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Journal of Applied Phycology ISSN 0921-8971Volume 24Number 4 J Appl Phycol (2012) 24:941-951DOI 10.1007/s10811-011-9715-y

Spatial and temporal variations ofChondrus crispus (Gigartinaceae,Rhodophyta) carrageenan content innatural populations from Galicia (NWSpain)Manuel García Tasende, Miguel Cid &M. Isabel Fraga

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Spatial and temporal variations of Chondrus crispus(Gigartinaceae, Rhodophyta) carrageenan content in naturalpopulations from Galicia (NW Spain)

Manuel García Tasende & Miguel Cid & M. Isabel Fraga

Received: 5 April 2011 /Revised and accepted: 12 August 2011 /Published online: 16 September 2011# Springer Science+Business Media B.V. 2011

Abstract Qualitative and quantitative differences in carra-geenan composition of gametophytes and tetrasporophytesof Chondrus crispus were observed in this study. Carra-geenans in gametophytes belong to the kappa family (κ-, ι-,ν- and μ-carrageenan). The dominant fractions were κ- andι-carrageenan (more than 50% of the total carrageenans). Intetrasporophytes, the presence of λ-carrageenan was con-firmed. Carrageenan content in gametophytes (37.4±1.68%DW) was higher than in tetrasporophytes (29.13±0.76%DW). Spatial and temporal variation in carrageenan contentin both life cycle phases appears to be related mainly toseawater and air temperatures, insolation, water movementand desiccation. The highest values of carrageenan contentwere recorded in those localities where higher values ofprecipitation, wind speed or water movement occurred. Abimodal temporal pattern on carrageenan content wasobserved. Fronds showed a high carrageenan content inspring and autumn. During these seasons, the content wasover 40% in gametophytes and 30% in the tetrasporo-phytes. In summer and winter, these values down in both

life cycle phases below 30%. In general the highestcarrageenan contents were related to highest seawatertemperatures. On the contrary, high air temperature andhigh insolation appeared to be unfavourable for carrageenanproduction. GLM models were obtained to predict carra-geenan production from natural C. crispus populations,along Galician coast.

Keywords Chondrus crispus . Gametophyte .

Tetrasporophyte . Carrageenan . Oceanographic variables .

Environmental variables

Introduction

Carrageenans are a complex family of water-solublegalactans produced by several red marine algae. They arepolysaccharides composed of alternating three-linked β-D-galactopyranose (G-units) and 4-linked α-galactopyranose(D-units) or four-linked 3,6-anhydro-galactopynose (DAunits) that form an ideal disaccharide repeating unit (Bixlerand Porse 2011; Campo et al. 2009). Different types ofcarrageenans are produced by chemical modification of thedisaccharide repeating unit with ester sulphates and with 3,6-anhydrogalactose.

Carrageenans are of great industrial interest and eco-nomically important because of their ability to increase theviscosity of solutions and to form thermoreversible gels(Bixler and Porse 2011; Campo et al. 2009; Yermak et al.1999). These compounds are used extensively as stabilisersin dairy products, because of their ability to react withproteins in milk; they are also commonly used as gelatinis-ing, stiffening and thickening agents in a variety of food,pharmaceutical, cosmetic and other products (Mathiesonand Tveter 1975; McHugh 2003).

M. García Tasende (*)Servizo de Planificación, Dirección Xeral de Ordenación eXestión dos Recursos Mariños, Consellería do Mar,Xunta de Galicia,CP 15703 Santiago de Compostela, Spaine-mail: manuel.garcia.tasende@xunta.es

M. CidInstituto de Ciencias Agrarias,Consejo Superior de Investigaciones Científicas,c/Serrano 115,CP 28006 Madrid, Spain

M. I. FragaDepartamento de Botánica, Facultade de Bioloxía,Universidade de Santiago,CP.15782 Santiago de Compostela, Spain

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The carrageenans of greatest commercial interest are theκ-, ι- and λ types. The κ- and ι-carrageenans are gelforming, whereas λ-carrageenan is a thickener/viscositybuilder. Chondrus crispus Stackhouse synthesises all threeof these types of carrageenans (Bixler and Porse 2011;Campo et al. 2009; McHugh 2003).

Black et al. (1965) observed important variations in therelative proportion of gelling and non-gelling carrageenansin members of the Gigartinaceae, although the analyseswere performed on mixtures of unspecified life cyclestages, which led to erroneous conclusions. Later, otherresearchers demonstrated that gametophytes and tetraspor-ophytes produce different types of carrageenans (Chen et al.1973; McCandless et al. 1983; Pickemere et al. 1973;Waaland 1975). Chondrus crispus gametophytes mainlyproduce κ- and ι-carrageenans, whereas the tetrasporo-phytes synthesise λ-carrageenans (McCandless and Craigie1974).

Spatial and temporal variations in carrageenan contenthave been recorded for different red seaweeds (Lutwick 1973;Durako and Dawes 1980; Reis et al. 2008), including C.crispus (McCandless and Craigie 1974; Fuller and Mathie-son 1972; Mathieson and Tveter 1975; Chopin and Floc'h1987; Chopin et al. 1987). Such variations are generallycaused by changes in the life cycle stages and environmentalconditions (Chopin 1985; Yermak et al. 2006).

Natural populations of C. crispus on the coast of Galicia(NW Spain) are traditionally harvested for industrialpurposes (Tasende and Rodríguez González 2003). Carra-geenan obtained from natural beds are limited and unable tosupply all industrial requirements, and the raw material iscurrently supplied by importing algae (≈2.500 t year−1) andby harvesting natural beds of C. crispus and Mastocarpusstellatus (Stackhouse) Guiry (≈250 t year−1).

We carried out a comparative study of the qualitative andquantitative carrageenan frond content in natural C. crispusbeds located in three sites representative of the threebiogeographic regions recognised in Galicia (OSPARCommission 2000). The study was conducted betweenDecember 2004 and November 2005. The main goal was todetermine which environmental factors have a majorinfluence on carrageenan content in C. crispus, in order toimprove the sustainable exploitation for the carrageenantrade.

Materials and methods

Three sites with similar C. crispus beds were selected alongthe Galician coast (NW Iberian Peninsula): Mougás (SantaMaría de Oia, 42°03′, 8°53′ W), Laxe (43°13′ N, 8°59′ W)and San Román (O Vicedo, 43°43′ N, 7°37′ W). Accordingto the OSPAR Commission (2000) these sites belong to the

Lusitanian province and are representative of the subtrop-ical subprovince (Mougás), the Atlantic-influenced sub-tropical/boreal transition subprovince (Laxe) and theeastern subtropical/boreal transition subprovince, whichhas the oceanographic characteristics of the Bay of Biscay(San Román, Fig. 1).

Laxe and San Román are exposed localities, withirregular shoreline and abrupt slopes of varying height.Both differ in the bedrock substract (granite in Laxe andshale in San Román) and the coast orientation (NW and N,respectively). On the other hand, Mougás is semi-exposedand facing to the west. The coastline is rocky (granite) andshallow with many intertidal pools.

Samples of gametophytes and tetrasporophytes of C.crispus were collected monthly at each of three samplingsites (see below) between December 2004 and November2005. Mature healthy fronds with cystocarps or tetraspor-angia were randomly collected, by hand, in the intertidalzone at low tide. The samples were washed with distilledwater to eliminate salt, debris and epiphytes. Gametophytesand tetrasporophytes were dried separately to constantweight at 60°C for approximately 16 h.

All samples used for carrageenan analysis were maturefronds with cystocarps or tetrasporangia, so that theobserved variation cannot be related to the developmentstage or age of the fronds.

Environmental parameters

Relevant geomorphological features, such as morphology,geology, orientation, exposure and continental influencewere recorded at the three sampling sites. The influence of

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Fig. 1 Location of sampling sites in the Lusitanian province andlimits of the subprovinces and of the surrounding biogeographicregions (OSPAR Commission 2000). 1 Mougás (Santa María de Oia,42°03′ N, 8°53′ W), 2 Laxe (43°13′ N, 8°59′ W) and 3 San Román (OVicedo, 43°43′ N, 7°37′ W)

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the following variables in the carrageenan content wasanalysed:

Weather variables: mean air temperature (AT, °C),maximum air temperature (AT max, °C), minimum airtemperature (AT min, °C), sunshine hours (SH, hours/month), insolation (I, monthly percentage of the dailysunshine hours in relation to the maximum potentialsunshine hours), precipitation (P, mm), wind speed(Ws, m s−1) and height wave (Hw, m).Oceanographic variables: seawater temperature (WT, °C),salinity (S, ‰) and water nutrient content (μmolmL−1): ammonium (NH4

+), orthophosphate (PO43−),

nitrate (NO3−) and total nitrogen (Nt). Total nitrogen

was calculated as the sum of nitrate and ammoniumnitrogen.

Weather data were obtained from 2004–2005 meteoro-logical reports from Meteogalicia, Xunta de Galicia (www.meteogalicia.es), and oceanographic data were provided byINTECMAR (www.intecmar.org, Xunta de Galicia).

Carrageenan extraction and purification

Samples of ten dried fronds (approximately 4 g) wererehydrated for a few minutes in distilled water. Then,rehydrated fronds were heated in distilled water (400 mL)at 85°C for 30 min before being ground with a homogeniserHeidolph DIAX 900 (24,000 rpm). The homogenisedmaterial was kept at 85°C for 4 h in order to solubilisethe polysaccharides. It was ground every 2 h within thisperiod. The hot solution obtained was vacuum filtered, firstthrough a thick canvas, in order to eliminate cellulosedebris, and then through glass microfibre filters (Filter-LabMFV 3, 110 mm). The filtrate was concentrated to a finalvolume of 200 mL with a rotary evaporator.

The polysaccharides were precipitated by adding twovolumes of cold ethanol, then pressure-filtered throughtulle, dried at 50°C overnight and ground to a powder.

The carrageenan extracts were subjected to threedifferent purification methods: (a) dialysis only; (b)hydrolysis with α-amylase and dialysis and (c) hydrolysiswith α-amylase, alkali treatment and dialysis.

Dialysis The extracts were dissolved in 20 mL of hotdistilled water and dialysed three times against water for36 h (Cole Parmer dialysis membrane 133-2900-29).

Floridean starch removal To hydrolyse the florideanstarch, 250 mg of carrageenan extract were dissolved in45 mL distilled water at 80°C and 5 mL 10 mM phosphatebuffer (pH 7) containing 150 units of α-amylase (Sigma α-Amylase from hog pancreas) were added. The sample was

incubated for 3 h at 37°C. The remaining polysaccharideswere precipitated by adding two volumes of cold ethanol.

Alkali treatment Proteins were denaturised with alkalihydrolysis in the presence of sodium borohydride tominimise degradation of the polysaccharide chain. Amixture of crude carrageenan extract (1.25 g), distilledwater (50 mL), sodium hydroxide (1.25 g) and sodiumborohydride (0.75 g) was heated at 80°C for 3 h, cooledand neutralised with acetic acid. The polysaccharides wereprecipitated by adding two volumes of cold ethanol. Thetreatment with hot aqueous alkali also converted μ- and ν-carrageenans into κ- and ι-carrageenan, respectively. Thepurified carrageenan extracts were lyophilised and storeduntil analysis.

1H NMR spectrometry of carrageenans

Samples were prepared from purified extracts by dissolvingthe lyophilised carrageenans in D2O (30 mg mL−1) at 80°C.1H NMR spectra were obtained at 65°C in a BrukerDRX500 spectrometer (500 MHz). Chemical shifts (δ)are relative to internal deuterated acetone (δ=2.225 ppm).Assignment of the chemical shift in the 1H NMR spectra ofthe native carrageenan types was based on reference spectraof commercial carrageenans (Sigma ι-carrageenan, Type V;κ-carrageenan Type III and λ-carrageenan Type IV), and onpreviously reported data (Usov 1984; Stortz et al. 1994;Van de Velde et al. 2002, 2004; Tojo and Prado 2003a, b).The relative abundance of carrageenans was calculatedfrom peak integral values of the 1H NMR spectra. Thepercentage of the different carrageenans was calculatedfrom the semipurified extracts. The alkali treatment wassuppressed to prevent transformation of the μ- and ν-carrageenans into ι- and κ-carrageenans, respectively.

FTIR spectroscopic analysis

The purified carrageenans (6–7 mg) were dissolved in20 mL of distilled water by heating at 80°C. Films wereprepared by drying the solution on a polyethylene disc at60°C and were analysed by FTIR spectroscopy (FTIRMattson Cygnus-100, resolution 0.125 cm−1).

Statistical analysis

The statistical analysis was carried out in two steps:

Temporal and spatial differences in the carrageenancontent of fronds. Spatial and temporal differences in thecarrageenan content of gametophytes and tetrasporophytes

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were analysed by one-way repeated-measures analyses ofvariance (ANOVAR). As sphericity was confirmed with theMauchly’s sphericity test (p>0.05), we used unadjusted pvalues for the univariate test.

Median values of κ-, ι-, μ- and ν-carrageenan content ingametophytes corresponding to the different sampling siteswere compared by one-way Kruskal–Wallis analyses ofvariance.

Analysis of relationships among environmental factors andcarrageenan content. In order to analyse the possiblecauses of the spatial variation in carrageenan yields,discriminant factorial analysis (DFA) was carried out toidentify those variables that were most effective indifferentiating sampling sites. Spearman correlation testswere performed (p<0.05) to assess relationships amongenvironmental variables and gametophyte and tetrasporo-phyte carrageenan yields.

Generalised linear models (GLM) were selected to explore thefactors affecting the variation in carrageenan production.GLM are an extension of linear models that enable incorpo-ration of non-normal distributions of the response variable andtransformation of the dependent variables to linearity (McCul-lagh and Nelder 1983). In the GLM it is assumed that theexpected value of a transformed response variable is relatedto a linear combination of explanatory variables.

The general form of GLM is:

g mð Þ ¼ btx

whereby a function link g(·) is used to achieve linearity in theparameters βt of the dependent variable x. A variancefunction (8) that relates the variance of the response variableto the mean is specified in a second part of the model:

varðY Þ ¼ 8V mð Þ; withm constant:According to Faraway (2004), the model of explanatory

variable selection was based on the lowest value of AkaikeInformation Criterion (AIC) found in the stepwise search,the model checking residual plots, the proportion ofdeviance explained (a generalisation of r2) and the p value.The goodness-of-fit of the models was assessed bycomparing their relative contribution to total devianceexplained. The model was fitted in R environment (RDevelopment Core Team, 2006) by use of a Gaussiandistribution with an identity link function.

Statistical analyses were conducted with SPSS 15 (SPSSInc, 2006) and R (Version 2.10.1, 2009). Data were testedfor normality (Shapiro–Wilk’s test) and variance homoge-neity (Levene’s test). The data were log transformed tomeet the required conditions of normality and variancehomogeneity for the variables.

Results

Qualitative carrageenan analysis

Carrageenan analysis by 1H NMR spectrometry Purifica-tion of the extracts removed some interfering substancesand enabled identification of several of the peaks. Compar-ative analysis among nonpurified extracts and α-amylase-treated extracts was used to identify the characteristic peakof floridean starch at 5.35 ppm. In the same way comparingthe alkali-treated and non-treated extracts, the peaks for ν-(5.52 ppm), μ- (5.25 ppm), κ- (5.11 ppm) and ι-carrageenans (5.32 ppm) were determined.

Carrageenan extracts from tetrasporophytes were highlyviscous even at high temperature and the 1H NMR spectrashowed no characteristic peaks. The purification processcarried out, analogous to purification of the gametophyteextract, improved the spectra to a certain extent, but did notenable identification of characteristic peaks.

Carrageenan analysis by FTIR The FTIR spectrum of thecarrageenans from gametophytes showed a strong absorp-tion band in the 930 cm−1 region due to the presence of 3,6-anhydro-D-galactose of κ- and ι-carrageenan. Moreover, thespectra displayed a band of high absorbance in the845 cm−1 region, associated with the D-galactose-4-sul-phate, typical of κ-carrageenan. A band of lower absor-bance was also observed in the 805 cm−1 region, associatedwith the presence of 3,6-anhydrogalactose-2-sulphate,which indicates the presence of lower quantities of ι-carrageenan. The ratio between 805 and 845 cm−1 absorp-tion bands was used to determine the degree of κ- and ι-hybridization. The characteristic absorption bands of ν- andμ-carrageenan were not detected because of insufficientresolution of the equipment.

The FTIR spectra of tetrasporophyte carrageenan dis-played broad absorption bands in 820–830 cm−1 regionassociated with 2- and 6-sulphate galactose, which arecharacteristic of λ-carrageenan.

Gametophyte carrageenan composition Carrageenans ingametophytes are a mixture of κ-, ι-, μ- and ν-carrageenans. The κ-carrageenans accounted for the64.1% (±1.83) of total carrageenan. There were nosignificant differences among the three sampling sites(Kruskal–Wallis analyses of variance, H=3.12, df=2, p=0.211), although a range of variation between 54% and74% was observed. Non-significant differences in the ι-carrageenan contents were also observed (Kruskal–Wallistest, H=1.66, df=2, p=0.436) which accounts for 23.5%(±0.88) of the total carrageenan. κ- and ι-carrageenans arethe most interesting compounds for the Galician carrageen-an industry. Both together represented the 87.6% (±1.19) of

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total carrageenan with a range of variation between 81.4%and 93.6%. μ- and ν-carrageenans are precursors of κ- andι-carrageenans, respectively. They are minor components inthe total carrageenan content in gametophytes. μ-Carrageenan represented the 8.12% (±0.63) and ν-carrageenan the 4.23% (±0.68).

Quantitative carrageenan analysis

Carrageenan content of gametophytes and tetrasporophytescollected at three sampling sites are shown in Fig. 2. Weobserved significant differences related to C. crispus lifecycle phases and time of year in which samples werecollected. Carrageenan content in gametophytes (37.4±1.68% DW) was significantly higher (Kruskal–Wallis test,H=25.19, p=0.000) than in tetrasporophytes (29.13±0.76% DW).

In gametophytes, significant differences among siteswere observed (ANOVAR, F=23.35, p=0.000). In SanRomán, fronds had significantly higher values (43.23±1.87% DW) than in Laxe (HSD Tukey test, p=0.005) andMougás (HSD Tukey test, p=0.001). Between these twosites significant differences were not observed, 35.96±1.94% DW and 32.77±1.27% DW, respectively. In tetra-sporophytes no significant differences among sites wereobserved (ANOVAR, F=1.05, p=0.406).

There is a similar pattern in the monthly meancarrageenan content in gametophytes and tetrasporophytes,with slight differences among sites. During the studyperiod, three peaks were observed in both phases of thelife cycle and for the three sites (Fig. 3). The maximumvalues in gametophytes were observed in December 2004–January 2005 (38.79±3.01% DW), May–June (42.75±3.08% DW) and November (45.08±4.95% DW). Intetrasporophytes, maximum values were also found inJanuary (30.17±2.67% DW), May–June (32.13±1.71%DW) and November (36.67±2.87% DW).

The December–January and November peaks corre-spond to the same period of maximum production thattakes place between late autumn and winter. Therefore, itseems that this is a bimodal pattern of production with twoperiods of maximum production, one in the late spring andearly summer and another during the autumn and earlywinter. Both periods were followed by periods of lowproduction. These variations appear to be linked totemporal variations in environmental conditions. The meanvalues of weather and oceanographic variables for theperiod December 2004–November 2005 are shown inFigs. 4 and 5.

According to the DFA results, there are significantdifferences (Lambda of Wilks test, p=0.000) among thethree sites along all study period, on the basis of theenvironmental and oceanographic variables analysed(Fig. 6). Wind speed, water nutrient content (NO3

−, Nt),salinity and height waves are positively correlated withdiscriminant function 1, which explains 70.7% of thevariance. Seawater temperature and maximum air temper-ature were also correlated with this first function, butnegatively. The second function, which explains 29.3% ofthe variance, is positively correlated with water nutrientcontent (PO4

3−), air temperature (AT, AT min), insolationand sunshine hours and negatively correlated with waternutrient content (NH4

+) and precipitation. Figures 4 and 5show the high temporal and spatial variation observed for

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environmental and oceanographic variables. In addition,most of these variables were correlated with each other,according to Spearman’s correlation coefficient analysisand any particular variable showed significant correlationwith carrageenan production, except for seawater tempera-ture that was significantly correlated with tetrasporophyte

carrageenan content (r=0.360, p=0.05). These results aredifficult to interpret and did not allow us to knowaccurately which interactions and ranges of these variableshad more influence on the carrageenan production.

In San Román and Laxe, the content of carrageenanincreased during winter (December–January) and during

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spring (May–June). This increase coincided with the periodwhen the air and water temperatures and the number ofsunshine raised. In summer, the carrageenan content infronds began to decline. During this period, water temper-ature and sunshine hours were constant, while maximum airtemperature exceeded 20°C. This trend continued untilNovember when the maximum air temperature was lowerthan 20°C. In this month, carrageenan content of game-tophytes and tetrasporophytes began to increase in bothsites. During the autumn, when a maximum of carrageenanproduction was observed in Laxe and San Román, thesunshine hours and water and air temperatures were lowest,but the rise of the wave power and the wind speed wasremarkable. This pattern was observed as well in game-tophytes as tetrasporophytes. In Mougás, however, therewas only an increase in carrageenan content in summer.

Nutrient levels were highly variable in time and space(Fig. 5). However, there was no clear relationship betweencarrageenan content and concentration of N and P inseawater. If we compare the results obtained in Novemberfor Laxe and San Román, we can see that for both sites thegametophyte carrageenan content was the same and quitehigh (51% DW), while the concentrations of P and N werevery different (Laxe: nitrate 39.4 μM L−1, ammonium2.84 μM L−1 and orthophosphate 0.81 μM L−1; SanRomán: nitrate 8.51 μM L−1, ammonium 1.58 μM L−1

and orthophosphate 0.43 μM L−1).GLM models that provided the best fits for both

gametophyte and tetrasporophyte carrageenan contents areshown in Table 1. The proportion of the deviance explainedby both models was 57.8% for gametophytes and 66.9% fortetrasporophytes, and the resulting residual plots enabled us

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to test if the residuals were evenly scattered, normallydistributed and if any particular data had substantial effecton the model results. Examination of the model residuals,which contain all the information in the data not explainedby the systematic part of the model, showed that theassumptions of the models were reasonable. Moreover, the

p values obtained by the χ2 test were also consistent forboth models. In both cases (gametophytes and tetraspor-ophytes), the carrageenan contents were linked to the airand seawater temperatures, sunshine hours and insolation(Fig. 7). However, in tetrasporophytes the effect of airtemperature was not significant, but it was included in themodel because it allows a lower AIC value than if onlyseawater temperature, sunshine hours and insolation areconsidered.

Discussion

Qualitative and quantitative differences in carrageenancomposition of gametophytes and tetrasporophytes of C.crispus were observed in this study. Gametophytes alwaysproduced more carrageenan than tetrasporophytes. Ingametophytes, carrageenans belong to the kappa family(κ-, ι-, ν- and μ-carrageenan), as previously reported byvarious authors (McCandless and Craigie 1974; Pereira andMesquita 2003). The dominant fractions were κ- and ι-carrageenan (more than 85% of the total carrageenans).Carrageenan fractions present in tetrasporophytes could notbe determined by 1HNMR, although the results of FTIRanalysis confirmed the presence of λ-carrageenan asobserved by different authors (McCandless et al. 1983;Correa-Diaz et al. 1990; Pereira and Mesquita 2003; Tojoand Prado 2003a).

Spatial and temporal variations on qualitative andquantitative carrageenan content in both phases may be

Fig. 6 Plot of discriminant scores for function 1 against function 2, todiscriminate the three sampling sites during the 12 months of study,according environmental and oceanographic variables (see text forexplanations)

Table 1 GLM models to predict gametophyte and tetrasporophyte carrageenan content

Estimate SE t value p IC95% (2.5%) IC95% (97.5%)

Gametophyte

(Intercept) 37.656 8.649 4.354 0.0001*** 20.705 54.607

WT 2.990 0.894 3.345 0.0021** 0.116 0.372

AT −2.142 0.792 −2.704 0.0110* −3.695 −0.589I −1.256 0.294 −4.268 0.0001*** 1.238 4.741

SH 0.244 0.065 3.729 0.0007*** −1.833 −0.679Tetrasporophyte

(Intercept) 22.280 6.042 3.688 0.000*** 10.439 34.122

WT 1.704 0.624 2.730 0.010* 0.481 2.928

AT −0.887 0.553 −1.603 0.119 −1.972 0.198

I −0.496 0.206 −2.411 0.022* −0.899 −0.093SH 0.094 0.046 2.052 0.048* 0.004 0.183

Gametophyte: null deviance—1,857.2, df 35; residual deviance—1,074, df 31; pseudo r2—57.82%; pseudo r2 ajusted—65.29%; AIC—236.42.Tetrasporophyte: null deviance—782.81, df 35; residual deviance—524.27, df 31; pseudo r2—66.97%; pseudo r2 adjusted—75.61%; AIC—210.59

AT air temperature, I insolation, SH hours of sunshine/month, WT seawater temperature

* p<0.05, ** p<0.01, *** p<0.001

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related to the biological roles of carrageenans: mechanical,hydration and electrochemical regulations (Carrington et al.2001). We have observed that any particular environmentalor oceanographic variable, as isolated variable, is directlylinked to carrageenan production in C. crispus naturalpopulations along Galician coast, but are complex inter-actions among different values of these variables that are

constantly changing, both in time and space, making verydifficult to determine what the conditions are that havemain influence on carrageenan production, for bothgametophytes and tetrasporophytes. At local scale, changesin oceanographic and weather conditions cause ups anddowns on carrageenan production. In the same way, thereare spatial differences on carrageenan yields because thehabitats from different localities, in which the naturalpopulations develop, are never identical.

Chopin et al. (1991) suggested that inorganic phos-phate may be an important regulator of the carbon flow inC. crispus toward either glucose and starch or galactoseand carrageenans. Fogg (1964), Dawes et al. (1974) andChopin (1986) showed that during the active growth ofseaweeds, the synthesis of proteins and other protoplasmicconstituents predominates, with little accumulation of cellwall material or reserve product; if nutrient suppliesbecome limited, the photosynthetic cycle may be divertedfrom synthesis of protein to synthesis of carrageenans(Chopin and Wagey 1999). In the present study, maximalcarrageenan contents were obtained at low and highconcentrations of N and P in seawater, so we did notobserve the “Neish effect” recorded by the above citedauthors in C. crispus cultures.

According Durako and Dawes (1980) and Reis et al.(2008), fronds exposed to stronger wave action produce morecarrageenan in order to increase their flexibility and ability toresist the waves, Thornber (2006) suggested that life cyclephases of intertidal species may differ in their ability towithstand wave forces. The variations in the resistance ofboth phases may be attributable to the qualitative composi-tion of carrageenans affecting the ability of the fronds toresist water movements. In accordance with this, in thisstudy, the highest carrageenan contents in gametophytes wereobtained in sites exposed to the open sea and windy, mainlyin autumn (San Román and Laxe). Although Mougás issemi-exposed to the open sea and windy site, lower values ofwind speed and wave height than Laxe and San Román wererecorded during autumn of 2005. In addition, in Mougásfronds were collected in tide pools that are more stablehabitats and more protected to wave action.

Temporal variations in carrageenan content can allowfronds to adapt to variations on the environmental andoceanographic factors. A bimodal temporal pattern wasobserved. Highest production values occurred in late springand autumn, and lower values in summer and late winter.The temporal carrageenan production trends along Galiciancoast seem to be mainly related to temperature (seawaterand air), sunshine hours and insolation. In general, thehighest carrageenan contents were related to highestseawater temperatures. On the contrary, highest air temper-ature and high insolation appeared to be unfavourable forcarrageenan production. There was an increase of the air

Gam

etop

hyte

car

rage

enan

con

tent

(%

DW

)

Hours of sunshine (h) Water temperature ( )

Hours of sunshine (h) Water temperature ( )

Irradiance (MJ/m)Air temperature ( C)

a Gametophyte

b Tetrasporophyte

Irradiance (MJ/m)Air temperature ( C)

Tet

rasp

orop

hyte

car

rage

enan

con

tent

(%

DW

)

Fig. 7 Estimated influence of air temperature, water temperature,insolation and hours of sunshine on a gametophyte and b tetraspor-ophyte carrageenan production, as estimated from the final GLMmodel (determined by stepwise selection and AIC)

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and water temperature and the daylight hours in spring andsummer. During these seasons the highest tides alsooccurred (Rasilla et al. 2004), which increase the periodof emersion of the fronds of intertidal beds. Maximumvalues of carrageenan content were reached just before themaximum values of air temperature and sunshine hoursoccurred. The hygroscopic capacity of the carrageenansallowed the plants to survive during periods of strongdesiccation (Percival and McDowell 1967).

In autumn and early winter, the air and watertemperature and the number of sunshine hours decreased,however the carrageenan content was similar to themaximum values observed in spring, or was even higherin tetrasporophytes, In this case it is evident that otherfactors had remarkable effects on carrageenan production,it could be factors included in the residuals of GLMmodels, like the storms that caused high water movementsand high rainfall that reduced the water salinity (RasillaÁlvarez et al. 2002).

According to the GLM models obtained in this study topredict carragenan production from natural C. crispuspopulations, along the Galician coast, it seems possible tomake predictions, with an acceptable level of reliability, onthe basis of a small number of environmental variables,namely temperature of seawater and air, insolation andmonthly sunshine hours. Information on these variables isfree and available in different public institutions. So, theresults of this study could be a useful tool for futureplanning of commercial extractions of C. crispus naturalresources, in order to maintain the sustainability of the bedsand to supply the industry demand.

Acknowledgements This study was funded by the Carrasea Project(www.carrasea.org) of the European programme Aquareg (Interreg III-C). We thank to María Soledad Saborido for her assistance in field andlaboratory work and to Christine Francis for help in revising theEnglish. We also thank to M. Doval and Y. Pazos (Intecmar, Xunta deGalicia) for providing us the oceanographic data.

References

Bixler HJ, Porse H (2011) A decade of change in the seaweedhydrocolloids industry. J App Phycol 23:321–335

Black WAP, Blakemore WR, Colquhoun JA, Dewar ET (1965) Theevaluation of some marine red algae as a source of carrageenanand of its κ- and λ-component. J Sci Food Agric 16:573–585

Campo VL, Kawano DF, da Silva DB, Carvalho I (2009) Carra-geenans: biological properties, chemical modifications andstructural analysis—a review. Carbohydr Polym 77:167–180

Carrington E, Grace SP, Chopin T (2001) Life history phase and thebiomechanical properties of the red alga Chondrus crispus(Rhodophyta). J Phycol 37:699–704

Chen LCM, McLachlan J, Neish AC, Shacklock PF (1973) The ratioof kappa-to-lambda-carrageenan in nuclear phases of the rhodo-

phycean algae Chondrus crispus and Gigartina stellata. J MarBiol Assoc UK 53:11–16

Chopin T (1985) Variations saisonnières de la nutrition phosphoréedes carraghénanes et de croissance chez deux formes de l’Alguerouge Chondrus crispus Stackhouse. Thèse de doctorat de 3ème

cycle, Université de Brest, FranceChopin T (1986) The red alga Chondrus crispus Stackhouse (Irish

moss) and carrageenans. A review. Can Tech Rep Fish Aquat Sci1514:69

Chopin T, Floc'h HJY (1987) Seasonal variations of growth in the redalga Chondrus crispus on the Atlantic French coast. I. A newapproach by fluorescence labelling. Can J Bot 65:1014–1018

Chopin T, Wagey T (1999) Factorial study of the effects ofphosphorus and nitrogen enrichments on nutrient and carrageen-an content in Chondrus crispus (Rhodophyceae) and on residualnutrient concentration in seawater. Bot Mar 42:23–31

Chopin T, Bodeau-Bellion C, Floc’h JY, Guittet E, Lallemand JY(1987) Seasonal study of carrageenan structures from femalegametophytes of Chondrus crispus Stackhouse (Rhodophyta).Hydrobiologia 151/152:535–539

Chopin T, Hanisak MD, Koehn FE (1991) Effects of seawaterphosphorus concentration on floridean starch content inAgardhiella subulata (C. Agardh) Kraft et Wynne (Rhodophy-ceae). Bot Mar 34:369–373

Correa-Diaz F, Aguilar-Rosas R, Aguilar-Rosas L (1990) Infraredanalysis of eleven carragenophytes from Baja California, Mexico.Hydrobiologia 204/205:609–614

Dawes CJ, Lawrence JM, Cheney DP, Mathieson AC (1974) Ecologicalstudies of floridian Eucheuma (Rhodophyta, Gigartinales). III.Seasonal variation of carrageenan, total carbohydrate, protein, andlipid. Bull Mar Sci 2:286–299

Durako MJ, Dawes CJ (1980) A comparative seasonal study of twopopulations of H. musciformis from the east and west coasts ofFlorida, USA. I. Growth and chemistry. Mar Biol 59:151–156

Faraway JJ (2004) Linear models with R. Chapman and Hall/CRC,Boca Raton, p 225

Fogg GE (1964) Environmental conditions and the pattern ofmetabolism in algae. In: Jackson D (ed) Algae and man. Plenum,New York, pp 77–85

Fuller SW, Mathieson AC (1972) Ecological studies of economicred algae. IV. Variations of carrageenan concentrationproperties in Chondrus crispus Stackhouse. J Exp Mar BiolEcol 10:49–58

Lutwick GD (1973) Seasonal variation of chemical composition ofChondrus crispus. Rep Project 3036 Nova Scotia ResearchFoundation

Mathieson AC, Tveter E (1975) Carrageenan ecology of Chondruscrispus stackhouse. Aquat Bot 1:25–43

McCandless EL, Craigie JS (1974) Reevaluation of seasonal factorsinvolved in carrageenan production by Chondrus crispus:carrageenans of carposporic plants. Bot Mar 17:125–129

McCandless EL, West JA, Guiry MD (1983) Carrageenan patterns inthe Gigartinaceae. Biochem Syst Ecol 11:175–182

McCullagh P, Nelder JA (1983) Generalized linear models. Chapman& Hall, London, p 511

McHugh DJ (2003) A guide to the seaweed industry. FAO FisheriesTechnical Paper 441:61–72

OSPAR Commission (2000) Quality Status Report 2000: Region IV—Bay of Biscay and Iberian Coast. OSPAR Commission, London.p 134+xiii

Percival E, McDowell RH (1967) Chemistry and enzymology ofmarine algal polysaccharides. London Academic, London,p 219

Pereira L, Mesquita JF (2003) Carrageenophytes of occidentalPortuguese coast: 1-spectroscopic analysis in eight carrageeno-phytes from Buarcos Bay. Biomol Eng 20:217–222

950 J Appl Phycol (2012) 24:941–951

Author's personal copy

Pickemere SE, Parsons MJ, Bailey RW (1973) Composition ofGigartina carrageenan in relation to sporophytes and gameto-phytes stages of the life cycle. Phytochemistry 12:2441–2444

Rasilla Álvarez D, García Codrón JC, Garmendia Pedraja C (2002)Los temporales de viento: propuesta metodológica para el análisis deun fenómeno infravalorado. In Cuadrat JM, Vicente SM and SazMA (eds) La información climática como herramienta de gestiónambiental, Zaragoza University Press, pp. 122–136

Rasilla D, García Codron JC, Hernández Gimena A (2004) Lasmareas atmosféricas en la costa norte de la Península Ibérica. InGarcía Codron JC, Diego Liaño C, Fernández de ArróyabeHernáez P, Garmendia Pedraja C, Rasilla Álvarez, D (eds) ElClima entre el Mar y la Montaña, Asociación Española deClimatología y Universidad de Cantabria, Serie A, No. 4,Santander, pp. 135–144

Reis RP, Yoneshigue-Valentin Y, Pereira dos Santos C (2008) Spatialand temporal variation of Hypnea musciformis carrageenan(Rhodophyta-Gigartinales) from natural beds in Rio de JaneiroState, Brazil. J Appl Phycol 20:1–8

Stortz CA, Bacon BE, Cherniak R, Cerezo AS (1994) High-fieldNMR, spectroscopy of cystocarpic and tetrasporic carrageenansfrom Iridaea undulosa. Carbohydr Res 261:317–326

Tasende MG, Rodríguez González LM (2003) Economic seaweeds ofGalicia (NW Spain). Thalassas 19:17–25

Thornber CS (2006) Functional properties of the isomorphic biphasicalgal life cycle. Integr Comp Biol 46:605–614

Tojo E, Prado J (2003a) A simple 1H NMR method for thequantification of carrageenans in blends. Carbohydr Polym53:325–329

Tojo E, Prado J (2003b) Chemical composition of carrageenan blendsdetermined by IR spectroscopy combined with a PLS multivar-iate calibration method. Carbohydr Res 338:1309–1312

Usov AI (1984) NMR Spectroscopy of red seaweed polysaccharides:agars, carrageenans and xylans. Bot Mar 27:189–202

Van de Velde C, Knutsen SH, Usov AI, Rollema HS, Cerezo AS(2002) 1H and 13C high resolution NMR spectroscopy ofcarrageenans: application in research and industry. Trends FoodSci Technol 13:73–92

Van de Velde C, Pereira L, Rollema HS (2004) The revised NMRchemical shift data of carrageenans. Carbohydr Res 339:2309–2313

Waaland JR (1975) Differences in carrageenan in gametophytes andtetrasporophytes of red algae. Phytochemistry 14:1359–1362

Yermak IM, Kim YH, Titlyanov EA, Isakov VV, Solov’eva T (1999)Chemical structure and gel properties of carrageenans from algaebeloning to the Gigartinaceae and Tichocarpaceae collected fromthe Russian Pacific coast. J Appl Phycol 11:41–48

Yermak IM, Barabanova A, Glazunov V, Isakov V, Hwan K, Soon S,Titlynova T, Solov’eva T (2006) Carrageenans from cystocarpicand sterile plants of Chondrus pinnulatus (Gigartinaceae,Rhodophyta) collected from the Russian Pacific coast. J ApplPhycol 18:361–368

J Appl Phycol (2012) 24:941–951 951

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