Seasonal functioning and dynamics of Caulerpa prolifera meadows in shallow areas: An integrated approach in Cadiz Bay Natural Park

Estuarine, Coastal and Shelf Science 112 (2012) 255e264

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Seasonal functioning and dynamics of Caulerpa prolifera meadows in shallow areas: An integrated approach in Cadiz Bay Natural Park Juan J. Vergara*, M. Paz García-Sánchez, Irene Olivé 1, Patricia García-Marín, Fernando G. Brun, J. Lucas Pérez-Lloréns, Ignacio Hernández Departamento de Biología, Area de Ecología, Facultad de Ciencias del Mar y Ambientales, Universidad de Cádiz, Campus de Excelencia Internacional del Mar (CEIMAR), Pol. Rio San Pedro, E-11510 Puerto Real, Cádiz, Spain

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 November 2011 Accepted 28 July 2012 Available online 8 August 2012

The rhizophyte alga Caulerpa prolifera thrives in dense monospecific stands in the vicinity of meadows of the seagrass Cymodocea nodosa in Cadiz Bay Natural Park. The seasonal cycle of demographic and biometric properties, photosynthesis, and elemental composition (C:N:P) of this species were monitored bimonthly from March 2004 to March 2005. The number of primary assimilators peaked in spring as consequence of the new recruitment, reaching densities up to 104 assimilators$m
2. A second peak was recorded in late summer, with a further decrease towards autumn and winter. Despite this summer maximum, aboveground biomass followed a unimodal pattern, with a spring peak about 400 g dry weight$m
2. In conjunction to demographic properties of the population, a detailed biometric analysis showed that the percentage of assimilators bearing proliferations and the number of proliferations per assimilator were maximal in spring (100% and c.a. 17, respectively), and decreased towards summer and autumn. The size of the primary assimilators was minimal in spring (May) as a result of the new recruitments. However, the frond area per metre of stolon peaked in early spring and decreased towards the remainder of the year. The thallus area index (TAI) was computed from two different, independent approaches which both produced similar results, with a maximum TAI recorded in spring (transient values up to 18 m2$m
2). The relative contribution of primary assimilators and proliferations to TAI was also assessed. Whereas the number of proliferations accounted for most of the TAI peak in spring, its contribution decreased during the year, to a minimum in winter, where primary assimilators were the main contributors to TAI. The present study represents the first report of the seasonal dynamics of C. prolifera in south Atlantic Spanish coasts, and indicates the important contribution of this primary producer in shallow coastal ecosystems. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: biomass C:N:P composition Caulerpa prolifera photosynthesis subterranean network TAI e thallus area index

1. Introduction The chlorophyte Caulerpa prolifera (Forsskål) J.V. Lamouroux, the only autochthonous Caulerpa species in the Mediterranean, is a clonal rhizophyte that inhabits soft bottom sediments in temperate and tropical waters (Lüning, 1990). C. prolifera is a non invasive species on south Atlantic Spanish coasts, as its presence has been dated back to XIX century by Clemente (see SeoaneCamba, 1965).

* Corresponding author. E-mail address: [email protected] (J.J. Vergara). 1 Present address: ALGAE-Marine Plant Ecology Research Group, Centre of Marine Sciences, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal. 0272-7714/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ecss.2012.07.031

In Cadiz bay this alga is anchored to soft sediments and occupies large shallow stands within the bay (Morris et al., 2009). However, its distribution is now wider than in previous decades, probably by outcompeting the seagrass Cymodocea nodosa at deeper waters due to the ongoing deterioration in light transparency. Field manipulative experiments have shown that Caulerpa prolifera is able to colonize empty spaces occupied by another seagrass, Halodule wrightii, via lateral expansion in Tampa Bay (Stafford and Bell, 2006). In addition, although some small patches are also able to thrive within intertidal pools in winter, when light is not stressful, the denser monospecific stands develop in shallow subtidal waters in a scattered landscape, cohabiting with the seagrass C. nodosa. Similar stable coexistence patterns for C. nodosa and the congeneric Caulerpa taxifolia have been described by Ceccherelli and Sechi (2002) under a variety of nutrient enrichment conditions. This alga becomes the predominant and almost exclusive species in the

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deepest soft bottom sediments of the bay (Morris et al., 2009). From satellite images, the percentage of shallow subtidal populations represents ca. 27% of C. prolifera populations in the bay (Morris, pers. comm.). Previous laboratory studies with Caulerpa prolifera showed that this species effectively responded to nutrient availability and light (Malta et al., 2005). In fact, this species can be characterized as nitrophilic, with a high internal N quota and requirements. Its morphology responded strongly to N supply, with belowground biomass accumulation (stolons and rhizoids) stimulated under N limitation and assimilator formation enhanced under N supply (Malta et al., 2005). With regard to light, although C. prolifera is quite phototolerant (Gacia et al., 1996), it behaves as a sciaphilic alga where growth rates and quantum yields (Fv/Fm) at saturating light were lower than those at dim light (Malta et al., 2005), with risk of photoinhibition in its natural habitat (Häder et al., 1997). The spatial distribution of Caulerpa prolifera in Cadiz bay, as well as the d15N isotopic signature (including N and C composition) has been previously studied by Morris et al. (2009) who revealed the role that tissue N can play as a tool in the management of nutrient inputs within shallow coastal zones. In addition, the dense meadows of this rhizophyte may be important in terms of their contribution to the primary productivity and biogeochemical cycles in the bay, as these meadows also play a role as sediment trap by altering the hydrodynamics of the water column (Hendricks et al., 2010). Against this background, the aim of this study was to study the population structure and dynamics of Caulerpa prolifera in the south Atlantic coast of Spain. Along an annual cycle (March 2004, March 2005), the number of assimilators, the aboveground and belowground biomass, the biometric properties of the population, photosynthesis-irradiance (P-E) curves, and the tissue C:N:P composition were monitored, together with physico-chemical variables. The study hypothesises the importance of the maximum spring development of C. prolifera and the dense belowground network in understanding the role of C. prolifera meadows as effective sediment traps (Hendricks et al., 2010). 2. Materials and methods 2.1. Study area and sampling procedure Dense and monospecific stands of the rhizophyte Caulerpa prolifera were monitored in Santibañez salt marsh in the inner part of Cadiz bay, southern Spain (36.47 N; 6.25 W) (Fig. 1). This population develops in shallow areas, at a depth of approximately 0.5e1 m below the lowest astronomical tide (LAT), mixed with patches of the seagrass Cymodocea nodosa. Other C. prolifera stands also thrive in deeper waters, however with lower biomass density (Morris et al., 2009). Rooted macrophytes cover a great extent of the bottom of the bay. C. prolifera is the dominant species in subtidal waters, while three seagrass species (C. nodosa, Zostera marina and Zostera noltii) cover the shallow subtidal and intertidal areas. For detailed information of the study area, see a previous description in Morris et al. (2009). 2.2. Physico-chemical data acquisition The solar radiation data set was provided by the Andalusian Agency of Energy from the meteorological stations adjacent to the study area. Mean daily air temperature was provided by the Spanish Agency of Meteorology, station of San Fernando, also near to the sampling area (36.27 N, 6.10 W). Seawater temperature was obtained from a buoy (Triaxys type, coastal network Puertos delEstado) located near the mouth of the Cadiz Bay (36.50 N,

6.33 W). As the register was hourly, the mean daily temperature was computed. Water samples for inorganic N and P nutrients were taken in the field every sampling date and measured in an automatic analyzer (model TRAACS 800), following the methods described in Grasshoff et al. (1983) slightly modified. 2.3. Sampling and processing of biological material The area was sampled bimonthly from March 2004 to March 2005 on 20th March and 3rd May (spring), 1st July and 30th August (summer), 15th November (autumn) and 10th February (winter). Three areas of 20  20 cm were randomly sampled with a metal frame, collecting all the aboveground (AG) and belowground (BG) biomass within the structure. Most of the sediment attached to samples was washed in situ, and plants were kept cool and transported to the laboratory within 1 h of collection. Once at the laboratory samples were further cleaned of remaining sediment with seawater. 2.4. Demographic and biometric measurements The photosynthetic biomass (AG) of Caulerpa prolifera is composed of fronds and can be divided into primary assimilators (those that arise directly from the stolon, henceforth assimilators) and proliferations that emerge from assimilators (or from primary proliferations, secondary ones). The BG biomass is composed of a subterranean network of cylindrical stolons with a number of rhizoid clusters. The number of primary assimilators was counted, and AG and BG biomass was split and dried separately in an oven at 60  C for 3 days. This dried algal material was ground for elemental C:N:P analysis (see below). The separation of AG and BG biomass is fundamentally operational, as some part of the stolons are located in the interface sediment-water and are green and with a certain degree of photosynthetic ability. In parallel, fresh algal samples were employed for biometric measurements. A variable number of thalli (n ¼ 8e10) bearing a stolon fragment with a variable number of assimilators were cleaned, placed between two acetate sheets, and scanned with a graduate ruler as a reference. These images were processed using a publicly available image analysis software (Image J, NIH, USA). The following variables were estimated: area of (individual) primary assimilators, frond (assimilators plus proliferations) area per metre stolon, % of proliferated assimilators, n proliferations per assimilator, and n assimilators or n rhizoid clusters per metre stolon. Two approaches were applied to estimate the individual assimilator area: i) by measuring length and width of the fronds and applying the ellipse formula, and ii) by application of image analysis from scanned images. Both estimates were highly correlated (r ¼ 0.99; p < 0.001; n ¼ 219), with a slope of 0.996 (data not shown). Therefore, both methods were adequate to measure the assimilator area, and accordingly, a simple measurement of the major and the minor axes of an ellipse was adequate to estimate the assimilator area of this species. A number of derived morphometric variables were also estimated from biometric measurements. The length of the stolon network per unit surface of the meadow (Lstolon; m stolon$m
2 meadow) was computed by dividing the density of primary assimilators found in the field (NA1; n assimilators$m
2), by the n primary assimilators per metre stolon (NAstolon; n assimilators$m
1), estimated in morphometric measurements.

Lstolon ¼ NA1 =NAstolon

(1)

The area covered by the stolon network (m2 stolon$m
2 sediment) was estimated by multiplying Lstolon by the mean width of

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Fig. 1. Caulerpa prolifera. Map of the field site in Cadiz Bay (SW Spain).

the stolon (1.1  0.2 mm). The volume occupied by the stolon network (m3 stolon$m
2 meadow) was computed by multiplying the Lstolon by the mean cross section area (0.95 mm2) of the stolon. The thallus Area index e TAI - (m2$m
2), a concept analogous to the leaf area index e LAI - for vascular plants (Hernández et al., 1997) was computed from two different, independent approaches. In a first approach, TAI1 was computed as the product of the total assimilator area per metre stolon (AM; m2 assimilator area$m
1 stolon) by the length of the stolon network (Lstolon; m stolon$m
2 meadow).

abundance of assimilators per unit area (NA1) by the number of proliferations per primary assimilator (NA2), the mean area of e individual e secondary assimilators (A2; m2), and the proportion of primary assimilators proliferated (P, 0e1). 2.5. Elemental C:N:P composition

In a second independent approach, TAI2 was computed by the following expression:

The C:N:P composition as well as the isotopic d13C and d15N abundance were measured on dried ground material. The C and N composition and isotopic signatures were measured at SAI (Servicio de Apoyo a la Investigación, University of A Coruña, Spain), as previously described (Morris et al., 2009). Total internal P was estimated as soluble reactive phosphorus (Murphy and Riley, 1962) following an acid digestion (Sommer and Nelson, 1972).

TAI2 ¼ TAIasim þ TAIprolif ¼ ðNA1 $A1 Þ þ ðNA1 $NA2 $A2 $PÞ

2.6. Photosynthetic measurements and pigment content

TAI1 ¼ AM$Lstolon

(2)

(3) where TAI2 is the sum of the contributions of assimilators (TAIasim) and proliferations (TAIprolif). The contribution of primary assimilators to TAI was estimated multiplying NA1 (n primary assimilators$m
2), by the mean area of e individual e primary assimilators (A1; m2). The second term is the contribution of primary proliferations, and it was calculated by multiplying the

Photosynthesis vs. irradiance (P-E) curves were obtained seasonally in plants collected at the study area. Mature assimilators of approximately 2 cm long were selected at different dates within each season (n ¼ 4). The P-E curves were conducted within one day of sampling. Net photosynthesis and dark respiration (Rd) rates were measured using a small incubation chamber (20 mL) connected to a thermostatic water bath (20  C) and to a computerized

A

30

25

-2

polarographic oxygen electrode (Hansatech Instruments, Norfolk, UK). Electrodes were calibrated with air and N2-saturated artificial seawater. Incubations were done in 11 mL of artificial seawater (salinity ¼ 30) prepared from a sea salt mixture (Marinemix professional, Wiegandt, Germany) supplemented with NaHCO3 (5 mM final concentration) to prevent CO2 limitation. Salinity was checked using a hand refractometer (Atago). Oxygen tension within the incubation chamber was maintained between 20 and 80% saturation by bubbling with N2 gas. One assimilator was positioned within the chamber perpendicular to the light beam and subjected to a set of 14 increasing photon flux densities (PFDs, from 0 to 700 mmol quanta m
2 s
1), plus a final incubation in darkness to measure respiration in light-acclimatized thalli (Rd). Light was provided by 36 red light emitting diodes (PAR, peak output above 635 nm, model LS3/LH36U, Hansatech Ltd, Norfolk, UK). At each PFD, oxygen concentration was measured during 7 min (except in darkness, 15 min), and the rates were computed within the last 5 min, once the slope was stabilized. Net maximum photosynthetic rates (Pmax) were obtained from the average maximum values above saturating irradiance. The photosynthetic efficiency (a) was estimated from the initial slope of the P-E curves by linear leastsquares regression analysis. Light saturation point (Ek) was computed as the ratio between maximum photosynthetic rate and photosynthetic efficiency (Pmax/a), and the light compensation point (Ec) as the intercept of the P-E curve with the abscise axis (eRd/a). Analogous assimilators to those used for P-E curves were cut and kept at
80  C for pigment determination. Pigment content was measured spectrophotometrically following pigment extraction of a homogenized tissue in 80% acetone at low temperature (Dennison, 1990). Chlorophyll a and b and total carotenoids in frond extracts were calculated using the equations of Lichtenthaler and Wellburn (1983).

Solar radiation (MJ · m )

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35 Air Seawater

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Temperature (ºC)

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25 20 15 10 5 0 Mar

May

Jul

Sep Month

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2.7. Statistics

Fig. 2. Seasonal changes of (A) solar radiation and (B) temperature in air and seawater.

The effect of seasonality on the biomass abundance (AG and BG), assimilator density, morphological and biometrical attributes, and photosynthesis, was tested by one-way ANOVAs. Data were log10 or root square transformed to satisfy analysis of variance assumptions (ShapiroeWilks’ test for normality and Levene’s test for homocedasticity). ANOVA was followed by a multiple comparison test (Tukey HSD). When ANOVA assumptions were not satisfied after data transformation, a KruskaleWallis non-parametric test was used (internal nutrient content data). Significance levels were considered at p  0.05. Differences in organic matter content of the sediment (measured by a standard combustion procedure in a 2.5 cm diameter  5 cm depth core), and isotope discrimination by AG or BG parts were checked by Student t-tests. In all cases, significance level was set at p  0.05. Statistical analyses were computed with SPSS and with R (R Development Core Team, 2010).

measured at 21 m depth, whereas C. prolifera populations thrive in shallower areas (around 1 m depth at low tide), where there is a high influence of air on seawater temperature. Therefore, temperature at the study site must be intermediate between the two data sets along the cycle: hotter than that registered in seawater in summer, and cooler than that in winter. The seawater inorganic nutrients ammonium and phosphate showed higher concentrations than nitrate and nitrite along the seasonal cycle (Fig. 3), peaking in spring (about 15 mM for ammonium and 2.3 mM for phosphate; Fig. 3A), and with a second, small pulse for ammonium in summer. In contrast, nitrate and nitrite concentrations were kept low, with a nitrate maximum of 1 mM in spring and autumn and almost undetectable levels in summer. Nitrite was always <0.3 mM (Fig. 3B).

3. Results

3.2. Demographic and biometric properties

3.1. Physico-chemical variables

Total biomass and density of primary assimilators followed similar trends along the year, with main spring maxima (ca. 600 g DW$m
2 and 104 assimilators$m
2, respectively) and secondary summer peaks (Fig. 4A, Table 1). Concerning biomass partitioning, maximum AG values were reached in spring, decreasing towards the rest of the year, while BG biomass showed a bimodal response, with maximal values in May and late summer and minimum values in winter (Fig. 4A). The summer maximum in assimilator density (Fig. 4B) was not matched by a maximum in AG biomass. The AG:BG ratio decreased markedly throughout the year, ranging between 2.8 (spring) and below 1 (winter) (Fig. 4B).

Solar radiation followed the typical seasonal trend for temperate climates, with maximal values in summer and minimal in winter (Fig. 2A), as previously shown in neighbouring areas (Hernández et al., 1997). Temperature also displayed a seasonal trend, with maxima in summer and minima in winter (Fig. 2B), being more damped in water than in air (mean seawater temperature of 19  3  C, with a range from 13.7  C to 24.4  C; mean air temperature of 17  5.3  C, with a range from 3.2  C to 31.9  C). However, it has to be taken into account that seawater temperature was

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Month Fig. 3. Seasonal changes of inorganic N and P nutrients in seawater (A) Ammonium and phosphate; (B) nitrate and nitrite. Data represent mean  SE (n ¼ 3).

The area of individual primary assimilators of Caulerpa prolifera was minimal in spring (May) matching the maximum AG biomass (Fig. 5A); therefore, in spring there was a high recruitment of new assimilators. Most of them were lost in summer, although the remaining ones reached larger sizes. According to the maximum recruitment of new primary assimilators in spring, the frond area per metre stolon decreased from March to May, and it was kept relatively constant throughout the rest of the year (Fig. 5B). Both, the degree of proliferation (% assimilators proliferated) and the number of proliferations per assimilator peaked in spring (c. a. 100% and a median about 17, respectively) and decreased along the seasonal cycle, with a partial recovering in late winter (Fig. 5C, D). The n of assimilators was always lower than the n rhizoid clusters per m stolon (Fig. 5E, F). Despite a maximum assimilator density in spring (May), the linear assimilator density (per m stolon) was minimal, which indicates a high development of the belowground stolon network at this period of the year. In fact, the estimated length of the stolon network was maximum in spring (May), reaching values up to 680 m$m
2 and decreasing strongly along the seasonal cycle (Fig. 6A). From the biometric and demographic measurements, several secondary variables were estimated. The TAI values, computed by two independent approaches, gave similar results except in early spring (Fig. 6A). Irrespective of the approach, it had extremely high (c. a. 18 m2$m
2) and transient values reached in spring (May),

May

Jul

Sep

Nov

Jan

0 Mar

Month Fig. 4. Caulerpa prolifera. Seasonal changes of (A) total biomass (AG þ BG) and assimilator density; (B) aboveground (AG) biomass (primary assimilators plus proliferations), belowground (BG) biomass (stolons and rhizoids) and AG:BG ratio. Data represent mean  SE (n ¼ 3).

followed by a decrease throughout the rest of year. Thus, it seems that the high degree of proliferations may oversaturate the biomass and, at short-term, achieve these high TAI values. The contribution of primary assimilators and proliferations to TAI is shown in Fig. 6B. Whereas the assimilators showed a maximum TAI close to 4, the proliferations accounted for most of the transient TAI increase in spring. Furthermore, from the mean diameter and the mean cross section area of the stolons, it was estimated that the maximum area covered by the stolon network was 0.75 m2 stolon$m
2 in spring, with a minimum of 0.05 m2 stolon$m
2 in winter. Similarly, the volume occupied by the stolon network was maximum in spring (0.65 L$m
2), while the minimum was reached in winter (0.04 L$m
2). This spring peak was attained with a BG biomass (stolons plus rhizoids) about 200 g DW$m
2, which corresponds to a fresh biomass of approximately 1.450 g FW$m
2. 3.3. Elemental C:N:P composition The internal C content decreased during the spring period of high growth (from March to May), recovering afterwards in AG parts (Fig. 7A, Table 2). As for C content, internal N in AG parts was always higher than in BG ones. The N content in AG and BG structures matched the early spring ammonium pulse (Fig. 7C) with a sharp decrease in May (specially in AG); the N content in AG biomass then increased and was kept at relatively high levels. In contrast, internal P increased from early spring to summer and decreased afterwards (Fig. 7E). The low values in autumn and

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Table 1 One way ANOVAs on the effect of time (seasonality) on different demographic and morphometric variables. Variables Total biomass

Sum of squares

df

Mean square

Between groups 5.69 5 1.14 Within groups 0.516 12 0.04 Total 6.21 17 Aboveground Between groups 8.35 5 1.67 biomass Within groups 0.488 12 0.041 Total 8.83 17 Belowground Between groups 66337 5 13267 biomass Within groups 21491 12 1791 Total 87828 17 AG:BG ratio Between groups 4.21 5 0.841 Within groups 0.44 12 0.036 Total 4.64 17 Assimilator Between groups 7.38 5 1.48 density Within groups 0.43 12 0.036 Total 7.81 17 Area of primary Between groups 13.38 5 2.68 assimilators Within groups 55.02 108 0.51 Total 68.40 113 Frond area per Between groups 10.32 5 2.07 metre stolon Within groups 9.56 54 0.18 Total 19.88 59 % Assimilators Between groups 12.33 5 2.47 proliferated Within groups 5.86 52 0.11 Total 18.19 57 99.17 5 19.83 No proliferations Between groups per assimilator Within groups 18.19 51 0.36 Total 117.36 56 No assimilators Between groups 9.90 5 1.98 per metre Within groups 7.99 52 0.15 stolon Total 17.89 57 0.11 5 0.022 No rizhoids per Between groups metre stolon Within groups 0.09 52 0.002 Total 0.20 57

F

p

26.49 <0.0001

41.08 <0.0001

7.41 <0.005

23.22 <0.0001

41.49 <0.0001

5.25 <0.0001

11.66 <0.0001

21.90 <0.0001

55.61 <0.0001

12.90 <0.0001

12.90 <0.0001

winter for internal P suggested that growth could be limited by this element, at least during part of the year. Overall, the C:N atomic ratio both was kept low in AG and BG biomass (between 12 and 13; Fig. 7B). In contrast, the C:P atomic ratio was relatively high, and decreased from early spring to summer in BG parts, suggesting a lack of P limitation during summer (Fig. 7D). There were three main results for N:P atomic ratio (Fig. 7F): firstly, algal N:P ratio varied seasonally; there was a decrease from spring to summer coinciding with the active growth period, recovering afterwards for AG but not for BG parts from late summer onwards. Secondly, N:P ratios were much higher in algal tissues than in seawater, reaching values as high as 60; this might indicate that Caulerpa prolifera is either able to use alternative N forms for nutrition from seawater and/or sediment (i. e. organic N compounds), and/or use preferentially N over P from the sediment nutrient pool to keep this disequilibrium. Finally, data also suggested a marked P limitation of growth in autumn and winter, especially in AG. The data from d13C and d15N showed consistent differences between AG and BG biomass without a clear seasonal trend. The d13C was
13.7  0.2 (SE) for AG and
12.6  0.1 (SE) for BG biomass (p < 0.05); with respect to d15N, it was 5.6  0.3 (SE) for AG and 3.5  0.3 (SE) for BG biomass (p < 0.05). The significant differences in the isotopic composition could also support the idea of a different N nutrition between AG and BG parts of C. prolifera. 3.4. Photosynthesis Photosynthesis was measured in assimilators, but not in stolons. Although some part of the stolon network can be green, with

a certain capacity for photosynthesis, its relative contribution to overall thalli photosynthesis will be very low, as TAI for assimilators (oscillating between 18 and 0.9 m2$m
2 in spring and winter) is ca. 20 times higher than TAI for stolons (between 0.75 and 0.05 m2$m
2 in spring and winter); in addition light reaching stolons will be highly attenuated through assimilator canopy (specially in spring) and further decreased by sediment particles mixed with the stolon network at sediment surface. The P-E curves also showed a seasonal trend. Despite an annual temperature pattern, incubations were performed at the same standard temperature (20 ) to make photosynthetic parameters comparable in terms of photoacclimation responses. Therefore, to extrapolate values such as Pmax and/or Rd to field conditions, temperature modifications should be considered. Maximum photosynthetic efficiency (a) was reached in autumn whereas the minimum was attained in winter (Table 3); similarly, the highest net Pmax was found in autumn (although it was not statistically significant). The Rd and Ek did not show any seasonal trend, whereas Ec was minimal in autumn (about 5.5 mmol photons$m
2 s
1) and maximal in winter (about 14 mmol photons$m
2 s
1). Overall, Ec and Ek were kept low throughout the seasonal cycle, without considering a possible in situ deviation by temperature effects. Chlorophyll a concentration was fairly constant along the year, and therefore, it was not responsible for changes in a and/or Pmax. 4. Discussion 4.1. Seasonal changes in the structure of Caulerpa prolifera meadows Caulerpa prolifera spreads widely in subtidal areas of the inner part of Cadiz Bay (Morris et al., 2009). Despite its importance in shallow ecosystems, prior to the present study there were few studies in south Atlantic Iberian coasts (Malta et al., 2005; Morris et al., 2009; Hendricks et al., 2010) and none focussed on the seasonal dynamics of the populations. Some comparative seasonal data are available from El Mar Menor, a Mediterranean lagoon (Terrados and Ros, 1995); however, this is a microtidal coastal lagoon compared to the mesotidal nature of Cadiz Bay. In this study, the seasonal cycles of physico-chemical variables together with demographic and morphological properties, photosynthesis and elemental composition have been monitored in shallow subtidal areas, where patchy meadows of C. prolifera-Cymodocea nodosa occur. The increase in biomass of C. prolifera, proliferations of primary assimilators, TAI and development of the belowground network started in early spring (March) and peaked in May, with a further decrease in summer towards the rest of the seasonal cycle. Terrados and Ros (1995) found a summer maximum for C. prolifera biomass in a Mediterranean coastal lagoon from southern Spain (El Mar Menor); however, this study was performed at deeper station (4 m) where the negative effects of summer irradiance and temperature on biomass were presumably dampened. The maximum of biomass reported by Terrados and Ros (1995) at a deeper, less illuminated area, was 3e4 times lower than that recorded in our study. In our case, summer seems to affect negatively C. prolifera abundance as at shallow stands, an excess of radiation can negatively affect the population. This species has been considered as a shade algae (Häder et al., 1997) as also shown in our study for Ek (mean annual value of 49  4 mmol photons m
2$s
1) and Ec (mean annual of 9.6  3.4 mmol photons m
2$s
1), despite the fact that populations from the same area showed very high Ek values (from 450 to 750 mmol photons m
2$s
1) based on rapid light curves of fluorescence, although estimates of Ek are not precise by this method (Malta et al., 2005). Low Ec and Ek values for this species have been also reported by other authors (Terrados and

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Nº rhizoids per metre stolon (m )

-1

Nº assimilators per metre stolon (m )

0.04

Nº proliferations per assimilator

% Assimilators proliferated

100

100 80 60 40 20 0

0.06

D

120

E

0.08

Mar

May

Jul Sep Month

Nov

Feb

120 100 80 60 40 20 0

Mar

May

Jul Sep Month

Nov

Feb

Fig. 5. Caulerpa prolifera. Seasonal changes of biometric properties. (A) Area of individual primary assimilators; (B) Frond (assimilator) area per metre stolon; (C) % proliferated assimilators; (D) no proliferations per assimilator; (E) no assimilators per metre stolon; and (F) no rhizoid clusters per metre stolon. Data are represented as box-plots, comprising between the smallest and the largest values excepting outliers in some figures (when the distance is higher than 1.5 the interquartile distance from the upper or lower quartile).

Ros, 1992; Robledo and Freile-Pelegrín, 2005). The winter decrease in seawater temperature seemed to affect negatively C. prolifera success in the bay, as found also by Terrados and Ros (1995) in a mediterranean lagoon. Optimal mild (20e30  C) temperatures for photosynthesis have been reported for this species, with negative effects on Pmax at 35  C; in contrast at low, winter temperatures (10e15  C), C. prolifera has a restricted or absent photosynthetic production (Terrados and Ros, 1992). In addition, seawater temperature at the study site must be cooler than seawater temperature recorded in winter especially at low tide. Actually, punctual observations of low seawater temperature (7e8  C) in winter days were recorded in the study site at low tide (unpubl. obs.). The spring peak in biomass development also resulted in a maximum network extension of about 680 m stolon$m
2 area. To our knowledge this is the first estimation of the complexity of this subterranean network. Assuming that this network is approximately bi-dimensional, it represents a spatial cover of 0.75 m2 stolons m
2 surface area, and a volumetric occupation of

0.65 L m
2 surface area. This complex buried network must play an important role for sediment retention, as suggested by Hendricks et al. (2010), and also for the abundance and diversity of benthic invertebrates in Caulerpa prolifera meadows (Rueda and Salas, 2003; López de la Rosa et al., 2006; Brun et al. unpublished data). In fact, the organic matter content of the sediment was significantly higher within the C. prolifera meadow than in the neighbouring subtidal Cymodocea nodosa meadows (annual mean of 10.5  0.9% DW and 8.2  0.9%DW, for C. prolifera and C. nodosa meadows, respectively, student’s t-test; p < 0.05), as found in the Mediterranean Balearic islands, where C. prolifera adversely affected seagrass (Posidonia oceanica) meadows through changes in sediment biogeochemistry (Holmer et al., 2009). Seasonal changes in biomass were driven mainly by changes in proliferations (% assimilators proliferated and number of proliferations per assimilator) rather than changes in the number of primary assimilators, as also observed by Terrados and Ros (1995). The transient, extremely high TAI spring peak is higher than typical TAI values reported for other photosynthetic

262

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Fig. 6. Caulerpa prolifera. Seasonal changes of (A) thallus area index (TAI) estimated according to two different methods (TAI1 and TAI2, see M&M section) and the dimension of the stolon network, estimated as stolon length per unit area; (B) In white columns, contribution of proliferations to the TAI; as well, contribution (%), of primary and secondary assimilators to TAI.

organisms. The optimum Leaf Area Index (LAI; m2$m
2) has been estimated to be about 4e5 in terrestrial ecosystems, although maximum values of 12e16 have been reported in forests (Margalef, 1974; Larcher, 1995). Middelboe and Binzer (2004) stated that while aquatic photosynthesis is often compared on the basis of thallus pieces or whole plants, community photosynthesis is scarcely investigated. These authors reported optimum TAIs for net photosynthesis in single macroalgal communities of 9.5 (Fucus serratus), 11.5 (Chordaria flagelliformis) and even 22.5 (Ahnfetia plicata), whereas for multispecies assemblages the optimum values can be even higher (Middelboe and Binzer, 2004). Blooms of Ulva spp. can easily reach values about 20, with a negative net production balance in the layers at the bottom of the canopy (Hernández et al., 1997). A maximum TAI about 7 was recorded in a deeper Caulerpa prolifera meadow from a Mediterranean lagoon Terrados and Ros (1995). Differences in morphological and photosynthetic traits have been found among Caribbean Caulerpa species, being habitat characteristics (hydrodynamic regime) and sun/shade adaptation the key factors affecting these traits (Collado-Vides and Robledo, 1999). Sand-Jensen et al. (2007) argued that species with thick

tissues, high LAI and erect orientation should dominate in high light shallow waters (e. g. tidal pools), whereas species with thin tissues, low LAI and prostrated thalli should dominate in deeper waters. Although C. prolifera has thin fronds, these are erected and clumped, forming a dense canopy with a multilayered structure for light interception, where proliferations sprout from primary assimilators. This particular arrangement would let C. prolifera to colonize successfully shallow and highly illuminated environments. When net photosynthesis decreased above optimum TAI for a single species, it is solved in terrestrial plants by shedding leaves, which is not possible in macroalgae forming one entire thallus (Sand-Jensen et al., 2007). However, in Caulerpa prolifera, shedding of proliferations can play a role in decreasing TAI. In fact, the maximum TAI attained in spring was mainly caused by proliferations, decreasing the number of assimilators proliferated and the number of proliferations per assimilator afterwards. In contrast to proliferations, the contribution of primary assimilators to the TAI values was always lower than 5. A shedding response has been observed in this species in response to nitrogen limitation, another stress factor for C. prolifera (Malta et al., 2005). Another approach to compare the TAI values obtained throughout the seasonal cycle is based on the total chlorophyll (a þ b) of the meadow, which has been estimated by two approaches. From a mean annual content of total chlorophyll of 1.9 mg$g
1 FW, the chlorophyll content of the meadow on area basis can be measured by multiplying chlorophyll concentration on DW basis (considering a ratio of 0.14 g DW$g
1 FW) by aboveground biomass (g DW$m
2). It yielded values ranging from 5.4 g Chl$m
2 (in spring) to 0.6 g Chl$m
2 (in winter). In a second approach, the total chlorophyll content of the meadow on area basis has been estimated by expressing chlorophyll content per unit of assimilator surface area (1.9 mg Chl g
1 FW divided by Caulerpa prolifera specific leaf area of 97.4 cm2$g
1 FW, from De los Santos et al., 2009, which yields a concentration of 195 mg Chl$m
2), and multiplying it by the estimated TAIs. It yielded values ranging from 3.6 g Chl$m
2 (in spring) to 0.13 g Chl$m
2 (in winter), which are in the same order of magnitude, and even lower than chlorophyll concentration calculated by the first approach (from aboveground chlorophyll content). The maximum values for chlorophyll concentration found are among or slightly over the top values found in several kinds of forests (from 2 to 3.5 g Chl$m
2; Larcher, 1995). 4.2. Inorganic nutrient dynamics A late winter pulse of ammonium and phosphate coincided with the onset of maximum proliferation of fronds, and preceded the maximum growth. In contrast, tissue C and N decreased in May, an indication of nutrient dilution in a period of a sustained high biomass growth (Stocker, 1980). Moreover, the low tissue P quotas suggested that, at least during autumn and winter, growth may be limited by P. During these months tissue P was lower than critical concentrations suggested for Caulerpa taxifolia (Delgado et al., 1996) and other macroalgae (Hernández et al., 2008). The P limitation is also suggested by the high N:P ratio in the AG biomass, clearly greater than typical values reported for macroalgae (49:1; Duarte, 1992). The isotopic signature from N and C was significantly different in AG and BG biomass, despite the coenocytic nature of this alga. It could indicate that N can be taken up from different sources (i. e. water vs. sediment), and/or can be of different nature (inorganic vs. organic). Recently, Van Engeland (2010) reported that the Caulerpa prolifera meadows from the Cadiz bay are able to use not only inorganic but also organic N sources. The differences between N: P

J.J. Vergara et al. / Estuarine, Coastal and Shelf Science 112 (2012) 255e264

A

B

40 AG BG

30

25

C

20 4

D

AG BG

12

8 400 AG BG

300

C:P atomic ratio

N (%DW)

AG BG

10

3.5

3

2.5

200

100

2

E

16

14

C:N atomic ratio

C (%DW)

35

263

F

0.3 AG BG

0 Mar 80

May

Jul

Sep

Nov

Jan

Mar

AG BG Seawater

N:P atomic ratio

60 P (%DW)

0.2

0.1

40

20

0 Mar

May

Jul

Sep

Nov

Jan

Mar

Month

0 Jan

Mar

May

Jul

Sep

Nov

Jan

Mar

Month

Fig. 7. Caulerpa prolifera. Seasonal changes of aboveground (AG, assimilators plus proliferations), and belowground (BG, stolons and rhizoids) internal C: N: P composition (left, A, C and E), and C: N, C: P, and N: P atomic ratios (right, B, D, F). The N:P ratio of inorganic nutrients in seawater is also represented (F). Data represent mean  SE (n ¼ 3).

ratios of the tissues and seawater can be explained by the fact that C. prolifera could take up nutrients from the sediment, as other authors stated (Chisholm et al., 1996), and/or the use of alternative dissolved organic nitrogen forms from seawater and sediments. This is also possible for P, as dissolved organic phosphorus is a relevant P source for macroalgae (Hernández et al., 2002). Van Engeland et al. (2011) showed, with a variety of 15N labelled substrates, the ability of C. prolifera to take up inorganic and organic N by AG and BG parts. Its high nutrient uptake potential from the water column and their retention in the sediment makes C. prolifera meadows efficient nutrient traps against eutrophication (Lloret et al., 2008). Differences in 13C signal between AB and BG parts may also imply the use of alternative sources of C apart from photosynthetic activity. However, Van Engeland et al. (2011) did not find a significant 13C uptake from organic sources in short-term in situ experiments in Caulerpa prolifera, in contrast to other macrophytes (Harrison et al., 2007; Mozdzer et al., 2010). This can be attributable

either to a selective breakdown of DOM by exo- or cell surface associated enzymes, or alternatively, to the high C background in macrophytes, which may cause a low signal-to-noise ratio (Van Engeland et al., 2011). These indications of different C and N partitioning, based on natural 13C and 15N abundance in field samples open a challenging subject of research. In conclusion, this study shows the important role of shallow subtidal Caulerpa prolifera meadows for primary production and biogeochemical cycles in south Atlantic Iberian shallow coastal lagoons, with the development of a highly complex structure both for the aboveground parts (i.e. high TAI values) and for the belowground ones (i.e. a highly complex subterranean network of stolons and rhizoids), which may be essential for light interception, primary production, nutrient cycles, community diversity, and sediment dynamics in shallow bays. This study constitutes an important data set, as a basis to upscale the contribution of C. prolifera population to the global production of Cadiz bay, and the fate of its high productivity.

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Table 2 Results of the non parametric KruskaleWallis tests for internal nutrient content.

%N

%C

%P

C:N

C:P

N:P

Variables

Chi-squared

df

p

Time-BG Time-AG Tissue Time-BG Time-AG Tissue Time-BG Time-AG Tissue Time-BG Time-AG Tissue Time-BG Time-AG Tissue Time-BG Time-AG Tissue

12.70 15.64 21.63 11.32 13.68 20.76 13.16 13.96 0.63 10.36 10.66 0.29 12.88 13.73 2.12 13.14 13.26 1.68

5 5 1 5 5 1 5 5 1 5 5 1 5 5 1 5 5 1

0.026 0.008 <0.0001 0.046 0.018 <0.0001 0.022 0.016 0.429 ns 0.066 ns 0.059 ns 0.591 ns 0.025 0.018 0.146 ns 0.022 0.021 0.195 ns

Table 3 Caulerpa prolifera. Parameters of the photosynthesis-irradiance (P-E) curves, and total chlorophyll (a þ b) concentration. Values are mean  SD. Different letters indicate significant differences (p < 0.05). Autumn Alpha (mmol O2 m
2 s
1/mmol quanta g
1 DW h
1) Pmax (mmol O2 g
1 DW h
1) Rd (mmol O2 g
1 DW h
1) Ec (mmol quanta m
2 s
1) Ek (mmol quanta m
2 s
1) Chl a þ b (mg g
1 FW)

Winter

Spring 8.8  0.7ab

Summer

12.4  3.8a

4.5  2.3b

574  259

221  112

380  79

319  32


62  31


57  21


87  26


74  26

5.5  2.9a 13.8  5.4b 10.0  3.3ab

6.9  0.7b

8.9  1.1ab

47.4  6.2 58.7  7.0 41.8  8.2 49.7  4.7 1.25  0.42 1.28  0.51 1.47  0.16 1.03  0.43

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