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Marine Environmental Research 101 (2014) 8e21

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Marine Environmental Research

journal homepage: www.elsevier .com/locate /marenvrev

Living in a coastal lagoon environment: Photosyntheticand biochemical mechanisms of key marine macroalgae

Marta García-S�anchez a, *, Nathalie Korbee b, Isabel María P�erez-Ruzafa c,Concepci�on Marcos a, F�elix L. Figueroa b, �Angel P�erez-Ruzafa a

a Departamento de Ecología e Hidrología, Facultad de Biología, Campus de Espinardo, Regional Campus of International Excellence “Campus MareNostrum”, Universidad de Murcia, Murcia 30100, Spainb Departamento de Ecología y Geología, Facultad de Ciencias, Universidad de M�alaga, M�alaga 29071, Spainc Departamento de Biología Vegetal I, Facultad de Biología, Universidad Complutense de Madrid, Madrid 28040, Spain

a r t i c l e i n f o

Article history:Received 11 April 2014Received in revised form22 July 2014Accepted 29 July 2014Available online 7 August 2014

Keywords:MacroalgaeCoastal lagoonChlorophyll fluorescencePhotosynthesisEcophysiologyAcclimation

* Corresponding author. Tel.: þ34 868 884326; faxE-mail address: martagarcia@um.es (M. García-S�a

http://dx.doi.org/10.1016/j.marenvres.2014.07.0120141-1136/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

The physiological status of Cystoseira compressa, Padina pavonica and Palisada tenerrima was studied byin vivo chlorophyll fluorescence, pigment content, stoichiometry (C:N), accumulation of UV photo-protectors and antioxidant activity; comparing their photosynthetic response in a coastal lagoon (MarMenor) and in Mediterranean coastal waters. In general, the specimens reached their highest ETRmax inspring in the Lagoon, but in summer in the Mediterranean, coinciding with their maximum biomasspeak. The species exhibited a dynamic photoinhibition. Except C. compressa, they showed a lowerdecrease in Fv/Fm and higher recovery rates in the Mediterranean populations when exposed to highirradiance. The higher salinity and temperature of the lagoon could impair the photoprotection mech-anisms. The acclimation to lagoon environments is species-specific and involves complex regulatorymechanisms. The results underline the importance of N in repair, avoidance, quenching and scavengingmechanisms. In general, Lagoon specimens showed higher pigment concentration. Although xantho-phylls play important photo-protective and antioxidant roles, the observed trend is more likely to beexplained by the higher temperatures reached in the lagoon compared to Mediterranean. Therefore thestudied photosynthetic and biochemical mechanisms can be effective not only for high irradiance, butalso for higher temperatures in a climate change scenario, but are highly dependent on nutrientavailability.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Coastal lagoons are part of a continuum between continentaland marine aquatic ecosystems and are characterised by beingshallow and relatively isolated from the open sea by coastal barriersthat provide some connecting channels or inlets, making themsubject to natural constraints (P�erez-Ruzafa et al., 2011a). In la-goons, the heterogeneity in environmental conditions may respondto a complex pattern of gradients, which contribute to the highbiodiversity and biological productivity housed in these environ-ments (Alongi, 1998; P�erez-Ruzafa et al., 2011a). Strong physicaland ecological gradients (UNESCO, 1981) make them dynamicsystems controlled and subsidized by physical energies. So, mostcoastal lagoons correspond to the type of coastal ecosystem that is

: þ34 868 883963.nchez).

characterised by frequent environmental disturbance and envi-ronmental fluctuations (Barnes, 1980; Kjerfve, 1994).

Submerged macrophytes living in coastal lagoons thus have tocope with large and frequent changes in their environment bymeans of morphological, physiological and life-cycle adaptations(Brock, 1986; Men�endez and Comin, 1989; Men�endez et al., 2002).Marine macroalgae in shallow waters need mechanisms for short-term acclimation to these fast changes. The most important arethose mechanisms involved in protecting PSII from photo-oxidativedamage. Carotenoids, the xanthophyll cycle and non-photochemical quenching (NPQ) are central constituents of suchprotection mechanisms (Andersson et al., 2006). Moreover, theaccumulation of other photoprotective and antioxidant com-pounds, such as UV screen photoprotectors with antioxidant ac-tivity, mycosporine-like amino acids (MAAs) and phenols, couldalso be important (Abdala-Díaz et al., 2006; Korbee et al., 2006).

The Mar Menor is a choked, relatively deep and hypersalinelagoon, with mean salinity ranging between 38.5 and 47.7 psu,

M. García-S�anchez et al. / Marine Environmental Research 101 (2014) 8e21 9

while the salinity in the Mediterranean ranges between 31.4 and40.7 psu. Although the lagoon is threatened by several pressures,with the detrimental impact on the natural community structureand dynamics increasing in recent decades, it still maintains highlevels of water quality and well-structured assemblages. Adescription of the hydrographic characteristics and the effects ofrecent human activities can be found in P�erez-Ruzafa et al. (1991;2005a). Spatial variations exist regarding isolation from the sea (orconfinement), and a vertical zonation of macrophytes and otherbenthic assemblages (P�erez-Ruzafa et al., 2007, 2008), as well as ahigh spatial heterogeneity and temporal fluctuations in water col-umn parameters (P�erez-Ruzafa et al., 2005b). Despite its relativeoligotrophy compared with other coastal lagoons, nutrient con-centrations in the Mar Menor are higher than in the studied coastalMediterranean area. A comparison between the environmentalconditions in the sampling stations can be found in P�erez-Ruzafaet al. (2007). Environmental stress gradients affecting algal as-semblages in the Mar Menor have been reported in a previousstudy (García-S�anchez et al., 2012).

While a specific lagoon flora does not exist and most salinecoastal lagoons contain typical coastal marine species (P�erez-Ruzafa et al., 2011b), there is a great heterogeneity in their spe-cies compositione very few species (7.3% of the total) are shared bymore than 10 lagoons in the Atlanto-Mediterranean area (P�erez-Ruzafa et al., 2011c). Despite this, coastal lagoons have well differ-entiated algal assemblages in relation to the open coastal sea inregard to the relative abundance and distribution of their species(P�erez-Ruzafa et al., 2008).

Several previous studies have focused on the effect of temper-ature, salinity and irradiance on the physiological response of somemacroalgal species (Connan and Stengel, 2011; Dawes et al., 1999;Eggert et al., 2007; Figueroa et al., 2014; Lüder et al., 2001;Men�endez et al., 2002; Zou and Gao, 2013). However, only someattempts have been made to evaluate the acclimation capacity ofthe same species living in different environments (Andersen et al.,2013; Eggert et al., 2006; Karsten et al., 1993; Ursi et al., 2003).Moreover, it is worth stressing that most of the previous studieswere based on culture experiments and not on field measurementssuch as those carried out in the present work.

In this study, we examine the photosynthetic and biochemicalmechanisms of three dominantmarine keymacroalgae living in theMar Menor lagoon, where species are exposed to wider thermalranges and higher salinity conditions than in marine Mediterra-nean ecosystems. Themain objective is to determinewhether thereare differences at the physiological and biochemical level betweenpopulations of the same species living in these two environmentswith different environmental characteristics: lagoonal stress vs theconditions found in Mediterranean coastal waters.

2. Material and methods

2.1. Study sites

The Mar Menor is a hypersaline coastal lagoon, located in thesoutheast of Spain (37� 420 N; 0� 470 W), with a surface area of135 km2, a mean depth of 3.6 m and maximum depth of about 6 m.A dense meadow of the seaweed Caulerpa prolifera (Forsskål) J.V.Lamouroux covers both the central area of the lagoon and theshallow muddy zones of low hydrodynamism, while in sandyshallow bottoms at depths of between 0 and 2 m, there are sparsepatches of the seagrass Cymodocea nodosa (Ucria) Asch. There aresome areas of natural rocky bottoms around the islands locatedwithin the lagoon and some calcareous and volcanic outcrops, inaddition to some artificial breakwaters, where photophilous rockyshore communities can grow (P�erez-Ruzafa et al., 2008).

Cabo de Palos study area (37� 380N; 0� 420W) is located close tothe Cabo de Palos-Islas Hormigas Marine Protected Area and rep-resents the northern boundary of the so called Almería-Or�an front.It also presents a great diversity of rocky assemblages and well-preserved seagrasses meadows, mainly composed by Posidoniaoceanica (L.) Delile and C. nodosa.

2.2. Biological material

Three macroalgae were selected to perform this study: Cys-toseira compressa (Esper) Gerloff and Nizamuddin (Fucales,Phaeophyceae), Padina pavonica (Linnaeus) Thivy (Dictyotales,Phaeophyceae) and Palisada tenerrima (Cremades) Serio, Cormaci,G. Furnari and Boisset (Ceramiales, Rhodophyta). Species werechosen on the basis of their key ecological role in rocky photo-philous habitats and for being present in both the Mar Menorlagoon and Mediterranean coastal systems.

The thick leathery C. compressa and P. tenerrima have complexmorphology and thallus with cortical and medullar cells, while thecorticated foliose P. pavonica presents a slightly calcified simplethallus.

C. compressa is present in the upper infralittoral zone of opencoastal waters in the Mediterranean, while in the Mar Menor itdominates the infralittoral zone of the confined area of the lagoon,with higher environmental stability. P. pavonica can be found in theinfralittoral zone both in the Mediterranean and in the Mar Menorlagoon. Finally, P. tenerrima inhabits the midlittoral zone alongMediterranean coasts, occupying the upper infralittoral in thelagoon.

2.3. Sample design

Specimens of C. compressa, P. pavonica and P. tenerrima werecollected from six locations (S.1eS.6). These sampling stations wererepresentative of three different environments: (1) the southernbasin of the Mar Menor, considered the most isolated and confinedarea (Lagoon: S.1, S.2), (2) the zone influenced by the Mediterra-nean Sea, close to the main channels through which water ex-change between the lagoon and the open sea takes place (Inlets:S.3, S.4) and (3) open coastal sites in Mediterranean Sea (Mediter-ranean: S.5, S.6) (Fig. 1). C. compressa was not found in the inletlocations while P. pavonica was only absent from one of the inletstations (S.4).

Biological samples were collected in two seasons, summer 2010and spring 2011, according to the period of maximal growth ratesand maximal biomass values for the selected species in both en-vironments (Hegazi, 1999; P�erez-Ruzafa et al., 2008, 2007).

Photosynthesis was measured in situ using a portable pulseamplitude modulation (PAM) fluorometer (Diving-PAM, Walz,Germany). Samples for biochemical analyses were collected andimmediately frozen in liquid nitrogen and stored at �80 �C toanalyse phenolic compounds, antioxidant activity and photosyn-thetic pigments. Samples for analysing mycosporine-like aminoacids (MAAs) and internal C and N were kept desiccated untilanalysis.

2.4. Environmental parameters

Temperature and salinity in the water column were recordedmonthly from January 2010 to December 2011 using a multi-parametric probe (YSI 6600), and samples for analysing nutrientconcentration were collected and transported to the laboratory indark and cold conditions. Nitrate (NO3

�), nitrite (NO2�), ammonium

(NH4þ) and orthophosphate (PO4

3�) were determined using an

Fig. 1. Map showing the location of the three different sampled areas and the sampling stations.

M. García-S�anchez et al. / Marine Environmental Research 101 (2014) 8e2110

automated wet chemistry analyser(AutoAnalyzer 3 HR, SEAL Ana-lytics) applying standard colorimetric procedures.

2.5. Light measurements

Global solar radiation for the study period was provided by theSpanish Meteorological Agency (AEMET), and photosysntheticallyactive radiation (PAR) was calculated from the data by applying acorrection factor, according to Jacovides et al. (2003). Lightextinction coefficients (Kd) of the water column were calculatedusing a Hyperspectral Irradiance Sensor for PAR (Ramses, TrioSGmbH, Oldenburg, Germany).

2.6. Photosynthetic activity as in vivo chlorophyll fluorescence

Minimum (Fo) and maximum (Fm) fluorescence were deter-mined after 15 min in darkness to obtain the maximal quantumyield ((Fm�Fo)/Fm) (Figueroa et al., 2003; Schreiber et al., 1995).

Rapid light curves (RLCs) can provide a reliable assessment ofphotosynthetic activity, reflecting the short-term light history ofalgae, and are designed to characterize a dynamic response in arapidly changing light environment in the field (Herlory et al.,2007; Ralph and Gademann, 2005). In order to determine the

optimal incubation time to reach steady state conditions of effec-tive quantum yield and ETR, algae were incubated under increasingintensities for 10, 15, 20, 30, 90 and 180 s at each actinic light. Nosignificant differences were found (data not shown) and so 20 s wasselected, which is also the most common incubation time used inthe literature.

RLCs were conducted in 10 mL incubation chambers, wherealgae were exposed for 20 s to eight incremental irradiances (20,66, 137, 224, 337, 469, 693, 942 mmol photons m�2 s�1) of actiniclight (internal Diving-PAM halogen lamp). The RLCs (n ¼ 4 persampling site) were obtained in order to determine the electrontransport rates (ETR) through PSII for each level of actinic light:

ETR�mmol electrons m�2s�1� ¼ ��

F 0m � F��

F 0m�$E$A$FII

(F0meF)/F0m estimates the effective quantum yield of PSII, E is theincident irradiance. A is the absorbance and FII is the fraction ofchlorophyll associated to photosystem II, being 0.8 in the case ofbrown macroalgae and 0.15 in red macroalgae (Grzymski et al.,1997; Johnsen, personal comment). The absorbance (A) wascalculated using the following equation (Beer et al., 2000):

A ¼ 1� T � R

M. García-S�anchez et al. / Marine Environmental Research 101 (2014) 8e21 11

Where T is the transmittance (T ¼ Et/Eo), Eo being the incidentirradiance and Et the transmitted irradiance with the algae locatedon the light sensor, estimated according to Figueroa et al. (2009).Reflectance (R) was measured with an integrating sphere (UNIKON900, KP 95-90136) according to Hegazi (1999) and values wereassumed to be negligible, except for P. pavonica, which had areflectance of 22%. Absorbance values (n ¼ 8) were 0.94 ± 0.03 forC. compressa, 0.60 ± 0.01 for P. pavonica and 0.92 ± 0.06 forP. tenerrima.

RLCs data were fitted to the model describe by Jassby and Platt(1976) to obtain values for the initial slope (aETR) and maximal ETR(ETRmax). Saturation irradiance (EkETR) was calculated asEkETR ¼ ETRmax/aETR. All RLCs were fitted using KaleidaGraph v 4.0.(Sinergy Software).

2.7. Non-photochemical quenching

Energy dissipation can also be driven by non-photochemicalquenching (NPQ), which was calculated using the fluorescenceparameters obtained during the RLCs (Fm and F'm) according toKlughammer and Schreiber (2008) as.

NPQ ¼ �Fm � F 0m

��F 0m

Maximal NPQ (NPQmax) and the initial slope of NPQ versusthe irradiance function (aNPQ) were obtained from the NPQversus irradiance function by using the fitting model proposedby Jassby and Platt (1976). The saturation irradiance for NPQ(EkNPQ) was calculated from the intercept between NPQm andaNPQ.

In the case of the red alga P. tenerrima, analysis of the variation ofthe fluorescence signal requires special caution due to the presenceof phycobilisomes. Nevertheless, NPQ was determined using thesame methodology as in previous works (Burdett et al., 2012;Eggert et al., 2007; Figueroa et al., 2014).

2.8. Internal carbon and nitrogen contents

Total internal C and N of algae samples were determined bycombustion, using an elemental analyser CNHS LECO-932 (Michi-gan, USA).

2.9. Photosynthetic pigments

The chlorophyll a (Chl a), chlorophyll c (Chl c) and carotenoidswere identified and quantified by high-performance liquid chro-matography (HPLC) following Lubi�an and Montero (1998) andGarcía-S�anchez et al. (2012). Pigments were extracted from algalsamples using 15 mg fresh weight (FW; n ¼ 3) in 1 mL of N, N-dimethylformamide (DMF) and maintained in darkness at 4 �C for12 h. Chlorophylls and carotenoids were identified using com-mercial standards (DHI LAB Products).

Phycobiliproteins for P. tenerrima (50 mg FW; n ¼ 3) wereextracted at 4 �Cwith 1.5 mL of 0.1 M phosphate buffer (pH 6.5) andcentrifuged at 4000 rpm for 30 min. Phycoerythrin and phycocy-anin concentrations were calculated following Sampath-Wiley andNeefus (2007). Results were expressed as mg g�1 dry weight (DW)after determining the fresh to dry weight ratio in the tissue.

2.10. Phenolic compounds

Samples of C. compressa and P. pavonica (250 mg FW; n ¼ 3)were ground with a mortar and pestle in sand at 4 �C, and extractedovernight in centrifuge tubes with 2.5 ml of 80% (v/v) methanol.The mixture was centrifuged at 4500 rpm for 20 min and the

supernatants were collected. Total phenolic compounds, expressedinmg g�1 DW, were determined using phloroglucinol as a standard,following Folin-Ciocalteu's method (Folin and Ciocalteu, 1927).

2.11. Mycosporine like amino acids (MAAs)

Samples (10e20mg DW) of P. tenerrimawere extracted for 2 h inscrew-capped centrifuge vials filled with 1 mL 20% aqueousmethanol (v/v) at 45 �C. Also the concentration and composition ofdifferent MAAs were analysedwith a Waters 600 HPLC system(Waters Cromatografía, Barcelona, Spain) according to Korbee-Peinado et al. (2004).

2.12. Antioxidant capacity: DPPH assay

The DPPH (2, 2-diphenyl-1-picrylhydrasyl) free-radical scav-enging assay was carried out in triplicate, according to the methodof Brand-Williams et al. (1995) in samples of C. compressa andP. pavonica. Briefly, 150 mL of each 80% methanolic extract, obtainedas explained in Section 2.11, were mixed with 150 mL of a 90%methanolic DPPH solution prepared daily at 1.27 mM. The reactionfinished after 30 min in the dark at room temperature, and theabsorbancewas read at 517 nm. The antioxidant activity of seaweedextracts was expressed as EC50 value (oxidation index), whichrepresents the concentration of the extract (mg DW alga ml�1)required to scavenge 50% of the DPPH in the reactionmixture. Thus,a lower EC50 means higher antioxidant activity.

2.13. Exposure-recovery experiments

The kinetics of exposure and recovery under strong sunlightwas studied in plants collected at around 11:00 h (local time).Plants were transferred from their natural setting to the surface,where they were exposed in situ to stronger natural solar irradi-ances than in their natural habitats. Algal samples from eachspecies were incubated for 2.5 h at midday (12:30e15:00 h; localtime) in 30 � 50 � 17 cm white plastic trays filled with seawater.Maximum quantum yield (Fv/Fm) of the algal samples was deter-mined in situ every 30 min during the exposure period. A recoveryphase of 3 h under dark conditions followed this period, afterwhich fluorescence was again determined at 15:30, 16:00, 17:00and 18:00 h. Water temperature in the trays and at the seashorewere measured during the exposure time using a HOBO U22Water Temp Pro v2 logger (Onset Computer Corporation, Massa-chusetts, USA). Water was periodically renewed to avoid anexcessive increase in temperature as well as oxygen or carbonlimitation.

Photoinhibition and recovery rate constants were calculated byfitting the Fv/Fm values (both during the exposure to high light andduring the recovery period) to the equations proposed by Hanelt(1998).

The inhibition phase is described by:

Yinh ¼ Pfast$eð�Kfast$tÞ þ Pslow$eð�Kslow$tÞ

Pfast þ Pslow ¼ 1 at t ¼ 0�t ¼ time

�

where Y represents the quantum yield, Pfast and Pslow represent theproportions of the fast and slow inhibition phase, respectively, andKfast and Kslow their respective rate constants.

The recovery phase is described by:

Yrec ¼ Fv�Fm �

hPfast$eð�Kfast$tÞ þ Pslow$e

ð�Kslow$tÞi

Fig. 2. Seasonal dynamics of temperature and salinity in the water column in the threestudy areas. Grey bars indicate sampling periods.

Fig. 3. Daily variation of photosynthetically active radiation (PAR) (mean ± SE) insummer 2010 and spring 2011.

M. García-S�anchez et al. / Marine Environmental Research 101 (2014) 8e2112

2.14. Data analysis

The assumption of data homogeneity (Cochran's test) wasverified and data were logarithmically transformed prior to anal-ysis. As the number of replicates for each of the considered factorswas unbalanced, the physiological variables and photosyntheticparameters for each species were analyzed using permutationalanalysis of variance (PERMANOVA) on Euclidean distances(Anderson, 2001). The experimental design consisted of two fixedfactors: “Zone”, with three levels (Lagoon, Inlet andMediterranean)and “Season” with two levels (Summer and Spring). A posterioripairwise comparisons test was applied for investigating significantterms. PRIMER 6 þ PERMANOVA v1.0.5 software was used for theanalysis (Anderson, 2005).

In order to determine general variation patterns between thephotosynthetic parameters and biochemical composition (pig-ments and stoichiometry) of the macroalgae species studied, wefollowed a multivariate approach (Ter Braak, 1999), usinga Redundancy Analysis (RDA). Only pigments common to all threespecies were included in the analysis. A preliminary PrincipalComponent Analysis (PCA) was previously performed andcompared with the RDA results. Ordinations were made usingCANOCO for Windows 4.5 (Ter Braak and Smilauer, 2002).

3. Results

3.1. Environmental parameters

Water temperature showed a marked seasonal pattern, withmaximum temperatures in July (27 �C in the Mediterranean and30 �C at the Inlet and Lagoon zones) and minimum in January andFebruary (13 �C in the Mediterranean and 11 and 9 �C at the Inletand Lagoon, respectively). The greatest differences in temperaturewere found in spring, when waters in the Mar Menor were 5 �Chigher than in the Mediterranean, while in summer these differ-ences fell to 3 �C. As a result, the thermal stress differed in the threestudied localities: algae living in the lagoon have to cope with anannual variation of 21 �C in the more confined zones and a slightlysmaller variation (19 �C) in the inlet zone. Whereas in the Medi-terranean the annual range is about 14 �C, with softer maximumand minimum temperatures (Fig. 2).

The greatest difference in salinity was found between theMediterranean, with an annual mean of 37.5 psu, and the lagoonenvironments (annual mean value of 43 psu). Salinity remainednearly constant throughout the year in open coastal waters,whereas both the lagoon and inlet zones showed peaks ofmaximum salinity in October (Fig. 2).

In general, waters were oligotrophic in the Mediterranean andhad slightly higher nutrient concentrations in the lagoon. Nutrientconcentrations were low, the annual mean NO3

� concentration inLagoonwas 0.73 ± 0.16 mM, while in the Inlet it was 0.26 ± 0.05 mMand in the Mediterranean 0.12 ± 0.02 mM NO2

� concentrations were0.06 ± 0.01 mM in Lagoon, 0.06 ± 0.01 mM in Inlet and0.05 ± 0.01 mM in the Mediterranean. NH4 mean values were2.20 ± 0.22 mM in Lagoon, 2.34 ± 0.22 mM in Inlet and2.25 ± 0.31 mM in Mediterranean. Finally, the mean PO4

3� concen-trations values were 0.08 ± 0.01 mM for Lagoon, 0.07 ± 0.01 mM forInlet and 0.05 ± 0.01 mM for Mediterranean.

Total Nitrogen in the water column was slightly higher inspring (3.03e3.66 mM) than in summer (1.43e1.69 mM) for all thelocalities. The opposite pattern was found in phosphate concen-tration, with values ranging between 0.1 and 0.11 mM in summerand, 0.04 and 0.05 mM in spring in both Lagoon and Inlet localities.For the Mediterranean, PO4

3� concentration did not differ betweenseasons.

3.2. Solar radiation

Fig. 3 exhibits the daily variation of photosysnthetically activeradiation (PAR) for the studied period (mean ± SE). PAR at 12:00 h(local time) had mean values of 1595 ± 67 mmol photons m�2 s�1 inspring and 1696 ± 10 mmol photons m�2 s�1 in summer. In bothseasons maxima were reached between 12:00 and 16:00 h (localtime). The accumulated daily dose of PAR for the 10 days prior tothe beginning of the study was 136.03 MJ m�2 in summer 2010 and99.54 MJ m�2 in spring 2011. Light extinction coefficients (Kd) ofthe water column for PAR showed a seasonal variation in the MarMenor, being higher in spring (0.46 m�1) than in summer(0.36 m�1). The Mediterranean waters presented higher trans-parency with a relatively constant Kd of 0.27 m�1.

Due to the shallowness of the selected localities, the speciesstudied received similar solar radiation in their natural habitats(80e90% of the incident radiation).

3.3. Photosynthetic activity as in vivo chlorophyll fluorescence

The photosynthetic parameters obtained from the rapid lightcurves (RLC) of the studied algae are shown in Table 1. Maximum

M. García-S�anchez et al. / Marine Environmental Research 101 (2014) 8e21 13

quantum yield (Fv/Fm) remained almost constant between thedifferent zones, with values of 0.60 ± 0.02 and 0.54 ± 0.01 forC. compresa and P. tenerrima, respectively. In the case of P. pavonica,spring samples from Lagoon and Inlet environments showed thelowest Fv/Fm values (0.49 ± 0.06 and 0.43 ± 0.03, respectively) andwere higher during summer.

The maximal photosynthetic capacity of C. compressa, measuredas ETRmax, was higher in the Mediterranean than in the Lagoon,particularly in summer. This was also the case for P. pavonica, butonly during summer since in spring there were no significant dif-ferences between zones. In contrast, P. tenerrima specimensinhabiting the lagoon had ETRmax values in spring(10.8 ± 0.9 mmol electrons m�2 s�1) that doubled those of speci-mens living in the inlet zones (5.52 ± 0.26 mmol electrons m�2 s�1)or in the Mediterranean (5.55 ± 0.45 mmol electrons m�2 s�1).

In general, the specimens of the Lagoon reached their highestETRmax values in spring, whereas in Mediterranean the valuestended to be higher in summer, although significant differenceswere only found for C. compressa.

In contrast to ETRmax, the values of photosynthetic efficiency(aETR) did not differ between zones or seasons for C. compressa. InP. tenerrima, inter-zonal differences were only found in spring,when values in Lagoon samples were higher (0.045 ± 0.002) thanthose in the Inlet zones (0.036 ± 0.002). P. pavonica had the highestvalues in Mediterranean during spring and at the Inlet zone insummer. Saturation irradiance (EkETR) values followed similar pat-terns to ETRmax for all three species.

3.4. Non-photochemical quenching

NPQmax tended to be higher in summer (p ¼ 0.06) inC. compressa. For the other species, there was a significant Sea-sonxZone interaction (Pseudo-F ¼ 7.559; df ¼ 2; p < 0.01 forP. pavonica; Pseudo-F ¼ 4.858; df ¼ 2; p < 0.05 for P.tenerrima), sothat the highest NPQmax values were found in different zones,

Table 1Maximum quantum yield (Fv/Fm), maximum Electron Transport Rate (ETRmax), photosynthquenching (NPQmax), the slope of the NPQ versus irrandiance (aNPQ) and the saturation irrameans ± SE (n¼ 6). Groups of homogeneous means obtained by the post hoc test are indicwhereas the asterisks are the differences among season.

Spring

Lagoon Inlets Medit.

(a) Cystoseira compressaFv/Fm 0.58 ± 0.05a e 0.60 ± 0.01aETRmax 57.35 ± 2.86b* e 82.74 ± 7.88aaETR 0.27 ± 0.01a e 0.24 ± 0.02aEK 210.25 ± 6.72b* e 348.58 ± 28.15aNPQmax 0.79 ± .24a e 0.66 ± .13aaNPQ 0.002 ± 0.0006a e 0.003 ± 0.001aEKNPQ 378.48 ± 28.41a e 262.24 ± 71.27a(b) Padina pavonicaFv/Fm 0.49 ± 0.06b 0.43 ± 0.03b 0.70 ± 0.02aETRmax 59.81 ± 13.66a* 35.27 ± 2.62a 44.19 ± 7.53aaETR 0.22 ± 0.03 ab 0.14 ± 0.01b 0.28 ± 0.02a*EK 264.39 ± 29.51a* 245.36 ± 18.24 ab 162.85 ± 34.03bNPQmax 0.73 ± 0.26b 0.30 ± 0.08b 1.68 ± 0.29a*aNPQ 0.001 ± 0.0003b 0.001 ± 0.0005 ab 0.0023 ± 0.0005aEKNPQ 706.10 ± 70.37a* 283.62 ± 58.09b 633.62 ± 68.58a*(c) Palisada tenerrimaFv/Fm 0.51 ± 0.02a 0.53 ± 0.03a 0.49 ± 0.03aETRmax 10.81 ± 0.99a* 5.52 ± 0.26b* 5.55 ± 0.45baETR 0.045 ± 0.002a 0.036 ± 0.002b 0.039 ± 0.008 abEK 239.56 ± 20.27a* 156.07 ± 11.49b* 155.84 ± 35.23 abNPQmax 0.42 ± 0.08b 0.68 ± 0.07a 0.44 ± 0.09 abaNPQ 0.002 ± 0.0006b 0.004 ± 0.0006a 0.004 ± 0.0007aEKNPQ 372.74 ± 110.42a 185.59 ± 19.08b 106.07 ± 5.11c

depending on the season. Both P. pavonica and P. tenerrima pre-sented a higher aNPQ in summer in the Lagoon. However,C. compressa, showed similar aNPQ values in all zones and seasons,as occurred for the EkNPQ values. In P. pavonica the saturating in-tensity for NPQ (EkNPQ) was two-fold higher in spring than insummer for both Lagoon and Mediterranean specimens. OnlyP. tenerrima samples in the Mediterranean showed significanthigher values in summer, although the highest saturation irradi-ances were found in the lagoon for both seasons. EkNPQ was higherthan EkETR for all the species as was expected, being evident a gapbetween the activation of the NPQ mechanism and the ETR curves(Table 1).

3.5. Internal carbon and nitrogen contents

As regards the nitrogen internal content, lagoon specimenspresented the highest values, especially in spring, coinciding withthe highest NO3

� concentration in thewater (Fig. 4). In the Inlets andthe Mediterranean area, internal N values were in some cases evenbelow the critical threshold for maximal growth (Fujita et al., 1989),especially in the case of P. pavonica. Among species, the highestvalues were measured in P. tenerrima (15e25 mg g�1 DW) and thelowest in P. pavonica (6e11mg g�1 DW). C. compressa values rangedfrom 9 to 15 mg g�1 DW and did not differ significantly betweenLagoon and Mediterranean.

Internal carbon content was also higher in the lagoon and inspring for C. compressa. In P. pavonica seasonal differences wereonly detected at the inlet, with higher values in spring. Finally, thevalues remained almost constant in P. tenerrima. Since the N con-centration varied much more than the C concentration, the C:Nratio showed an inverse relationship with the N concentration inthe three species (C. compressa r ¼ �0.912; n ¼ 14; p < 0.001;P. pavonica r ¼ �0.828; n ¼ 19; p < 0.001 and P. tenerrimar ¼ �0.918; n ¼ 22; p < 0.001).

etic efficiency (aETR), saturation light intensity (EkETR), maximum non-photochemical

diance for NPQ (EkNPQ) for (a) C. compressa, (b) P. pavonica and (c) P. tenerrima. Data are

ated by different letters (lower case letters for Spring and capital letters for Summer),

Summer

Lagoon Inlet Medit.

0.57 ± 0.02A e 0.65 ± 0.04A42.97 ± 2.60B e 123.13 ± 4A*0.28 ± 0.01A e 0.26 ± 0.01A

157.52 ± 12.32B e 474.19 ± 38.11A*1.16 ± 0.16A e 1.01 ± 0.03A

0.004 ± 0.0006A e 0.003 ± 0.0003A326.36 ± 10.76A e 372.54 ± 10.76A

0.62 ± 0.02B 0.71 ± 0.01A* 0.66 ± 0.04AB22.33 ± 1.67B 32.65 ± 4.73A 38.65 ± 3.24A0.19 ± 0.01B 0.23 ± 0.02A* 0.19 ± 0.01AB

115.88 ± 8.92B 147.53 ± 29.26AB 201.90 ± 12.10A0.77 ± 0.10B 1.38 ± 0.14A* 0.84 ± 0.18AB

0.003 ± 0.0008A* 0.004 ± 0.0003A* 0.002 ± 0.0005A372.66 ± 82.11A 390.20 ± 52.16A 377.25 ± 31.03A

0.58 ± 0.02A* 0.56 ± 0.02A 0.56 ± 0.04A6.96 ± 0.43A 3.61 ± 0.68B 7.08 ± 0.50A

0.046 ± 0.004A 0.043 ± 0.003A 0.045 ± 0.005A156.97 ± 12.98A 85.10 ± 15.70B 165.25 ± 15.93A

0.87 ± 0.11A* 0.59 ± 0.07B 0.79 ± 0.14AB0.003 ± 0.0005A* 0.003 ± 0.0005A 0.004 ± 0.0009A

266.29 ± 14.50A 201.75 ± 16.58B 218.51 ± 26.37AB*

05

101520253035

SPRING SUMMER

A A A b b

a *

Lagoon Inlets Mediterranean

B)

05

101520253035

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a * b* c * A

B B

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101520253035

SPRING SUMMER

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N (

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g-1 D

W)

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A B

a

a C:N

A) B) C)

D) E) F)

G) H) I)

a A A A

b* a

a a ab A A

B

a* b A A

Fig. 4. Internal N content (mg g�1 DW), C content (mg g�1 DW) and C:N ratio for C. compressa (A, D, G), P. pavonica (B, E, H) and P. tenerrima (C, F, I). Data are mean values ± SE(n ¼ 6). Letters indicate differences among zones in Spring (lower case letters) and Summer (capital letters). Asterisks denote the differences between seasons.

Table 2Pigment concentration for (a) C. compressa, (b) P. pavonica and (c) P. tenerrima. Data are means ± SE (n ¼ 6). Groups of homogeneous means obtained by the post hoc test areindicated by different letters (lower case letters for Spring and capital letters for Summer), whereas the asterisks are the differences among season.The xanthophyll cycle (XC)pool size is the sum of (violaxanthin þ anteraxanthin þ zeaxanthin; VAZ). P(t) present in traces; Fx ¼ Fucoxanthin. Chl a (chlorophyll a), Chl c (chlorophyll c), PE (phyco-erythrin) and PC (phycocyanin) expressed as mg g�1 DW. Other pigments expressed as mg g�1 DW.

Spring Summer

Lagoon Inlets Medit. Lagoon Inlet Medit.

(a) Cystoseira compressaChl a 2.47 ± 0.44a e 1.81 ± 0.32a 2.54 ± 0.14A e 1.91 ± 0.10BChl c2 þ Fx 1.35 ± 0.24a e 0.85 ± 0.10a 1.32 ± 0.08A e 0.96 ± 0.04B(Chl c2 þ Fx) :Chla 0.51a e 0.41b 0.52A e 0.50BNeoxanthin P(t) e P(t) P(t) e P(t)Violaxanthin 107.7 ± 20.4a e 94.6 ± 14.57a 112 ± 6.9A e 91.9 ± 4.7AAnteraxanthin 93.2 ± 20.7a e 71.4 ± 8.5a 77.4 ± 5.3A e 50.7 ± 3.5BZeaxanthin 19.4 ± 2.9a e 14.4 ± 2.75a 27.9 ± 2.6A e 15.2 ± 3.2BXC pool (V þ A þ Z) 220.3 ± 43a e 180.5 ± 20.9a 217.3 ± 9.6A e 157.8 ± 1.8Bb�carotene 3.7 ± 0.6a e 3.8 ± 0.82a 3.9 ± 0.3A e 2.6 ± 0.3B% De-epoxidation 50.7 ± 1a e 48.5 ± 3a 48.6 ± 2A e 41.7 ± 3A(b)Padina pavonicaChl a 1.90 ± 0.14a 1.42 ± 0.06b 1.44 ± 0.21b 1.74 ± 0.06A 1.38 ± 0.13B 1.33 ± 0.15BChl c2 þ Fx 0.73 ± 0.05a 0.53 ± 0.06b 0.61 ± 0.10 ab 0.78 ± 0.03A 0.68 ± 0.06A 0.64 ± 0.10A(Chl c2 þ Fx) :Chla 0.38a 0.37a 0.43a 0.45B* 0.49A* 0.45ABNeoxanthin P(t) P(t) P(t) P(t) P(t) P(t)Violaxanthin 111.7 ± 19.1a 94.8 ± 12.4 ab 63.2 ± 7.1b 124.1 ± 11.4A 89.7 ± 9.9AB 76.5 ± 8.2BAnteraxanthin 96.8 ± 12.4a 67.2 ± 7.6a 72.5 ± 12a 84.5 ± 5.6A 53.6 ± 4.4B 61.7 ± 6.2BZeaxanthin 43.1 ± 5.8a 26 ± 7.9a 42 ± 9.2a* 46.7 ± 6.8A 22.8 ± 1.6B 10.5 ± 1.8CXC pool (V þ A þ Z) 251.6 ± 33.7a 188 ± 5.6a 177.7 ± 25.1a 255.2 ± 15.3A 166.2 ± 15.3B 148.7 ± 14.1Bb�carotene P(t) P(t) P(t) P(t) P(t) P(t)% De-epoxidation 56.5 ± 3a 49.3 ± 7a 63.4 ± 3a* 51.4 ± 3A 46.2 ± 1A 48.0 ± 2A(c) Palisada tenerrimaChl a 3.03 ± 0.27a* 1.94 ± 0.43b 2.54 ± 0.17 ab 2.30 ± 0.18A 2.69 ± 0.16A 2.94 ± 0.25APE 11.51 ± 1.47a* 5.58 ± 1.52b 9.42 ± 0.41 ab 7.73 ± 0.9A 9.49 ± 1.47A 10.81 ± 1.48APC 10.65 ± 1.25a* 6.75 ± 1.91a 12.54 ± 0.47a 2.93 ± 0.32B 9.67 ± 1.42A 12.79 ± 2.11A(PE þ PC): Chl a 7.7a* 6.0a 8.7a 4.6B 7.1A 8.5AFucoxanthin 0.082 ± 0.033a 0.077 ± 0.028a P (t) 0.069 ± 0.013A 0.047 ± 0.010A 0.011 ± 0.001B*Violaxanthin 8 ± 3a 4.7 ± 2.6a* P (t) 1.9 ± 0.6B P (t) 5.9 ± 1.7A*Anteraxanthin 10.7 ± 2a 5 ± 1b 5.9 ± 0.4 ab 10 ± 1.6AB 6.1 ± 1.6B 12.3 ± 1.8A*Zeaxanthin 422.3 ± 38a 289.3 ± 69.9a 327.7 ± 25.3a 429.2 ± 20.3A 467.9 ± 49.4A 466.1 ± 20.9A*XC pool (V þ A þ Z) 0.424 ± 0.038a 0.299 ± 0.07a 0.334 ± 0.026a 0.442 ± 0.018A 0.478 ± 0.048A 0.485 ± 0.022A*Luteine 6.8 ± 0.7a 5.1 ± 0.08a 8.9 ± 2.5a 5.4 ± 1.4B 11.4 ± 1.1AB* 13.9 ± 3.3Ab�carotene 6.8 ± 1.4a 4.1 ± 1.4a 5.1 ± 0.3a 10.2 ± 1.7A 7.2 ± 1.8A* 13.1 ± 2.5A*

M. García-S�anchez et al. / Marine Environmental Research 101 (2014) 8e2114

0

10

20

30

40

50

60

SPRING SUMMER

a*

B

a

A

A

Lagoon Inlets Mediterranean

Tota

l phe

nolic

con

tent

(m

g PE

g-1

DW

)M

AA

s (m

g g-

1 D

W)

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ab*

b*a

B

A

C

0.0

0.5

1.0

1.5

2.0

SPRING SUMMER

a

bb

BB

A

C

Fig. 5. Total phenolic content in (A) Cystoseira compressa and (B) Padina pavonicaexpressed as phloroglucinol equivalents (mg PE g�1 DW). Mycosporine-like amino-acids (MAAs) concentration (mg g�1 DW) in Palisada tenerrima (C). Data are expressedas mean value ± SE (n ¼ 6). Letters indicate differences among zones in Spring (lowercase letters) and Summer (capital letters). Asterisks denote the differences betweenseasons.

M. García-S�anchez et al. / Marine Environmental Research 101 (2014) 8e21 15

3.6. Photosynthetic pigments composition

The photosynthetic pigment content of the three species wascompared in the different environments in spring and summer(Table 2). The chlorophyll a (Chla) content presented the highestvalues in the Lagoon and only differed between the two analysedseasons, being higher in spring, in the case of P. tenerrima. Light-harvesting pigments in brown algae (fucoxanthin þ chlorophyll c)related to Chl a were more abundant in the lagoon in summer forC. compressa, while no differences were detected for P. pavonica.

The pigment content related to the xanthophyll cycle(violaxanthin þ antheraxanthin þ zeaxanthin ¼ VAZ) in brownalgae was higher in the lagoon than in the other zones duringsummer. Moreover, C. compressa showed a positive correlationbetween this VAZ pool and NPQmax (r ¼ 0.508; p < 0.05; n ¼ 17). Inthe case of P. tenerrima, which also contained xanthophyll cyclepigments, the total pool did not differ between zones but increasedsignificantly in the Mediterranean in summer. This species showeda seasonal pattern in the concentration of phycobiliproteins(Phycoerythrin (PE) þ phycocyanin (PC)) but only for the lagoonsamples, with higher values in spring (Table 2). Regarding the(PE þ PC):Chla ratio, it was significantly lower during summer inthe Lagoon, mainly due to the strong drop in PC value (from10.65 ± 1.25 to 2.93 ± 9.67 mg g�1 DW). A positive correlation wasfound between the photosynthetic pigment content (Chla and PE)and ETRmax (r ¼ 0.484; p < 0.01; n ¼ 33 Chl a versus ETRmax;r ¼ 0.370; p < 0.05; n ¼ 33 PE versus ETRmax).

3.7. Phenolic content

In summer, the total phenolic content in lagoon specimens ofC. compressa (Fig. 5a) and P. pavonica (Fig. 5b) decreased by 50e60%compared to the spring values, while in the case of Mediterraneansamples no significant differences were detected. Regarding thedifferent zones, Inlets had the lowest values andMediterranean thehighest.

3.8. MAAs

In P. tenerrima, the concentration of all mycosporine-like ami-noacids (MAAs) showed significance differences between zones(Pseudo-F¼ 4.29; df¼ 2; p < 0.05). They followed the same patternin spring and summer, with the concentration being highest in theInlets zones (Fig. 5c).

Four major MAAs were identified (asterina-330, palythinol,porphyra-334 and palythine), while shinorine contributed less than2% to the total amount of MAAs. No seasonal changes were found inthe MAA composition.

3.9. Antioxidant activity

Antioxidant activity dropped in summer for Lagoon specimensof C. compressa (Fig. 6a) and for the Inlets specimens of P. pavonica,as indicated by the higher EC50 values. The EC50 index was nega-tively correlated with the phenolic content in P. pavonica(r ¼ �0.599; p < 0.01; n ¼ 29) (Fig. 6b) and C. compressa(r ¼ �0.739; p < 0.001; n ¼ 24).

3.10. Exposure-recovery

The exposure-recovery kinetics in C. compressa showed a similarpattern in all localities. After 2.5 h of exposure, Fv/Fm decreased~40% and recovered to 90% in this species (Table 3). On the otherhand, Mediterranean populations of the two species showed alower decrease in Fv/Fm (30% P. pavonica; 26% P. tenerrima) but

higher recovery rates (100% P. pavonica; 79% P. tenerrima) thanLagoon or Inlets counterparts (Table 3, Fig. 7). Moreover, the in-dividuals of the three species growing in Mediterranean watersstarted to recover during the full light exposure period, as reflectedby the negative Kslow. This behaviour, with negative Kslow, was alsofound in P. tenerrima populations in the lagoon and in P. pavonicafrom the Inlet (Table 4). Of the three species studied, P. tenerrimapresented the lowest percentage of recovery of Fv/Fm after 3 h indarkness, maximal values not reaching 80%.

3.11. Relationship between photosynthetic parameters andbiochemical variables

The results for RDA ordination were consistent with those ob-tained from a previous PCA (data not shown), as ordination plots forboth analyses were very similar (Fig. 8). The RDA ordinationexplained 45.6% of the variance of photosynthetic data and showeda clear separation between species based on their photosyntheticresponse and biochemical composition. Taxonomical featuresseemed to be more important than seasonal or environmentalpatterns. Brown algae were grouped together, with higher photo-synthetic capacity (ETRmax), efficiency (aETR) and non-photochemical quenching (NPQmax) than the red alga studied.Most of the variancewas explained by the first axis, which is relatedwith Zeaxanthin, Antheraxanthin and Chl a concentrations. Rather

0.0

0.5

1.0

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3.0

3.5

EC

50(m

g D

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L-1

)

aaA *

B

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1.0

1.5

2.0

2.5

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3.5

SPRING SUMMER

B

a

bab

A*

CB

Lagoon Inlets Mediterranean

Fig. 6. DPPH radical scavenging activity expressed as oxidation index EC50 in (A)Cystoseira compressa and (B) Padina pavonica given in mg DW mL�1. Data are meanvalues ± SE (n ¼ 6). Letters indicate differences among zones in Spring (lower caseletters) and Summer (capital letters). Asterisks denote the differences betweenseasons.

M. García-S�anchez et al. / Marine Environmental Research 101 (2014) 8e2116

than Chl a, it seems that, besides the role of specific pigments foreach species which were not included in the analysis, otheraccessory pigments are related with the higher photosyntheticfeatures of brown algae. Secondly, there was a seasonal differenti-ation in axis II, with summer samples tending towards the positivepart, which is related to the higher non-photochemical quenchingcapacity (NPQmax and aNPQ) and spring samples tending towardsthe negative part of the axis.

4. Discussion

We studied three different species, two brown algaeC. compressa and P. pavonica and one red alga P. tenerrima, all rep-resentatives of midlitoral and infralitoral photophilic communitiesin the Mediterranean region and common to lagoon and marineopen coasts. All of them were found in all the sampling stations inthe lagoon and the open sea, except that no C. compressawas foundin the Inlets zones. All the sampling stations receive similar solar

Table 3Fv/Fm decrease after 2.5 h of exposure to full light and its recovery after 3 h indarkness. Values are normalized and expressed as percentage of initial valuesmeasured before light treatment (mean ± SE; n ¼ 8).

Species Initial Fv/Fm %Decrease %Recovery

C. compressaLagoon 0.66 ± 0.03 43 ± 4 86 ± 4Mediterranean 0.53 ± 0.03 35 ± 3 95 ± 5P. pavonicaLagoon 0.49 ± 0.02 40 ± 5 85 ± 6Inlet 0.52 ± 0.03 41 ± 5 84 ± 11Mediterranean 0.54 ± 0.02 30 ± 6 100 ± 4P. tenerrimaLagoon 0.52 ± 0.03 57 ± 7 74 ± 6Inlet 0.51 ± 0.01 40 ± 5 67 ± 6Mediterranean 0.49 ± 0.04 26 ± 4 79 ± 2

radiation at the studied depth although the extinction coefficient inthe Mar Menor shows marked seasonality, with lower values insummer than in spring, probably due to the top-down control ofthe trophic web exerted by jellyfish, which reach their maximumpopulation level in this season (P�erez-Ruzafa et al., 2002).

The lagoon and coastal sea locations showed differences insalinity, temperature and nutrient concentrations. Nevertheless,the Inlet zones (particularly S.3) cannot be considered as an ecotonebetween open coastal and lagoon communities characterized by anaverage value for the environmental factors, but as a lagoon loca-tion with unstable conditions that are only tolerated by somespecies. This instability is related to the short-term (hours to days)scale determined by the changes in direction of the current at theinlet (with a mean periodicity of 6 h) (Ar�evalo, 1988) and thealternating influence of Mediterranean and lagoon conditions(García-S�anchez et al., 2012; P�erez-Ruzafa et al., 2008). These highfrequency fluctuations can be more stressful than seasonal varia-tions, although the latter have greater amplitude. This fact couldaffect the growth of C. compressa in these localities, which wouldpartly explain the absence of this species.

On a large spatial scale, ocean temperatures generally dictatepatterns of seaweed distribution (Lüning, 1990). It has beendescribed how different species and even different populations of agiven species may have different optimal temperatures for photo-synthesis (Davison et al., 1991) and growth (Breeman and Pakker,1994). The temperature tolerance of seaweeds can depend on ge-netic adaptation and on their phenotypic acclimation to tempera-ture fluctuations (Kuebler et al., 1991).

In general, maximum biomass in rocky shore communities ofmacroalgae is reached in summer or autumn in the Mediterraneanbut in spring in the case of confined lagoons (De Biasi et al., 2003;Hegazi, 1999). Moreover, as regards the temporal dynamics of thestudied species, there was a small time lag in the development ofthe species in the different environments, with C. compressa andP. tenerrima having maximal biomass in spring for the Mar Menorlagoon, and in summer in the Mediterranean. Similar results wereobtained by other authors for C. compressa in the Mediterranean(Falace et al., 2005; G�omez Garreta et al., 1982). On the other hand,P. pavonica had the highest biomass values in summer in all cases.Reflecting these biomass patterns, a good correlation betweenETRmax and biomass was found for C. compressa and P. tenerrima,but not for P. pavonica, for which ETRmax values were higher inspring although maximal biomass occurred in summer. These re-sults could be related with a negative effect of temperature on thephotosynthetic apparatus during summer, with temperaturesreaching 31 �C in the lagoon localities, as photosynthesis is affectedby temperature (Padilla-Gami~no and Carpenter, 2007). In general,the higher ETRmax found in the lagoon in spring is directly corre-lated with a higher internal nitrogen content, i.e., there is a higherflux of electrons in nitrogen uptake. Seasonality did not influencethe photosynthetic efficiency (aETR) in any of the species, whichmay be related to the maintenance of photosynthetic activity.

The C:N ratio indicates the metabolic potential to synthesizebiomolecules (Altamirano et al., 2000; Weykam et al., 1996).Different internal N contents were found in the three studiedspecies. In general, the Mediterranean Rhodophyceae are charac-terized by higher N concentrations and lower C:N ratios, whereasPhaeophyceae have higher C:N ratios (Mouri~no et al., 1981). Med-iterraneanwaters have a lower N content than lagoon ones and thisis reflected in the higher internal N content of the Lagoon speci-mens of P. pavonica and P. tenerrima. It seems that the studiedspecies take N from the surroundings during spring, when thewater N concentration is higher, and accumulate it as a reserves forother periods, since we observed a consumption of internal Nduring summer. It has been said that the spring growth period will

Max

imum

qua

ntum

yie

ld (

Fv/

F m)

0

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Time (min)

LagoonMediterranean

InletsLagoon

Mediterranean

InletsLagoon

Mediterranean

Fig. 7. Photoinhibition and recovery of optimal quantum yield (Fv/Fm) in (A) Cystoseiracompressa, (B) Padina pavonica and (C) Palisada tenerrima. Thalli were first exposed tostrong light during 2.5 h (white bar) and subsequently to darkness (black bar)(means ± SE, n ¼ 8).

Table 4Rate constant (Kslow) and proportion (Pslow) for the slow component and for the fast comenvironments (following Hanelt, 1998).

Inhibition

Pfast Kfast Pslow Kslo

C. compressa Lagoon 0.091 0.323 0.470 0.0Mediterranean 0.387 0.019 0.147 �0.0

P. pavonica Lagoon 0.191 0.058 0.302 0.0Inlet 0.370 0.024 0.152 �0.0Mediterranean 0.406 0.015 0.123 �0.0

P. tenerrima Lagoon 0.338 0.022 0.182 �0.0Inlet 0.145 0.077 0.367 0.0Mediterranean 0.361 0.022 0.135 �0.0

Fig. 8. Redundancy analysis (RDA) carried out on photosynthetic parameters usingbiochemical composition as explanatory variables. Samples were denote d with blackcircle (Cystoseira compressa), white circle (Padina pavonica) and grey triangle (Palisadatenerrima) with the corresponding data label (Sp ¼ spring; Su ¼ summer; numbercorrespond to the sampling station). Photosynthetic parameters were representedwith dashed-arrows and biochemical composition was signed out with solid arrows.

M. García-S�anchez et al. / Marine Environmental Research 101 (2014) 8e21 17

sustain growth for some time after the depletion of externalnutrient supplies (Naldi and Wheeler, 1999). C. compressa followedthe same pattern but not statistically significant. On the other handP. pavonica, especially in the Mediterranean, is likely to be N-limited, with a tissue N content under concentration for maximummacroalgal growth (1.5%) (Fujita et al., 1989). The lower values insummer could be related with the additional N cost of repair,avoidance, quenching and scavenging mechanisms (Raven, 2011).In fact, it seems that these mechanisms allowed this species tomaintain the photosynthetic efficiency and maximal capacity inthis period.

The only seasonal variation in Chl a concentration was found inLagoon samples, with higher values in spring than in summer,probably due to the higher N availability, as explained in Bonomiet al. (2010). Otherwise, Lagoon specimens showed a tendencytoward higher Chl a values than Mediterranean specimens, rein-forcing the above idea. The (Chlc þ Fx):Chla ratio for brown algaeseems to increase in summer, although a reduction in antenna size

ponent (Kfast, Pfast) of the inhibition kinetics for the species studied in the different

Recovery

w r2 Pfast Kfast Pslow Kslow r2

03 0.922 1.000 0.016 1.000 0.016 0.67005 0.992 0.599 0.077 1.000 0.011 0.937

00 0.989 0.599 0.078 0.814 0.008 0.88905 0.990 0.016 0.011 0.727 0.007 0.98106 0.895 1.000 0.014 1.000 0.014 0.945

02 0.983 0.000 0.019 0.462 0.004 0.89702 0.982 0.000 0.022 0.291 0.002 0.87606 0.966 0.599 0.077 0.238 0.003 0.188

M. García-S�anchez et al. / Marine Environmental Research 101 (2014) 8e2118

in summer may be expected. It might be suggested therefore thatthese pigments are uncoupled from the reaction centres, absorbingexcess light and consequently playing a photoprotective role. InP. tenerrima, specimens from the different zones presented similarlevels of phycobiliproteins (PBs). Decrease in phycoerithrin (PE) anda sharp decrease in phycocianin (PC) was recorded in summersamples from the Lagoon. We suggest that these pigments could beacting as an N reservoir, and that during summer N is used tosynthesize molecules involved in repair and photoprotectionmechanisms (Roy, 2000; Talarico and Maranzana, 2000).

There is a clear dependence of the maximum extent of NPQ onenvironmental factors, such as depth distribution, seasonality ordifferent marine habitats (Goss and Jakob, 2010). With some ex-ceptions, there was a general tendency to higher NPQmax values insummer, which is consistent with the higher solar radiation andtemperatures reached in this period and which could potentiallyaffect the photosynthetic apparatus. In addition, there was an in-crease in NPQ efficiency (aNPQ) during summer in the lagoon,resulting in an earlier activation of the NPQ mechanism.P. tenerrima presented the highest seasonal differences for thisparameter, with NPQmax values two fold higher in summer, both forthe lagoon and the Mediterranean.

Although P. tenerrima presented the lowest photosynthetic ca-pacity, the non-photochemical values reachedwere similar to thosefrom the other species. Zeaxanthin (Zx) is the major carotenoid inthis species, in agreement with the carotenoid profile described forthe Rhodomelaeacea group, to which it belongs (Esteban et al.,2009; Schubert et al., 2006). The uncertain presence of a func-tional xanthophyll cycle in red algae (Andersson et al., 2006;Schubert et al., 2006) does not exclude a photoprotective functionfor Zx, which may play a variety of functions that can be of adaptivevalue in intertidal environments with large diurnal light fluctua-tions due to tides and aerial exposure (Esteban et al., 2009;Schubert et al., 2011). Hence, a photoprotective role could beargued for Zx, since Zx-dependent NPQ has been described (Gosset al., 2006), which is also supported by the positive correlationfound between Zx and NPQmax (r ¼ 0.363; p < 0.05; n ¼ 32). Ashappens with high irradiance, a temperature-induced increase inthylakoid membrane dynamics facilitates the diffusion of viola-xanthin molecules and conversion into antheraxanthin and zeax-anthin (Latowski et al., 2002). Zx may also act like cholesterol, witha structural function in the lipid layer itself, protecting the thyla-koid membranes in high temperatures (Latowski et al., 2002; Tardyand Havaux, 1997). Thus, it could be important by lowering thepenetration of reactive oxygen species (ROS) inside the thylakoid(Muller, 2001). Our findings show a summer accumulation ofxanthophyll cycle-related pigments in both brown algae from theLagoon, with Zx concentrations two-fold higher in C. compressa andeven four-fold in the case of P. pavonica, coinciding with the highesttemperatures. Therefore, although seaweeds minimize photo-inhibition and the destruction of their photosynthetic apparatusduring strong irradiance periods by accumulating more of thepigments involved in the xanthophyll cycle (Gevaert et al., 2002),the observed trend is more likely to be explained by the highertemperatures reached in the lagoon compared with the Mediter-ranean zones, as the radiation received in both sites in each seasonwas similar.

On the other hand, fucoxanthin (Fx), which may have strongantioxidant properties (Fung et al., 2013; Le Tutour et al., 1998;Moriet al., 2004) appears in larger amounts in lagoonal individuals ofP. tenerrima, but is only present in trace amounts or in quantitiesfour to six times lower in the Mediterranean. Its presence couldenhance cell viability against oxidative damage (Heo et al., 2008). Itcould be suggested that algae exposed to those extreme conditionsare more photoprotected due to their higher Fx content.

In response to intense solar radiation, organisms have evolvedcertain photoprotective mechanisms, such as the accumulation ofmycosporine-like aminoacids (MAAs) in red algae, and carotenoidsand phenolic compounds in green and brown algae, respectively.The content of MAAs in P. tenerrimawas higher at the Inlet stationscompared to Lagoon and Mediterranean. In all cases the contentvaried between 0.5 and 1 mg g�1 DW, which is similar to thefindings for other species of Rhodomelaceae (Korbee-Peinado,2004). It seems that exposure of this species to fluctuating condi-tions, characterized by different temperatures and salinities, couldenhance their MAA content.

A higher phenolic content was found in spring time than insummer, in contrast to the results obtained by Abdala-Díaz et al.(2006) and Connan (2004). In our case, it seems that phenoliccompounds were more related to nutrient status, since a positivecorrelationwas found between internal N and the phenolic contentin the case of P. pavonica (r ¼ 0.546; p ¼ 0.015; n ¼ 19). Althoughthey are not an N-compound, N may enhance the accumulation ofphenolic compounds in some brown algae (Pavia and Toth, 2000)as well as in Ulva rigida (Cabello-Pasini et al., 2011). Additionally,the decrease in the total phenolic content observed in summer wasmore noticeable in the Lagoon and Inlet zones than in the Medi-terranean. We suggest that higher temperature could induce therelease of these compounds to the surrounding waters, as has beenproposed in several studies (Celis-Pl�a et al., 2014; Koivikko et al.,2005; Swanson and Druehl, 2002). The lower phenolic contentfound at the Inlet could be related with the higher inhabitant depthand thus, lower irradiance, at this location. Accordingly, a lowerantioxidant capacity would be needed. The clear relationship be-tween phenolic amounts and antioxidant capacity in these brownalgae was validated by the results.

Different disturbing factors, such as pollution events, couldaffect the oxidative stress of algae and therefore the antioxidantcapacity. In the case of theMarMenor, thewater columnwas highlyoligotrophic until the 1990s, when a change in agricultural prac-tices led to an increase in the nutrient inputs into the lagoon.However, homeostatic mechanisms through the pelagic food-webstill maintained the water quality and low Chl a concentration inthe water column (P�erez-Ruzafa et al., 2002, 2005a,b). No dystro-phic crises or algal blooms have ever been reported in the MarMenor. Besides the incipient eutrophication process started in the1990s, the main source of pollution has always come from miningactivities for silver, lead and zinc, in the mountain range of LaUnion, parallel to the southwestern Mediterranean coast of thestudy area. This lasted from the time of Carthaginian and Romandomination until the wastes were diverted to the Mediterranean inthe 1950s although such activity ceased in the 1990s. Heavy metalscan increase oxidative stress in algae (Coll�en et al., 2003). However,although the sediments in the southwestern basin of the MarMenor show high contents of heavy metals, concentrations indissolved form in the water column are very low, while the heavymetal content of the particulate matter is much lower than that ofthe sediments, with concentrations close to those of the Mediter-ranean Sea bottom sediment (Dassenakis et al., 2010; unpublisheddata), so this is not expected to affect the assemblages on rockybottoms of the studied areas. Other potential sources of pollutionare polycyclic aromatic hydrocarbons and persistent organochlo-rines linked to agricultural practices, but their effects are local andrestricted to the mouth of the main watercourses (P�erez-Ruzafaet al., 2000; Le�on et al., 2013).

Finally, the reduction of PSII maximum efficiency (Fv/Fm) whenalgae are exposed to high irradiances and its recovery in shaded ordark conditions can be used as a good indicator of the sensitivity oforganisms to short-term environmental stress conditions (Hanelt,1992). The studied species exhibited dynamic photoinhibition,

M. García-S�anchez et al. / Marine Environmental Research 101 (2014) 8e21 19

which was expected taking into account that they are adapted tolive in intertidal environments with high irradiance levels (Hanelt,1996). The lower recovery of Fv/Fm (67e80%) observed inP. tenerrima even after 3 h in darkness could be explained by thepermanently activated energy dissipation mechanism present inzeaxanthin-predominant species that provides effective photo-protection, but it may be a disadvantage under subsaturating lightconditions because it cannot quickly suppress its quenching activityand may result in a significant reduction in photosynthesis(Schubert et al., 2011). Although the xanthophyll pool was higher inLagoon specimens, individuals from the Mediterranean showed aless pronounced decrease in Fv/Fm and higher recovery rates, exceptfor C. compressa. Moreover, the recovery of Fv/Fm started during thefull light exposure period in individuals of the species growing intheMediterranean. In this sense, moderate heat and salt stress havebeen reported to stimulate the photoinhibition of PSII (Murataet al., 2007; Takahashi and Murata, 2008). Since natural waterconditions were maintained during the course of this field exper-iment, the observed differences in temperature and salinity, whichwere less stressful in the Mediterranean, could explain thisbehaviour. Hence, the higher water salinity and temperature in theMar Menor could impair the photoprotection mechanisms of thesespecies.

5. Conclusions

Macrophyte assemblages in the Mar Menor lagoon are notcomposed of species that are unique to this environment, but ofpopulations that are able to acclimate to their particular environ-mental conditions. Adaptations to the lagoon are more likely toconsist of taking advantage of the more favourable conditions thatoccurred in the lagoon during spring rather than in other seasons,as well as of withstanding the extreme conditions in summer orwinter. Our results show that acclimation to lagoon environmentsinvolves complex regulatory mechanisms and that there is nocommon pattern for the acclimation of the three studied macro-algae to lagoon environments since their responses are species-specific.

In this sense, P. tenerrima is the species most adapted tolagoon environmental conditions. Specimens from the lagoon inspring showed the highest maximal photosynthetic capacity(ETRmax) and the greatest concentration of Chl a and phycobili-proteins, coinciding with the higher nutrient concentration, highirradiance and favourable temperatures for growth during thisseason. In addition, ETRmax values in summer are very similar tothose found in the Mediterranean, even at temperatures above30 �C in the lagoon. This species is present throughout the yearin the lagoon, and is dominant in the upper infralittoral zone,where it reaches a bigger size and has more biomass than in theMediterranean.

Secondly, P. pavonica benefits from spring conditions in thelagoon, showing higher ETRmax values and Chl a concentrationsalthough its photosynthetic capacity drops in summer, with lowervalues than in the Inlet or Mediterranean zones. It cannot with-stand winter conditions so it disappears from the Lagoon and theInlet location by the end of autumn and develops again in spring(P�erez-Ruzafa, 1989). In contrast, in the Mediterranean waters it ispresent throughout the year.

Finally, C. compressa seems to be the least adapted/acclimated tolagoon conditions. Although it is able to grow is present throughoutthe year in the Mar Menor, its physiological status is not as good asin the Mediterranean populations. Moreover, compared with itsdominant role in Mediterranean rocky shores, its presence in thelagoon is restricted only to some zones where both its size andbiomass are lower.

The results underline the importance of N in repair, avoidance,quenching and scavenging mechanisms. Phycoerithrin and phyco-cianin pigments might act as an N reservoir, which is used insummer to synthesize molecules involved in repair and photo-protection mechanisms. Xanthophyll pigments play an importantphotoprotective and antioxidant roles. However, the observedtrend is more likely to be explained by the higher temperaturesreached in the lagoon compared with the Mediterranean zonesrather than by irradiance. Therefore, the studied photosyntheticand biochemical mechanisms seem to be effective not only againsthigher irradiance, but also at higher temperatures (important in aclimate change scenario), but are highly dependent on nutrientavailability.

Acknowledgements

This study was financed by the ECOLIFE Project (CGL08-05407-C03) of the Ministry of Education and Science of Spain and by theproject “Sistema de Monitorizaci�on Costera para el Mar Menor”(Plan de Ciencia y Tecnología de la Regi�on de Murcia 2007e2010)(Consejería de Universidades, Empresa e Investigaci�on). Thanks aredue to all colleagues who helped in field sampling and laboratorywork, especially to G. Hern�andez, P. Rodríguez, B. Domínguez, N.Navarro and J. Jofre. We want to thank to three anonymous re-viewers their kind and stimulating comments about the ms.

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