Physiological response and photoacclimation capacity of Caulerpa prolifera (Forsskål) J.V. Lamouroux and Cymodocea nodosa (Ucria) Ascherson meadows in the Mar Menor lagoon (SE Spain)

Marine Environmental Research 79 (2012) 37e47

Contents lists available at SciVerse ScienceDirect

Marine Environmental Research journal homepage: www.elsevier.com/locate/marenvrev

Physiological response and photoacclimation capacity of Caulerpa prolifera (Forsskål) J.V. Lamouroux and Cymodocea nodosa (Ucria) Ascherson meadows in the Mar Menor lagoon (SE Spain) Marta García-Sánchez a, *, Nathalie Korbee b, Isabel Ma Pérez-Ruzafa c, Concepción Marcos a, Belén Domínguez b, Félix L. Figueroa b, Ángel Pérez-Ruzafa a a

Departamento de Ecología e Hidrología, Facultad de Biología, Campus Espinardo, Regional Campus of International Excellence “Campus Mare Nostrum”, Universidad de Murcia, Murcia 30100, Spain Departamento de Ecología y Geología, Facultad de Ciencias, Universidad de Málaga, Málaga 29071, Spain c Departamento de Biología Vegetal I, Facultad de Biología, Universidad Complutense de Madrid, Madrid 28040, Spain b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 January 2012 Received in revised form 28 April 2012 Accepted 3 May 2012

The macroalga Caulerpa prolifera colonized the Mar Menor coastal lagoon after the enlargement of the main inlet in 1972, coexisting now with the previous Cymodocea nodosa meadows. The physiological response and the photoacclimation capacity of both species were studied. For this purpose in vivo chlorophyll a fluorescence, photoprotective mechanisms and oxidative stress were measured in both species in summer 2010 and exposure-recovery experiments were conducted to determine the acclimation capacity of both species. The results suggest that C. prolifera behaves as a shade-adapted species with a low photoprotective capacity, light being one of the main factors governing its distribution in the lagoon. The high photosynthetic capacity and lack of photoinhibition found in C. nodosa suggest that this species is highly photoprotected. It also possesses a high concentration of lutein and a high deepoxidation degree, related to a much higher NPQmax value. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Caulerpa prolifera Chlorophyll fluorescence Coastal lagoon Cymodocea nodosa Photoprotection mechanism Oxidative stress

1. Introduction Traditionally, in the Mar Menor lagoon, primary production due to benthic vegetation was more important than that due to planktonic (Ros, 1987). This benthic vegetation is predominantly made up of macrophyte meadows, the most important species in the functioning of the lagoon being the chlorophycean Caulerpa prolifera (Forsskål) J.V. Lamouroux and the seagrass Cymodocea nodosa (Ucria) Ascherson (Terrados and Ros, 1991). Until the 1970s, the Mar Menor was oligotrophic, and primary productivity was mainly due to C. nodosa, the dominant macrophyte in soft bottoms. Since the enlargement of the Estacio inlet in 1972, the Mar Menor has gradually lost its lagoon characteristics and increasingly resembles the Mediterranean Sea (Pérez-Ruzafa et al., 1991, 2005). More particularly, there has been a substantial decrease in salinity levels and a smoothing of the more extreme temperatures, especially the lower ones, which has had a marked influence on the growth and expansion of C. prolifera, which can not * Corresponding author. Tel.: þ34 868 884326; fax: þ34 868 883963. E-mail address: [email protected] (M. García-Sánchez). 0141-1136/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.marenvres.2012.05.001

withstand temperatures below 10
C (Meinesz, 1979). In fact, there is no mention of the presence of C. prolifera in the Mar Menor in studies carried out prior to dredging El Estacio inlet (Lozano, 1954; Pérez-Ruzafa et al., 1987, 1989; Simonneau, 1972). During the early 1980s, the bottom of the Mar Menor was covered by a mixed meadow of C. nodosa and C. prolifera (PérezRuzafa et al., 1989; Terrados and Ros, 1991), which by the end of the decade, covered almost three-quarters of the lagoon. The monoespecific beds of C. prolifera had been increasing until the 90s and remain constant since then (Pérez-Ruzafa et al., 2012). Meanwhile, the distribution of C. nodosa became mainly restricted to small patches in sandy shallower areas (Pérez-Ruzafa et al., 2005, 2012). According to these studies C. nodosa has a greater biomass in shallow zones (<2 m) than in deeper zones during the summer period. In contrast, C. prolifera has a greater biomass in deep zones than in shallow ones (Pérez-Ruzafa et al., 2012). Several causes have been suggested for this change: the increasing demographic stress on the lagoon (mainly domestic pollution and fisheries), the construction of marine sport harbors, the dredging of parts of the lagoon bottoms, the filling-in of some beaches, the input of terrigenous allochtonous materials, the

38

M. García-Sánchez et al. / Marine Environmental Research 79 (2012) 37e47

opening of the inlets and so on (Pérez-Ruzafa et al., 1989, 1991, 2006). Both the dredging of the lagoon and, in particular, the deepening of the shallow inlets, has largely modified the bottom topography and, accordingly, the movements of the lagoon water masses have also been altered. The replacement of Mediterranean seagrasses like C. nodosa by Caulerpa species may affect the structure and function of the ecosystem. Moreover, the substitution of C. nodosa and the colonization of unvegetated bottoms by a continuous and dense bed of C. prolifera changes substratum characteristics and fish species composition (Pérez-Ruzafa et al., 2006). The same process reduces native species richness and alters ecological processes (Piazzi and Balata, 2008), resulting in several negative effects like the gradual decrease in fisheries that has occurred with Mugilidae species in the Mar Menor (Pérez-Ruzafa et al., 1987; Perez-Ruzafa and Marcos, 1987). These changes in vegetation cover in the Mar Menor have involved changes in the sediment nature, producing siltation and an increase in the organic matter content of the sediments. At the same time, dissolved oxygen in the water at the bottom level decreases nearly to zero in muddy areas with dense plant cover (Pérez-Ruzafa et al., 1989, 2005). Macrophytes are sensitive to enhanced solar radiation, including short wavelengths (UVB, 280e315 nm), and its effects may be expressed as the photoinhibition of photosynthesis (Bischof et al., 1998; Dring et al., 1996; Figueroa et al., 1997; Hanelt et al., 1993), DNA damage (Pakker et al., 2000a, 2000b; van de Poll et al., 2001; Wiencke et al., 2000), inhibition of the activity of enzymes involved in nutrient transport and carbon assimilation (Bischof et al., 2000; Gómez and Figueroa, 1998), and finally decreased growth (Aguilera et al., 1999; Altamirano et al., 2000; Grobe and Murphy, 1998; Pang et al., 2001). Because macrophytes are sessile organisms, they have to acclimate (or adapt) to the prevailing light conditions in their natural habitats (Figueroa et al., 2003). They have developed mechanisms to protect their photosynthetic apparatus in order to cope with increasing levels of UV and PAR radiation (Häder et al., 1996). The synthesis of UV-absorbing compounds, DNA repair mechanisms and other photoprotective processes, such as dynamic photoinhibition, allow macrophytes to survive under increasing irradiances levels (Bischof et al., 2006; Häder and Figueroa, 1997; Korbee-Peinado et al., 2004). Despite the information available concerning the physiological response and protective mechanisms against stress factors in macrophytes, little has been reported for the two studied species. A few studies provide reliable data about photosynthesis (Beer et al., 1998; Enríquez et al., 2004; Malta et al., 2005; Olesen et al., 2002; Silva and Santos, 2003; Terrados and Ros, 1991, 1992, 1993, 1995), and others on pigment composition (Casazza and Mazzella, 2002; Hegazi et al., 1998, 2000) and salinity adaptation (FernándezTorquemada and Sánchez-Lizaso, 2011; Pagès et al., 2010; Sandoval-Gil et al., 2012). The main goal of the present work was to describe the physiological response and the photoacclimation capacity of C. nodosa and C. prolifera under solar radiation. This basic knowledge is very important as it contributes to our understanding of the ecology of these macrophyte assemblages, which play a major role as primary producers in the ecosystem studied.

ranges from 39 to 47 and annual water temperature ranges from 9 to 31
C. The bottom of the lagoon is mainly composed of mud with high organic matter content (up to 28% in central areas covered by the C. prolifera meadows) (Pérez-Ruzafa et al., 2012). Specimens of C. prolifera and C. nodosa were collected from seven different sampling stations in the Mar Menor lagoon (Fig. 1), which are representative of the environmental heterogeneity of this ecosystem, including more confined sites, locations influenced by the Mediterranean Sea, sites exposed to agricultural and urban wastes as well as those characterized by their good ecological status. The purpose was to encompass the whole variability present in the lagoon. Within each locality, C. prolifera and C. nodosa meadows are distributed according to the organic matter content and size of sediment particles, with C. nodosa meadows on sandy bottoms with a low organic matter content and C. prolifera on mud with high organic matter content (García-Sánchez et al., 2012). Depths of around 1.5e2 m determine the boundary for the dominance of each species, C. nodosa maintaining mono-specific

2. Materials and methods 2.1. Sampling sites and plant material The Mar Menor is a hypersaline coastal lagoon, located in the southeast of Spain, with a surface area of 135 km2, a mean depth of 3.6 m and maximum depth of about 6 m. The salinity of the lagoon

Fig. 1. Location of the Mar Menor lagoon on the Mediterranean coast, and location of the sampling sites.

M. García-Sánchez et al. / Marine Environmental Research 79 (2012) 37e47

meadows in shallow areas while C. prolifera dominates in deeper areas (Pérez-Ruzafa et al., 2012). Therefore, plants were collected by scuba diving from an average depth of 2.5 m for C. prolifera meadow and <1 m depth for C. nodosa meadow. To account for intra-station variability, three samples of each species were collected at each sampling station for analytic determinations. During the study period mean salinity was 41  1.8 and the mean water temperature 28  1.5
C. The environmental conditions prevailing during the sampling period, as well as parameters characterizing C. prolifera and C. nodosa meadows, are summarized in Table 1 and Table 2, respectively. More detailed information about environmental characteristic of each sampling site and the methodology used is reported in García-Sánchez et al. (2012). The measurements were carried out in early July 2010, which is the time for maximal growth rates and maximal biomass values for both species (Terrados and Ros, 1991). Samples of both species were collected and immediately freezing in liquid nitrogen to analyze phenolic compounds, DPPH, photosynthetic pigments and lipid peroxidation. Samples were stored at 80
C until analyses. Samples for C and N determinations were kept desiccated until analysis. In all cases, only leaves from C. nodosa and fronds for C. prolifera were used for the analyses.

Table 2 Minimum, maximum and mean values  SD (n ¼ 7) of parameters characterizing Caulerpa prolifera and Cymodocea nodosa meadows. Depths (shallow and deep) were selected according to the natural boundaries for the dominance of each species (see Materials and Methods section). Shallow (<1.5 m) Min

Max

Deep (>1.5 m)

Mean  SD

Depth range (m) 0.5 1.5 0.7 Organic matter 0.06 0.35 0.18 in sediment (%) 0.0 2.8 1.3 Caulerpa prolifera biomass (g DW/m2) Cymodocea nodosa 77.3 382.5 210.2 biomass (g DW/m2)

 0.4  0.1  1.1  127.8

Min 1.5 0.10

Max 2.8 2.76

109.0

366.7

0.0

1.2

Mean  SD 2.1  0.4 1.11  0.92 205.4  94.9 0.2  0.5

224, 337,469, 693, 942, 1418 mmol photons m2 s1). After an initial quasi-dark measurement (w1.5 mmol photons m2 s1) to provide estimates of minimum (Fo) fluorescence, a saturating flash was applied to obtain the maximal fluorescence level from fully reduced PSII reaction center (Fm) and Fv/Fm was obtained (n ¼ 4). RLCs were constructed by calculating ETR through PSII for each level of actinic light:

 
0 0 ETR mmol electrons m2 s1 ¼ Fm  F=Fm $E$A$FII

2.2. Measurement of solar radiation Solar radiation was measured at three wavelength bands (UVB ¼ 280e315 nm, UVA ¼ 315e400 nm and PAR ¼ 400e700 nm) using two Hyperspectral Irradiance Sensors for UV and PAR (Ramses, TrioS GmbH, Oldenburg, Germany). The average irradiances during the experimental period at 12:00 GMT were 398.3  20.4 W m2, 45.5  1.8 W m2 and 1.1  0.01 W m2 for PAR, UVA and UVB radiation, respectively. The light attenuation coefficients were 0.6 m1, 1.1 m1 and 3.9 m1 for PAR, UVA and UVB, respectively. Taking into account that the average depth at which C. nodosa grows in the Mar Menor is 2.1 m, the average irradiances for this species in its natural habitat are 29% and 5% of incident PAR and UVA, respectively. As the average depth for C. prolifera in the lagoon is 3.3 m the irradiances for this species are 14% and 4% of the incident PAR and UVA, respectively. Less than 1% of UVB reaches either meadow at these depths. 2.3. Photosynthetic activity as in vivo chlorophyll fluorescence In vivo chlorophyll a fluorescence of photosystem II (PSII) was determined using a portable pulse amplitude modulation fluorometer (Diving-PAM, Waltz GmbH, Effeltrich, Germany). After 15 min of dark adaptation, a rapid light curve (RLC) was initiated, involving a 20-s exposure to 9 incremental irradiances (20, 66, 137, Table 1 Environmental variables recorded during the sampling period. Minimum, maximum and mean values  SD (n ¼ 7) of the sampling stations.

Water column NH3 (mmol/L) NO2 (mmol/L) NO3 (mmol/L) DIN (mmol/L) PO4 (mmol/L) Water temperature (
C) Salinity pH Dissolved oxygen (mg/L) Suspended solids (g/L) Air conditions Air temperature (
C)

39

Minimum

Maximum

Mean  SD

0.1 0.0 0.0 0.2 0.1 26.7 40.2 8.4 5.1 0.03

5.6 0.3 3.2 8.8 0.1 30.8 41.9 8.6 12.3 0.05

2.0 0.1 1.4 3.5 0.1 28.0 41.1 8.5 8.7 0.04

24.9

28.4

26.9  1.4

         

2.0 0.1 1.6 3.5 0.0 1.5 0.6 0.1 2.5 0.01

where F0 m is the maximal fluorescence induced with a saturating white light pulse, F is the steady-state fluorescence in the light and F0 m  F/F0 m estimates the effective quantum yield of PSII. E is the incident irradiance, A is the absorptance and FII is the fraction of chlorophyll associated to photosystem II. In the case of green macroalgae and marine angiosperms, FII is 0.5 (Grzymski et al., 1997). The absorptance A ¼ 1  (Et/Eo), was calculated from the light transmitted through a piece of each species (Et) placed on a cosine-corrected PAR sensor (Licor 192 SB, Li-Cor, Lincoln, NE, USA) connected to a data-logger (Licor-1000), and Eo is the incident irradiance in the absence of the macrophytal piece. The absorptance measured was 0.86  0.05 for C. nodosa and 0.92  0.03 for C. prolifera. RLC data were fitted to the model describe by Jassby and Platt (1976) to obtain values for the initial slope (aETR) and maximal ETR (ETRmax) in C. nodosa, and to the model of Platt and Gallegos (1980) for C. prolifera as photoinhibition was observed. The inhibition term (b) was calculated for the latter species. 2.4. Non-photochemical quenching Y(NO), which reflects the fraction of energy that is passively dissipated in the form of heat and fluorescence, mainly due to closed PSII reaction centers, was calculated as follows (Klughammer and Schreiber, 2008):

YðNOÞ ¼ F=Fm Y(NPQ), which corresponds to the fraction of energy dissipated in the form of heat via a regulated photoprotective NPQ mechanism (Klughammer and Schreiber, 2008), was calculated as:


0 YðNPQ Þ ¼ F=Fm  YðNOÞ Then, non-photochemical quenching (NPQ) was calculated as follows:

NPQ ¼ YðNPQ Þ=YðNOÞ Y(NPQ)max and NPQmax were calculated using the Jassby and Platt (1976) equation for both species.

40

M. García-Sánchez et al. / Marine Environmental Research 79 (2012) 37e47

The Y(NO)max was calculated using a modified Jassby and Platt (1976) equation for C. prolifera:

YðNOÞmax ¼ YOmax $ðtanhða$E=YOmax ÞÞ þ Y0 where YOmax is the light-saturated value for this variable, tanh is the hyperbolic tangent function, a is the slope at low irradiance, E is the incident irradiance and Y0 determines the point at which the function crosses the y-axis. As the Y(NO) values were similar for the whole light curve obtained for C. nodosa, the Y(NO)max was assumed to be the maximal value found for each curve. 2.5. Photosynthetic pigments Chlorophyll a (chl a) and chlorophyll b (chl b) contents were determined spectrophotometrically, while carotenoids were identified and quantified by high-performance liquid chromatography (HPLC). Both analyses were made by extracting pigments from plants (15 mg fresh weight, FW) using 1 mL of N,Ndimethylformamide (DMF) and maintained in darkness at 4
C for 12 h. After centrifugation at 4000 rpm for 10 min (Labofuge 400R, Heraeus, Kendro Laboratory Products, Langenselbold, Germany) each supernatant was used to measure chlorophyll spectrophotometrically. The chlorophyll concentrations were calculated using Wellburn (1994) equations. Additionally, the extracts were filtered (0.2 mm) and the carotenoid composition and concentrations were determined by HPLC as described by Lubián and Montero (1998). Three replicates were measured for each species and site. Carotenoids were identified using commercial standards (DHI LAB Products), except for siphonein and siphonaxanthin, which were identified using secondary standards according to their retention times and absorption spectra as indicated in Hegazi et al. (1998). Pigment contents were expressed as mg g1 dry weight (DW) after determining the fresh to dry weight ratio in the tissue (6.2 and 6.0 for C. prolifera and C. nodosa, respectively). The de-epoxidation degree (in %) was calculated as follows:

de-epoxidationð%Þ ¼ ðA=V þ AÞ$100 where A ¼ antheraxanthin and V ¼ violaxanthin 2.6. Phenolic compounds Samples of C. prolifera and C. nodosa (0.25 g FW, n ¼ 3) were ground with a mortar and pestle in sand at 4
C, and extracted overnight 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, expressed in mg g1 DW, were determined using gallic acid as a standard following FolineCiocalteu’s method (Folin and Ciocalteu, 1927).

against plant extract concentration (mg DW/mL) in order to obtain the EC50 value (oxidation index), which represents the concentration of the extract (mg/mL) required to scavenge 50% of the DPPH in the reaction mixture. Ascorbic acid was used as positive control. 2.8. Lipid peroxidation As an indication of oxidative stress, lipid peroxidation was studied from the content of thiobarbituric acid reactive compounds (TBARs), mainly malondialdehyde (MDA) (Salama and Pearce, 1993). Pieces of thallus (0.3 g FW, n ¼ 3) were ground in sea sand, mixed with 1.5 mL 0.1% trichloroacetic acid (TCA), and centrifuged at 4000 rpm for 30 min. 100 mL of supernatant was mixed with 900 mL of 0.5% TBA prepared with 20% TCA and boiled for 30 min. Then, the absorbance at 512 nm was measured. An extinction coefficient of 150 M1 cm1 was used to determine the lipoperoxide content. 2.9. Internal carbon and nitrogen contents Total internal C and N were determined by combustion using a CNHS LECO-932 (Michigan, USA) elemental analyzer. 2.10. Exposure-recovery experiments The kinetics of exposure and recovery under strong sunlight was studied in plants collected at around 11:00 GMT. Plants were transferred from their natural setting to the surface, where they were exposed in situ for 2.5 h to natural solar irradiances stronger than in their natural habitats. Incubations were made on the shore at midday (12:00 GMT), using white trays in which water was renewed periodically to avoid any increase in temperature. A recovery phase of 2 h under dark conditions followed this exposure period. The Fv/Fm was measured every 30 min during both exposure and recovery periods. 2.11. Statistical analysis All the collected samples were pooled to study the general physiological pattern of both species in the Mar Menor lagoon. The assumption of normality and homogeneity data were verified and data were square root or double squared root transformed when necessary. Differences between C. prolifera and C. nodosa concerning photosynthetic parameters, pigment content, oxidation indices, lipoperoxides and internal C and N were tested by T-Student test. For phenolic compounds and de-epoxidation degree there was no homogeneity of variance, even after transformation of the data, so a non-parametric ManneWhitney test was performed. SPSS Statistics v.19 was used for the analyses. 3. Results

2.7. Antioxidant activity

3.1. Photosynthetic activity as in vivo chlorophyll fluorescence

The DPPH (2, 2-diphenyl-1-picrylhydrasyl) free-radical scavenging assay was carried out in triplicate, according to the method of Brand-Williams et al. (1995). Briefly, 150 mL of each methanolic extract were mixed with 0.5 mL of a 90% methanolic DPPH solution prepared daily at 1.27 mM. The reaction was complete after 30 min in the dark at room temperature, and the absorbance was read at 517 nm. The calibration curve made with DPPH was used to calculate the remaining concentration of DPPH in the reaction mixture after incubation. Values of DPPH concentration (mM) were plotted

The ETR versus irradiance curve was very different for C. prolifera and C. nodosa (Fig. 2, Table 3). The latter showed no photoinhibition even at 1500 mmol photons m2 s1, while C. prolifera showed high photoinhibition at irradiances higher than w100 mmol photons m2 s1. Moreover, C. nodosa had a maximal ETR (ETRmax) that was more than 3 times higher than that corresponding to C. prolifera (p < 0.001). However, the photosynthetic efficiency (aETR) of the seagrass was half that of the macroalga (p < 0.001). Thus, the saturation light intensity was 6 times higher (p < 0.001).

M. García-Sánchez et al. / Marine Environmental Research 79 (2012) 37e47

A

2

20

1.5

15 1

10 5 0 0

200

400

600

800

1000 1200 1400 1600

PAR (µmol photons m -2 s-1) Fig. 2. Electron transport rate (ETR) versus irradiance curves for Cymodocea nodosa (square) and Caulerpa prolifera (triangle). Data are means  SE (n ¼ 7).

Y(NO), Y(NPQ), NPQ

ETR (µmol e - m-2 s-1)

25

41

0.5 0

B

2 1.5 1

3.2. Non-photochemical quenching The RLCs also provide data on the evolution of the Y(NO), Y(NPQ) and NPQ, which also showed very different patterns between both species (Fig. 3a,b). C. nodosa presented higher Y(NO) values at low irradiances (0e70 mmol photons m2 s1) but high values for Y(NPQ) and consequently for NPQ at mild and high irradiances (range 200e1500 mmol photons m2 s1). In the case of C. prolifera, both Y(NPQ) and NPQ were very low. In fact, the NPQmax for C. nodosa was 8 times higher than for C. prolifera (p < 0.001).

0.5 0 0

200

400

600

800

PAR (µmol photons m -2 s-1) Fig. 3. Light intensity response curves of Y(NO) (black rhombus), Y(NPQ) (gray square) and NPQ (black triangle) in (A) Cymodocea nodosa and (B) Caulerpa prolifera. Data are means  SE (n ¼ 7).

3.3. Photosynthetic pigments The chl a (p < 0.01) and chl b (p < 0.001) contents were much higher in C. prolifera than in C. nodosa. The carotenoids common to both species were neoxanthin, violaxanthin, antheraxanthin, lutein and b-carotene. Two further carotenoids were detected in C. prolifera, siphonein and siphonaxanthin, which were not present in C. nodosa. In general, the content of the carotenoids common to both was significantly lower in C. prolifera than in C. nodosa (Table 4). Nonetheless, no significant differences were found in the total violaxanthineantheraxanthinezeaxanthin pool (p ¼ 0.694). It is also important to highlight that C. nodosa had a very high concentration of lutein (p < 0.001) and showed a higher degree of de-epoxidation (p < 0.01) (Fig. 4a). 3.4. Phenolic compounds and antioxidant activity The oxidation index EC50 calculated by the DPPH radical scavenging method was higher for C. prolifera that for C. nodosa (Fig. 4b), indicating that the latter species has a higher antioxidant capacity. Since the total phenolic content calculated using the FolineCiocalteu’s method was much higher in C. prolifera than in C. nodosa (Fig. 4c, p < 0.01), it seems that the total phenol content is Table 3 Electron Transport Rate (ETR) versus irradiance curve parameters, maximal ETR (ETRmax, mmol e m2 s1), photosynthetic efficiency (aETR), photoinhibition term (b) and the saturation light intensity (Ek) (mmol photons m2 s1). And the maximal Y(NO), Y(NPQ) and NPQ for Cymodocea nodosa and Caulerpa prolifera. Data are means  SE (n ¼ 7). Significance level: ***p < 0.001.

ETRmax

aETR b

Ek Y(NO)max Y(NPQ)max NPQmax

Cymodocea nodosa

Caulerpa prolifera

20.1  1.7 *** 0.14  0.01 e 146.7  12.9 *** 0.47  0.02 0.51  0.02 *** 1.42  0.18 ***

6.2 0.26 0.05 23.5 0.89 0.14 0.18

      

0.8 0.01 *** 0.01 2.7 0.02 *** 0.01 0.02

negatively correlated with the antioxidation capacity, which is quite the opposite to what might be expected. 3.5. Lipid peroxidation Lipoperoxides did not significantly differ between C. prolifera (48  3 nmol g1 DW) and C. nodosa (41  3 nmol g1 DW) (p ¼ 0.318). 3.6. Internal carbon and nitrogen contents The internal C content was similar between species (Fig. 5a, p ¼ 0.305). However, C. prolifera showed a higher total internal N content (Fig. 5b, p < 0.01) than C. nodosa and hence a lower C/N ratio (Fig. 5c, p < 0.05). 3.7. Exposure-recovery experiments The decay in Fv/Fm after exposure to high irradiances for 2.5 h was less pronounced in C. nodosa (17%) than in C. prolifera (60%). Table 4 Pigment concentrations (mg g1 DW) for Cymodocea nodosa and Caulerpa prolifera. Chlorophylls (Chl a, Chl b). ND ¼ not detected. Data are means  SE (n ¼ 7). Significance levels: *p < 0.05; **p < 0.01; ***p < 0.001. Cymodocea nodosa Chl a Chl b Chl b/chl a Siphonein Neoxanthin Violaxanthin Antheraxanthin Siphonaxanthin Lutein b-carotene

4.48 1.66 0.32 ND 0.11 0.14 0.08 ND 0.59 0.01

 0.52  0.33  0.05  0.01 *  0.01  0.01 **  0.05 ***  0.001 ***

Caulerpa prolifera 23.16 12.62 0.65 0.03 0.08 0.20 0.04 0.03 0.05 0.002

         

3.02 ** 1.50 *** 0.07 * 0.004 0.01 0.02 0.01 0.01 0.01 0.0003

42

M. García-Sánchez et al. / Marine Environmental Research 79 (2012) 37e47

2.0

** EC50 (mg DW mL-1)

A

30 25 20 15 10 5 0

B

1.5 1.0 0.5 0.0

40 35

TPC (mg GAE g-1DW)

de-epoxidation %

40 35

**

C

30 25 20 15 10 5 0

Fig. 4. (A) Percentage of de-epoxidation, (B) DPPH radical scavenging activity expressed as oxidation index EC50 given in mg DW mL1 and (C) Total phenolic content (TPC) of methanolic extracts expressed as gallic acid equivalents (mg GAE g1 DW) for Cymodocea nodosa (white bar) and Caulerpa prolifera (black bar). Data are means  SE (n ¼ 7). Significance levels: **p < 0.01.

Furthermore, the latter species did not recover it photosynthetic capacity (recovery phase) after 2 h in dark conditions (Fig. 6).

to south, mainly covering the deepest areas of the lagoon (PérezRuzafa et al., 1989, 2012). Meanwhile, the seagrass C. nodosa has shown a gradual but continuous decrease in mean biomass in the entire lagoon, especially in deeper areas (Pérez-Ruzafa et al., 2012). The low ETRmax and high aETR, accompanied by low Ek and high photoinhibition at mild irradiance levels, point to the shadeadapted plant pattern of C. prolifera. In fact, this species shows optimal growth in the Mar Menor lagoon at depths greater than 1.5 m. Similar results for the same species were found by Terrados

4. Discussion For three decades, the gradual expansion of C. prolifera in the Mar Menor lagoon has confined C. nodosa meadows mainly to sparse patches in sandy shallow zones. Since the introduction of this species in the lagoon in the early 1970s, the macroalga has spread from north

40 35

A C (mg g-1 DW)

N (mg g-1 DW)

400 350 300 250 200 150 100 50 0

14 12

C

B

**

30 25 20 15 10 5 0

*

C/N

10 8 6 4 2 0 1

Fig. 5. (A) Internal C content (mg g DW), (B) internal N content (mg g1 DW) and (C) C:N ratio for Cymodocea nodosa (white bar) and Caulerpa prolifera (black bar). Data are means  SE (n ¼ 7). Significance levels: *p < 0.05; **p < 0.01.

M. García-Sánchez et al. / Marine Environmental Research 79 (2012) 37e47

Exposure

Recovery

120

% Fv/Fm

100 80 60 40 20 0 0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

Time (h) Fig. 6. Percentage of maximal quantum yield (Fv/Fm) related to initial value during exposure and recovery periods for Cymodocea nodosa (square) and Caulerpa prolifera (triangle). Data are means  SE (n ¼ 3).

and Ros (1992) and for other Caulerpa spp. from the Gulf of Mexico and the Caribbean (Driscoll, 2004; Gacia et al., 1996; Robledo and Freile-Pelegrin, 2005). Species that are low light acclimated could experience a drastic drop in photosynthetic activity when exposed to extremely higher light regimes (Häder et al., 1996). The ability for dynamic photoinhibition during exposure to high light conditions, as well as the general degree of acclimation of photosynthesis to different light regimes, may influence the upper depth distribution of algae (Hanelt, 1996). Apparently, two different molecular mechanisms are involved in the photoinhibition response. Thus, during rapid light acclimation, the reaction can be described as a combination of two different processes: one of a relative proportion with a slow rate constant and a second of a relative proportion with a fast rate constant at a given time (t) (Hanelt, 1998). In our study, when C. prolifera was exposed to increased irradiances for 2.5 h at midday a severe drop in photosynthetic activity (high photoinhibition) was found. The response for the first 30 min of exposure to high irradiance was a decrease up to 58% in Fv/Fm (fast reaction), while the following 2 h caused a slight reduction up to 40.4% (slow reaction). Additionally, no recovery was observed even after 2 h in darkness. This decrease in the photosynthetic activity observed for C. prolifera may be caused by photodamage rather than by dynamic photoinhibition, which is a quickly reversible process (Krause and Weis, 1991). This was not the case for C. nodosa, which exhibited a slight decrease (17.2%) in the same light conditions as for C. prolifera and recovery up to 87.3% reflecting a high ability for dynamic photoinhibition. Thus, based on its high ETRmax and low photosynthetic efficiency together with high Ek and no photoinhibition even at high irradiances, C. nodosa showed a typical sun-adapted behavior. The estimated photosynthetic parameters obtained for C. nodosa in this study were lower than those found by Beer et al. (1998) in the Mediterranean (ETRmax ca. 60 mmol e m2 s1) and by Silva and Santos (2003) in Ria Formosa (ETRmax ca. 69.9  9.2 mmol e m2 s1; aETR ca. 0.4). Once macrophytes absorb light, they have ways of getting rid of excess energy, by means of non-photochemical mechanisms that quench singlet-excited chlorophylls and harmlessly dissipate excess excitation energy as heat (Müller et al., 2001). In this context, complementary PSII quantum yields calculated from simple fluorescence parameters measured by PAM fluorometry have been proposed recently (Klughammer and Schreiber, 2008). In our study, we found a totally different behavior for C. prolifera and C. nodosa in terms of Y(NO) and Y(NPQ) parameters. The first species showed markedly high values of Y(NO), mostly at the cost of Y(NPQ). This can be explained by damage or non-activation of the NPQ-generating reactions, particularly due to the MehlereAscorbateePeroxidase

43

cycle (Asada, 1999), which is mainly responsible for the generation of a transthylakoidal DpH after inactivation of CO2 dependent electron flow. In fact, if NPQ ¼ Y(NPQ)/Y(NO), low NPQ is indicative of severe damage to the photoprotective reactions, which can be expected to lead to secondary damage by photoinhibition. Opposite results were found for C. nodosa, which was characterized by high values of NPQ, indicating active photoprotective mechanisms. These mechanisms are highly related with the xanthophyll cycle (Demmig-Adams and Adams, 1996). Otherwise, sun-adapted macrophytes typically exhibit a larger pool of xanthophyll cycle pigments as well as greater ability to convert violaxanthin to antheraxanthin and zeaxanthin rapidly under high light (Demmig-Adams and Adams, 1996). As regards the pigments involved in this cycle, zeaxanthin was not found in C. nodosa or C. prolifera in our study, which agrees with the observations of Hegazi (1999) for the same species. The absence of zeaxanthin in C. prolifera could be related with the depth (>1.5 m) at which this species lives in the Mar Menor, taking into account that other authors have only found zeaxanthin in Caulerpa spp. sampled no deeper than 0.3 m (Raniello et al., 2006). A truncation of the ViolaxanthineAntheraxanthineZeaxanthin (VAZ) cycle might occur, as has been reported in other studies (Gilmore and Yamamoto, 2001; Goss et al., 1998; Raven and Geider, 2003). In fact, antheraxanthin may have a zeaxanthin-independent function in quenching excess energy (Müller et al., 2001), such as the conversion of violaxanthin into antheraxanthin, which seems to be directly involved in the photoprotection of species exposed to light stress (Raniello et al., 2006). In our study, C. nodosa showed a higher de-epoxidation degree (36%) than C. prolifera (16%). In addition to the above-mentioned VAZ cycle, interconversion based on luteinesiphonaxanthin seems to operate in some species, as the latter is biosynthetically related with and derived from lutein (Raniello et al., 2006). Two pigments typical of Caulerpales, siphonaxanthin and siphonein (Raniello et al., 2006; Walton et al., 1970), were both identified in C. prolifera. Another role of these carotenoids could be the conversion of siphonaxanthin into siphonein by oxidation. An important role of siphonaxanthin in the acclimation to low light of deep environments of Caulerpa racemosa var. cylindracea has been described in Raniello et al. (2006). Some carotenoids, such as lutein, also play a fundamental role in energy dissipation, since they are able to quench the excitation of chl a in the reaction centers (Esteban et al., 2009; Raniello et al., 2006). Such is the case with lutein, to which a photoprotective function has been attributed (Müller et al., 2001). Lutein was the most abundant carotenoid in C. nodosa (0.59 mg g1 DW) but was present at very low concentrations in C. prolifera (0.06 mg g1 DW). The pigment composition of C. prolifera obtained in our study was within the same range that others reported by Raniello et al. (2004) and Hegazi (1999). It has been shown that macrophytes are able to adjust pigment levels and ratios, allowing the organism to use the available light more efficiently (Riechert and Dawes, 1986). Pigment changes affect absorptance values, in fact, absorptance is expected to vary considerably, both interspecifically and intraspecifically. One example of such variation is the difference between the absorptance determined by Beer et al. (1998) and by Silva and Santos (2003) for C. nodosa collected in different locations. In general, the chlorophyll content tends to be higher in macroalgae than in seagrasses, so that the results obtained for both C. prolifera and C. nodosa in this study were in agreement with this assumption and were similar to those described by Hegazi (1999). The higher chl b/ chl a ratio found in C. prolifera is also characteristic of a shadeadapted species. However, the chlorophyll content in our samples was double that those reported by Robledo and Freile-Pelegrin (2005) in C. prolifera at Yucatan (Mexico), a difference that is

44

M. García-Sánchez et al. / Marine Environmental Research 79 (2012) 37e47

probably related to the different environmental factors of both collection sites. The polyphenols of seaweeds, commonly found in brown algae (Targett and Arnold, 1998), could assist the algae to overcome oxidative stress, while playing a putative adaptive role in defense against grazers, such as marine herbivores (Altena and Steinberg, 1992). There is also some evidence of the accumulation of other phenolic compounds, namely coumarins, with their high UVabsorption properties, in green macroalgae like Caulerpa and Dasycladus (Kubitzki, 1987; Pérez-Rodríguez et al., 1998, 2001). The presence of phenols in seagrasses was also reported in Posidonia oceanica (Agostini et al., 1998; Cuny et al., 1995; Kartal et al., 2009; Vergés et al., 2011), Zostera marina (Vergeer et al., 1995) and Zostera noltii (Zapata and McMillan, 1979). To the best of our knowledge this is the first time that the concentration of phenols in C. nodosa has been reported. Although Cariello et al. (1979) detected phenols in C. nodosa (4-hydroxy-3-methoxybezoic acid (vanillic acid), 1,3,5trihydroxybenzene (phloroglucinol) and 3,4-dihydroxycinnamic acid (caffeic acid)), no concentration of these compounds was included in the work. In our study, the total phenol content was much higher in C. prolifera than in C. nodosa. In the literature, a higher phenol content has been mentioned for Caulerpa spp., even higher than for some brown algae such as Padina spp. or Dictyota spp. (Matanjun et al., 2008; Zubia et al., 2007). C. racemosa showed values of 40.4  1.1 mg g1 DW (Matanjun et al., 2008), and C. prolifera a maximum of 81.3  5.2 mg g1 DW (Zubia et al., 2007). However, we found a lower phenol content than those mentioned above (33.0  2.8 mg g1 DW). Although no specific phenol content has been published for Caulerpa spp., coumarins are known to appear in siphonous green algae and their role in plug formation processes in relation to injury has been described (Pérez-Rodríguez et al., 2003). This evidence could support the idea that the phenols found in C. prolifera play a role in its propagation strategy and their role as plug is suggested by the concentrations found in our study, which seems to be more related with this function than with light intensity (Abdala-Díaz et al., 2006). The published concentrations for P. oceanica (18e40 mg g1 DW) and Z. marina (w33 mg g1 DW) are 2e3-fold higher than those found for C. nodosa (12.1  0.79 mg g1 DW) in this study. C. prolifera showed a higher EC50 than C. nodosa, which implies a lower antioxidant capacity for the first species despite its higher phenol content. Based on the lower phenol content of C. nodosa, it seems that other compounds could be conferring an antioxidant capacity, probably carotenoids such as lutein or b-carotene, which have protective functions in reaction centers (Takaichi, 2011). According to Duarte (1992), macroalgae are characterized by lower concentrations of C than angiosperms; however, our data point to no significant differences between the two species studied. Both the C and N content in leaves exceeded the average values recorded for macroalgae and seagrasses (Duarte, 1992). The N content of C. prolifera was higher than that of C. nodosa and it was over the critical concentration for maximum macroalgal growth (1.5% DW; Fujita et al., 1989). The seagrass showed a higher C:N ratio than C. prolifera, which indicated a better nutritional status for the seaweed at the study site. These results were consistent with those reported by Enríquez et al. (1995, 2004) and Olesen et al. (2002) for these two species. Oxidative stress was determined by measuring lipid peroxidation markers. In this respect, these compounds were similar for both species, although different roles might exist. In C. prolifera the light intensity in their natural habitats may not be high enough to cause severe oxidative damage, and so we suggest that lipoperoxides could play another physiological role related with the release of H2O2, which may function as a defense against herbivores and epiphytes, or allochemical indirect competition with other

species as has been proposed by Choo et al. (2005). In fact, the increase in lipoperoxides levels in Caulerpa taxifolia when they were epiphytized was attributed to this mechanism rather than a role as a marker of oxidative damage (Box et al., 2008). Since lipoperoxides are expressed as a function of the total protein content of the samples in the above study, it was not possible to compare the absolute amount of these compounds with that obtained for C. prolifera in this study. Contreras et al. (2005) found a concentration of 20 nmol g1 DW lipoperoxides in the Phaeophyceae Scytosiphon lomentaria in northern Chile, which is half that obtained in our study for both species. Although no report on lipoperoxides in C. nodosa or in any other seagrasses was found in the literature, we suggest that the lipoperoxides could play a role as markers of oxidative stress in this species, as they live in a high light environment. Heavy metal toxicity could be related to oxidative stress induced in living systems either by increasing concentrations of reactive oxygen species or by reducing cellular antioxidant capacity (Pinto et al., 2003). In primary producers, these effects may result in the inhibition of chlorophyll production, photosynthesis and growth (Baumann et al., 2009). Data of heavy metals in both species are provided by a previous study realized by Sanchiz et al. (2000). The bioaccumulation of Hg, Pb and Zn was higher in C. nodosa leaves than in C. prolifera fronds from the Mar Menor, and was even more than three times higher in the case of Pb. However, no conclusions about the correlation between oxidative stress and contaminant level could be made since no heavy metal was measured in our study. The tolerance of C. nodosa to hypersaline stress has been demonstrated by several studies and no effect of salinity increments on the growth and survival of this seagrass have been found (Sandoval-Gil et al., 2012). Pagès et al. (2010) showed that, at least in the short term, C. nodosa is able to tolerate salinity levels up to 54, without any apparent damage. Moreover, compared with those from Mediterranean, plants from the Mar Menor lagoon showed lower sensitivity to increases in salinity (Fernández-Torquemada and Sánchez-Lizaso, 2011). In fact, when C. nodosa was introduced in the lagoon the salinity range was 52e54, which is higher than in subsequent conditions which allowed C. prolifera to settle (salinity ranges 42e45) (Pérez-Ruzafa et al., 2005). These conditions could have provided pre-existing C. nodosa populations higher stress tolerance, evolving antioxidant defense mechanisms, consistent with the higher photoprotective capacity observed by us in this species. It has been reported that higher antioxidant contents and antioxidant enzyme activities are associated with higher stress tolerance (Choo et al., 2004; Lu et al., 2006). In the case of Caulerpa spp, very few experimental works study the response of C. taxifolia in different salinities (Theil et al., 2007; West and West, 2007). These studies were mainly carried out in hyposaline conditions (<35) that are lower than those in our study area and therefore, a proper discussion in this sense can not be made. Our results suggest that light flux is one of the main factors controlling the spatial distribution of C. prolifera in the lagoon and that it limits its growth in shallow zones. The decline in water column transparency avoids high solar radiation reaching the bottoms and so photoinhibition might be lower in C. prolifera, as has also been described by Häder et al. (1997). This could have favored the faster spread of monoespecific C. prolifera meadows, as occurred in the 1980s, in the north basin of the lagoon, which suffered turbidity episodes during early summer, coinciding with the highest irradiance period (Gilabert, 1992). The lower biomass found for C. prolifera in shallow areas, as well as its generally positive relationship with depth and suspended solids concentration (Pérez-Ruzafa et al., 2012), reinforce the idea that the excess of light could be a major limiting factor for this species.

M. García-Sánchez et al. / Marine Environmental Research 79 (2012) 37e47

However, other abiotic factors such as hydrodynamism, could also affect the growth of this species in shallow waters. It has been described that increased hydrodynamism (>30 cm s1) reduces Caulerpa spp. photosynthetic activity (Driscoll, 2004). In contrast, for C. nodosa the current velocity thresholds for the decline of this species are higher (>60 cm s1) (Cabaço et al., 2010) and, in addition, its internal oxygen status is enhanced by moderate water flow (7 cm s1) (Binzer et al., 2005). In deep areas, light scarcity could limit C. nodosa biomass, according to Terrados and Ros (1991). Nevertheless, although monoespecific C. nodosa meadows are restricted to shallow areas, scatter plants are present at depths greater than 4 m (Pérez-Ruzafa et al., 2012). Even though recent trophic conditions in the Mar Menor have increased the light attenuation coefficient, data show that irradiance above the light compensation point (Ic ¼ 28 mmol photons m2 s1, Terrados and Ros, 1991) could reach more than 5 m depth, even during the months of lower irradiance (average of 3.9 h per day at 6 m depth). Other factors like starch reserves, respiration rates and, therefore, carbon balances would be involved in net photosynthesis rates, which would limit the depth distribution of this species (Taiz and Zeiger, 2010). It is known that marine angiosperms require high energy due both to high respiration rates and the low efficiency of CO2 incorporation (Madsen and Sand-Jensen, 1991; Invers et al., 2001). Also, significant depth-related changes in photosynthetic parameters in C. nodosa have been described, consisting of a higher photosynthetic efficiency, leading to a low light compensation point near the depth limit, even though respiration rates remain constant (Olesen et al., 2002). Therefore, the whole-plant carbon balance should be calculated for the deeper plants and a more detailed study, taking into account seasonal and spatial variability, should be performed. The regression of C. nodosa preceded the deterioration of the water quality in the Mar Menor lagoon, meaning that other factors could be involved in its regression. An excess of organic matter and carbonates in sediments (Pérez, 1989), nutrient limitation (Terrados and Ros, 1993) or interactive effects with other environmental factors (temperature or sediment hypoxia) have been described as provoking a cumulative stress that curtailis the longterm health of seagrass meadows (Koch et al., 2007). Additionally, some competition with C. prolifera could be involved, as suggested by the negative relationship between both species detected by Pérez-Ruzafa et al. (2012) may be in terms of increasing organic carbon and silt in the sediment, as well as sulfide content. In this context, it has been found that caulerpenyne extracts of C. racemosa, directly applied to the leaves of the seagrass C. nodosa, trigger alterations in photosynthesis (Raniello et al., 2007) and other works reflect the negative interaction between C. prolifera and other seagrass species (Halodule wrightii) (Stafford and Bell, 2006; Taplin et al., 2005).

4.1. Conclusion In conclusion, from the results obtained in this work, we could suggest that C. prolifera behaves as a shade-adapted species with a low photoprotective capacity, light being one of the main factors that control its distribution in the lagoon. However, C. nodosa seems to be highly photoprotective and its distribution does not seem to be directly related to the light environment. It possesses a high concentration of lutein and a high de-epoxidation degree, related to a much higher NPQmax value. Additionally, the decay of Fv/Fm after exposure to strong irradiance for 2.5 h was less pronounced in C. nodosa. Moreover, C. prolifera did not recover its photosynthetic capacity (recovery phase) even after 2 h under dark conditions.

45

Acknowledgments This study was partly supported by the projects “Sistema de Monitorización Costera para el Mar Menor” (Plan de Ciencia y Tecnología de la Región de Murcia 2007e2010) (Consejería de Universidades, Empresa e Investigación) and Project CGL08-05407-C03 of the Ministry of Education and Science. Thanks are due to all colleagues who helped in field sampling work, especially to Gabriel Hernández, Nelso Navarro and Joselyn Jofre.

References Abdala-Díaz, R., Cabello-Pasini, T., Pérez-Rodríguez, A., Álvarez, E., Conde, R.M., Figueroa, F.L., 2006. Daily and seasonal variations of optimum quantum yield and phenolic compounds in Cystoseira tamariscifolia (Phaeophyta). Marine Biology 148, 459e465. Agostini, S., Desjobert, J.M., Pergent, G., 1998. Distribution of phenolic compounds in the seagrass Posidonia oceanica. Phytochemistry 48 (4), 611e617. Aguilera, J., Karsten, U., Lippert, H., Vögele, B., Philipp, E., Hanelt, D., Wiencke, C., 1999. Effects of solar radiation on growth, photosynthesis and respiration of marine macroalgae from the Arctic. Marine Ecology Progress Series 191, 109e119. Altamirano, M., Flores-Moya, A., Conde, F., Figueroa, F.L., 2000. Growth seasonality, photosynthetic pigments, and carbon and nitrogen content in relation to environmental factors, a field study of Ulva olivascens. Phycologia 39, 50e58. Altena, V., Steinberg, P.D., 1992. Are differences in the responses between North American and Australasian marine herbivores to phlorotannins due to differences in phlorotannin structure? Biochemical Systematics and Ecology 20, 493e499. Asada, K., 1999. The water e water cycle in chloroplasts, scavenging of active oxygens and dissipation of excess photons. Annual Review of Plant Physiology and Plant Molecular Biology 50, 601e639. Baumann, H.A., Morrison, L., Stengel, D.B., 2009. Metal accumulation and toxicity measured by PAM e chlorophyll fluorescence in seven species of marine macroalgae. Ecotoxicology and Environmental Safety 72, 1063e1075. Beer, S., Vilenkin, B., Weil, A., Veste, M., Susel, L., Eschel, A., 1998. Measuring photosynthetic rates in seagrass by pulse amplitude modulated (PAM) fluorometry. Marine Ecology Progress Series 174, 293e300. Binzer, T., Borum, J., Pedersen, O., 2005. Flow velocity affects internal oxygen conditions in the seagrass Cymodocea nodosa. Aquatic Botany 83, 239e247. Bischof, K., Hanelt, D., Tüg, H., Karsten, U., Brouwer, P.E.M., Wiencke, C., 1998. Acclimation of brown algal photosynthesis to ultraviolet radiation in Arctic coastal waters (Spitsbergen, Norway). Polar Biology 20, 388e395. Bischof, K., Hanelt, D., Wiencke, C., 2000. Effects of ultraviolet radiation on photosynthesis and related enzyme reactions of marine macroalgae. Planta 211, 555e562. Bischof, K., Gómez, I., Molis, M., Hanelt, D., Karsten, U., Lüder, U., Roleda, M., Zacher, K., Wiencke, C., 2006. Ultraviolet radiation shapes seaweed communities. Reviews of Environmental Science and Biotechnology 51, 141e166. Box, A., Sureda, A., Terrados, J., Pons, A., Deudero, S., 2008. Antioxidant response and caulerpenyne production of the alien Caulerpa taxifolia (Vahl) epiphytized by the invasive algae Lophocladia lallemandii (Montagne). Journal of Experimental Marine Biology and Ecology 364, 24e28. Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of a free radical method to evaluate antioxidant activity. Lebensm-Wiss U-Technology 28, 25e30. Cabaço, S., Ferreira, O., Santos, R., 2010. Population dynamics of the seagrass Cymodocea nodosa in Ria Formosa lagoon following inlet artificial relocation. Estuarine, Coastal and Shelf Science 87, 510e516. Cariello, L., Zanetti, L., De Estefano, S., 1979. Phenolic compounds from marine phanerogames, Cymodocea nodosa and Posidonia oceanica. Comparative Biochemistry and Physiology 62B, 159e161. Casazza, G., Mazzella, L., 2002. Photosynthetic pigment composition of marine angiosperms, preliminary characterization of Mediterranean seagrasses. Bulletin of Marine Science 71 (3), 1171e1181. Choo, K., Snoeijs, P., Pedersén, M., 2004. Oxidative stress tolerance in the filamentous green algae Cladophora glomerata and Enteromorpha ahlneriana. Journal of Experimental Marine Biology and Ecolology 298, 111e123. Choo, K.S., Nilsson, J., Pedersén, M., Snoeijs, P., 2005. Photosynthesis, carbon uptake and antioxidant defence in two coexisting filamentous green algae under different stress conditions. Marine Ecology Progress Series 292, 127e138. Contreras, L., Moenne, A., Correa, J., 2005. Antioxidant responses in Scytosiphon lomentaria (Phaeophyceae) inhabiting copper-enriched coastal environments. Journal of Phycology 41, 1184e1195. Cuny, P., Serve, L., Jupin, H., Boudouresque, C.F., 1995. Water soluble phenolic compounds of the marine phanerogam Posidonia oceanica in a Mediterranean area colonised by the introduced chlorophyte Caulerpa taxifolia. Aquatic Botany 52, 237e242. Demmig-Adams, B., Adams, W.W., 1996. The role of xanthophyll cycle carotenoids in the protection of photosynthesis. Trends in Plant Science 1 (1), 21e26. Dring, M.J., Wagner, A., Boeskov, J., Lüning, K., 1996. Sensitivity of intertidal and subtidal red algae to UVA and UVB radiation, as monitored by chlorophyll

46

M. García-Sánchez et al. / Marine Environmental Research 79 (2012) 37e47

fluorescence measurements, influence of collection depth, and season, and length of irradiance. European Journal of Phycology 31, 293e302. Driscoll, M., 2004. Effects of Hydrodynamic Regime on Photosynthesis in the Green Alga Caulerpa. MSc thesis. University of South Florida, 77 pp. Duarte, C.M., 1992. Nutrient concentration of aquatic plants, patterns across species. Limnology and Oceanography 37 (4), 882e889. Enríquez, S., Duarte, C.M., Sand-Jensen, K., 1995. Patterns in the photosynthetic metabolism of Mediterranean macrophytes. Marine Ecology Progress Series 119, 243e252. Enríquez, S., Marba, N., Cebrián, J., Duarte, C.M., 2004. Annual variation in leaf photosynthesis and leaf nutrient content of four Mediterranean seagrasses. Botanica Marina 47, 295e306. Esteban, R., Martínez, B., Fernández-Marín, B., Becerril, J.M., García-Plazaola, J.I., 2009. Carotenoid composition in Rhodophyta, insights into xanthophyll regulation in Corallina elongata. European Journal of Phycology 44 (2), 221e230. Fernández-Torquemada, Y., Sánchez-Lizaso, J.L., 2011. Responses of two Mediterranean seagrasses to experimental changes in salinity. Hydrobiologia 669 (1), 21e33. Figueroa, F.L., Salles, S., Aguilera, J., Jiménez, C., Mercado, J., Viñegla, B., FloresMoya, A., Altamirano, M., 1997. Effects of solar radiation on photoinhibition and pigmentation in the red alga Porphyra leucosticta. Marine Ecology Progress Series 151, 81e90. Figueroa, F.L., Nygård, C., Ekelund, N., Gómez, I., 2003. Photobiological characteristics and photosynthetic UV responses in two Ulva species (Chlorophyta) from southern Spain. Journal of Photochemistry and Photobiology B, Biology 72, 35e44. Folin, O., Ciocalteu, V., 1927. On tyrosine and tryptophane determinations in proteins. Journal of Biological Chemistry 12, 239e243. Fujita, R.M., Wheeler, P.A., Edwards, R.L., 1989. Assessment of macroalgal nitrogen limitation in a seasonal upwelling region. Marine Ecology Progress Series 53, 293e303. Gacia, E., Littler, M.M., Littler, D.S., 1996. The relationships between morphology and photosynthetic parameters within the polymorphic genus Caulerpa. Journal of Experimental Marime Biology and Ecology 204, 209e224. García-Sánchez, M., Pérez-Ruzafa, I.M., Marcos, C., Pérez-Ruzafa, A., 2012. Suitability of benthic macrophyte indices (EEI, E-MaQI and BENTHOS) for detecting anthropogenic pressures in a Mediterranean coastal lagoon (Mar Menor, Spain). Ecological Indicators 19, 48e60. Gilabert, J., 1992. Análisis del Ecosistema Planctónico del Mar Menor. Ciclo anual, distribución de tamaños y red trófica. PhD thesis, Universidad de Murcia, 147 pp. Gilmore, A.M., Yamamoto, H.Y., 2001. Time-resolution of the antheraxanthin- and DpH-dependent chlorophyll a fluorescence components associated with photosystem II energy dissipation in Mantoniella squamata. Photochemistry and Photobiology 74 (2), 291e302. Gómez, I., Figueroa, F.L., 1998. Effects of solar UV stress on chlorophyll fluorescence kinetics of intertidal macroalgae from southern Spain, a case study in Gelidium species. Journal of Applied Phycology 10, 285e294. Goss, R., Bohme, K., Wilhelm, C., 1998. The xanthophyll cycle of Mantoniella squamata converts violaxanthinin to antheraxanthin but not to zeaxanthin, consequences for the mechanism of enhanced non-photochemical energy dissipation. Planta 205, 613e621. Grobe, C.W., Murphy, T.M., 1998. Solar ultraviolet-B radiation effects on growth and pigment composition of the intertidal alga Ulva expansa (Setch.) S and G. (Chlorophyta). Journal of Experimental Marine Biology and Ecolology 225, 39e51. Grzymski, J., Johnsen, G., Sakshaug, E., 1997. The significance of intracellular selfshading on the biooptical properties of brown and green macroalgae. Journal of Phycology 33, 408e414. Häder, D.P., Figueroa, F.L., 1997. Photoecophysiology of marine macroalgae. Journal of Photochemistry and Photobiology 66, 1e14. Häder, D.P., Lebert, M., Mercado, J., Aguilera, J., Salles, S., Flores-Moya, A., Jiménez, C., Figueroa, F.L., 1996. Photosynthetic oxygen production and PAM fluorescence in the brown alga Padina pavonica measured in the field under solar radiation. Marine Biology 127, 61e66. Häder, D.P., Porst, M., Herrmann, H., Schäfer, J., Santas, R., 1997. Photosynthesis of mediterranean green alga Caulerpa prolifera measured in the field under solar irradiation. Journal of Photochemistry and Photobiology B, Biology 37, 66e73. Hanelt, D., Huppertz, K., Nultsch, W., 1993. Daily course of photosynthesis and photoinhibition in marine macroalgae investigated in the laboratory and in the field. Marine Ecology Progress Series 97, 31e71. Hanelt, D., 1996. Photoinhibition of photosynthesis in marine macrolagae. Scientia Marina 60 (l1), 243e248. Hanelt, D., 1998. Capability of dynamic photoinhibition in Arctic macroalgae is related to their depth distribution. Marine Biology 131, 361e369. Hegazi, M.M., Pérez-Ruzafa, A., Almela, L., Candela, M.E., 1998. Separation and identification of chlorophylls and carotenoids from Caulerpa prolifera, Jania rubens and Padina pavonica by reversed phase high-performance liquid chromatography. Journal of Chromatography A 829, 153e159. Hegazi, M.M., Pérez-Ruzafa, A., Pérez-Ruzafa, I.M., Candela, M.E., 2000. Temporal variation in the pigment composition of Caulerpa prolifera (Forsskal) Lamouroux meadows in the Mar Menor lagoon (SE Spain). Egyptian Journal of Phycology 1, 63e70. Hegazi, M., 1999. Composición pigmentaria y propiedades ópticas de la vegetación submarina costera del Mediterráneo Occidental. PhD thesis, Universidad de Murcia, 348 pp.

Invers, O., Zimmerman, R.C., Alberte, R.S., Pérez, M., Romero, J., 2001. Inorganic carbon sources for seagrass photosynthesis: an experimental evaluation of bicarbonate use in species inhabiting temperate waters. Journal of Experimental Marine Biology and Ecology 265 (2), 203e217. Jassby, A.D., Platt, T., 1976. Mathematical formulation of the relationship between photosynthesis and light for phytoplankton. Limnology and Oceanography 21 (4), 540e547. Kartal, M., Abu-Asaker, M., Senol, F.S., Atici, T., Sener, B., 2009. Antioxidant and anticholinesterase assets and Liquid ChromatographyeMass Spectrometry preface of various fresh-water and marine macroalgae. Pharmacognosy Magazine 5, 291e297. Klughammer, C., Schreiber, U., 2008. Complementary PS II quantum yields calculated from simple fluorescence parameters measured by PAM fluorometry and the Saturation Pulse method. PAM Application Notes 1, 27e35. Koch, M.S., Schopmeyer, S.A., Kyhn-Hansen, C., Madden, C.J., Peters, J.S., 2007. Tropical seagrass species tolerance to hypersalinity stress. Aquatic Botany 86 (1), 14e24. Korbee-Peinado, N., Abdala-Díaz, R., Figueroa, F., Helbling, W., 2004. Ammonium and UV radiation stimulate the accumulation of mycosporine-like aminoacids in Porphyra columbina (Rhodophyta) from Patagonia, Argentina. Journal of Phycology 40, 248e259. Krause, G.H., Weis, E., 1991. Chlorophyll fluorescence and photosynthesis, the basics. Annual Review of Plant Physiology (Plant Molecular Biology) 42, 313e349. Kubitzki, K., 1987. Phenylpropanoid metabolism in relation to land plant origin and diversification. Journal of Plant Physiology 131, 17e24. Lozano, F., 1954. Una campaña de prospección pesquera en Mar Menor (Murcia). Boletín del Instituto Español de Oceanografía 66, 1e34. Lu, I.F., Sung, M.S., Lee, T.M., 2006. Salinity stress and hydrogen peroxide regulation of antioxidant defense system in Ulva fasciata. Marine Biology 150, 1e15. Lubián, L.M., Montero, O., 1998. Excess light-induced violaxanthin cycle activity in Nannochloropsis gaditana (Eustigmatophyceae), Effects of exposure time and temperature. Phycologia 37, 16e23. Madsen, T.V., Sand-Jensen, K., 1991. Photosynthetic carbon assimilation in aquatic macrophytes. Aquatic Botany 41 (1e3), 5e40. Malta, E.J., Ferreira, D.G., Vergara, J.J., Pérez-lloréns, J.L., 2005. Nitrogen load and irradiance affect morphology, photosynthesis and growth of Caulerpa prolifera ( Bryopsidales, Chlorophyta ). Marine Ecology Progress Series 298, 101e114. Matanjun, P., Mohamed, S., Mustapha, Noordin M., Muhammad, K., Ming, C.H., 2008. Antioxidant activities and phenolics content of eight species of seaweeds from north Borneo. Journal of Applied Phycology 20 (3), 67e373. Meinesz, A., 1979. Contribution à l’ètude des Caulerpales (Chlorophytes). PhD thesis, Université de Nice, 262 pp. Müller, P., Li, X.P., Niyogi, K.K., 2001. Non-photochemical quenching. A response to excess light energy. Plant Physiology 125, 1558e1566. Olesen, B., Enríquez, S., Duarte, C.M., Sand-Jensen, K., 2002. Depth-acclimation of photosynthesis, morphology and demography of Posidonia oceanica and Cymodocea nodosa in the Spanish Mediterranean Sea. Marine Ecology Progress Series 236, 89e97. Pagès, J.F., Pérez, M., Romero, J., 2010. Sensitivity of the seagrass Cymodocea nodosa to hypersaline conditions: a microcosm approach. Journal of Experimental Marine Biology and Ecology 386, 34e38. Pakker, H., Beekman, C.A.C., Breeman, A.M., 2000a. Efficient photoreactivation of UVBR-induced DNA damage in the sublittoral macroalga Rhodymenia pseudopalmata (Rhodophyta). European Journal of Phycology 35, 109e114. Pakker, H., Martins, R., Boelen, P., Buma, A.G.J., Nikaido, O., Breeman, A.M., 2000b. Effects of temperature on the photoreactivation of ultraviolet-B-induced DNA damage in Palmaria palmata (Rhodophyta). Journal of Phycology 36, 334e341. Pang, S., Gómez, I., Lüning, K., 2001. The red macroalgae Delesseria sanguinea as a UVB-sensitive model organisms, selective growth reduction by UVB in outdoor experiments and rapid recording of growth rate during and after UV pulses. European Journal of Phycology 36, 207e216. Pérez, M., 1989. Fanerógamas marinas en sistemas estuáricos, producción, factores limitantes y algunos aspectos del ciclo de nutrientes. PhD thesis, Universidad de Barcelona, 244 pp. Pérez-Rodríguez, E., Gómez, I., Karsten, U., Figueroa, F.L., 1998. Effects of UV radiation on photosynthesis and excretion of UV-absorbing compounds of Dasycladus vermicularis (Dasycladales, Chlorophyta) from southern Spain. Phycologia 37 (5), 379e387. Pérez-Rodríguez, E., Aguilera, J., Gómez, I., Figueroa, F.L., 2001. Excretion of coumarins by the Mediterranean green alga Dasycladus vermicularis in response to environmental stress. Marine Biology 139 (4), 633e639. Pérez-Rodríguez, E., Aguilera, J., Figueroa, F.L., 2003. Tissular localization of coumarins in the green alga Dasycladus vermicularis (Scopoli) Krasser, a photoprotective role? Journal of Experimental Botany 54 (384), 1093e1100. Perez-Ruzafa, A., Marcos, C., 1987. Los sustratos arenosos y fangosos del Mar Menor (Murcia), su cubierta vegetal y su posible relación con la disminución del mújol en la laguna. In: Cuaderno Marisquero, vol. 11. Publicaciones Técnicas, pp. 111e123. Pérez-Ruzafa, A., Marcos, C., Pérez-Ruzafa, I.M., Ros, J.D., 1987. Evolución de las características ambientales y de los poblamientos del Mar Menor (Murcia, SE de España). Anales de Biología 12 (Biología ambiental, 3), 53e65. Pérez-Ruzafa, A., Ros, J.D., Marcos, C., Ballester, R., Pérez-Ruzafa, I.M., 1989. Distribution and biomasa of the macrophyte beds in a hypersaline coastal lagoon (the Mar Menor, SE Spain), and its recent evolution following major environmetal changes. International Workshop on Posidonia Beds. In: Bouderesque, C.F.,

M. García-Sánchez et al. / Marine Environmental Research 79 (2012) 37e47 Meisnesz, A., Fresi, E., Gravez, V. (Eds.). GIS Posidonie Publication, Fr.2, pp. 49e62. Pérez-Ruzafa, A., Marcos, C., Ros, J., 1991. Environmental and biological changes related to recent human activities in the Mar Menor. Marine Pollution Bulletin 23, 747e751. Pérez-Ruzafa, A., Marcos, C., Gilabert, J., 2005. The ecology of the Mar Menor coastal lagoon, a fast-changing ecosystem under human pressure. In: Gönenç, I.E., Wolflin, J.P. (Eds.), Coastal Lagoons, Ecosystem Processes and Modeling for Sustainable Use and Development. CRC Press, Boca Ratón, Florida, pp. 392e422. Pérez-Ruzafa, A., García-Charton, J.A., Barcala, E., Marcos, C., 2006. Changes in benthic fish assemblages as a consequence of coastal works in a coastal lagoon, the Mar Menor (Spain, western Mediterranean). Marine Pollution Bulletin 53, 107e120. Pérez-Ruzafa, A., Marcos, C., Bernal, C.M., Quintino, V., Freitas, R., Rodrigues, A.M., García-Sánchez, M., Pérez-Ruzafa, I.M., 2012. Cymodocea nodosa vs. Caulerpa prolifera: causes and consequences of a long term history of interaction in macrophyte meadows in the Mar Menor coastal lagoon (Spain, Southwestern Mediterranean). Estuarine, Coastal and Shelf Science. doi:10.1016/j.ecss.2012.04.004. Piazzi, L., Balata, D., 2008. The spread of Caulerpa racemosa var. cylindracea in the Mediterranean Sea: an example of how biological invasions can influence beta diversity. Marine Environmental Research 65, 50e61. Pinto, E., Signaud-Kutner, T.C.S., Leitao, M.A.S., Okamoto, O.K., Morse, D., Colepicolo, P., 2003. Heavy metal-induced oxidative stress in algae. Journal of Phycology 39, 1008e1018. Platt, T., Gallegos, C.L., 1980. Modelling primary production. In: Falkowski, P.G. (Ed.), Primary Productivity in the Sea. Plenum Press, New York, pp. 339e362. Raniello, R., Lorenti, M., Brunet, C., Buia, M.C., 2004. Photosynthetic plasticity of an invasive variety of Caulerpa racemosa in a coastal Mediterranean area, light harvesting capacity and seasonal acclimation. Marine Ecology Progress Series 271, 113e120. Raniello, R., Lorenti, M., Brunet, C., Buia, M.C., 2006. Photoacclimation of the invasive alga Caulerpa racemosa var. cylindracea to depth and daylight patterns and a putative new role for siphonaxanthin. Marine Ecology 27, 20e30. Raniello, R., Mollo, E., Lorenti, M., Gavagnin, M., Buia, M.C., 2007. Phytotoxic activity of caulerpenyne from the Mediterranean invasive variety of Caulerpa racemosa: a potential allelochemical. Biological Invasions 9, 361e368. Raven, J.A., Geider, R.J., 2003. Adaptation, acclimation and regulation in algal photosynthesis. In: Larkum, A.W., Douglas, S.E., Raven, J.A. (Eds.), Photosynthesis in Algae. Kluwer Academic Publishers, Netherlands, pp. 385e412. Riechert, R., Dawes, C.J., 1986. Acclimation of the green alga Caulerpa racemosa var. uvifera to light. Botanica Marina 29 (6), 533e538. Robledo, D., Freile-Pelegrin, Y., 2005. Seasonal variation in photosynthesis and biochemical composition of Caulerpa spp. (Bryopsidales, Chlorophyta ) from the Gulf of Mexico. Phycologia 44 (3), 312e319. Ros, J.D., 1987. El Mar Menor, fisiografía y biota acuática de una laguna hipersalina y su evolución reciente. In: Bases científicas para la protección de los humedales en España. Real Academia de Ciencias Exactas, Físicas y Naturales, Madrid, pp. 237e256. Salama, A.M., Pearce, R.S., 1993. Ageing of cucumber and onion seeds, phospholipase D, lipoxygenase activity and changes in phospholipid content. Journal of Experimental Botany 44, 1253e1265. Sanchiz, C., García-Carrascosa, A.M., Pastor, A., 2000. Heavy metal contents in softbottom marine macrophytes and sediments along the Mediterranean coast of Spain. Marine Ecology 21 (1), 1e16. Sandoval-Gil, J.M., Marín-Guirao, L., Ruiz, J.M., 2012. Tolerance of Mediterranean seagrasses (Posidonia oceanica and Cymodocea nodosa) to hypersaline stress: water relations and osmolyte concentrations. Marine Biology. doi:10.1007/ s00227-012-1892-y.

47

Silva, J., Santos, R., 2003. Daily variation patterns in seagrass photosynthesis along a vertical gradient. Marine Ecology Progress Series 257, 37e44. Simonneau, J., 1972. Mar Menor, évolution sédimentologique et géochimique récente du remplissage. Thèse doctorale, Université de Montpellier. France. 172 pp. Stafford, N.B., Bell, S.S., 2006. Space competition between seagrass and Caulerpa prolifera (Forsskaal) Lamouroux following simulated disturbances in Lassing Park, FL. Journal of Experimental Marine Biology and Ecology 333, 49e57. Taiz, L., Zeiger, E., 2010. Plant Physiology, fifth ed. Sinauer Associates, Sunderland, MA. Takaichi, S., 2011. Carotenoids in algae, distributions, biosyntheses and functions. Marine Drugs 9, 1101e1118. Taplin, K.A., Irlandi, E.A., Raves, R., 2005. Interferente between the macroalga Caulerpa prolifera and the seagrass Halodule wrightii. Aquatic Botany 83, 175e186. Targett, N.M., Arnold, T.M., 1998. Predicting the effects of brown algal phlorotannins on marine herbivores in tropical and temperate oceans. Journal of Phycology 34, 195e205. Terrados, J., Ros, J.D.,1991. Production dynamics in a macrophyte-dominated ecosystem, the Mar Menor coastal lagoon (SE Spain). Oecologia Aquatica 10, 255e270. Terrados, J., Ros, J.D., 1992. The influence of temperature on seasonal variation of Caulerpa prolifera (Forsskal) Lamouroux photosynthesis and respiration. Journal of Marine Biology and Ecology 162, 199e212. Terrados, J., Ros, J.D., 1993. Limitación por nutrientes del crecimiento de Cymodocea nodosa (Ucria) Ascherson en sedimentos carbonatados en el Mar Menor, Murcia, SE de España, vol. 11. Publicación Especial del Instituto Español de Oceanografía, pp. 9e14. Terrados, J., Ros, J.D., 1995. Temperature effects on photosynthesis and depth distribution of the seagrass Cymodocea nodosa (Ucria) Ascherson in a Mediterranean Coastal Lagoon, the Mar Menor (SE Spain). Marine Ecology 16 (2), 133e144. Theil, M., Westphalen, G., Collings, G., Cheshire, A., 2007. Caulerpa taxifolia responses to hyposalinity stress. Aquatic Botany 87, 221e228. van de Poll, W.H., Eggert, A., Buma, A.G.J., Breeman, A.M., 2001. Effects of UV-Binduced DNA damage and photoinhibition on growth of temperate marine red macrophytes, habitat-related differences in UV-B tolerance. Journal of Phycology 37, 30e37. Vergeer, L.H.T., Aarts, T.L., de Groot, J.D., 1995. The ‘wasting disease’ and the effect of abiotic factors (light intensity, temperature, salinity) and infection with Labyrinthula zosterae on the phenolic content of Zostera marina shoots. Aquatic Botany 52, 35e44. Vergés, A., Alcoverro, T., Romero, J., 2011. Plant defences and the role of epibiosis in mediating within-plant feeding choices of seagrass consumers. Oecologia 166, 381e390. Walton, T.J., Britton, G., Goodwin, T.W., 1970. The structure of siphonaxanthin. Phytochemistry 9, 2545e2552. Wellburn, A.R., 1994. The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. Journal of Plant Physiology 144, 307e313. West, E.J., West, R.J., 2007. Growth and survival of the invasive alga, Caulerpa taxifolia, in different salinities and temperatures: implications for coastal lake management. Hydrobiologia 577, 87e94. Wiencke, C., Gómez, I., Pakker, H., Flores-Moya, A., Altamirano, M., Hanelt, D., Bischof, K., Figueroa, F.L., 2000. Impact of UV radiation on viability, photosynthetic characteristics and DNA of brown algal zoospores, implications for depth zonation. Marine Ecology Progress Series 197, 217e229. Zapata, O., McMillan, C.,1979. Phenolic acids in seagrasses. Aquatic Botany 7, 307e317. Zubia, M., Robledo, D., Freile-Pelegrin, Y., 2007. Antioxidant activities in tropical marine macroalgae from the Yucatan Peninsula, Mexico. Journal of Applied Phycology 19, 449e458.