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

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Estuarine, Coastal and Shelf Science

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Seasonal functioning and dynamics of Caulerpa prolifera meadows in shallowareas: 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 HernándezDepartamento de Biología, Area de Ecología, Facultad de Ciencias del Mar y Ambientales, Universidad de Cádiz, Campus de Excelencia Internacional del Mar (CEIMAR), Pol. Rio SanPedro, E-11510 Puerto Real, Cádiz, Spain

a r t i c l e i n f o

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

Keywords:biomassC:N:P compositionCaulerpa proliferaphotosynthesissubterranean networkTAI e thallus area index

* Corresponding author.E-mail address: juanjose.vergara@uca.es (J.J. Verga

1 Present address: ALGAE-Marine Plant EcologyMarine Sciences, Universidade do Algarve, Campus dPortugal.

0272-7714/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.ecss.2012.07.031

a b s t r a c t

The rhizophyte alga Caulerpa prolifera thrives in dense monospecific stands in the vicinity of meadows ofthe seagrass Cymodocea nodosa in Cadiz Bay Natural Park. The seasonal cycle of demographic andbiometric properties, photosynthesis, and elemental composition (C:N:P) of this species were monitoredbimonthly from March 2004 to March 2005. The number of primary assimilators peaked in spring asconsequence of the new recruitment, reaching densities up to 104 assimilators$m�2. A second peak wasrecorded in late summer, with a further decrease towards autumn and winter. Despite this summermaximum, aboveground biomass followed a unimodal pattern, with a spring peak about 400 g dryweight$m�2. In conjunction to demographic properties of the population, a detailed biometric analysisshowed that the percentage of assimilators bearing proliferations and the number of proliferations perassimilator were maximal in spring (100% and c.a. 17, respectively), and decreased towards summer andautumn. The size of the primary assimilators was minimal in spring (May) as a result of the newrecruitments. However, the frond area per metre of stolon peaked in early spring and decreased towardsthe remainder of the year. The thallus area index (TAI) was computed from two different, independentapproaches which both produced similar results, with a maximum TAI recorded in spring (transientvalues up to 18 m2$m�2). The relative contribution of primary assimilators and proliferations to TAI wasalso assessed. Whereas the number of proliferations accounted for most of the TAI peak in spring, itscontribution decreased during the year, to a minimum in winter, where primary assimilators were themain contributors to TAI. The present study represents the first report of the seasonal dynamics ofC. prolifera in south Atlantic Spanish coasts, and indicates the important contribution of this primaryproducer in shallow coastal ecosystems.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

The chlorophyte Caulerpa prolifera (Forsskål) J.V. Lamouroux, theonly autochthonous Caulerpa species in the Mediterranean, isa clonal rhizophyte that inhabits soft bottom sediments intemperate and tropical waters (Lüning, 1990). C. prolifera is a noninvasive species on south Atlantic Spanish coasts, as its presencehas been dated back to XIX century by Clemente (see Seoane-Camba, 1965).

ra).Research Group, Centre ofe Gambelas, 8005-139 Faro,

All rights reserved.

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

J.J. Vergara et al. / Estuarine, Coastal and Shelf Science 112 (2012) 255e264256

deepest soft bottom sediments of the bay (Morris et al., 2009). Fromsatellite images, the percentage of shallow subtidal populationsrepresents ca. 27% of C. prolifera populations in the bay (Morris,pers. comm.).

Previous laboratory studies with Caulerpa prolifera showed thatthis species effectively responded to nutrient availability and light(Malta et al., 2005). In fact, this species can be characterized asnitrophilic, with a high internal N quota and requirements. Itsmorphology responded strongly to N supply, with belowgroundbiomass accumulation (stolons and rhizoids) stimulated under Nlimitation and assimilator formation enhanced under N supply(Malta et al., 2005). With regard to light, although C. prolifera isquite phototolerant (Gacia et al., 1996), it behaves as a sciaphilicalga where growth rates and quantum yields (Fv/Fm) at saturatinglight were lower than those at dim light (Malta et al., 2005), withrisk of photoinhibition in its natural habitat (Häder et al., 1997).

The spatial distribution of Caulerpa prolifera in Cadiz bay, as wellas the d15N isotopic signature (including N and C composition) hasbeen previously studied by Morris et al. (2009) who revealed therole that tissue N can play as a tool in the management of nutrientinputs within shallow coastal zones. In addition, the densemeadows of this rhizophyte may be important in terms of theircontribution to the primary productivity and biogeochemical cyclesin the bay, as these meadows also play a role as sediment trap byaltering the hydrodynamics of the water column (Hendricks et al.,2010).

Against this background, the aim of this study was to study thepopulation structure and dynamics of Caulerpa prolifera in thesouth Atlantic coast of Spain. Along an annual cycle (March 2004,March 2005), the number of assimilators, the aboveground andbelowground biomass, the biometric properties of the population,photosynthesis-irradiance (P-E) curves, and the tissue C:N:Pcomposition were monitored, together with physico-chemicalvariables. The study hypothesises the importance of themaximum spring development of C. prolifera and the densebelowground network in understanding the role of C. proliferameadows 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 Caulerpaproliferawere monitored in Santibañez salt marsh in the inner partof Cadiz bay, southern Spain (36.47� N; 6.25� W) (Fig. 1). Thispopulation develops in shallow areas, at a depth of approximately0.5e1 m below the lowest astronomical tide (LAT), mixed withpatches of the seagrass Cymodocea nodosa. Other C. prolifera standsalso thrive in deeper waters, however with lower biomass density(Morris et al., 2009). Rootedmacrophytes cover a great extent of thebottom of the bay. C. prolifera is the dominant species in subtidalwaters, while three seagrass species (C. nodosa, Zostera marina andZostera noltii) cover the shallow subtidal and intertidal areas. Fordetailed information of the study area, see a previous description inMorris et al. (2009).

2.2. Physico-chemical data acquisition

The solar radiation data set was provided by the AndalusianAgency of Energy from the meteorological stations adjacent to thestudy area. Mean daily air temperature was provided by theSpanish Agency of Meteorology, station of San Fernando, also nearto the sampling area (36.27� N, 6.10� W). Seawater temperaturewasobtained from a buoy (Triaxys type, coastal network Puertos del-Estado) located near the mouth of the Cadiz Bay (36.50� N,

6.33� W). As the register was hourly, the mean daily temperaturewas computed. Water samples for inorganic N and P nutrients weretaken in the field every sampling date and measured in an auto-matic analyzer (model TRAACS 800), following the methodsdescribed in Grasshoff et al. (1983) slightly modified.

2.3. Sampling and processing of biological material

The area was sampled bimonthly from March 2004 to March2005 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 metalframe, collecting all the aboveground (AG) and belowground (BG)biomass within the structure. Most of the sediment attached tosamples was washed in situ, and plants were kept cool and trans-ported to the laboratory within 1 h of collection. Once at thelaboratory samples were further cleaned of remaining sedimentwith seawater.

2.4. Demographic and biometric measurements

The photosynthetic biomass (AG) of Caulerpa prolifera iscomposed 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 primaryproliferations, secondary ones). The BG biomass is composed ofa subterranean network of cylindrical stolons with a number ofrhizoid clusters. The number of primary assimilators was counted,and AG and BG biomass was split and dried separately in an oven at60 �C for 3 days. This dried algal material was ground for elementalC:N:P analysis (see below). The separation of AG and BG biomass isfundamentally operational, as some part of the stolons are locatedin the interface sediment-water and are green and with a certaindegree of photosynthetic ability.

In parallel, fresh algal samples were employed for biometricmeasurements. A variable number of thalli (n ¼ 8e10) bearinga stolon fragment with a variable number of assimilators werecleaned, placed between two acetate sheets, and scanned witha graduate ruler as a reference. These images were processedusing a publicly available image analysis software (Image J, NIH,USA). The following variables were estimated: area of (individual)primary assimilators, frond (assimilators plus proliferations) areaper metre stolon, % of proliferated assimilators, n� proliferationsper assimilator, and n� assimilators or n� rhizoid clusters permetre stolon. Two approaches were applied to estimate theindividual assimilator area: i) by measuring length and width ofthe fronds and applying the ellipse formula, and ii) by applicationof image analysis from scanned images. Both estimates werehighly correlated (r ¼ 0.99; p < 0.001; n ¼ 219), with a slope of0.996 (data not shown). Therefore, both methods were adequateto measure the assimilator area, and accordingly, a simplemeasurement of the major and the minor axes of an ellipse wasadequate to estimate the assimilator area of this species.

A number of derived morphometric variables were also esti-mated from biometric measurements. The length of the stolonnetwork per unit surface of the meadow (Lstolon; m stolon$m�2

meadow) was computed by dividing the density of primaryassimilators found in the field (NA1; n� assimilators$m�2), by the n�

primary assimilators per metre stolon (NAstolon; n� assim-ilators$m�1), estimated in morphometric measurements.

Lstolon ¼ NA1=NAstolon (1)

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

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

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the stolon (1.1 � 0.2 mm). The volume occupied by the stolonnetwork (m3 stolon$m�2 meadow) was computed by multiplyingthe Lstolon by the mean cross section area (0.95 mm2) of the stolon.

The thallus Area index e TAI - (m2$m�2), a concept analogous tothe leaf area index e LAI - for vascular plants (Hernández et al.,1997) was computed from two different, independentapproaches. In a first approach, TAI1 was computed as the productof the total assimilator area per metre stolon (AM; m2 assimilatorarea$m�1 stolon) by the length of the stolon network (Lstolon; mstolon$m�2 meadow).

TAI1 ¼ AM$Lstolon (2)

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

TAI2 ¼ TAIasim þ TAIprolif ¼ ðNA1$A1Þ þ ðNA1$NA2$A2$PÞ(3)

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

abundance of assimilators per unit area (NA1) by the number ofproliferations per primary assimilator (NA2), the mean area of eindividual e secondary assimilators (A2; m2), and the proportion ofprimary assimilators proliferated (P, 0e1).

2.5. Elemental C:N:P composition

The C:N:P composition as well as the isotopic d13C and d15Nabundance were measured on dried ground material. The C and Ncomposition and isotopic signatures weremeasured at SAI (Serviciode Apoyo a la Investigación, University of A Coruña, Spain), aspreviously described (Morris et al., 2009). Total internal P wasestimated as soluble reactive phosphorus (Murphy and Riley, 1962)following an acid digestion (Sommer and Nelson, 1972).

2.6. Photosynthetic measurements and pigment content

Photosynthesis vs. irradiance (P-E) curves were obtainedseasonally in plants collected at the study area. Mature assimilatorsof approximately 2 cm long were selected at different dates withineach season (n¼ 4). The P-E curves were conducted within one dayof sampling. Net photosynthesis and dark respiration (Rd) rateswere measured using a small incubation chamber (20 mL) con-nected to a thermostatic water bath (20 �C) and to a computerized

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J.J. Vergara et al. / Estuarine, Coastal and Shelf Science 112 (2012) 255e264258

polarographic oxygen electrode (Hansatech Instruments, Norfolk,UK). Electrodes were calibrated with air and N2-saturated artificialseawater. Incubations were done in 11 mL of artificial seawater(salinity ¼ 30) prepared from a sea salt mixture (Marinemixprofessional, Wiegandt, Germany) supplemented with NaHCO3(5 mM final concentration) to prevent CO2 limitation. Salinity waschecked using a hand refractometer (Atago). Oxygen tensionwithinthe incubation chamber was maintained between 20 and 80%saturation by bubbling with N2 gas. One assimilator was positionedwithin the chamber perpendicular to the light beam and subjectedto a set of 14 increasing photon flux densities (PFDs, from 0 to700 mmol quanta m�2 s�1), plus a final incubation in darkness tomeasure respiration in light-acclimatized thalli (Rd). Light wasprovided by 36 red light emitting diodes (PAR, peak output above635 nm, model LS3/LH36U, Hansatech Ltd, Norfolk, UK). At eachPFD, oxygen concentration was measured during 7 min (except indarkness, 15 min), and the rates were computed within the last5 min, once the slope was stabilized. Net maximum photosyntheticrates (Pmax) were obtained from the average maximum valuesabove saturating irradiance. The photosynthetic efficiency (a) wasestimated from the initial slope of the P-E curves by linear least-squares regression analysis. Light saturation point (Ek) wascomputed as the ratio between maximum photosynthetic rate andphotosynthetic efficiency (Pmax/a), and the light compensationpoint (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 cutand kept at �80 �C for pigment determination. Pigment contentwas measured spectrophotometrically following pigment extrac-tion of a homogenized tissue in 80% acetone at low temperature(Dennison,1990). Chlorophyll a and b and total carotenoids in frondextracts were calculated using the equations of Lichtenthaler andWellburn (1983).

2.7. Statistics

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

3. Results

3.1. Physico-chemical variables

Solar radiation followed the typical seasonal trend for temperateclimates, with maximal values in summer and minimal in winter(Fig. 2A), as previously shown in neighbouring areas (Hernándezet al., 1997). Temperature also displayed a seasonal trend, withmaxima in summer and minima in winter (Fig. 2B), being moredamped in water than in air (mean seawater temperature of19 � 3 �C, with a range from 13.7 �C to 24.4 �C; mean air temper-ature of 17� 5.3 �C, with a range from 3.2 �C to 31.9 �C). However, ithas to be taken into account that seawater temperature was

measured at 21 m depth, whereas C. prolifera populations thrive inshallower areas (around 1 m depth at low tide), where there isa high influence of air on seawater temperature. Therefore,temperature at the study site must be intermediate between thetwo data sets along the cycle: hotter than that registered inseawater in summer, and cooler than that in winter.

The seawater inorganic nutrients ammonium and phosphateshowed higher concentrations than nitrate and nitrite along theseasonal cycle (Fig. 3), peaking in spring (about 15 mM for ammo-nium and 2.3 mM for phosphate; Fig. 3A), and with a second, smallpulse for ammonium in summer. In contrast, nitrate and nitriteconcentrations were kept low, with a nitrate maximum of 1 mM inspring and autumn and almost undetectable levels in summer.Nitrite was always <0.3 mM (Fig. 3B).

3.2. Demographic and biometric properties

Total biomass and density of primary assimilators followedsimilar trends along the year, with main spring maxima (ca. 600 gDW$m�2 and 104 assimilators$m�2, respectively) and secondarysummer peaks (Fig. 4A, Table 1). Concerning biomass partitioning,maximum AG values were reached in spring, decreasing towardsthe rest of the year, while BG biomass showed a bimodal response,with maximal values in May and late summer andminimumvaluesin winter (Fig. 4A). The summer maximum in assimilator density(Fig. 4B) was not matched by amaximum in AG biomass. The AG:BGratio decreasedmarkedly throughout the year, ranging between 2.8(spring) and below 1 (winter) (Fig. 4B).

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Fig. 4. Caulerpa prolifera. Seasonal changes of (A) total biomass (AG þ BG) andassimilator density; (B) aboveground (AG) biomass (primary assimilators plus prolif-erations), belowground (BG) biomass (stolons and rhizoids) and AG:BG ratio. Datarepresent mean � SE (n ¼ 3).

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

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The area of individual primary assimilators of Caulerpa proliferawas minimal in spring (May) matching the maximum AG biomass(Fig. 5A); therefore, in spring there was a high recruitment of newassimilators. Most of them were lost in summer, although theremaining ones reached larger sizes. According to the maximumrecruitment of new primary assimilators in spring, the frond areaper metre stolon decreased from March to May, and it was keptrelatively constant throughout the rest of the year (Fig. 5B). Both,the degree of proliferation (% assimilators proliferated) and thenumber of proliferations per assimilator peaked in spring (c. a.100%and a median about 17, respectively) and decreased along theseasonal cycle, with a partial recovering in late winter (Fig. 5C, D).The n� of assimilators was always lower than the n� rhizoid clustersper m stolon (Fig. 5E, F). Despite a maximum assimilator density inspring (May), the linear assimilator density (per m stolon) wasminimal, which indicates a high development of the belowgroundstolon network at this period of the year. In fact, the estimatedlength of the stolon network was maximum in spring (May),reaching values up to 680m$m�2 and decreasing strongly along theseasonal cycle (Fig. 6A).

From the biometric and demographic measurements, severalsecondary variables were estimated. The TAI values, computed bytwo independent approaches, gave similar results except in earlyspring (Fig. 6A). Irrespective of the approach, it had extremely high(c. a. 18 m2$m�2) and transient values reached in spring (May),

followed by a decrease throughout the rest of year. Thus, it seemsthat the high degree of proliferations may oversaturate the biomassand, at short-term, achieve these high TAI values. The contributionof primary assimilators and proliferations to TAI is shown in Fig. 6B.Whereas the assimilators showed a maximum TAI close to 4, theproliferations accounted for most of the transient TAI increase inspring.

Furthermore, from the mean diameter and the mean crosssection area of the stolons, it was estimated that the maximum areacovered 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, thevolume 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$m�2, which corresponds toa fresh biomass of approximately 1.450 g FW$m�2.

3.3. Elemental C:N:P composition

The internal C content decreased during the spring period ofhigh growth (from March to May), recovering afterwards in AGparts (Fig. 7A, Table 2). As for C content, internal N in AG parts wasalways higher than in BG ones. The N content in AG and BGstructuresmatched the early spring ammonium pulse (Fig. 7C) witha sharp decrease in May (specially in AG); the N content in AGbiomass then increased and was kept at relatively high levels. Incontrast, internal P increased from early spring to summer anddecreased afterwards (Fig. 7E). The low values in autumn and

Table 1One way ANOVAs on the effect of time (seasonality) on different demographic andmorphometric variables.

Variables Sum ofsquares

df Meansquare

F p

Total biomass Between groups 5.69 5 1.14 26.49 <0.0001Within groups 0.516 12 0.04Total 6.21 17

Abovegroundbiomass

Between groups 8.35 5 1.67 41.08 <0.0001Within groups 0.488 12 0.041Total 8.83 17

Belowgroundbiomass

Between groups 66337 5 13267 7.41 <0.005Within groups 21491 12 1791Total 87828 17

AG:BG ratio Between groups 4.21 5 0.841 23.22 <0.0001Within groups 0.44 12 0.036Total 4.64 17

Assimilatordensity

Between groups 7.38 5 1.48 41.49 <0.0001Within groups 0.43 12 0.036Total 7.81 17

Area of primaryassimilators

Between groups 13.38 5 2.68 5.25 <0.0001Within groups 55.02 108 0.51Total 68.40 113

Frond area permetre stolon

Between groups 10.32 5 2.07 11.66 <0.0001Within groups 9.56 54 0.18Total 19.88 59

% Assimilatorsproliferated

Between groups 12.33 5 2.47 21.90 <0.0001Within groups 5.86 52 0.11Total 18.19 57

No proliferationsper assimilator

Between groups 99.17 5 19.83 55.61 <0.0001Within groups 18.19 51 0.36Total 117.36 56

No assimilatorsper metrestolon

Between groups 9.90 5 1.98 12.90 <0.0001Within groups 7.99 52 0.15Total 17.89 57

No rizhoids permetre stolon

Between groups 0.11 5 0.022 12.90 <0.0001Within groups 0.09 52 0.002Total 0.20 57

J.J. Vergara et al. / Estuarine, Coastal and Shelf Science 112 (2012) 255e264260

winter for internal P suggested that growth could be limited by thiselement, at least during part of the year.

Overall, the C:N atomic ratio both was kept low in AG and BGbiomass (between 12 and 13; Fig. 7B). In contrast, the C:P atomicratio was relatively high, and decreased from early spring tosummer in BG parts, suggesting a lack of P limitation duringsummer (Fig. 7D). There were three main results for N:P atomicratio (Fig. 7F): firstly, algal N:P ratio varied seasonally; there wasa decrease from spring to summer coinciding with the activegrowth period, recovering afterwards for AG but not for BG partsfrom late summer onwards. Secondly, N:P ratios were much higherin algal tissues than in seawater, reaching values as high as 60; thismight indicate that Caulerpa prolifera is either able to use alterna-tive N forms for nutrition from seawater and/or sediment (i. e.organic N compounds), and/or use preferentially N over P from thesediment nutrient pool to keep this disequilibrium. Finally, dataalso suggested a marked P limitation of growth in autumn andwinter, especially in AG.

The data from d13C and d15N showed consistent differencesbetween AG and BG biomass without a clear seasonal trend. Thed13C was �13.7 � 0.2 (SE) for AG and �12.6 � 0.1 (SE) for BGbiomass (p< 0.05); with respect to d15N, it was 5.6� 0.3 (SE) for AGand 3.5 � 0.3 (SE) for BG biomass (p < 0.05). The significantdifferences in the isotopic composition could also support the ideaof 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 tooverall thalli photosynthesis will be very low, as TAI for assimilators(oscillating between 18 and 0.9 m2$m�2 in spring and winter) is ca.20 times higher than TAI for stolons (between 0.75 and0.05 m2$m�2 in spring and winter); in addition light reachingstolons will be highly attenuated through assimilator canopy(specially in spring) and further decreased by sediment particlesmixed with the stolon network at sediment surface. The P-E curvesalso showed a seasonal trend. Despite an annual temperaturepattern, incubations were performed at the same standardtemperature (20�) to make photosynthetic parameters comparablein terms of photoacclimation responses. Therefore, to extrapolatevalues such as Pmax and/or Rd to field conditions, temperaturemodifications should be considered. Maximum photosyntheticefficiency (a) was reached in autumn whereas the minimum wasattained in winter (Table 3); similarly, the highest net Pmax wasfound in autumn (although it was not statistically significant). TheRd and Ek did not show any seasonal trend, whereas Ec wasminimal in autumn (about 5.5 mmol photons$m�2 s�1) andmaximal in winter (about 14 mmol photons$m�2 s�1). Overall, Ecand Ek were kept low throughout the seasonal cycle, withoutconsidering a possible in situ deviation by temperature effects.Chlorophyll a concentration was fairly constant along the year, andtherefore, it was not responsible for changes in a and/or Pmax.

4. Discussion

4.1. Seasonal changes in the structure of Caulerpa proliferameadows

Caulerpa prolifera spreads widely in subtidal areas of the innerpart of Cadiz Bay (Morris et al., 2009). Despite its importance inshallow ecosystems, prior to the present study there were fewstudies in south Atlantic Iberian coasts (Malta et al., 2005; Morriset al., 2009; Hendricks et al., 2010) and none focussed on theseasonal dynamics of the populations. Some comparative seasonaldata are available from El Mar Menor, a Mediterranean lagoon(Terrados and Ros, 1995); however, this is a microtidal coastallagoon compared to themesotidal nature of Cadiz Bay. In this study,the seasonal cycles of physico-chemical variables together withdemographic and morphological properties, photosynthesis andelemental composition have been monitored in shallow subtidalareas, where patchy meadows of C. prolifera-Cymodocea nodosaoccur. The increase in biomass of C. prolifera, proliferations ofprimary assimilators, TAI and development of the belowgroundnetwork started in early spring (March) and peaked in May, witha further decrease in summer towards the rest of the seasonal cycle.Terrados and Ros (1995) found a summer maximum for C. proliferabiomass in a Mediterranean coastal lagoon from southern Spain (ElMar Menor); however, this study was performed at deeper station(4 m) where the negative effects of summer irradiance andtemperature on biomass were presumably dampened. Themaximum of biomass reported by Terrados and Ros (1995) ata deeper, less illuminated area, was 3e4 times lower than thatrecorded in our study. In our case, summer seems to affect nega-tively C. prolifera abundance as at shallow stands, an excess ofradiation can negatively affect the population. This species has beenconsidered as a shade algae (Häder et al., 1997) as also shown in ourstudy 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), despitethe fact that populations from the same area showed very high Ekvalues (from 450 to 750 mmol photons m�2$s�1) based on rapidlight curves of fluorescence, although estimates of Ek are notprecise by this method (Malta et al., 2005). Low Ec and Ek values forthis species have been also reported by other authors (Terrados and

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Fig. 5. Caulerpa prolifera. Seasonal changes of biometric properties. (A) Area of individual primary assimilators; (B) Frond (assimilator) area per metre stolon; (C) % proliferatedassimilators; (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, comprisingbetween 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).

J.J. Vergara et al. / Estuarine, Coastal and Shelf Science 112 (2012) 255e264 261

Ros, 1992; Robledo and Freile-Pelegrín, 2005). The winter decreasein seawater temperature seemed to affect negatively C. proliferasuccess in the bay, as found also by Terrados and Ros (1995) ina mediterranean lagoon. Optimal mild (20e30 �C) temperatures forphotosynthesis have been reported for this species, with negativeeffects on Pmax at 35 �C; in contrast at low, winter temperatures(10e15 �C), C. prolifera has a restricted or absent photosyntheticproduction (Terrados and Ros, 1992). In addition, seawatertemperature at the study site must be cooler than seawatertemperature recorded in winter especially at low tide. Actually,punctual observations of low seawater temperature (7e8 �C) inwinter days were recorded in the study site at low tide (unpubl.obs.).

The spring peak in biomass development also resulted ina maximum network extension of about 680 m stolon$m�2 area. Toour knowledge this is the first estimation of the complexity of thissubterranean network. Assuming that this network is approxi-mately bi-dimensional, it represents a spatial cover of0.75 m2 stolons m�2 surface area, and a volumetric occupation of

0.65 L m�2 surface area. This complex buried network must play animportant role for sediment retention, as suggested by Hendrickset al. (2010), and also for the abundance and diversity of benthicinvertebrates 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 significantlyhigher within the C. prolifera meadow than in the neighbouringsubtidal 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 Mediter-ranean Balearic islands, where C. prolifera adversely affected sea-grass (Posidonia oceanica) meadows through changes in sedimentbiogeochemistry (Holmer et al., 2009).

Seasonal changes in biomass were driven mainly by changes inproliferations (% assimilators proliferated and number of prolif-erations per assimilator) rather than changes in the number ofprimary assimilators, as also observed by Terrados and Ros(1995). The transient, extremely high TAI spring peak is higherthan typical TAI values reported for other photosynthetic

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

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organisms. The optimum Leaf Area Index (LAI; m2$m�2) has beenestimated to be about 4e5 in terrestrial ecosystems, althoughmaximum values of 12e16 have been reported in forests(Margalef, 1974; Larcher, 1995). Middelboe and Binzer (2004)stated that while aquatic photosynthesis is often compared onthe basis of thallus pieces or whole plants, community photo-synthesis is scarcely investigated. These authors reportedoptimum TAIs for net photosynthesis in single macroalgalcommunities of 9.5 (Fucus serratus), 11.5 (Chordaria flagelliformis)and even 22.5 (Ahnfetia plicata), whereas for multispeciesassemblages the optimum values can be even higher (Middelboeand Binzer, 2004). Blooms of Ulva spp. can easily reach valuesabout 20, with a negative net production balance in the layers atthe bottom of the canopy (Hernández et al., 1997). A maximumTAI about 7 was recorded in a deeper Caulerpa prolifera meadowfrom a Mediterranean lagoon Terrados and Ros (1995). Differ-ences in morphological and photosynthetic traits have beenfound among Caribbean Caulerpa species, being habitat charac-teristics (hydrodynamic regime) and sun/shade adaptation thekey 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 highlight shallow waters (e. g. tidal pools), whereas species with thintissues, low LAI and prostrated thalli should dominate in deeperwaters. Although C. prolifera has thin fronds, these are erectedand clumped, forming a dense canopy with a multilayeredstructure for light interception, where proliferations sprout fromprimary assimilators. This particular arrangement would letC. prolifera to colonize successfully shallow and highly illumi-nated environments.

When net photosynthesis decreased above optimum TAI fora 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, sheddingof proliferations can play a role in decreasing TAI. In fact, themaximum TAI attained in spring was mainly caused by prolifera-tions, decreasing the number of assimilators proliferated and thenumber of proliferations per assimilator afterwards. In contrast toproliferations, the contribution of primary assimilators to the TAIvalues was always lower than 5. A shedding response has beenobserved in this species in response to nitrogen limitation, anotherstress factor for C. prolifera (Malta et al., 2005).

Another approach to compare the TAI values obtainedthroughout the seasonal cycle is based on the total chlorophyll(a þ b) of the meadow, which has been estimated by twoapproaches. From a mean annual content of total chlorophyll of1.9 mg$g�1 FW, the chlorophyll content of the meadow on areabasis can be measured by multiplying chlorophyll concentration onDW basis (considering a ratio of 0.14 g DW$g�1 FW) by above-ground biomass (g DW$m�2). It yielded values ranging from 5.4 gChl$m�2 (in spring) to 0.6 g Chl$m�2 (in winter). In a secondapproach, the total chlorophyll content of the meadow on areabasis has been estimated by expressing chlorophyll content per unitof assimilator surface area (1.9 mg Chl g�1 FW divided by Caulerpaprolifera specific leaf area of 97.4 cm2$g�1 FW, from De los Santoset al., 2009, which yields a concentration of 195 mg Chl$m�2),and multiplying it by the estimated TAIs. It yielded values rangingfrom 3.6 g Chl$m�2 (in spring) to 0.13 g Chl$m�2 (in winter), whichare in the same order of magnitude, and even lower than chloro-phyll concentration calculated by the first approach (from above-ground chlorophyll content). The maximum values for chlorophyllconcentration found are among or slightly over the top valuesfound in several kinds of forests (from 2 to 3.5 g Chl$m�2; Larcher,1995).

4.2. Inorganic nutrient dynamics

A latewinter pulse of ammonium and phosphate coincidedwiththe onset of maximum proliferation of fronds, and preceded themaximum growth. In contrast, tissue C and N decreased in May, anindication of nutrient dilution in a period of a sustained highbiomass growth (Stocker, 1980). Moreover, the low tissue P quotassuggested that, at least during autumn and winter, growth may belimited by P. During these months tissue P was lower than criticalconcentrations suggested for Caulerpa taxifolia (Delgado et al.,1996) and other macroalgae (Hernández et al., 2008). The P limi-tation 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 fromN and Cwas significantly different inAG and BG biomass, despite the coenocytic nature of this alga. Itcould 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 Caulerpaprolifera meadows from the Cadiz bay are able to use not onlyinorganic but also organic N sources. The differences between N: P

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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, Cand 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).

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ratios of the tissues and seawater can be explained by the fact thatC. prolifera could take up nutrients from the sediment, as otherauthors stated (Chisholm et al., 1996), and/or the use of alternativedissolved organic nitrogen forms from seawater and sediments.This is also possible for P, as dissolved organic phosphorus isa relevant P source for macroalgae (Hernández et al., 2002). VanEngeland et al. (2011) showed, with a variety of 15N labelledsubstrates, the ability of C. prolifera to take up inorganic and organicN by AG and BG parts. Its high nutrient uptake potential from thewater column and their retention in the sedimentmakes C. proliferameadows efficient nutrient traps against eutrophication (Lloretet al., 2008).

Differences in 13C signal between AB and BG parts may alsoimply the use of alternative sources of C apart from photosyntheticactivity. However, Van Engeland et al. (2011) did not find a signifi-cant 13C uptake from organic sources in short-term in situ experi-ments 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 surfaceassociated enzymes, or alternatively, to the high C background inmacrophytes, which may cause a low signal-to-noise ratio (VanEngeland et al., 2011). These indications of different C and N par-titioning, based on natural 13C and 15N abundance in field samplesopen a challenging subject of research.

In conclusion, this study shows the important role of shallowsubtidal Caulerpa prolifera meadows for primary production andbiogeochemical cycles in south Atlantic Iberian shallow coastallagoons, with the development of a highly complex structure bothfor the aboveground parts (i.e. high TAI values) and for thebelowground ones (i.e. a highly complex subterranean network ofstolons and rhizoids), which may be essential for light interception,primary production, nutrient cycles, community diversity, andsediment dynamics in shallow bays. This study constitutes animportant data set, as a basis to upscale the contribution of C. pro-lifera population to the global production of Cadiz bay, and the fateof its high productivity.

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

Variables Chi-squared df p

% N Time-BG 12.70 5 0.026Time-AG 15.64 5 0.008Tissue 21.63 1 <0.0001

% C Time-BG 11.32 5 0.046Time-AG 13.68 5 0.018Tissue 20.76 1 <0.0001

% P Time-BG 13.16 5 0.022Time-AG 13.96 5 0.016Tissue 0.63 1 0.429 ns

C:N Time-BG 10.36 5 0.066 nsTime-AG 10.66 5 0.059 nsTissue 0.29 1 0.591 ns

C:P Time-BG 12.88 5 0.025Time-AG 13.73 5 0.018Tissue 2.12 1 0.146 ns

N:P Time-BG 13.14 5 0.022Time-AG 13.26 5 0.021Tissue 1.68 1 0.195 ns

Table 3Caulerpa prolifera. Parameters of the photosynthesis-irradiance (P-E) curves, andtotal chlorophyll (a þ b) concentration. Values are mean � SD. Different lettersindicate significant differences (p < 0.05).

Autumn Winter Spring Summer

Alpha (mmol O2 m�2

s�1/mmol quanta g�1

DW h�1)

12.4 � 3.8a 4.5 � 2.3b 8.8 � 0.7ab 6.9 � 0.7b

Pmax (mmol O2 g�1

DW h�1)574 � 259 221 � 112 380 � 79 319 � 32

Rd (mmol O2 g�1

DW h�1)�62 � 31 �57 � 21 �87 � 26 �74 � 26

Ec (mmol quanta m�2

s�1)5.5 � 2.9a 13.8 � 5.4b 10.0 � 3.3ab 8.9 � 1.1ab

Ek (mmol quanta m�2 s�1) 47.4 � 6.2 58.7 � 7.0 41.8 � 8.2 49.7 � 4.7Chl a þ b (mg g�1 FW) 1.25 � 0.42 1.28 � 0.51 1.47 � 0.16 1.03 � 0.43

J.J. Vergara et al. / Estuarine, Coastal and Shelf Science 112 (2012) 255e264264

Acknowledgements

This research has been supported by the projects EVAMARIA(CTM2005-00395/MAR) and IMACHYDRO (CTM2008-00012/MAR)of the Spanish Ministry of Science and Innovation (MiCiNN).

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