Marine macrophytes are vertically distributed according to their ability to optimize their photosynthetic performance. We assessed the photo-physiological performance of the seagrass Cymodocea nodosa and the green seaweed Caulerpa prolifera at varying depth at Gran Canaria Island (Canary Islands, eastern Atlantic). The biomass of C. nodosa decreases with depth, while the opposite occurs for C. prolifera. Photochemical responses of both macrophytes were measured in shallow (5 m) and deep (20 m) waters at two times via chlorophyll a fluorescence and internal content of photoprotective pigments and antioxidant activity. We additionally carried out a reciprocal transplant experiment by relocating shallow and deep vegetative fragments of both macrophytes to assess their short-term photo-physiological acclimation. Overall, C. nodosa behaves as a ‘light-plant’, including a larger optimum quantum yield and ETRmax under scenarios of high photosynthetically active radiation and a larger antioxidant activity. In contrast, C. prolifera is a ‘shade-adapted’ plant, showing a larger carotene content, particularly in shallow water. Deep-water C. nodosa and C. prolifera are more photochemically efficient than in shallow water. The alga C. prolifera shows a rapid, short-term acclimation to altered light regimes in terms of photosynthetic efficiency. In conclusion, decreased light regimes favour the photosynthetic performance of the green alga when both species coexist.
RESUMEN
Los macrófitos marinos se distribuyen verticalmente de acuerdo a sus capacidades para optimizar su rendimiento fotosintético. Evaluamos el rendimiento foto-fisiológico de la fanerógama marina Cymodocea nodosa y el alga verde Caulerpa prolifera a diferentes profundidades en la isla de Gran Canaria (Islas Canarias, Atlántico oriental). La biomasa de C. nodosa decrece con la profundidad, mientras que para C. prolifera ocurre lo contrario. Las respuestas foto-químicas de ambos macrófitos se midieron en aguas someras (5 m) y profundas (20 m), en dos tiempos, a través de la fluorescencia de la clorofila a y los contenidos internos en pigmentos fotoprotectores y la actividad antioxidante. Además, ejecutamos un experimento de trasplante recíproco, recolocando fragmentos vegetativos de ambos macrófitos entre aguas someras y profundas para determinar su aclimatación a corto plazo. En general, C. nodosa se comporta como “planta de sol”, con mayor rendimiento cuántico óptimo y ETRmax bajo escenarios de alta radiación PAR y mayor actividad antioxidante. Contrariamente, C. prolifera es una “planta de sombra”, mostrando mayor cantidad de carotenos, en particular a poca profundidad. Ejemplares profundos de ambos macrófitos son más eficientes foto-químicamente que los de aguas someras. El alga C. prolifera muestra una aclimatación rápida, a corto plazo, de su eficiencia fotosintética ante cambios en el régimen luminoso. En conclusión, regímenes depauperados lumínicamente favorecen el rendimiento fotosintético del alga verde.
A variety of marine macrophytes (e.g. seagrasses and macroalgae) typically coexist in the same habitat; their distribution, however, may fluctuate under varying environmental conditions (e.g. light, nutrients), operating at a range of spatial and temporal scales (Lüning 1990). Marine macrophytes acclimate and adapt their photosynthetic apparatus to prevailing irradiance conditions (Betancor et al. 2015), which define species’ niche requirements, including photoprotective mechanisms under high levels of UV and photosynthetically active radiation (PAR) (Häder et al. 1997). In turn, zonation patterns (or vertical distribution) of marine macrophytes are often related to their ability to resist high radiation stress (Hanelt 1998), e.g. upper-shore species are more resistant to elevated solar UV. In addition to their physiological responses, marine macrophytes show a certain degree of morphological flexibility that reflect long-term adaptations to varying environmental scenarios, including decreased light with depth in subtidal habitats (Olesen et al. 2002).
Under varying environmental scenarios, ecophysiological approaches that integrate estimates of light absorption efficiency, pigment contents and mechanisms to dissipate excess energy (Hanelt 1998) provide useful insight to assess changes in the distribution of marine macrophytes. This is particularly pertinent under scenarios of global change, in which the intensity and frequency of anthropogenic perturbations are increasing. For example, increasing turbidity in coastal areas alters the dominance and functioning of seascapes dominated by submersed vegetation, particularly those constituted by marine angiosperms (seagrasses) (Duarte et al. 2008, Silva et al. 2013). Understanding the physiological response of seagrasses and accompanying seaweeds to altered light regimes is therefore important (Collier et al. 2012).
Cymodocea nodosa (Ucria) Ascherson is a seagrass distributed across the entire Mediterranean Sea and the contiguous eastern Atlantic coasts, including the oceanic archipelagos of Madeira and the Canary Islands (Tuya et al. 2013a). Meadows constituted by C. nodosa are found on shallow sandy substrates throughout the Canary Islands (Barberá et al. 2005), supporting diverse invertebrate and fish assemblages (Tuya et al. 2006, Gardner et al. 2013, Tuya et al. 2013b). Across its distribution range, declines in the presence of C. nodosa may result in increases in the biomass of green algae, particularly those of the genus Caulerpa, including native (Lloret et al. 2005, García-Sánchez et al. 2012) and non-native species (Ceccherelli and Cinelli 1997). Species within the genus Caulerpa usually show rapid growth in high nutrient conditions (Lapointe et al. 2005). Caulerpa prolifera (Forsskål) J.V. Lamouroux is a native species from continental Europe, occurring in the Mediterranean Sea and the adjacent Atlantic Ocean. This coenocytic alga is a slow-growing, nitrophilic species with considerable morphological plasticity (Collado-Vides 2002, Malta et al. 2005), which may be connected with a large physiological plasticity when environmental conditions change (Malta et al. 2005). This alga has a prostrate axis (stolon) with rhizoids and upright axes (fronds) that are potentially independent units; the whole plant can regenerate after a frond or stolon is lost (Collado-Vides 2002). In the Canary Islands, the presence of C. nodosa has decreased in the last two decades, particularly in certain areas, correlating with a significant increase in the abundance of C. prolifera (Tuya et al. 2013a, Tuya et al. 2014). In this region, C. nodosa typically inhabits shallower waters than C. prolifera, though the two macrophytes can be found intermixed (Tuya et al. 2013a). In the Mar Menor (Mediterranean Sea), C. nodosa is mainly restricted to shallow-water areas (<1 m), while slightly deeper areas (2-5 m) are dominated by mono specific meadows of C. prolifera (García-Sánchez et al. 2012). It has been demonstrated that C. nodosa typically behaves as a ‘light-plant’, whereas C. prolifera behaves as a ‘shade-plant’, based on the differences in the way PAR is absorbed, the pigment composition, and the subsequent physiological mechanisms for dissipating excess light energy between the two macrophytes (Malta et al. 2005, García-Sánchez et al. 2012).
Previous studies have demonstrated morphological plasticity with depth of both C. nodosa (Olesen et al. 2002) and C. prolifera (Collado-Vives 2002). In this study, we assessed the photo-physiological performance of both macrophytes between a shallow and a deep-water stratum (5 vs 20 m depth), which involve significant changes in light regimes. This bathymetric gradient is considerably larger than those previously considered to compare the photo-physiology of both C. nodosa (0.4 vs 3.8 m, Olesen et al. 2002; 0.5 vs 5 m, García-Sánchez et al. 2012) and C. prolifera with depth (0.5 vs 5 m, García-Sánchez et al. 2012). We followed a two-step strategy. First, the photo-physiological performance—here quantified through photochemical responses via in vivo chlorophyll a fluorescence of photosystem II, as well as the contents in carotenes (widely recognized photoprotective pigments) and antioxidant activity—of the two macrophytes was directly measured from shallow and deep-water specimens at two times. Second, we carried out a reciprocal transplant experiment on the same two occasions by relocating shallow (5 m) and deep (20 m) vegetative fragments of both macrophytes. Subsequently, the photo-physiological performance of both macrophytes was assessed as an indicator of short-term acclimation to chronic changes in light regimes. Integration of results from both approaches provides insight on the photophysiological performance of these two co-occurring macrophytes and potential responses under altered light regimes.
MATERIALS AND METHODS Reciprocal transplants: field work
This study was performed at Gran Canaria Island, a subtropical island in the centre of the Canary Islands (eastern Atlantic, 28°N). Sea surface temperature typically varies between 18°C in winter and 24ºC in summer (Tuya et al. 2014). Vegetative fragments of C. nodosa (horizontal rhizomes ca. 10-15 cm long, including 5-7 shoots and associated leaves) and ramets of C. prolifera, including stolons, rhizoids and fronds (ca. 20-25 cm long), were collected by hand by SCUBA divers at 5 and 20 m depth at Gando Bay (27°55′49″N, 15°22′1″W). Neither species is typically found in shallower water in the study region, which is exposed to large oceanic swells that prevent long-term persistence. Collections of both macrophytes were immediately used to assess photochemical responses, as a way to infer their physiological status (see below). On the day of collection, we additionally carried out a reciprocal transplant experiment, in which 10-15 C. nodosa and C. prolifera vegetative fragments collected at 5 m were transplanted at 20 m depth and vegetative fragments collected at 20 m were transplanted at 5 m depth (hereafter called ‘allochthonous’ transplants). To control for potential artefacts during manipulations, C. nodosa and C. prolifera fragments from both depths were re-transplanted at the same depth (i.e. procedural controls, hereafter called ‘autochthonous’ transplants). All transplants were then subjected to the same manipulations. During transplants, the below-ground compartments of both macrophytes were gently buried inside the sediment by hand; to facilitate retention of below-ground tissues, plastic meshes covering horizontal rhizomes and stolons were secured to the bottom by metal stakes (Fig. 1). In all cases, the erect parts of both macrophytes (seagrass leaves and C. prolifera fronds) come out from the mesh to capture light with minimum perturbation. This experimental approach was repeated twice to evaluate the temporal consistency of results; the first started on 6 February 2014 and the second started on 15 May 2014. The duration of each transplant set varied slightly as a result of logistical constraints; on the first occasion (February 2014), transplants were maintained for 11 days; on the second occasion (May 2014), transplants were maintained for 14 days. During both periods, the amount of PAR reaching the bottom was measured by using Onset HOBO U12 4-channel external data loggers at 5 and 20 m depth, respectively.
Experimental transplantation of vegetative fragments of C. nodosa and C. prolifera on a shallow-water soft bottom (5 m depth) at Gando Bay (Gran Canaria Island).
Bathymetric changes in macrophyte distribution and environmental drivers
To facilitate further interpretation of results, we described changes in the biomass of both C. nodosa and C. prolifera with depth at the study site. Samples (n=5) were collected through a 0.0125 m–2 core at 5, 10, 15 and 20 m in Gando Bay at both sampling times. All vegetation was dried (24 h at 70°C) and the dry weight was calculated. Additionally, sediment samples (n=2 cores, ca. 100 g) were collected, in February 2014, at 5, 10, 15 and 20 m to work out differences in the mean particle diameter (D50), the total N and P content and the organic matter content of sediments with depth. After thawing, sediment samples were sieved through a 0.5-mm sieve, and the fraction <0.5 mm was oven-dried at 90°C for 24 h. This fraction was then dry-sieved at 0.5 ϕ intervals, down to 1.0 ϕ (0.5 mm). The <0.5 mm fraction was freeze-dried and analysed on a Coulter LS130 laser sizer. The laser sizer results were combined with the dry sieve results to give the full particle size distribution. The method of Walkley and Black (1934) was used to calculate the organic matter content of sediments via rapid dichromate oxidation. Total N was determined following the Kjeldahl method (Bradstreet 1965) and total P was determined using a spectrophotometric method (Murphy and Riley 1962).
Photochemical responses
At the start (initial collections) and at the end of the transplantation experiments, between n=6 and 8 C. nodosa leaves (12-15 cm long with no necrosis signs) and n=6 and 8 C. prolifera fronds (5-8 cm long) from each depth and manipulation treatment were used to estimate their photochemical status. In vivo chlorophyll a fluorescence of Photosystem II (PSII) was assessed through a portable pulse amplitude modulation fluorometer (Diving-PAM, Waltz GmbH, Germany). After 15 min of dark adaptation (‘relaxed state’), the minimum (basal) fluorescence was measured (Fo) and the maximum fluorescence (Fm) was obtained immediately after applying a saturated pulse of actinic light (2,350 µmol photons m–2 s–1, 0.8 s). The optimum quantum yield was therefore calculated as Fv/Fm=Fm–Fo/Fm, which is an indicator of physiological stress (Maxwell and Johnson 2000). A rapid light curve (RLC) was initiated, involving 20 s of exposure to 9 successive irradiances, including 20, 66, 137, 224, 337, 469, 693, 942 and 1418 µmol photons m–2 s–1. RLCs were then obtained by calculating the electron transport rate (ETR) through the PSII for each level of actinic light through the formula:
ETR (µmol electrons m–2 s–1) = (ΔF/F′m) × E × A × FII
where ‘E’ is the irradiance; ‘A’ is the absorptance of each macrophyte (0.88±0.02 for C. nodosa and 0.93±0.03 for C. prolifera, calculated using the method of García-Sánchez et al. 2012); and ‘FII’ is the fraction of chlorophyll associated with the PSII (0.5 for green seaweeds, according to Grzymski et al. 1997). RLCs were fitted through the model provided by Jassby and Platt (1976) to obtain the initial slope of the curve (αETR, i.e. the photosynthetic efficiency), the saturation irradiance (Ek) and the maximal ETR (ETRmax); the model of Platt and Gallegos (1980) was applied when photo-inhibition was detected.
Carotenes and antioxidant activity
The carotene content was determined spectro-photometrically. The analyses were carried out by extracting pigments from plants (ca. 20 mg dry weight [DW], n=3) using 1 ml of saturation solution of acetone 90% + C4Mg4O12 and maintaining them in darkness at 4°C for 12 h. After centrifugation at 4000 rpm for 20 min, each supernatant was used to measure carotenes in a spectrophotometer at an absorption of 480 and 750 nm. The carotene concentration, expressed as mg g–1 DW, was calculated using the equation provided by Parsons and Strickland (1963).
Thalli of C. nodosa and C. prolifera (ca. 0.25 g FW, n=3) were ground with a mortar and a 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 4000 rpm (30 min) and the supernatants were collected (Sigma 2-16PK, Göttingen, Germany). The DPPH (2,2-diphenyl-1-picrylhydrasyl) free-radical scavenging assay was carried out in triplicate, according to the method of Blois (1958). Briefly, 150 µL of each methanolic extract was mixed with 1.5 mL of 90% methanol and 150 µL of DPPH solution prepared daily at 1.27 mM. The reaction was complete after 30 min in darkness at room temperature, and the absorbance was registered 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 (µM) were plotted against plant extract concentration (mg DW mL–1) to obtain the EC50 value (oxidation index), which represents the concentration of the extract (mg mL–1) required to scavenge 50% of the DPPH in the reaction mixture. Ascorbic acid was used as a positive control.
Statistical analyses
A three-way ANOVA tested for differences in the biomass of C. nodosa and C. prolifera across depth; the model included the factors ‘Species’ (fixed factor), depth (fixed factor) and ‘Time’ (random factor). Linear regression models tested whether the mean particle diameter (D50), the mean total N and P content and the mean organic matter content of sediments varied with depth. To test for initial differences (‘start’ collections) in physiological responses between macrophytes (C. nodosa vs C. prolifera), depths (5 vs 20 m) and times (February vs May 2014), three-way ANOVAs were carried out. Each model incorporated the factors ‘Species’ (fixed factor), depth (fixed factor) and ‘Time’ (random factor). A three-way ANOVA tested separately for each macrophyte for significant differences in physiological responses between depths (two levels: 5 vs 20 m), times (two levels: February vs May) and the origin of the transplant (autochthonous = procedural control vs. allochthonous) at the end of the transplants. The model incorporated the same factors as above, in addition to ‘Origin’ (fixed factor with two levels). For all ANOVAs, data were transformed to avoid heterogeneous variances (see Tables 1, 2 and 3 for specific transformations); Cochran’s C test was used in this sense. If no transformation rendered homogeneous variances, the significance level was set at 0.01 rather than the 0.05 level to avoid increasing a type I error (Underwood 1997). Pairwise comparisons were carried out via Student-Newman-Keul (SNK) tests wherever necessary.
RESULTSPAR patterns, sediment characteristics and vegetated biomass with depth
In February 2014, integrated mean PAR values at 5 m were ca. 2.57 times larger than at 20 m (Fig. 2A; means =124.50 vs 48.40 μ mol photons m–2 s–1), whereas in May 2014 they were ca. 2.24 times larger (Fig. 2B; means =251.72 vs 111.89 μ mol photons m–2 s–1). In general, maximum daily peaks in PAR increased from February to May 2014; for example, at 5 m, peaks increased from <1300 µmol photons m–2 s–1 to ~2000 µmol photons m–2 s–1. Sediments were dominated by fine sands at all depths (Appendix 1).
Incident PAR (mmol photons m–2 s–1) patterns through experimentation at 5 and 20 m depth in February (A) and May 2014 (B).
The biomass of both macrophytes varied between depth strata following different trends (‘Sp×De’, F3,64= 66.89, P<0.0001, Fig. 3). The biomass of the seagrass C. nodosa was larger at 5 and 10 m than at 15 and 20 m (SNK tests, Fig. 3). The biomass of C. prolifera showed a minimum at 5 m and similar values at 10, 15 and 20 m (SNK tests, Fig. 3). These patterns were consistent through times (‘Species×Time’, F1,64=0.19, P=0.6657, ‘De×Ti’, F3,64=0.11, P=0.9556), reinforcing the generality of these observations.
Changes in the biomass of C. nodosa and C. prolifera between depth strata at Gando Bay (Gran Canaria Island). Error bars are +SE of means (n=5). Times were pooled, as a result of lack of significant effects. Different letters above bars denote significant differences for each macrophyte.
Differences in initial photo-physiological responses
The seagrass C. nodosa showed a larger optimum quantum yield (Fv/Fm) than C. prolifera, particularly in May 2014 (‘Ti×Sp’, P=0.001, Table 1; Fig. 4), independently of depth (all terms involving ‘De’, P>0.05, Table 1; Fig. 4). Similarly, C. nodosa showed a significantly larger ETRmax than C. prolifera in May 2014 (‘Ti×Sp’, P=0.0008, Table 1; Fig. 4), regardless of depth (all terms involving ‘De’, P>0.05, Table 1; Fig. 5). The seagrass C. nodosa also had a larger αETR than C. prolifera (‘Sp’, P<0.00001, Table 1; Fig. 6), which was particularly accentuated in May 2014 (‘Ti×Sp’, P=0.001, Table 1; Fig. 6). Both macrophytes were more photosynthetically efficient, i.e. larger αETR at 20 than 5 m depth (‘De’, P=0.0230, Table 1; Fig. 6). Appendix 2 includes means and SE of means of all photosynthetic parameters. The carotenoid content was significantly larger in C. prolifera than in C. nodosa(‘Sp’, P=0.001, Table 1; Fig. 7). While C. nodosaincreased its carotene content from 5 to 20 m depth at both times, C. prolifera decreased it (‘De×Sp’, P<0.00001, Table 1; Fig. 7). The antioxidant activity of C. nodosa was significantly greater than that of C. prolifera (‘Sp’, P=0.0001, Table 1; Fig. 8).
Three-way ANOVAs testing the effect of ‘Time’ (Ti), ‘Depth’ (De) and ‘Species’ (Sp) on the optimum quantum yield of chlorophyll a fluorescence (Fv/Fm), the maximum electron transport rate (ETRmax), the photosynthetic efficiency calculated from ETR-E relationship (αETR), the carotenoid content and the antioxidant activity (EC50) of Cymodocea nodosa and Caulerpa prolifera. Significant values are highlighted in bold (P<0.05).
Fv/Fm
ETRmax
αETR
Carotenoids
EC50
No transformation
C=0.3781 (n.s.)
No transformation
C=0.1811 (n.s.)
No transformation C=0.3261 (n.s.)
Ln(x+1), C=0.5953 (P<0.05)
Ln(x+1)
C=0.7288 (P<0.01)
MS
F
P
MS
F
P
MS
F
P
MS
F
P
MS
F
P
Ti
0.0000
0.04
0.8502
14.8712
1.88
0.1898
0.0000
0.04
0.8502
0.0006
0.08
0.7753
0.0076
2.97
0.1039
De
0.0017
79.25
0.0712
2.5585
241.73
0.0409
0.0017
6.32
0.0230
0.0167
2.18
0.1592
0.0017
0.66
0.4298
Sp
0.0202
4.78
0.2732
452.34
3.32
0.3197
0.0202
76.48
0.0000
0.2114
27.63
0.0001
0.1466
57.35
0.0000
Ti×De
0.0000
0.08
0.7813
0.0106
0.00
0.9713
0.0000
0.08
0.7813
0.0103
1.34
0.2639
0.0091
3.57
0.0769
Ti×Sp
0.0042
16.01
0.0010
136.393
17.20
0.0008
0.0042
16.01
0.0010
0.0298
3.89
0.0660
0.0105
4.11
0.0596
De×Sp
0.0000
0.15
0.7660
4.5920
0.35
0.6601
0.0000
0.12
0.7364
0.3046
39.83
0.0000
0.0014
0.54
0.4727
Ti×De×Sp
0.0002
0.79
0.3868
13.1394
1.66
0.2163
0.0002
0.79
0.3868
0.0012
0.15
0.7003
0.0049
1.9
0.1871
Residual
0.0003
7.9290
0.0003
0.0076
0.0026
Optimum quantum yield of chlorophyll a fluorescence of the seagrass Cymodocea nodosa and the green alga Caulerpa prolifera at 5 and 20 m depth in February 2014 (A) and May 2014 (B). Error bars are +SE of means (n=6). Different letters above bars denote significant differences.
The maximum electron transport rate (ETRmax) of the seagrass Cymodocea nodosa and the green alga Caulerpa prolifera at 5 and 20 m depth in February 2014 (A) and May 2014 (B). Error bars are +SE of means (n=6). Different letters above bars denote significant differences.
Photosynthetic efficiency calculated from ETR-E relationship (αETR) of the seagrass Cymodocea nodosa and the green alga Caulerpa prolifera at 5 and 20 m depth in February 2014 (A) and May 2014 (B). Error bars are +SE of means (n=6). Different letters above bars denote significant differences.
Carotenoids content of the seagrass Cymodocea nodosa and the green alga Caulerpa prolifera at 5 and 20 m depth in February 2014 (A) and May 2014 (B). Error bars are +SE of means (n=6). Different letters above bars denote significant differences.
Antioxidant activity (EC50 index) of the seagrass Cymodocea nodosa and the green alga Caulerpa prolifera at 5 and 20 m depth in February 2014 (A) and May 2014 (B). Error bars are +SE of means (n=6). The lower the EC50 index, the larger the antioxidant activity. Different letters above bars denote significant differences.
Differences in photo-physiological responses at the end of transplants
The optimum quantum yield of C. nodosa did not differ either between depth strata or between the origin of the vegetative fragment (all terms involving ‘De’ and ‘Or’, P>0.05, Table 2; Fig. 9A, B); significant differences only occurred between times (‘Ti’, P<0.00001, Table 2; Fig. 9A, B), including a decay in the optimum quantum yield in May in comparison with February (2014). The optimum quantum yield of C. prolifera varied between depths inconsistently from time to time (‘Ti×De’, P=0.0011, Table 3; Fig. 9C, D): in February 2014, the optimum quantum yield was larger for specimens collected at 20 than at 5 m, whereas in May 2014 no significant differences were detected (SNK tests, Fig. 9C, D). Thalli of C. prolifera increased the optimum quantum yield when transplanted from 5 to 20 m, while the opposite pattern was detected when thalli from 20 m were transplanted at 5 m (Fig. 9C, D). The ETRmax of both macrophytes was larger in May than in February 2014 (‘Ti’, P<0.01, Tables 2 and 3; Fig. 10), regardless of depth and the origin of the vegetative fragments. The photosynthetic efficiency of C. nodosa (αETR) varied exclusively between times; larger αETRvalues were observed in May (2014) (‘Ti’, P<0.00001, Table 2; Fig. 11A, B). Irrespective of times, however, C. prolifera showed a larger photosynthetic efficiency at 20 than at 5 m (‘De’, P=0.0066, Table 3; Fig. 11C, D). Thalli of C. prolifera increased their photosynthetic efficiency when transplanted from 5 to 20 m, while the opposite pattern was observed for thalli transplanted from 20 to 5 m (Fig. 11C, D); however, this pattern was not detected statistically (all terms involving ‘De’ and ‘Or’, P>0.05, Table 3; Fig. 11C, D). Appendix 3 includes means and SE of means of all photosynthetic parameters. The carotene content of C. nodosa differed between depths according to the origin of vegetative fragments from time to time (‘Ti×De×Or’, P=0.0004, Table 3; Fig. 12A, B). The magnitude of differences between depth levels depended on the origin (‘De×Or’, P=0.08, Table 3; Fig. 12C, D); differences in the carotene content between allochthonous and autochthonous fragments were more accentuated at 5 than at 20 m depth (SNK tests, Fig. 12C, D). The carotene content of C. prolifera differed exclusively between times (‘Ti’, P=0.00025, Table 3; Fig. 12C, D); a decrease in the carotene content was observed with increasing depth, but it was not statistically significant (‘De’, P=0.2084, Table 3; Fig. 12C, D). The antioxidant activity of both macrophytes varied between depths according to times, regardless of the origin of the vegetative fragments (‘Ti×De’, P<0.00001, Tables 2 and 3; Fig. 13); differences in the antioxidant activity between 5 and 20 m depth were significant in February but not in May 2014 (SNK tests, Fig. 13).
Three-way ANOVAs testing the effect of ‘Time’ (Ti), ‘Depth’ (De) and ‘Origin’ (Or) on the optimum quantum yield of chlorophyll a fluorescence (Fv/Fm), the maximum electron transport rate (ETRmax), the photosynthetic efficiency calculated from ETR-E relationship (αETR), the carotenoid content and the antioxidant activity (EC50) of Cymodocea nodosa at the end of the reciprocal transplants. Significant values are highlighted in bold (P<0.05).
Fv/Fm
ETRmax
αETR
Carotenoids
EC 50
No transformation C=0.3084 (n.s.)
No transformation C=0.2602 (n.s.)
No transformation
C=0.2849 (n.s.)
No transformation C=0.1730 (n.s.)
Ln(x+1)
C=0.4114 (P<0.05)
MS
F
P
MS
F
P
MS
F
P
MS
F
P
MS
F
P
Ti
0.0217
23.00
0.0000
1866.33
94.49
0.0000
0.0272
29.07
0.0000
0.0038
0.18
0.6757
0.0268
10.59
0.0023
De
0.0001
0.08
0.8264
31.7493
231.81
0.0418
0.0004
4.32
0.2855
0.0674
0.19
0.7410
0.1944
3.33
0.3190
Or
0.0003
0.19
0.7411
7.5986
2.03
0.3899
0.0032
2.74
0.3460
0.0244
1.36
0.4510
0.0001
0.37
0.6531
Ti×De
0.0009
0.98
0.3275
0.1370
0.01
0.9341
0.0001
0.09
0.7650
0.3634
17.09
0.0002
0.0583
23.02
0.0000
Ti×Or
0.0015
1.57
0.2170
3.7498
0.19
0.6654
0.0012
1.26
0.2678
0.0179
0.84
0.3640
0.0003
0.12
0.7313
De×Or
0.0008
1.29
0.4592
11.1554
0.21
0.7268
0.0036
14.47
0.1637
0.1158
0.37
0.6539
0.0022
0.24
0.7086
Ti×De×Or
0.0006
0.68
0.4159
53.2945
2.70
0.1083
0.0003
0.27
0.6066
0.3169
14.90
0.0004
0.0090
3.57
0.0661
Residual
0.0009
19.7517
0.0009
0.0213
0.0025
Optimum quantum yield of chlorophyll a fluorescence of the seagrass Cymodocea nodosa (A, B) and the green alga Caulerpa prolifera (C, D) at 5 and 20 m depth in February (A, C) and May 2014 (B, D). Error bars are +SE of means (n=6). Different letters above bars denote significant differences for each macrophyte.
Three-way ANOVAs testing the effect of ‘Time’ (Ti), ‘Depth’ (De) and ‘Origin’ (Or) on the optimum quantum yield of chlorophyll a fluorescence (Fv/Fm), the maximum electron tranposrt rate (ETRmax), the photosynthetic efficiency calculated from ETR-E relationship (αETR), the carotenoid content and the antioxidant activity (EC50) of Caulerpa prolifera at the end of the reciprocal transplants. Significant values are highlighted in bold (P<0.05).
Fv/Fm
ETRmax
αETR
Carotenoids
EC50
No transformation, C=0.2621 (n.s.)
Ln(x+1),
C=0.4585 (P<0.05)
No transformation, C=0.3211 (n.s.)
No transformation, C=0.2425 (n.s.)
Ln(x+1),
C=0.5852 (P<0.01)
MS
F
P
MS
F
P
MS
F
P
MS
F
P
MS
F
P
Ti
0.0018
0.12
0.7319
1.5804
11.03
0.0019
0.0000
0.05
0.8324
0.7868
10.43
0.0025
2.9795
106.67
0.0000
De
0.0103
0.06
0.8520
0.6397
87.29
0.0679
0.0088
9165.25
0.0066
0.4619
8.67
0.2084
1.0153
0.27
0.6955
Or
0.0105
16.65
0.1530
0.0806
0.38
0.6493
0.0002
11.90
0.1796
0.6239
17.36
0.1500
0.0288
1.14
0.4787
Ti×De
0.1833
12.45
0.0011
0.0073
0.05
0.8222
0.0000
0.00
0.9522
0.0533
0.71
0.4058
3.7779
135.26
0.0000
Ti×Or
0.0006
0.04
0.8370
0.2136
1.49
0.2292
0.0000
0.07
0.7927
0.0359
0.48
0.4940
0.0252
0.90
0.3477
De×Or
0.0601
9.67
0.1981
0.0010
0.04
0.8742
0.0010
2.14
0.3816
0.0287
52.54
0.0873
0.0555
0.41
0.6363
Ti×De×Or
0.0062
0.42
0.5196
0.0238
0.17
0.6859
0.0005
1.77
0.1912
0.0005
0.01
0.9327
0.1344
4.81
0.0341
Residual
0.0147
0.1432
0.0003
0.0754
0.0279
The maximum electron transport rate (ETRmax) of the seagrass Cymodocea nodosa (A, B) and the green alga Caulerpa prolifera (C, D) at 5 and 20 m depth in February 2014 (A, C) and May 2014 (B, D). Error bars are +SE of means (n=6). Different letters above bars denote significant differences for each macrophyte.
Photosynthetic efficiency calculated from ETR-E relationship (αETR) of the seagrass Cymodocea nodosa (A, B) and the green alga Caulerpa prolifera (C, D) at 5 and 20 m depth in February 2014 (A, C) and May 2014 (B, D). Error bars are +SE of means (n=6). Different letters above bars denote significant differences for each macrophyte.
Carotenoids content of the seagrass Cymodocea nodosa (A, B) and the green alga Caulerpa prolifera (C, D) at 5 and 20 m depth in February 2014 (A, C) and May 2014 (B, D). Error bars are +SE of means (n=6). Different letters above bars denote significant differences for each macrophyte.
Antioxidant activity (EC50 index) of the seagrass Cymodocea nodosa (A, B) and the green alga Caulerpa prolifera (C, D) at 5 and 20 m depth in February 2014 (A, C) and May 2014 (B, D). Error bars are +SE of means (n=6). The lower the EC50 index, the larger the antioxidant activity. Different letters above bars denote significant differences for each macrophyte.
DISCUSSION
Our results have suggested a connection between the patterns of vertical distribution of the seagrass C. nodosa (i.e. decreased biomass with depth) and the green alga C. prolifera (i.e. increased biomass with depth) with their respective photochemical adaptations at the study site in Gran Canaria Island. Overall, C. nodosa behaves as a ‘light-plant’ relative to C. prolifera, which follows a ‘shade-plant’ pattern. This result is similar to that of a study comparing the photo-physiology of both macrophytes within a Mediterranean coastal lagoon (García-Sánchez et al. 2012). This outcome has implications in the way light is absorbed by both macrophytes, as well as in the quantity and quality of pigment contents, and in ways to ameliorate/dissipate the excess of light energy reaching photosynthetic tissues (Malta et al. 2005, García-Sánchez et al. 2012).
Our results demonstrated that the seagrass C. nodosa has a consistently larger optimum quantum yield (Fv/Fm), ETRmax and photosynthetic efficiency than the green alga C. prolifera, particularly when PAR values are larger (May in comparison with February 2014). Evidence for this, moreover, was provided by direct measurements from collected thalli and at the end of the reciprocal transplants. This outcome is a clear indication of adaptation of the seagrass to larger irradiances and therefore optimization of light absorption in this time (May). Not surprisingly, this is particularly pertinent in this season, as this seagrass experiences rapid growth in spring-summer in the study region (Tuya et al. 2006), as well as in a range of sites across the Mediterranean Sea (e.g. Terrados and Ros 1992). Biomass accumulation (e.g. seagrass leaf growth and rhizome production) is metabolically based on increased photosynthetic rates and therefore high C demands. For C. nodosa, there is a positive correlation between gross primary production and ETR at the PSII, except under stressful conditions (e.g. low nutrients levels and very high PAR), as usually occurs for C4 plants, including C. nodosa (Cabello-Pasini et al. 2015). In turn, decreased irradiances reaching the photosynthetic apparatus of C. nodosa, for example during fertilization events, negatively affect the production of photochemical energy by C. nodosa (Tuya et al. 2015). This seagrass showed increased photosynthetic potential (ETRmax) and photochemical energy conversion efficiency (α) under increased irradiances, i.e. at 5 in comparison with 20 m, and in May in comparison with February (2014). Under decreased irradiances (e.g. at 20 in comparison with 5 m depth), this seagrass shows increased photochemical energy conversion efficiency (α); this is a common strategy of marine angiosperms when they are subjected to chronic light deprivation (Collier et al. 2012), including C. nodosa (Silva et al. 2013).
The green alga C. prolifera has adapted its photosynthetic apparatus to prevailing light regimes, e.g. a larger photosynthetic efficiency at 20 than at 5 m depth. A similar pattern has been described for other Caulerpa species, such as Caulerpa racemosa in the Mediterranean Sea (Raniello et al. 2006, Bernardeau-Esteller et al. 2011). Not only deep-water (20 m depth) thalli of C. prolifera are more adapted to low irradiances: this alga also showed short-term acclimation under varying light regimes, i.e. when reciprocal transplants of vegetative fragments were carried out. When fragments from 5 m were relocated at 20 m depth, these fragment became more photo-chemically efficient (i.e. larger Fv/Fm and αETR), while the opposite occurred for deep-water fragments (20 m) relocated at 5 m. This photo-physiological versatility of C. prolifera provides an additional, short-term advantage of this alga under varying environmental scenarios. Due to their lateral (clonal) growth, Caulerpa spp. often experience spatial variation in a range of environmental conditions and resources (Collado-Vides 2002). As a result, species within the genus Caulerpa show morphological plasticity, including varying patterns of internode production and length of fronds, e.g. for a more efficient light use, which affect the overall appearance of the plant (Collado-Vides 2002).
The seagrass and the green alga seem to have different physiological mechanisms to achieve photo-protection. The exposition of C. nodosa to high PAR seems to explain the larger antioxidant activity of the seagrass in comparison with C. prolifera, as a way to dissipate excess light energy. This has been described in the Mediterranean Sea, where C. nodosa typically live in shallow water (García-Sánchez et al. 2012). The green alga showed a larger content of carotenes than the seagrass, particularly in shallow water (i.e. under high irradiances), most likely to protect its photosynthetic machinery from light excess. In turn, when vegetative fragments of C. prolifera from 5 m were relocated at 20 m, the alga decreased its carotene content. In the particular case of the seagrass, however, the carotene content increased with depth; this is most likely a response to increase light harvesting by photosynthetic antennas under scenarios of low PAR. In turn, leaves of flowering plants have the capacity to adapt the carotene content either to improve light harvesting or to protect the photosynthetic machinery from light excess (Matsubara et al. 2009). These results, however, should be taken with caution, as we have not analysed the specific carotenes associated with each macrophyte. Moreover, we cannot link differences in antioxidant activity with varying quality and quantity of carotenes (e.g. b-carotene and lutein) and other chemical ways of excess light dissipation.
A range of human-induced perturbations may reduce the transparency of coastal waters, including sedimentation (e.g. associated with construction of infrastructures and coastal runoff) and fertilization (Burkholder et al. 2007). Decreased light regimes, where C. nodosa and C. prolifera coexist in subtidal soft bottoms, seem to clearly favour the alga in comparison with the seagrass, at least from a physiological point of view. The results of this study are therefore added to a body of literature that demonstrates the need to maintain coastal waters with a good quality status to promote seagrass conservation.
ACKNOWLEDGEMENTS
This study was partially supported by the EU project ECOSERVEG, within the BEST initiative (Voluntary Scheme for Biodiversity and Ecosystem Services in Territories of the EU Outermost Regions and Oversees Countries and Territories, Grant nº 07.032700/2012/635752/SUB/B2). F. Tuya was supported by the MINECO ‘Ramón y Cajal’ programme. We thank T. Sánchez for his help during fieldwork. Two anonymous reviewers provided positive feedback on a previous draft of this paper.
REFERENCES Barberá C. Tuya F. Boyra A. Spatial variation in the structural parameters of Cymodocea nodosa seagrass meadows in the Canary Islands: a multiscaled approach200548122126http://dx.doi.org/10.1515/BOT.2005.021 Bernardeau-Esteller J. Marín-Guirao L. Sandoval-Gil J.M. Photosynthesis and daily metabolic carbon balance of the invasive Caulerpa racemosa var. cylindracea (Chlorophyta: Caulerpales) along a depth gradient 201175803810http://dx.doi.org/10.3989/scimar.2011.75n4803 Betancor S. Domínguez B. Tuya F. Photosynthetic performance and photoprotection of Cystoseira humilis (Phaeophyceae) and Digenea simplex (Rhodophyceae) in an intertidal rock pool 20151211625http://dx.doi.org/10.1016/j.aquabot.2014.10.008 Blois M. Antioxidant determinations by the use of a stable free radical 195818111991200 Bradstreet R.B.1965 New York Academic Press Burkholder J.M. Tomasko D. Touchette B.W.Seagrasses and eutrophication 20073504672http://dx.doi.org/10.1016/j.jembe.2007.06.024 Cabello-Pasini J.M. Abdala-Díaz R. Macías-Carranza V, Effect of irradiance and nitrate levels on the relationship between gross photosynthesis and electron transport rate in the seagrass Cymodocea nodosa. 20154193105http://dx.doi.org/10.7773/cm.v41i2.2499 Ceccherelli G. Cinelli F. Short-term effects of nutrient enrichment of the sediment and interactions between the seagrass Cymodocea nodosa and the introduced green alga Caulerpa taxifolia in a Mediterranean bay. 1997217165177http://dx.doi.org/10.1016/S0022-0981(97)00050-6 Collado-Vides L. Morphological plasticity of Caulerpa prolifera (Caulerpales, Chlorophyta) in relation to growth form in a coral reef lagoon. 200245123129http://dx.doi.org/10.1515/BOT.2002.013 Collier C.J. Waycott M. Giraldo Ospina A. Responses of four Indo-West Pacific seagrass species to shading 201265342354http://dx.doi.org/10.1016/j.marpolbul.2011.06.017 Duarte C.M. Dennison W.C. Orth R.J. The charisma of coastal ecosystems. 200831233238http://dx.doi.org/10.1007/s12237-008-9038-7 García-Sánchez S. Korbee N. Pérez-Ruzafa I.M. 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).2012793747http://dx.doi.org/10.1016/j.marenvres.2012.05.001 Gardner A. Tuya F. Lavery P.S. Habitat preferences of macroinvertebrate fauna among seagrasses with varying structural forms.2013439143151http://dx.doi.org/10.1016/j.jembe.2012.11.009 Grzymski J. Johnsen G. Sakshaug E. The significance of intracellular self-shading on the bio-optical properties of brown, red and green macroalgae. 199733408414http://dx.doi.org/10.1111/j.0022-3646.1997.00408.x Häder D.P. Porst M. Herrmann H. Photosynthesis of mediterranean green alga Caulerpa prolifera measured in the field under solar irradiation 1997376673http://dx.doi.org/10.1016/S1011-1344(96)07338-1 Hanelt D. The capability for dynamic photoinhibition in Arctic macroalgae isrelated to their depth distribution 1998131361369http://dx.doi.org/10.1007/s002270050329 Jassby A. Platt T. Mathematical formulation of the relationship between photosynthesis and light for phytoplankton 197621540547http://dx.doi.org/10.4319/lo.1976.21.4.0540 Lapointe B.E. Barile P.J. Littler M.M. Macroalgal blooms on southeast Florida coral reefs: II. Cross - shelf discrimination of nitrogen sources indicates widespread assimilation of sewage nitrogen 2005411061122http://dx.doi.org/10.1016/j.hal.2005.06.002 Lloret J. Marin A. Marin-Guirao L. Changes in macrophytes distribution in a hypersaline coastal lagoon associated with the development of intensively irrigated agriculture 200548828842http://dx.doi.org/10.1016/j.ocecoaman.2005.07.002 Lüning K.1990 New York, NY. Wiley-Interscience, Malta E.J. Ferreira D.G. Vergara J.J.. Nitrogen load and irradiance affect morphology, photosynthesis and growth of Caulerpa prolifera (Bryopsidales, Chlorophyta)2005298101114http://dx.doi.org/10.3354/meps298101 Matsubara S. Krause G.H. Aranda J. Sun-shade patterns of leaf carotenoid composition in 86 species of neotropical forest plants 2009362036http://dx.doi.org/10.1071/FP08214 Maxwell K. Johnson G. Chlorophyll fluorescence—a practical guide 200051659668http://dx.doi.org/10.1093/jexbot/51.345.659 Murphy J. Riley J.P. A modified single solution method for the determination of phosphate in natural waters 1962273136http://dx.doi.org/10.1016/S0003-2670(00)88444-5 Olesen B. Enríquez S. Duarte C.M. Depth-acclimation of photosynthesis, morphology and demography of Posidonia oceanica and Cymodocea nodosa in the Spanish Mediterranean Sea. 20022368997http://dx.doi.org/10.3354/meps236089 Parsons T.R. Strickland J.D.H. Discussion of spectrophometric determination of marine-plant pigments, with revised equations for ascertaining chlorophyll-a and carotenois 196321105156http://dx.doi.org/10.1007/978-1-4684-3890-1_19 Platt T. Gallegos I. Modelling primary production Falkowski P.G.1980 Plenum 339351http://dx.doi.org/10.1007/978-1-4684-3890-1_19 Raniello R. Lorenti M. Brunet C. Photoacclimation of the invasive alga Caulerpa racemosa var. cylindracea to depth and daylight patterns and a putative new role for siphonoxanthin 2006272030http://dx.doi.org/10.1111/j.1439-0485.2006.00080.x Silva J. Barrote J. Costa M.M. Physiological responses of Zostera marina and Cymodocea nodosa to light-limitation stress 20138 e81058http://dx.doi.org/10.1371/journal.pone.0081058 Terrados J. Ros J.D. Growth and primary production of Cymodocea nodosa (Ucria) Ascherson in a Mediterranean coastal lagoon: The Mar Menor (SE Spain). 1992436374http://dx.doi.org/10.1016/0304-3770(92)90014-A Tuya F. Martín J.A. Luque A. Seasonal cycle of a Cymodocea nodosa seagrass meadow and of the associated ichthyofauna at Playa Dorada (Lanzarote, Canary Islands, eastern Atlantic). 200632695704 Tuya F. Hernández-Zerpa H. Espino F. Drastic decadal decline of the seagrass Cymodocea nodosa at Gran Canaria (Eastern Atlantic): interactions with the green alga Caulerpa prolifera201310516http://dx.doi.org/10.1016/j.aquabot.2012.10.006 Tuya F. Viera-Rodríguez M.A. Guedes R. Seagrass responses to nutrient enrichment depend on clonal integration, but not flow-on effects on associated biota. 20134902335http://dx.doi.org/10.3354/meps10448 Tuya F. Ribeiro-Leite L. Arto-Cuesta N. Decadal changes in the structure of Cymodocea nodosa seagrass meadows: Natural vs. human influences. 20141374149http://dx.doi.org/10.1016/j.ecss.2013.11.026 Tuya F. Betancor S. Viera-Rodríguez M.A. Effect of chronic versus pulse perturbations on a marine ecosystem: integration of functional responses across organization levels 20151814551471http://dx.doi.org/10.1007/s10021-015-9911-8 Underwood A.J.1997 Cambridge, UK, Cambridge Univ. Press Walkley A. Black J.A. An examination of the Degtjareff method for determining soil organic matter and a proposed modification of the chromic titration method 1934372938http://dx.doi.org/10.1097/00010694-193401000-00003APPENDICES
Sediment characteristics across depth at Gando Bay (Gran Canaria Island). Only the Ptotal showed a significant change with depth.
5 m
10 m
15 m
20 m
Mean
SD
Mean
SD
Mean
SD
Mean
SD
Ntotal (mg kg–1)
405.50
19.09
251.00
39.60
307.50
60.10
403.50
177.48
Ptotal (mg kg–1), R2=0.58, P<0.01
1375.20
1669.20
29.95
9.12
57.20
11.46
55.25
41.65
Organic matter (%)
1.05
0.07
0.90
0.14
0.75
0.07
0.95
0.07
D50
0.14
0.00
0.33
0.25
0.19
0.02
0.14
0.00
Type of sediment
Fine sands
Fine sands
Fine sands
Fine sands
Fine sands
Fine sands
Fine sands
Fine sands
Photosynthetic parameters of Cymodocea nodosa and Caulerpa prolifera at the start of transplants (means and SE are included, n=6).
Time
Species
Depth
Fv/Fm
SE
ETRmax
SE
αETR
SE
Ek
SE
February (2014)
C. nodosa
5 m
0.824
0.003
16.490
1.760
0.097
0.009
173.156
24.342
February (2014)
C. prolifera
5 m
0.683
0.021
11.972
1.354
0.061
0.009
196.070
13.459
February (2014)
C. nodosa
20 m
0.823
0.007
16.583
1.956
0.112
0.006
150.331
25.888
February (2014)
C. prolifera
20 m
0.807
0.009
13.278
1.509
0.084
0.008
157.577
7.494
May (2014)
C. nodosa
5 m
0.747
0.007
21.394
1.826
0.118
0.016
183.970
17.979
May (2014)
C. prolifera
5 m
0.484
0.111
10.298
1.828
0.041
0.002
244.082
37.737
May (2014)
C. nodosa
20 m
0.772
0.011
24.361
1.711
0.141
0.006
171.441
5.138
May (2014)
C. prolifera
20 m
0.507
0.155
8.554
0.703
0.048
0.009
184.985
25.912
Photosynthetic parameters of Cymodocea nodosa and Caulerpa prolifera at the end of transplants (means and SE are included, n=6).