680.00.977Co (Re × Sh (Re)  × Te × Li)
Res64187.76  120.75   
Cochran's test C = 0.1110, P > 0.05C = 0.1200, P > 0.05 
SNK  Re × Te × Li 
   Col × Uns – Wal > Por 
   War × Uns – Por > Wal 
   War × Sha – Wal > Por 
   Por × Uns – War > Col 
   Wal × Sha – War > Col 
   All Re at all Te – Uns > Sha 
Figure 5.

Percentage of pre-stress Fv/Fm lost as a result of stress in Fucus vesiculosus tissues – TrialFves1 (a) and TrialFves2 (b) – and in Fucus spiralis tissues – TrialFspi1 (c) and TrialFspi2 (d) – from Portuguese and Welsh, UK, populations. Smaller graphs represent significant interactions of fix factors on each trial: TrialFves1, effect of light intensity (a'); TrialFves2, effect of light intensity (b'); TrialFspi1, effect of light intensity (c'). Error bars, ±1SE; lowercase letters indicate comparison of responses of fucoid specimens from different geographical regions when exposed to equal air temperature and solar radiation intensity, while uppercase letters indicate comparison of responses of fucoid specimens from the same geographical regions when exposed to equal solar radiation intensity but also to different air temperatures. Significant differences: *, < 0.05; **, < 0.01. UNS, unshaded (white bars where not labelled); SHA, shaded (shaded bars where not labelled).

After a 16 h period of recovery

Percentage of pre-stress Fv/Fm exhibited 16 h after stress

Although Fv/Fm values improved from the values observed after stress, only algae exposed to low light intensities achieved complete or high degrees of recovery (Fig. 6). In high-light treatments, mean Fv/Fm values (mean ± SE; n = 48) after 16 h only corresponded to 71.8 ± 8.7% (TrialFves1) and 66.7 ± 9.4% (TrialFves2) in F. vesiculosus and 78.7 ± 6.7% (TrialFspi1) and 74.5 ± 7.5% (TrialFspi2) in F. spiralis of observed pre-stress levels (other factors pooled), respectively. In all trials there was a significant effect of light (Table 4, Fig. 6). This effect was consistent across regions and shores for F. spiralis, but varied by region for F. vesiculosus (Fig. 6). Specimens of F. vesiculosus collected in Portugal, which had previously been stressed in high-light conditions, achieved significantly greater degrees of recovery than individuals from Wales. The mean differences between F. vesiculosus specimens previously subjected to high-light conditions from Portugal and Wales were 20.2% and 11.5% in TrialFves1 and TrialFves2, respectively (Table 4; SNK of Re × Li, Fig. 6). Similarly, F. vesiculosus specimens collected in Portugal also achieved significantly greater degrees of recovery than individuals from Wales when subjected to low-light conditions (other factors pooled), although the effect sizes were smaller (TrialFves1, 1.7%; and TrialFves2, 2.6%) (Fig. 6).

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 in Fucus spiralis tissues (TrialFspi3 and TrialFspi4)
Sourcedf F. vesiculosus F ratio vs
TrialFves1TrialFves2
MS F P MS F P
Re13.32E+06117.2 0.008 1.45E+0625.1 0.038 Sh (Re)
Sh (Re)228295.970.20.81057801.300.10.869Co (Re × Sh (Re)  × Te × Li)
Te11.31E+069.60.0907.29E+054.30.175Te × Sh (Re)
Li12.33E+0788754.4 < 0.001 2.02E+0719928.2 < 0.001 Li × Sh (Re)
Co (Re × Sh (Re)  × Te × Li)161.33E+051.10.4094.07E+053.4 < 0.001 Res
Re × Te128166.210.20.6943.69E+052.20.279Te × Sh (Re)
Re × Li12.17E+068265.2 < 0.001 4.86E+05479.0 0.002 Li × Sh (Re)
Te × Sh (Re)21.37E+051.00.3801.71E+050.40.664Co (Re × Sh (Re)  × Te × Li)
Li × Sh (Re)2262.290.00.9981015.470.00.998Co (Re × Sh (Re)  × Te × Li)
Te × Li12.98E+054.10.1801806.350.00.910Li × Te × Sh (Re)
Re × Te × Li122 609.670.30.63343 770.770.40.594Li × Te × Sh (Re)
Li × Te × Sh (Re)272 467.410.60.5891.11E+050.30.765Co (Re × Sh (Re)xTe × Li)
Res641.25E+05  1.19E+05   
Cochran's test  C = 0.1944, P > 0.05C = 0.1975, P > 0.05 
SNK Re × LiRe × Li 
  All Li – Por > WalAll Li – Por > Wal 
  All Re – Sha > UnsAll Re – Sha > Uns 
Sourcedf F. spiralis F ratio vs
TrialFspi1TrialFspi2
MS F P MS F P
  1. Complex post hoc SNK tests of significant differences are presented. Bold indicates P < 0.05.

  2. Re, geographical region (Por, Portugal; Wal, Wales); Sh, shore (Via, Viana do Castelo; Car, Carreço; Cwy,Porth Cwyfan; Cem, Cemlyn Bay); Te, air temperature (Col, cold; War, warm); Li, light intensity (Uns, unshaded; Sha, shaded); Co, container; Res, residual.

Re12.31E+084.40.1713.50E+0619.3 0.048 Sh (Re)
Sh (Re)25.24E+070.40.6561.81E+051.00.401Co (Re × Sh (Re)  × Te × Li)
Te11.14E+0917.40.0532.41E+063.90.187Te × Sh (Re)
Li11.52E+101259.3 < 0.001 4.65E+07278.7 0.004 Li × Sh (Re)
Co (Re × Sh (Re)  × Te × Li)161.21E+081.40.1531.87E+050.70.785Res
Re × Te13.67E+070.60.53233565.230.10.837Te × Sh (Re)
Re × Li11.58E+071.30.3721.55E+069.30.093Li × Sh (Re)
Te × Sh (Re)26.54E+070.50.5926.18E+053.30.063Co (Re × Sh (Re)  × Te × Li)
Li × Sh (Re)21.21E+070.10.9061.67E+050.90.429Co (Re × Sh (Re)  × Te × Li)
Te × Li19.25E+0889.2 0.011 1.26E+061.40.363Li × Te × Sh (Re)
Re × Te × Li151692.210.00.9506.30E+050.70.496Li × Te × Sh (Re)
Li × Te × Sh (Re)21.04E+070.10.9189.23E+054.9 0.021 Co (Re × Sh (Re)  × Te × Li)
Res648.41E+07  2.68E+05   
Cochran's test  C = 0.1937, P > 0.05C = 0.1951, P > 0.05 
SNK Te × LiLi × Te × Sh(Re) 
  Uns – Col > WarVia × Uns – Col > War 
  All Te – Sha > UnsCwy × Uns – Col > War 
   Uns × Col – Via > Car 
   Uns × War – Cem > Cwy 
   All Sh (Re) × all TE (except Via × Col) – Sha > Uns 
Figure 6.

Percentage of pre-stress Fv/Fm exhibited 16 h after stress in Fucus vesiculosus tissues – TrialFves1 (a) and TrialFves2 (b) – and in Fucus spiralis tissues – TrialFspi1 (c) and TrialFspi2 (d) – from Portuguese (POR) and Welsh (WAL), UK, populations. Smaller graphs represent significant effects or significant interactions of fix factors on each trial: Trial Fves1, interaction of geographical region with light intensity (a'); TrialFves2, interaction of geographical region with light intensity (b'); TrialFspi1, interaction of air temperature with light intensity (c'). Error bars, ±1SE; lowercase letters indicate comparison of responses of fucoid specimens from different geographical regions (TrialFves1 and TrialFves2) or different air temperatures (TrialFspi1) when exposed to equal solar radiation intensity. Significant differences: **, < 0.01. COL, cold; WAR, warm; uns, unshaded (white bars where not labelled); sha, shaded (shaded bars where not labelled).

Variation in air temperatures during the stress period also affected photophysiological state after recovery in F. spiralis but not in F. vesiculosus (Table 4). Fv/Fm values of F. spiralis were greater in cold than in warm treatments, but only under certain conditions of light and/or shores from which specimens were collected (Table 4: TrialFspi1, SNK of Te × Li; TrialFspi2, SNK of Li × Te × 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 temperature (from the surface to a depth of 700 m) averaging 0.5 and 0.1°C, respectively (IPCC, 2007). Can the observed decline in the abundance of fucoid species in southern Europe and contractions in adjacent areas be linked with an increasing 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 are the local populations adapted to deal with those amounts of stress?

Solar irradiance is considered a major influence on distribution of plants at local and regional scales (Austin & Van Niel, 2011). An increase in solar radiance has been noted in the Iberian Peninsula over recent decades, owing to a decrease in cloud cover and an increase in sunshine duration (Calbo & Sanchez-Lorenzo, 2009; Sanchez-Lorenzo et al., 2009, 2013). The importance of solar radiance for algae has previously been shown by studies describing patterns 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 photosynthetic algae species. In our study, the photophysiological response of fucoid algae to stressful conditions indicates that harsh summer conditions can influence the physiological capacity of F. vesiculosus and F. spiralis. Specimens of both species exposed to high light intensities during emersion in our trials lost a greater photosynthetic capacity and showed increased frond temperature and desiccation when compared with specimens subjected to shaded treatments, following the results also observed by Martinez et al. (2012) for F. serratus. This is especially evident when high intensities of solar radiation reach the fronds, as specimens subjected to such treatments were not able to physiologically recover 100% of their photosynthetic capacities even after 16 h in hydrated conditions (a period approximately twice that experienced in the field). When subjected to low light intensities, both fucoid species were capable, after the recovery period in hydrated conditions, of regaining a photosynthetic performance very similar to that shown before the application of stress. These results demonstrate the importance of ameliorated conditions and the impact that stronger solar radiation can have on the photosystem of fucoid algae. The fact that the Fv/Fm values of specimens subjected to high light intensity were not completely re-established suggests damage of the electron transport chain by oxidation or denaturalization of pigments and proteins at the PSII, probably as a result of the insufficient capacity of photoprotection mechanisms (Nishiyama et al., 2011; Takahashi & Badger, 2011). Excessive radiation can affect concentrations of reactive oxygen species that prevent repair of PSII machinery, leading to decreased photosynthetic activity (Collen & Davison, 2001; Nishiyama et al., 2011), which ultimately could limit the rates of resource acquisition, growth, reproductive capacity 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 of fucoid species under emerged conditions were also clear in our study, similar to previous work in southern Europe (Zardi et al., 2011; Martinez et al., 2012). Results from both species indicate that warmer air conditions, such as those observed during hot summer days throughout southern Europe, promote desiccation and lead to the decline of Fv/Fm values observed after stress, limiting the photosynthetic recovery success over time. The influence of air temperature variation was most pronounced on the control of frond temperature, contributing to tissue overheating during the experiments. This is critical, as increased tissue temperature can lead to a reduction in growth rates and to changes in the chemical composition of algae, owing to its effects on protein denaturation, kinetics of cellular enzymes, membrane stability and active influence on membrane transport (Lobban & Harrison, 1997; Chen et al., 2012; Martinez et al., 2012).

Our experimental design allowed us to establish that solar radiation and air temperature act additively for the values tested. It should be pointed out that maximal air temperature and solar radiation, naturally reached in Portugal, can be higher than those used in our experiment and therefore impacts on physiological performance may be greater than those observed. Interactions in some conditions still appeared, but not as a generalized phenomenon, and where present were associated with a response of low magnitude. Therefore, we propose that solar radiation and air temperature can be seen as important additive, rather than interactive, summer emersion stresses acting on F. vesiculosus and F. spiralis populations. This idea is supported by Martinez et al. (2012), who also showed no synergistic effects acting on F. serratus populations at southern limits. This is a particularly relevant point, as additivity of abiotic stresses is normally assumed in the development of species distribution models (SDMs) (Darling & Cote, 2008), which attempt to predict species biogeographic shifts in response to climatic change.

Our results further indicate that the ability to tolerate amunts of summer emersion stress is dependent on the species concerned, with results reflecting, as we hypothesised, their position in the intertidal. As proposed by Ji et al. (2005), it seems that it is the ability to withstand desiccation stress that determines the distribution of intertidal seaweeds on the shore. Previous work has shown that tolerance to climatic variability and the capacity to tolerate desiccation is greater in F. spiralis than in fucoid species living on lower areas of the shore (Schonbeck & Norton, 1978; Dring & Brown, 1982). Dring & Brown (1982) showed that F. spiralis specimens were capable of a complete recovery of photosynthetic levels, even when subjected to tissue water loss of 80–90%, a result similar to those presented here for specimens of both species subjected to shaded conditions. Comparison of the results of our experiments, run separately on different fucoid species, seems to support our initial expectation, as F. spiralis specimens, when subjected to an environment with high air temperature and strong solar irradiance, were able to recover photosynthetic performance more efficiently than specimens of F. vesiculosus, despite displaying similar levels of desiccation and frond overheating after stress application. These results, comparable to those of Zardi et al. (2011), are in accordance with the vertical distribution of these two fucoid species on the shore and could form part of the explanation as to why F. vesiculosus has been reported to be retracting in areas adjacent to Portugal, such as Morocco and northern Spain, while F. spiralis has not.

Our comparative experimental approach shows that, within the same fucoid species, specimens from southern and central populations can have distinct physiological tolerances and performances when exposed to similar abiotic stresses. Harsh conditions of high air temperature and solar radiation vary between regions and occur more frequently in southern than in northern European regions (validated by mean photosynthetically available radiation and mean air temperature data from NASA Aqua (http://disc.sci.gsfc.nasa.gov/giovanni) – AIRS standard & MODIS-Aqua missions, respectively). Our results suggest that the photosynthetic systems of specimens from northern populations of F. vesiculosus were significantly more affected than the ones from southern populations, a result that contrasts with previous physiological studies that showed no regional differentiation in the physiological response of F. vesiculosus (Pearson et al., 2009; Zardi et al., 2013). These contrasting results may be explained by the nature of the stress provided in our experiments, which allowed for investigation of the additive effects of temperature and solar radiance. When strong light intensities were applied in our experiments, the effect of these stressful summer emersion conditions was felt on all specimens, but the effects were more marked in Welsh than in Portuguese populations. This was particularly evident for values of Fv/Fm sustained by F. vesiculosus specimens after 16 h of recovery from stress. For F. spiralis, differences between central and southern populations, observed in Fv/Fm values immediately after stress, are not so obvious and consistent, reflecting the greater physiological resilience of this species (Schonbeck & Norton, 1978; Dring & Brown, 1982; Chapman, 1995). Differences in physiological capacity among northern and southern populations may reflect phenotypic plasticity of species or the strong effect of natural selection by environmental factors on genetically distinct fixed traits. These alternatives cannot be distinguished by our study and require additional work using a true common garden approach that eliminates long-term carryover effects, supplemented by molecular approaches, to determine if this is a case of phenotypic plasticity or genotype × environment interactions, which are the essence of local adaption (Scheiner, 1993; Rutter & Fenster, 2007; Bergmann et al., 2010). Despite greater resistance to stress of southern algae, the fact that F. vesiculosus and F. spiralis specimens from Portuguese populations are clearly physiologically stressed and do not recover for a considerable time period may have important consequences. The conditions of solar radiation and air temperature used in our experiments are experienced regularly and often on consecutive tides in Portugal. Environmental stress may therefore have a significant nonlethal effect on Portuguese algae, with consequent negative effects on fitness. Such nonlethal effects have been observed in F. vesiculosus through a reduction in reproductive capacity in Iberian populations (Viejo et al., 2011; Ferreira, 2012) and no doubt contribute to the generally low level of recruitment and adult abundance in southern Europe.

In summary, the present study further clarifies which proximate ecological processes may cause the observed decline of some fucoid species near their southern limits of distribution (Ballantine, 1961; Hawkins & Hartnoll, 1983; Jenkins et al., 2008; Ferreira, 2012). Biological processes, such as grazing pressure, competition and facilitation, have been shown to influence fucoid abundance (Jenkins et al., 1999b, 2005; Arrontes et al., 2004; Coleman et al., 2006). However, the experiments performed here clearly demonstrate that physical stress, mainly exposure to the additive effects of high air temperatures and high intensities of solar radiation caused by emersion on warm cloudless summer days, has an important nonlethal effect on photophysiology, frond temperature and desiccation of specimens, affecting their physiological performance and potentially leading to reductions in abundance of F. vesiculosus and F. spiralis. Our results also demonstrate that the ability to tolerate summer emersion stress varies with both species identity and geographical origin of populations. Greater resilience to emersion stress at the studied area is apparent in F. spiralis, the species living higher on the shore. Furthermore, we show how fucoid populations inhabiting geographical regions with distinct climatic backgrounds can have differential photosynthetic capacities and consequently show different degrees of resistance when exposed to equal amounts of physical stress. This observation, combined with the fact that genetic differentiation has been shown in populations exposed to different physical conditions (Billard et al., 2005), highlights the importance of considering differences between populations, especially when analysing effects of climate change or local anthropogenic stresses.

The capacity to identify the multiple drivers and comprehend their possible effects on fucoid species abundance in southern European regions is essential for understanding and predicting the consequences of future climatic variations on the biogeographical ranges and intertidal primary productivity. Given the importance of these fucoid species as intertidal ecosystem engineers and primary producers (Hawkins et al., 2009), the acquisition of such information is essential for successful projections of future broad ecological changes on the intertidal ecosystem (Darling & Cote, 2008). Therefore, we propose future studies to focus on the clarification of the processes controlling the distribution of ecologically important species, as such knowledge will allow advanced predictions of the consequences of eventual climatic alterations on the intertidal ecosystem balance.

Acknowledgements

Our research was supported by a PhD grant from Fundação para a Ciência e Tecnologia (FCT) (SFRH/BD/41541/2007) awarded to J.G.F. and by the FCT project PHYSIOGRAPHY (PTDC/MAR/105147/2008 cofunded by FEDER through the program COMPETE-QREN). Support to B.M. was provided by the project CGL2010-19301 funded by the Spanish Ministry of Science. We would also like to thank Dr Bruno Jesus and Dr Martin Skov for their expert advice; all the members of the LCB from CIIMAR, Porto, especially Alba Trilla, for their valuable help during the experimental period; and finally Ian Nicolas for his indispensable technical support. This manuscript was improved by editorial comments from anonymous reviewers.

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