Journal of Applied Phycology (2019) 31:665–682 https://doi.org/10.1007/s10811-018-1532-0

Geographic variation in biochemical and physiological traits of the red seaweeds chamissoi and Gelidium lingulatum from the south east Pacific coast

Karina Véliz1,2,3 & Nancy Chandía 2,3 & Ulf Karsten4 & Carlos Lara 5 & Martin Thiel3,6,7

Received: 16 November 2017 /Revised and accepted: 28 May 2018 /Published online: June 20184 # Springer Science+Business Media B.V., part of Springer Nature 2018

Abstract Diverse phenotypic characteristics have evolved in seaweeds to cope with environmental stress, but these traits can vary among populations of the same species especially if these are distributed along environmental gradients. In this study, natural populations of the carrageenophyte Chondracanthus chamissoi and the agarophyte Gelidium lingulatum from a latitudinal gradient along the Chilean coast (between 20° S and 41° S) were compared. We determined physiological and biochemical traits in field and culture samples. Sulfated polysaccharide contents ranged from 15.4 to 52.7% dry weight (DW) in C. chamissoi and from 10.9 to 25.1% DW in G. lingulatum. Carrageenan amounts were higher in gametophytes than tetrasporophytes and were also, depending on life cycle phase, negatively correlated with the geographic variation of temperature, photosynthetically active radiation (PAR), and chlorophyll a (Chl a), whereas agar showed no significant correlation with these variables. The UV-absorbing mycosporine-like amino acids (MAAs) shinorine and palythine in both species ranged from 0.8 to 6.8 mg g−1 DW and these contents were positively correlated to PAR and Chl a levels at the sampling site. In C. chamissoi variation among populations in their photosynthetic characteristics, pigment concentrations, antioxidant capacity, and MAA contents were persistent after acclimation under common-garden conditions, suggesting ecotypic differentiation in this species. Contrary, G. lingulatum seems to have a more generalist strategy because differences after cultivation were observed only in some photosynthetic parameters and phycobiliprotein concentration. This study confirms that intraspecific differences in phenotypic traits along the same geographic area are strongly dependent on species and life cycle phases.

Keywords Rhodophyta . Carrageenans . Agar . Mycosporine-like amino acids . Ecotypes . Chile

Introduction * Karina Véliz [email protected] The biosynthesis of chemical compounds in benthic sea- weeds, as in other sessile organisms, is an important adaptive 1 Doctorado en Biología y Ecología Aplicada, Universidad Católica strategy to deal with environmental variability. Among those, del Norte, Coquimbo, Chile sulfated cell wall polysaccharides (carrageenans and agar) and 2 Laboratorio de Moléculas Bioactivas, Universidad Católica del UV-absorbing mycosporine-like amino acids (MAAs) are two Norte, Coquimbo, Chile different classes of important compounds biosynthesized by 3 Departamento de Biología Marina, Facultad de Ciencias del Mar, red seaweeds, which are involved in primary and secondary Universidad Católica del Norte, Larrondo 1281, Coquimbo, Chile metabolic functions, respectively (Carreto and Carignan 2011; 4 Institute of Biological Sciences, Applied Ecology and Phycology, Ficko-Blean et al. 2015; Lee et al. 2017a). University of Rostock, Albert-Einstein-Strasse 3, 18057 Rostock, Germany Carrageenans and agar mainly perform a structural func- tion as cell wall components. Both polysaccharides are com- 5 Centro de Investigación en Recursos Naturales y Sustentabilidad (CIRENYS), Universidad Bernardo O’Higgins, Av. Viel 1497, posed of a linear backbone of galactose residues linked by Santiago, Chile alternating β-1,3 and α-1,4 glycosidic bonds, being the 4- α 6 Centro de Estudios Avanzados en Zonas Áridas (CEAZA), Av. linked -galactose residues of the D-series in carrageenans Ossandon 877, Coquimbo, Chile and of the L-series in agar. However, this regular structure 7 Millennium Nucleus Ecology and Sustainable Management of becomes complex by the substitution of hydroxyl groups with Oceanic Island (ESMOI), Coquimbo, Chile sulfate, and by the presence of the α-1,4-(3,6)- 666 J Appl Phycol (2019) 31:665–682 anhydrogalactose (3,6-AG) moiety in the galactan chain of carbohydrates and nitrogenous compounds is differently (Lahaye 2001; van de Velde 2008). The chemical variation regulated by nutrient availability (Macler 1986; Bird 1988; in carrageenans and agar determines differences in their gel- Collén et al. 2004), an inverse trend between the contents of like physicochemical properties. Consequently, their addition- sulfated polysaccharides and MAAs could be expected ac- al functions, which include the provision of flexibility against cording to nutrient concentration in the natural habitats of hydrodynamic stress (Carrington et al. 2001), resistance to seaweeds. algal endophyte attack (Bouarab et al. 1999), and cellular ion- The quantity and quality of seaweed compounds can also ic regulation by selective cationic exchange (Mariani et al. vary among populations of the same species depending on 1990), can also be modified. geographical variation of selective factors. Genetic or ecotypic The MAAs are a family of intracellular nitrogenous metab- differentiation, as result of divergent selection, is more prob- olites that function as photoprotective sunscreens (Karsten able in species distributed along environmental gradients and 2008; Bischof and Steinhoff 2012). Different types of with a low genetic connectivity (Sanford and Kelly 2011). MAAs vary in the nitrogen substituent bound to their Although some studies have examined the variation in the cyclohexenone or cyclohexenimine chromophore, which in- production of carrageenans and agar regarding the site of sea- fluences the maximum of UVabsorption in the range of 310 to weed origin (e.g., Hurtado et al. 2011; Pereira and van de 360 nm, as well as their antioxidant capacity (Carreto and Velde 2011; Tasende et al. 2012, 2013), no consistent geo- Carignan 2011). Currently, in marine organisms, there are graphic patterns have been identified to date, maybe due to about 23 MAAs identified, being shinorine, palythine, the restricted spatial scale used which does not include the asterina-330, mycosporine-glycine, palythinol, and entire species geographic range. In the case of MAAs, their porphyra-334 the most commonly reported MAAs from red intraspecific variation has been mainly analyzed considering seaweeds (Karsten et al. 1998a, b). coastal bathymetric gradients, reporting an inverse relation- The biosynthesis of carrageenans, agar, and MAAs is af- ship between MAA contents and depth of seaweed collection fected by several factors acting at different levels. In the first (Karsten and Wiencke 1999; Hoyer et al. 2001, 2002; place, the type of cell wall polysaccharide is determined by Huovinen et al. 2004). phylogenetic relationships among species, as well as by the The coast of Chile (18° S to 56° S) is located along the life cycle phase in the specific case of carrageenans (Craigie South Eastern (SE) Pacific temperate coast. It is characterized 1990;Chopinetal.1999). MAA composition also appears to by a latitudinal gradients of sea surface temperature (SST) be genetically defined according to studies carried out in sea- (Ramos-Rodriguez et al. 2012) and solar radiation (Vernet et weeds (Hoyer et al. 2002; Karsten 2008). Based on the total al. 2009), but also by mesoscale variation (10’sto100’sof MAA amounts and the induction patterns after exposure to km) in oceanographic and atmospheric nearshore conditions different radiation conditions, red can be classified in related to coastal upwelling (Hernández et al. 2012; Tapia et three groups: type I—unable to biosynthesize MAAs, type al. 2014). There is little information about the adaptive phys- II—MAAs inducible in variable concentrations, and type iological and biochemical mechanisms of seaweed species III—permanently high MAA contents. While type I typically inhabiting latitudinal gradients along this coast. This contrasts represents deep-water seaweeds, types II and III species grow with the high commercial and social importance of seaweeds from the upper and mid sublittoral zone up to the supra- and in Chile, which is the main producer of red seaweeds in South eulittoral zone (Hoyer et al. 2002). America for the phycocolloid industry (Hayashi et al. 2014). The content and composition of carrageenans and agar are Among the most important commercial species, also determined by the interaction of several environmental Chondracanthus chamissoi isasourceofcarrageenans factors such as light, temperature, nutrient supply, salinity, and (Wang et al. 2012; Véliz et al. 2017), whereas Gelidium water movement. These factors can influence the availability lingulatum produces agar (Matsuhiro and Urzúa 1991). and allocation of necessary resources for carbohydrate biosyn- Since both species have a wide geographic range along the thesis due to their effects on photosynthesis (Macler 1986, SE Pacific coast where their populations are exposed to con- 1988;Bird1988; Fournet et al. 1999; Goulard et al. 2001), trasting environmental conditions, there is a high probability or they can alter the physiological conditions of seaweeds that intraspecific differences in their physiological and/or bio- depending on the species’ tolerance to limitation or disruptive chemical traits could have evolved as response to local habitat stress (Collén et al. 2004; Lee et al. 2017b; Navarro et al. conditions. 2017). In the case of MAAs, it is well known that the biosyn- In this study, the geographic variation of sulfated cell wall thesis of these nitrogen-containing metabolites depends on polysaccharides and UV-absorbing MAAs of the red sea- nutrient availability (Korbee et al. 2005a; Huovinen et al. weeds C. chamissoi and G. lingulatum were evaluated, con- 2006;Barufietal.2011), and on irradiance and spectral com- sidering populations from different latitudes along the Chilean position (Karsten and Wiencke 1999; Korbee et al. 2005b; coast. Furthermore, the sampled populations of each species Torres et al. 2016). On the other hand, since the biosynthesis were maintained in laboratory cultures under the same abiotic J Appl Phycol (2019) 31:665–682 667 conditions during 6 to 9 months before characterizing their coast (Fig. 1, Table 1). In the case of G. lingulatum, nine physiological (photosynthetic performance measured as max- locations were sampled (29° S to 41° S; ~ 1300 km) (Fig. 1, imum quantum yield of photosystem II and electron transport Table 1). rate) and biochemical (photosynthetic pigments, MAAs, anti- Field samples used for MAA analysis were cleaned from oxidant capacity) traits, in order to determine persistent phe- epibionts and stored in individual plastic bags filled with silica notypic differences that could be related to ecotypic gel, whereas the samples used for polysaccharide characteri- differentiation. zation and subsequent cultivation were transported in cooling boxes with gelpack to the Marine Botany Laboratory, Coquimbo (29° 58′ S) within 24 h after collection. Material and methods In the laboratory both species were identified using mor- phological traits as described by Hoffmann and Santelices (1997) and Calderon et al. (2010). The isomorphic phases Species and sites of collection were identified according to the presence of reproductive structures, with female gametophytes bearing cystocarps and Chondracanthus chamissoi (C. Agardh) Kützing tetrasporophytes bearing tetrasporangia. The determination of () is distributed from Paita, Perú (5° S) to MAAs and sulfated polysaccharides was carried out on female Chiloé, Chile (42° S), but also is found on the coasts of gametophytes and tetrasporophytes of C. chamissoi,whereas Japan, Korea, and France (Yang et al. 2015). The geographic in G. lingulatum only tetrasporophytes were used because this range of Gelidium lingulatum Kützing (Gelidiales) has been was the predominant life cycle phase in this species (Table 1). described from Antofagasta (23° S) to Tierra del Fuego (56° S) by Ramírez and Santelices (1991). However, a recent in- vestigation has suggested that this species does not occur Values of environmental parameters north of 29° S (López et al. 2017). during the sampling period of seaweeds Individuals of C. chamissoi and G. lingulatum were col- lected during austral summer 2016 (January/February) from Data of plankton chlorophyll a (Chl a) concentration natural populations in the subtidal zone (3–4-m depth) of (mg m−3), sea surface temperature (SST: °C), and photosyn- semi-protected bays and in the mid-low intertidal zone of thetically active radiation (PAR: μmol photons m−2 s−1)for wave-exposed rocky shores, respectively. The samples of C. each sampling site were obtained by satellite data from chamissoi were collected from six localities (20° S to 41° S; ~ MODIS Aqua level 3, which are freely available from the 2300 km) covering its distributional range along the Chilean NASA website (https://oceancolor.gsf.nasa.gov/cgi/I3). A

Fig. 1 Map of sampling sites of Chondracanthus chamissoi (black circles) and Gelidium lingulatum (gray circles) along the Chilean coast and their corresponding environmental conditions during the seaweed collection. Values of photosynthetically active radiation (PAR), sea surface temperature (SST), and chlorophyll a (Chl a)represent seasonal composites for the austral summer of 2016 (January and February) extracted from MODIS satellite data 668 J Appl Phycol (2019) 31:665–682

Table 1 Geographic location of sampling sites of Chondracanthus chamissoi and Gelidium lingulatum. The codes assigned to identify each sampling site, coordinates, and the life cycle phase used in each analysis are indicated

Species Location Code Latitude Longitude Life cycle phase used in each activity

SP MAAs Cultivation*

C.chamissoi Iquique IQU 20° 14′ S70°08′ WG+TG+TG Mejillones MEJ 23° 04′ S70°29′ WG+TG+TG Caldera CAL 27° 40′ S70°50′ WG+TG+TG La Herradura LHE 29° 59′ S71°23′ WG+TG+TG Coliumo COL 36° 32′ S72°57′ WG+TG+TG Lechagua LEC 41° 52′ S73°52′ WG+TG+TG G. lingulatum La Pampilla LPA 29° 57′ S71°21′ WT T T Punta Talca PTA 30° 52′ S71°41′ WT T Maitencillo MAI 32° 37′ S71°25′ WT T T Curañipe CUR 35° 50′ S72°38′ WT T T Lebu LEB 37° 43′ S73°39′ WT T T Pucatrihue PUC 40° 31′ S73°42′ WT T Huar Huar HUA 41° 18′ S73°50′ WT T Mar Brava MBR 41° 56′ S74°01′ WT T T Cucao CUC 42° 40′ S74°07′ WT T

G female gametophyte, T tetrasporophyte, SP sulfate polysaccharide, MAAs mycosporine-like amino acids from field samples

*Samples maintained under cultivation were used to determine maximum quantum yield of PSII (Fv/Fm), photosynthetic pigments, MAAs, and antioxidant capacity spatial resolution of 4 km was used from seasonal composites their vegetative propagation through creeping axes. One averaged over the sampling period (January and February 2000-L outdoor tank was used and inside this tank five rect- 2016) (Fig. 1). Data of plankton Chl a were estimated using angular containers with shells (one container for each popula- the OC-3 algorithm (O’Reilly et al. 2000). Since Chl a con- tion) were placed. The standard cultivation included continu- centrations are a sensitive indicator of phytoplankton primary ous flow of seawater (2 L min−1) and permanent aeration. productivity in the water column their values were related to Sun-shading nets were put over the tanks in order to reduce nutrient loads in coastal waters (Boyer et al. 2009). the incident photosynthetically active photon flux density During the sampling period of seaweeds, SST showed a (PPFD) to about 50–150 μmol photons m−2 s−1. significant and negative correlation to latitude (Spearman’s Temperature inside the tanks was measured in triplicate at coefficient = − 0.93, p < 0.05). In the case of PAR and Chl a 12:00 am every day and values ranged from 17.9 ± 0.4 °C values, no significant correlations between both environmen- (February) to 14.5 ± 0.3 °C (September). tal variables and latitude were observed (Spearman’scoeffi- Before measuring their physiological and biochemical cient = − 0.17, p > 0.05, respectively). characteristics, both species were maintained under indoor laboratory conditions for 1 month; this was done to eliminate General culture conditions and prevent epiphyte contamination on thallus surfaces. Apical segments of 2–3-cm length, free of epiphytic algae, Female gametophytes of C. chamissoi from six populations were cut from individual thalli of C. chamissoi and G. (Table 1) and tetrasporophytes of G. lingulatum from five lingulatum.About10–20 segments per thallus were placed populations (Table 1) were maintained in outdoor tanks during in independent glass flasks (1 flask per thallus) with 700 mL 5 and 8 months, respectively. For C. chamissoi, thalli without of autoclaved seawater enriched with half strength von Stosch reproductive structures with an independent holdfast disc medium, according to Bulboa et al. (2008). The cultures were were maintained in three 1000-L outdoor tanks. In each tank, maintained with continuous aeration in a temperature- six rectangular containers (one container for each population) controlled room at 14 ± 0.5 °C. A photoperiod of 12:12 h made with a PVC frame and plastic mesh were placed, and (light:dark) and a PPFD of 60 ± 5 μmol photons m−2 s−1 were within these containers, the thalli were kept in suspension used. The cultivation medium was renewed every 3 days. provided by continuous aeration. In the case of G. lingulatum, For both outdoor and indoor laboratory acclimation of sea- stoloniferous thalli composed of several erect axes were fixed weeds, C. chamissoi was maintained for a total period of over scallop shells (20 shells for each population) to allow 6 months under identical laboratory conditions whereas G. J Appl Phycol (2019) 31:665–682 669

lingulatum was maintained for 9 months. Common-garden using the turbidimetric method of gelatin-BaCl2 (Dodgson acclimation has been used to distinguish between genetic and Price 1962)withNa2SO4 used as the standard. All mea- and plastic responses in seaweeds (e.g., Gerard 1988), because surements were made with ten replicates. this period of acclimation reduces the influence of the envi- ronment on phenotypic differences among individuals from Determination of mycosporine-like amino acids different populations (reviewed in Sanford and Kelly 2011). Field and culture samples were used for MAA determination Carrageenan extraction and purification in both species (Table 1), with five individuals of about 10– 11 mg of dry weight (DW) from each population and condi- Ten gametophytes and ten tetrasporophytes from each popu- tion (field or culture). The samples were extracted in 1 mL lation of C. chamissoi were analyzed (Table 1). Samples were 25% aqueous methanol (v/v)for2hinscrew-cappedcentri- washed with distilled water and then dried to constant weight fuge vials put in a water bath at 45 °C. After centrifugation at at 50 °C. Dried and ground samples were pre-treated with 5000×g for 5 min, 800 μL of the supernatant was evaporated methanol and acetone (1:1) in a Soxhlet extractor in order to to dryness in a rotavapor under vacuum. Dried extracts were eliminate pigments and lipids. For the polysaccharide extrac- re-dissolved in 800 μL distilled water and vortexed for 30 s, tion, the samples were placed in distilled water (50 mL g−1)at followed by filtration through a 0.22-μm MCM membrane 90 °C for 3 h with continuous stirring, according to Matsuhiro filter. The MAAs were separated by high-performance liquid et al. (2005). The algal residue was removed by centrifugation chromatography (HPLC) (Agilent Technologies, 1100 Series, and the supernatant was dialyzed against distilled water for USA) according to Karsten et al. (1998b), using a stainless- 72 h using cellulose membranes (Spectra/Por MWCO: steel Phenomenex Synergi 4 μ fusion RP column (C18, 4 μm, 3500 Da, Spectrum Laboratories Inc.). The dialyzed solution 250 × 3 mm I.D) with a pre-column (RP-18 guard cartridge was concentrated in a vacuum rotary evaporator and poured 4 × 3.0 mm I.D.). The mobile phase was 2.5% aqueous meth- over ethanol (1:5). The precipitate was separated by centrifu- anol (v/v)plus0.1%aceticacid(v/v) in water, run isocratically gation, dissolved in the minimum volume of distilled water at a flow rate of 0.5 mL min−1 at room temperature. and freeze dried. Carrageenan contents were calculated as a Identification of MAAs was done by absorption spectra, re- percentage (%) of algal dry weight. tention time and by co-chromatography with secondary stan- dards obtained from samples of C. chamissoi and G. Agar extraction and purification lingulatum previously analyzed at the University of Rostock, Germany. Ten tetrasporophytes from each population of G. lingulatum were analyzed (Table 1). The field samples were washed with Determination of photosynthetic performance distilled water and dried to constant weight at 50 °C. Then, the by chlorophyll fluorescence thalli were ground in a mortar and treated with methanol and acetone (1:1) in a Soxhlet extractor. The samples were stirred In vivo chlorophyll fluorescence of photosystem II (PSII) was with distilled water (50 mL g−1) at 95 °C for 3 h for agar measured in ten gametophytes of C. chamissoi and ten extraction according to Matsuhiro and Urzúa (1991). The so- tetrasporophytes of G. lingulatum per population maintained lution was filtered through cotton tissue and the solid residue under cultivation (Table 1). For this, a portable pulse ampli- was re-extracted twice under the same conditions. The solu- tude modulated fluorometer (PAM 2500, Heinz Walz GmbH, tion (with the filtrate obtained from the three extractions) was Germany) was used following the nomenclature described by maintained at room temperature until it became a gel (12 h) Enríquez and Borowitzka (2010). Maximum quantum yield and then frozen overnight. This freeze-thaw cycle was repeat- (Fv/Fm) was measured in apical segments incubated for ed three times. The remaining gel was washed twice with 15 min in the dark. A 5 s far-red light pulse (30 μmol photons ethanol and then was dissolved in distilled water and freeze m−2 s−1 at 735 nm) was applied before and after dark acclima- dried. The agar yield (%) was calculated as the percentage of tion in order to fully oxidize the electron transport chain. algal dry weight. To compare variation in photosynthetic efficiency and ca- pacity among populations of both species, photosynthesis ver- Chemical composition of carrageenans and agar sus irradiance curves (P-I curves) were determined. Three (C. chamissoi)tofour(G. lingulatum) individuals for each popu- The total sugar content was determined by the phenol-sulfuric lation were exposed to 9 different irradiances of actinic red acid method using D-galactose as standard (Dubois et al. light (650 nm) between 0 and 500 μmol photons m−2 s−1.The 1956). The content of 3,6-anhydrogalactose was quantified duration of exposure to each irradiance level was 7 min, which by the resorcinol method using fructose as standard (Yaphe was necessary to reach photosynthetic steady-state. After each and Arsenault 1965). The sulfate content was determined exposure to actinic light, a saturating pulse was applied to 670 J Appl Phycol (2019) 31:665–682

measure effective quantum yield (ΔF/F’m). Then the electron mortar with liquid nitrogen and the phycobiliproteins extract- transport rate (ETR) was calculated using the equation: ed in 1.5 mL of 0.1 M phosphate buffer (pH = 6.8). The ex- tracts were incubated for 12 h at 4 °C and then centrifuged for ¼ Δ = 0 Â Â Â : ETR F F m IPAR A 0 5 15 min at 4500×g. The concentration of phycoerythrin (PE) and phycocyanin (PC) (mg g−1 fresh weight) was determined where IPA R is the irradiance of the actinic light, A is the ab- spectrophotometrically using the equations described by Beer sorptance of the sample, and 0.5 is the fraction of absorbed and Eshel (1985). quanta directed to PSII (Schreiber and Neubauer 1990). The absorptance of samples was measured following the method Antioxidant capacity described by Mercado et al. (1996). ETR values were plotted against irradiance of actinic light, and the P-I parameters were Five gametophytes of C. chamissoi and ten tetrasporophytes fitted to the equation of Platt et al. (1980): of G. lingulatum per population were obtained from lab cul- tures (Table 1) and their total antioxidant capacity was evalu- ETR ¼ ETR ðÞ1−expðÞ−α Â I=ETR s s ated. For this, the scavenging effect of their extracts was mea-

 expðÞ−β  I=ETRs sured for the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (Brand-Williams et al. 1995). The seaweed extracts were ob- tained according Tala et al. (2016)bymixinggroundfrozen where ETR is the maximum potential electron transport rate s samples (~ 50 mg fresh weight) with 1 mL 70% ethanol, in the absence of photoinhibition under optimal light, α is the which were then incubated in a water bath at 50 °C for initial slope of the P-I curve and defines the electron transport 60 min. The mixture was centrifuged and 300 μLofsuperna- efficiency at low irradiance, β is the photoinhibition constant, tant were mixed with 700 μL DPPH (50 mg L−1 70% meth- and I is the irradiance. − − anol). The decrease in absorbance was determined at 0 and Maximal ETR (ETR : μmol ē m 2 s 1) was calculated as max 30 min at 517 nm. Reduction of the DPPH was calculated as a β=α percentage of antioxidant capacity from the equation [1- (A / ETRmax ¼ ETRs  ðÞÂα=ðÞα þ β ðÞβ=ðÞα þ β F AI)] × 100, where AF is the absorbance of algal extract after 30 min of reaction with DPPH and AI is the initial absorbance Finally, the saturation irradiance for electron transport (Ek: μmol photons m−2 s−1) was calculated as the intercept be- at time 0 (Molyneux 2004). tween α and the ETRmax values. Statistical analyses During the measurements of Fv/Fm and P-I curves, algal samples were mounted at a distance of 5 mm from the end of the fiber optic probe of the fluorometer and inserted into a Yield and chemical composition of carrageenans and MAA custom-made opaque cuvette filled with seawater. The mea- contents from field samples of C. chamissoi were analyzed surements of P-I curves per each population of G. lingulatum with two-way ANOVAs to evaluate the effects of collection and C. chamissoi were carried out between 9:30 and 12:30 h sites and life cycle phases. Contents and chemical composi- during consecutive days in a temperature-controlled room at tion of agar and MAA levels of field samples of G. lingulatum 14 ± 0.5 °C. were analyzed with one-way ANOVAs in order to determine the effects of origin of seaweeds. Levels of MAAs in field and culture samples considering different populations were ana- Determination of photosynthetic pigments lyzed by a two-way ANOVA. Contents of Chl a,carotenoids,

phycobiliproteins, antioxidant capacity, Fv/Fm,ETRmax, α,Ek, Ten gametophytes of C. chamissoi and ten tetrasporophytes of were evaluated with a one-way multivariate ANOVA G. lingulatum per population were taken from lab cultures (MANOVA)in order to determine the effects of collection site (Table 1) and stored frozen at − 20 °C for later analysis of for each species. The assumptions of normal distribution and pigments. For the Chl a extraction, 1.5 mL of N,N- homogeneity of variances were tested using the Levene tests dimethylformamide (DMF) was added to each sample. After and Shapiro-Wilk W, respectively. Percentage data (carra- an incubation period of 24 h at 4 °C in darkness, the extinc- geenans and agar yield, antioxidant capacity) were arcsine tions of the extract were measured in a scanning UV-visible transformed, and logarithmic transformation was employed spectrophotometer (Rayleigh, model UV-1601, China). with contents of photosynthetic pigments and MAAs when Dichromatic equations described by Inskeep and Bloom necessary to comply with ANOVA assumptions. A posteriori (1985) were used for calculations of Chl a concentration Tukey’s test was used when the levels of the factor showed (mg g−1 fresh weight), whereas the methodology of Henley significant differences (p < 0.05). Spearman’s correlation tests and Dunton (1995) was used for total carotenoid estimation (p < 0.05) were carried out to assess the relationship among (mg g−1 fresh weight). The frozen samples were ground in a environmental variables (Chl a, SST, PAR) and contents of J Appl Phycol (2019) 31:665–682 671 sulfated polysaccharides and MAAs obtained from field sam- ples. All statistical analyses were run using the R statistical software (R Development Core Team, 2016).

Results

Carrageenan contents of C. chamissoi ranged from 15.4 ± 7.1 to 52.7 ± 6.5% DW (Fig. 2(a)). These values were significant- ly different among life cycle phases (gametophytes had more carrageenans than tetrasporophytes) and among populations (Table 2). In the gametophytes, the higher carrageenan con- tents were observed in the samples from CAL (27° S), LEC (41° S), and IQU (20° S) (Fig. 2(a)). In the case of tetrasporophytes, the samples from the northernmost sites, IQU (20° S) and MEJ (23° S), showed the lowest contents, whereas an increase in the amount of this polysaccharide was observed towards higher latitudes (Fig. 2(a), Table 3). There were also negative correlations between carrageenan amounts of tetrasporophytes and SST and Chl a, and between gameto- phyte carrageenan and PAR (Table 3). The chemical composition of the carrageenan of C. chamissoi also varied significantly among life cycle phases and populations (Table 2). The percentage of 3,6-AG was higher in the gametophytes than in tetrasporophytes (Fig. 2(b)), with an opposite tendency for the amounts of sulfates (Fig. 2(c)). Among populations, gametophytes and tetrasporophytes from IQU (20° S) and CAL (27° S) had carrageenans with higher levels of 3,6-AG and sulfates than samples from LHE (29° S) and LEC (41° S) (Fig. 2(b, c)). In both life cycle phases, there were negative correlations be- tween the amounts of 3,6-AG and latitude (Table 3), as well as positive correlations between the amount of 3,6-AG and SST (in the gametophytes) and PAR (in the tetrasporophytes) Fig. 2 Carrageenan characteristics of gametophytes and tetrasporophytes (Table 3). of Chondracanthus chamissoi collected at different sites along the Chilean coast. (a) carrageenan contents (% of dry weight), (b) Agar contents of G. lingulatum ranged from 10.9 ± 2.1 to percentages of anhydrogalactose (3,6-AG), and (c) sulfates (as Na2SO4) 25.1 ± 2.5% DW with significant differences among popula- in the carrageenans (100% represent the total carrageenans). Values are tions (Fig. 3(a), Table 2). The highest amount was observed in mean ± SD (n = 10). Different letters indicate significant differences ’ samples from MAI (32° S) whereas the lowest level was reg- among populations from Tukey stest(p < 0.05) istered in LPA (29° S) (Fig. 3(a)). With respect to the chemical composition of agar, differences among populations were ob- in LHE (29° S) and LEC (41° S) (Fig. 4(a)). In both life cycle served only in the percentages of 3,6-AG, with higher levels in phases, a positive correlation was observed between the total samples from LEB (37° S) than in CUR (35° S) (Fig. 3(b), MAA content and levels of PAR and Chl a (Table 3). Two Table 2). No correlations between agar characteristics and MAA types were identified in C. chamissoi,shinorine,and environmental variables at the sampling site were observed palythine (Fig. 4(b)). Generally, shinorine was the predomi- (Table 3). nant MAA with a relative composition ranging from 65 to Total contents of MAAs ranged from 0.8 ± 0.1 to 3.3 ± 83% of total levels, except in gametophytes from LEC (41° 0.5 mg g−1 DW in field samples of C. chamissoi (Fig. 4(a)), S) where shinorine contents were similar to palythine (Fig. with significant differences among life cycle phases and pop- 4(b)). ulations (Table 4). Gametophytes from CAL (27° S) and COL The samples of C. chamissoi after 6 months of cultivation (36° S) had higher MAA contents than tetrasporophytes. had lower amounts of MAAs than field samples (Fig. 4(c); Among populations, the highest level of MAAs was observed Table 4), but significant differences among populations in the in COL (36° S), whereas the lowest contents were registered total levels of MAAs were persistent after laboratory 672 J Appl Phycol (2019) 31:665–682

Table 2 Results of ANOVAs on contents and chemical composition of sulfated polysaccharides considering the effects of (a) life cycle phases and sampling site in Chondracanthus chamissoi and (b) the effect of sampling site in Gelidium lingulatum

DF Contents (% DW) 3,6-AG (%) Sulfates* (%)

FP FPFP

(a) C. chamissoi Sampling site 5 12.10 <0.001 14.18 <0.001 11.85 <0.001 Life cycle phases 1 362.59 <0.001 469.75 <0.001 42.43 <0.001 Sites × phases 5 15.48 <0.001 2.21 0.06 0.86 0.51 Error 108 (b) G. lingulatum Sampling site 4 15.01 <0.001 2.65 0.04 2.42 0.06 Error 45

Italic p values are significant

*Sulfate contents determined as Na2SO4 acclimation of the gametophytes (Fig. 4(c); Table 4). Higher contributing to 70–79% of the total MAA (Fig. 5(b)). When levels of these compounds were observed in samples from the samples from cultures were analyzed, lower amounts of southernmost COL (36° S) and LEC (41° S) in comparison MAAs were measured in comparison with field samples with IQU (20° S), MEJ (23° S), and LHE (29° S) (Fig. 4(c)). (Fig. 5(c); Table 4). On the other hand, no significant differ- The level of shinorine was also higher than palythine in the ences among populations were observed in the MAA contents gametophytes from culture (Fig. 4(d)). and composition after acclimation of G. lingulatum under The levels of MAAs in G. lingulatum collected from the common-garden conditions (Fig. 5(c), d; Table 4). field ranged from 2.6 ± 0.6 to 6.8 ± 1.3 mg g−1 DW (Fig. 5(a)). Photosynthetic performance was characterized in samples Significant differences among populations were observed of C. chamissoi and G. lingulatum after cultivation by mea- (Table 4), with the highest levels in seaweeds from CUR suring parameters derived from P-I curves such as electron (35° S) whereas the lowest amounts were observed in the transport efficiency at low irradiance (α), maximal electron samples from MAI (32° S) (Fig. 5(a)). A positive correlation transport rate (ETRmax), and saturation irradiances (Ek) was observed between the MAA contents and PAR and Chl a (Table 5). These variables were significantly different among (Table 3). With respect to MAA composition, shinorine was populations of C. chamissoi (MANOVA test, Wilks’s also the predominant MAA type in G. lingulatum, Lambda = 0.04, F(15,28) =3.74, p < 0.01, Table 5)andG.

Table 3 Spearman’s correlation coefficients for comparisons between species (a) Chondracanthus chamissoi (gametophytes and abiotic conditions and contents and chemical composition of sulfated tetrasporophytes) and (b) Gelidium lingulatum (tetrasporophytes), both polysaccharides, and contents of mycosporine-like amino acids in the collected from different sites along the Chilean coast

Latitude SST PAR Chl a

GT GTG TGT

(a) C. chamissoi Carrageenan contents − 0.09 0.71** 0.04 − 0.71** − 0.44** − 0.22 − 0.25 − 0.47** 3,6-AG − 0.38* − 0.41** 0.34* 0.28 − 0.01 0.33* 0.15 0.24 Sulfates − 0.16 − 0.39* 0.11 0.30 0.11 0.23 0.06 0.22 MAA contents − 0.18 − 0.26 0.24 0.38 0.43* 0.54* 0.58** 0.73** (b) G. lingulatum Agar contents nd 0.28 nd − 0.28 nd 0.11 nd − 0.10 3,6-AG nd 0.13 nd − 0.13 nd − 0.21 nd − 0.18 Sulfates nd − 0.39* nd 0.39* nd 0.17 nd − 0.31 MAA contents nd − 0.03 nd 0.03 nd 0.34* nd 0.36*

*p <0.01,**p < 0.001 (statistical significance) SST sea surface temperature, PAR photosynthetically active radiation, Chl a chlorophyll a contents from satellite data, G female gametophytes, T tetrasporophytes, 3,6-AG anhydrogalactose (%), MAA mycosporine-like aminoacids (mg g−1 DW), nd no determined J Appl Phycol (2019) 31:665–682 673

Wilks’s Lambda = 0.29, F(24,106) = 1.89, p = 0.01, Table 6). ThevaluesofFv/Fm were higher in samples from LEB (37° S) in comparison to MBR (41° S), whereas lower electron transport efficiencies were recorded in seaweeds from LPA (29° S) and CUR (35° S) (Table 5). In the case of phycobiliproteins, the seaweeds from LEB (37° S) had lower amounts of these pigments than MAI (32° S) and CUR (35° S) (Table 5).

Discussion

This study shows geographic variation, no related to the lati- tudinal gradient along the SE Pacific coast, in the contents and composition of sulfated polysaccharides and UV-absorbing MAAs in the seaweeds C. chamissoi and G. lingulatum.In addition, physiological traits also varied among populations after acclimation to laboratory conditions, especially in C. chamissoi.

Geographic variation of sulfated polysaccharides

Carrageenans of C. chamissoi were found in higher amounts than previously reported by Pereira et al. (2009)(24.6%DW) and Véliz et al. (2017) (19.2 to 35.4% DW). This variation is probably due to the larger geographic area included herein. However, the differences between life cycles phases from La Herradura with respect to the yield and composition of carrageenans reported by Véliz et al. (2017) were also ob- served in the present study. In fact, the higher production of carrageenans by the gametophytic phase of C. chamissoi has also been reported in the related seaweeds Chondracanthus teedei var. lusitanicus (Pereira and Mesquita 2004), Gigartina skottsbergii (Buschmann et al. 2001), and crispus Fig. 3 Agar characteristics of tetrasporophytes of Gelidium lingulatum (Tasende et al. 2012). Additionally, the variation in the chem- collected at different sites along the Chilean coast. (a) Agar contents (% of ical composition between phases is in agreement with the dry weight), (b) percentages of 3,6-anhydrogalactose, and (c) sulfates (as production of the hybrid kappa/iota-carrageenans by gameto- Na2SO4) in agar (100% represent the total agar). Values are mean ± SD phytes and xi/theta-carrageenans by tetrasporophytes (n = 10). Different letters indicate significant differences among popula- tions from Tukey’stest(p <0.05) (Jouanneau et al. 2011,Wangetal.2012). Even though this biochemical variation between gametophytes and tetrasporophytes is a common character in species belonging lingulatum (MANOVA test, Wilks’s Lambda = 0.12, to the family (Chopin et al. 1999;Lahaye2001;

F(12,35) =3.60, p <0.01Table 5). Furthermore, maximum van de Velde 2008), its adaptive significance is not well un- quantum yield of PSII (Fv/Fm), Chl a, phycobiliproteins (PE derstood. For instance, there is some evidence that gameto- and PC), and antioxidant capacity were significantly influ- phytes, with carrageenans belonging to the kappa family, enced by sites of seaweed collection in C. chamissoi could be more resistant to hydrodynamic stress and endophyte

(MANOVA test, Wilks’s Lambda = 0.09, F(30,198) = 5.55, p attack than tetrasporophytes with lambda carrageenans < 0.0001, Table 6), with higher percentages of antioxidant (Bouarab et al. 1999; Carrington et al. 2001), but further in- activity in samples from COL (35° S) and LEC (41° S) in vestigation is needed in this area. comparison with northernmost populations (Table 5). In the An increase in the carrageenan yield towards high latitudes case of G. lingulatum Fv/Fm, electron transport efficiencies was observed in the tetrasporophytes of C. chamissoi,whereas and the phycobiliprotein contents were significantly different in the gametophytes, no latitudinal tendency was identified. In among samples collected at different sites (MANOVA test, addition, the amount of carrageenans was correlated with 674 J Appl Phycol (2019) 31:665–682

Fig. 4 Levels (mg g−1 of dry weight) and relative composition (%) of composition in gametophytes from culture. Data are means ± SD (n =5). mycosporine-like amino acids (MAAs) in field and culture samples of Different letters indicate significant differences among populations from Chondracanthus chamissoi. (a) Contents and (b) composition in gameto- Tukey’stest(p <0.05) phytes and tetrasporophytes collected from field, and (c) contents and (d) different environmental variables depending on life cycle and Urzúa 1991). Although the agar content of G. lingulatum phases (Table 3), which could indicate that each phase has a varied among populations, this variation was not correlated differential biosynthesis of this polysaccharide in response to with the latitude or local environmental variables. Likewise, the local environmental conditions. Likewise, in a previous Freile-Pelegrín et al. (1996) did not find any correlation be- study, Tasende et al. (2012) reported differences in the carra- tween environmental conditions (SST and water motion) and geenan yield of C. crispus from different locations along the agar yield of Peterocladia capillacea from several sites at the Galician coast, but this geographic variation was observed northern rocky shore of Gran Canaria Island, Spain. In G. only in the gametophytic phase. In fact, in a recent study, robustum, however, the geographical variation of its agar yield Navarro et al. (2017) reported differential dynamics of carra- was inversely correlated with the equatorward increase of the geenan biosynthesis between phases of Mazzaella net primary production along the western coast of Baja laminarioides cultivated under different light qualities, with California Peninsula (Hurtado et al. 2011). higher carrageenan contents in the gametophytes exposed to The chemical composition of sulfated polysaccharides also high irradiances in comparison to tetrasporophytes. varied among the populations of C. chamissoi and G. With respect to the agar of G. lingulatum, its amounts were lingulatum, but the magnitude of this variation and their rela- within the range of a previous report from central Chile (~ 16 tionship with environmental variables was dependent on the to 27% DW), and for other Chilean species such as G. chilense species. For instance, while in C. chamissoi the levels of 3,6- (~ 12 to 15% DW) and G. rex (~ 15 to 36% DW) (Matsuhiro AG of their polysaccharide exhibited a negative correlation J Appl Phycol (2019) 31:665–682 675

Table 4 Results of ANOVAson mycosporine-like amino acid (MAAs) gametophytes of C. chamissoi, (d) sites of collection in samples of (mg g−1 of dry weight) considering the effects of (a) sites of collection and Gelidium lingulatum obtained from the field, (e) sites of collection in life cycle phases in C. chamissoi obtained from the field, (b) sites of samples of G. lingulatum obtained from lab cultures, and (f) sites of collection in gametophytes of C. chamissoi obtained from lab cultures, collection and type of samples (field versus culture samples) in G. (c) sites of collection and type of samples (field versus culture samples) in lingulatum

DF Total MAAs Palythine Shinorine

FP FP FP

(a) C. chamissoi from field Sites of collection 5 45.62 <0.001 103.79 <0.001 35.03 <0.001 Life cycle phases 1 55.02 <0.001 85.69 <0.001 29.35 <0.001 Sites x Phases 5 3.26 <0.05 9.73 <0.001 2.05 0.08 Error 48 (b) C. chamissoi from cultures Sites of collection 5 56.03 <0.001 38.71 <0.001 58.51 <0.001 Error 24 (c) C. chamissoi field vs. culture samples Sites of collection 5 40.19 <0.001 111.15 <0.001 22.69 <0.001 Types of samples 1 428.68 <0.001 330.69 <0.001 325.79 <0.001 Sites x types of samples 5 18.61 <0.001 57.94 <0.001 19.03 <0.001 Error 48 (d) G. lingulatum from field samples Sites of collection 8 9.86 <0.01 7.11 <0.01 9.04 <0.01 Error 36 (e) G. lingulatum from culture samples Sites of collection 4 0.52 0.72 1.14 0.36 0.95 0.46 Error 20 (f) G. lingulatum field vs. culture samples Sites of collection 4 12.73 <0.001 4.82 <0.001 7.12 <0.001 Types of samples 1 84.64 <0.001 56.12 <0.001 43.84 <0.001 Sites × type of samples 4 12.42 <0.001 3.59 <0.05 9.78 <0.001 Error 40

Italic p values are significant

with latitude and positive relationship to SST and PAR, in G. At present, no clear geographic pattern has been identified lingulatum the presence of 3,6-AG was not correlated with for the production of sulfated polysaccharides in red seaweeds any environmental condition. Since the chemical composition (e.g., Pereira and van de Velde 2011; Hurtado et al. 2011; of these polysaccharides determines the physicochemical Tasende et al. 2012, 2013), probably due to the different ways properties and consequently the biological traits of seaweeds in which environmental factors can influence carrageenan and (Craigie 1990; Ficko-Blean et al. 2015), it is possible that the agar biosynthesis (Table 7). Briefly, environmental conditions differential geographic variation between species is due to can modify the amounts of carbon precursors and energy specific adaptive requirements imposed by local habitat con- available for polysaccharide production through their effects ditions. The samples of C. chamissoi were obtained from on photosynthesis and the physiological state of seaweeds subtidal beds while the G. lingulatum thalli were collected (Macler 1986; Bird 1988; Collén et al. 2004). Furthermore, from exposed intertidal habitats. Furthermore, other environ- environmental cues may induce differential gene expression mental conditions such as osmotic stress due to salinity chang- of enzymes involved in carbohydrate metabolism (Ficko- es, desiccation and degree of wave exposure can affect the Blean et al. 2015; Lee et al. 2017a). In addition, phylogenetic chemical composition of sulfated cell wall polysaccharides relationships, life cycle phases, and ecotypic differentiation in intertidal species (reviewed in Lee et al. 2017b), which, also affect carrageenan and agar biosynthesis (Chopin et al. however, were not measured in this study. 1999; Lee et al. 2017b; Véliz et al. 2017). 676 J Appl Phycol (2019) 31:665–682

Fig. 5 Levels (mg g−1 of dry weight) and relative composition (%) of composition in tetrasporophytes from culture. Data are means ± SD mycosporine-like amino acids (MAAs) in field and culture samples of (n = 5). Different letters indicate significant differences among popula- Gelidium lingulatum. (a) Contents and (b) composition in tions from Tukey’stest(p <0.05) tetrasporophytes collected from field, and (c) contents and (d)

Geographic variation in MAAs from field samples The MAA levels quantified in this study are in the range of previous reports for temperate red seaweeds from southern In this study, the MAAs shinorine and palythine were identi- Chile (1.0 to 10.6 mg g−1 DW) (Huovinen et al. 2004), as well fied in C. chamissoi and G. lingulatum, being the first report as from southern Spain (0.2 to 7.8 mg−1 DW) (Karsten et al. for the former species. These types of MAAs are the most 1998b). The intertidal species G. lingulatum had substantially commonly found in temperate red seaweeds (Huovinen et al. higher MAA levels than the subtidal samples of C. chamissoi, 2004). In other Gelidium species, such as G. sesquipedale and probably because G. lingulatum is exposed to much higher G. pusillum from southern Spain, asterina-330 and palythinol UV radiation in its intertidal habitat. Both species have the have also been observed, albeit in trace amounts (Karsten et al. capability of adjusting their levels of UV-absorbing com- 1998b). It has been suggested that MAA composition is a pounds to environmental conditions, because higher MAA species-specific trait instead of being a character under envi- levels were observed in field samples in comparison with ronmental control (Boedeker and Karsten 2005; Karsten et al. seaweeds from culture. Since UV radiation is an important 2005). This finding is in agreement with the results of the factor determining the upper limit of seaweed distribution on present study, because there was no intraspecific variation in marine rocky shores, the production of MAAs is a fundamen- MAA composition neither for C. chamissoi nor for G. tal mechanism in red seaweeds exposed to high solar radiation lingulatum, with geographical differences only in the relative in the intertidal and shallow subtidal zone (Bischof et al. 2006; predominance of each MAA. Karsten 2008; Bischof and Steinhoff 2012). Contrary, it is less J Appl Phycol (2019) 31:665–682 677

Table 5 Physiological and chemical variables determined in culture samples of (a) Chondracanthus chamissoi and (b) Gelidium lingulatum collected from different sites along the Chilean coast

Samples Fv/Fm α ETRmax Ek Chl a PE PC Carotenoids Antiox Cap

(a) C. chamissoi IQU 0.57 ± 0.02b 0.35 ± 0.06ab 4.74 ± 1.21a 13.43 ± 2.83ab 0.92 ± 0.37ab 0.60 ± 0.25ab 0.68 ± 0.11bc 0.32 ± 0.14 16.18 ± 2.27c MEJ 0.59 ± 0.03ab 0.32 ± 0.03ab 5.04 ± 0.59a 15.79 ± 1.92a 1.06 ± 0.29a 0.93 ± 0.42a 1.17 ± 0.27a 0.40 ± 0.28 16.22 ± 2.67c CAL 0.59 ± 0.01ab 0.30 ± 0.03ab 2.98 ± 0.12b 9.96 ± 0.99b 0.95 ± 0.31a 0.85 ± 0.40ab 0.50 ± 0.26c 0.28 ± 0.24 17.39 ± 3.16bc LHE 0.60 ± 0.02a 0.39 ± 0.02b 3.66 ± 0.19ab 9.34 ± 0.70b 0.59 ± 0.23b 0.50 ± 0.31b 0.52 ± 0.12bc 0.21 ± 0.12 20.37 ± 3.76abc COL 0.56 ± 0.03b 0.33 ± 0.02ab 3.65 ± 0.07ab 11.23 ± 1.03ab 0.78 ± 0.19ab 0.63 ± 0.23ab 0.76 ± 0.20bc 0.27 ± 0.19 21.32 ± 4.90ab LEC 0.60 ± 0.02a 0.25 ± 0.05a 2.79 ± 0.64b 11.08 ± 2.24ab 0.86 ± 0.28ab 0.86 ± 0.15a 0.68 ± 0.13bc 0.43 ± 0.17 22.49 ± 4.04a (b) G. lingulatum LPA 0.49 ± 0.04ab 0.39 ± 0.03b 6.71 ± 0.55 17.16 ± 1.43 7.90 ± 2.04 2.48 ± 1.16ab 1.79 ± 0.93b 2.20 ± 0.69 13.83 ± 3.16 MAI 0.52 ± 0.05ab 0.48 ± 0.04a 5.83 ± 1.15 12.15 ± 2.09 7.95 ± 2.42 3.69 ± 1.27a 2.97 ± 0.92a 1.96 ± 0.85 16.48 ± 3.90 CUR 0.52 ± 0.03ab 0.36 ± 0.02b 6.78 ± 2.14 18.94 ± 6.51 8.16 ± 1.87 3.80 ± 1.05a 2.96 ± 0.77a 2.16 ± 0.71 13.66 ± 3.70 LEB 0.54 ± 0.03a 0.44 ± 0.03a 5.15 ± 1.12 11.59 ± 1.70 7.91 ± 1.92 2.01 ± 1.29b 1.71 ± 0.84b 2.11 ± 0.72 13.63 ± 4.51 MBR 0.47 ± 0.05b 0.48 ± 0.01a 6.61 ± 0.34 13.66 ± 0.88 7.74 ± 1.50 3.32 ± 0.91ab 2.86 ± 0.78ab 1.89 ± 0.60 16.77 ± 2.95

Different lower case letters indicate significant differences among populations from Tukey’sHSDtest(p < 0.05) IQU Iquique, MEJ Mejillones, CAL Caldera, LHE La Herradura, COL Coliumo, LEC Lechagua, LPA La Pampilla, MAI Maitencillo, CUR Curañipe, LEB Lebu, MBR Mar Brava, Fv/Fm maximum quantum yield, α electron transport efficiency at low irradiance, ETRmax maximal electronic transport rate −2 −1 −2 −1 −1 −1 (μmol ē m s ), Ek saturation irradiance for electron transport (μmol photons m s ), Chl a chlorophyll a (mg g FW), PE phycoerythrin (mg g FW), PC phycocyanin (mg g−1 FW), carotenoids (mg g−1 FW), Antiox Cap antioxidant capacity measures as reduction of the diphenyl-1-picrilhydrazil (%). Values shown are means ± standard deviation known how the latitudinal gradients of UV radiation might The natural levels of PAR and Chl a at the site of seaweed affect the biochemistry and ultimately the geographical distri- collection were positively correlated with the total amounts of bution of littoral seaweed species. MAAs in both species. The highest level of these UV-

Table 6 Results of one-way ANOVA comparing the effects of sampling lingulatum. (Previous MANOVA tests were carried out and their Wilks’s site (populations) on physiological and chemical variables measured in Lambda values are presented in the result section) culture samples of (a) Chondracanthus chamissoi and (b) Gelidium

Variable Factor (a) C. chamissoi (b) G. lingulatum

DF FP DF FP

α Population 5 4.15 < 0.05 416.74<0.001 Error 12 15 ETRmax Population 5 6.86 < 0.01 4 1.22 0.34 Error 12 15 Ek Population 5 5.35 < 0.01 4 3.32 0.04 Error 12 15 Fv/Fm Population 5 6.06 <0.001 43.79< 0.05 Error 54 35 Chl a Population 5 3.49 < 0.01 4 0.12 0.98 Error 54 35 PE Population 5 3.62 < 0.01 44.39< 0.01 Error 54 35 PC Population 5 13.59 <0.001 45.09< 0.01 Error 54 35 Carotenoids Population 5 1.73 0.14 4 0.31 0.87 Error 54 35 Antiox Cap Population 5 6.00 <0.001 4 1.64 0.18 Error 54 35

Italic p values are significant −2 −1 Fv/Fm maximum quantum yield, α electron transport efficiency at low irradiance, ETRmax maximal electron transport rate (μmol ē m s ), Ek saturation irradiance for electron transport (μmol photons m−2 s−1 ), Chl a chlorophyll a (mg g−1 FW), PE phycoerythrin (mg g−1 FW), PC phycocyanin (mg g−1 FW), carotenoids (mg g−1 FW), Antiox Cap antioxidant capacity measures as reduction of the diphenyl-1-picrilhydrazil (%) 678 J Appl Phycol (2019) 31:665–682

Table 7 Environmental factors with impact on the production of sulfated polysaccharides and MAAs mycosporine-like amino acids in red seaweeds

Compound Species Metabolic process/physiological response involved in compound Relationship between factors Reference production and compound production

PAR BL UV T N S W

Agar Gracilaria sp. Agar content is dependent on total carbohydrate metabolism and −−−−Bird 1988b allocation of resources to meet physiological requirements Gracilaria Agar content is dependent on gene expression of UDP-glucose nr − Chang et al. lemaneiformis pyrophosphorylase 2014b Gracilaria Seaweed nitrogen status produces a differential induction of − Collén et al. tenuistipitata starch-synthesizing and starch-degrading enzymes 2004b Gelidium Allocation of resources to amino acid synthesis inhibit carbon flux − Macler 1986b coulterii to agar Gelidium Probable adaptation of seaweed to environmental conditions nr nr nr This study lingulatum Carrageenans Chondrus Biosynthesis of carrageenans to improve seaweed flexibility to + Carrington et crispus hydrodynamic stress al. 2001a Chondrus Allocation of resources to carrageenan synthesis depending of − Chopin and crispus nitrogen requirement for growth Wagey 1999c Kappaphycus Disruptive stress decreases carrageenan content and gel strength − Eswaran et al. alvarezii 2001b Solieria Carrageenan synthesis is related to floridean starch degradation + − Fournet et al. chordalis 1999b Soliera chordalis UDP-glucose pyrophosphorylase activity in inhibit under osmotic − Goulard et al. stress 2001b Mazzaella Carrageenans are biosynthesized as photoprotective mechanism + Navarro et al. laminarioides depending on life cycle phase 2017c Hypnea Carrageenans are biosynthesized in response to environmental −−+Reisetal. musciformis stressful conditions 2008a Chondrus Adaptation of seaweeds to environmental change by carrageenan − +nr+ Tasendeetal. crispus synthesis 2012a Chondracanthus Probable adaptation of seaweed to environmental conditions −−−This study chamissoi MAAs Gracilaria MAAs are biosynthesized as photoprotective mechanism +++Barufietal. tenuistipitata depending on nitrate supply 2011b Grateloupia MAAs are biosynthesized as photoprotective mechanism +++Huovinenetal. lanceola depending on ammonium supply 2006b Chondrus Potential activation of a photoreceptors for blue light and UV-A + + Franklin et al. crispus radiation 2001b Porphyra Potential activation of a non-photosynthetic blue light + + Korbee et al. leucostica photoreceptor 2005bb Palmaria Induction of photoprotective compounds is more effective under + + Karsten and palmata full solar spectrum Wiencke 1999b Gracilaria High PAR induce accumulation of MAAs + Torres et al. tenuifrons 2016b Chondracanthus MAA contents related to field levels of PAR and plankton + nr + This study chamissoi chlorophyll a Gelidium lingulatum

Letters on reference: a data from field samples, b data from samples under indoor laboratory cultivation, c data from samples under outdoor cultivation PAR photosynthetic active radiation, BL blue light, UV ultraviolet radiation, T temperature, N nutrients supply, S salinity, W water movement, nr no relationship identified J Appl Phycol (2019) 31:665–682 679 absorbing compounds were quantified in the samples from some populations of G. lingulatum (LPA and MAI) and C. CUR (35° S) and COL (36° S), respectively, both sites being chamissoi (COL and LEC), the amounts of MAAs and sulfat- characterized by high levels of both environmental conditions ed polysaccharides appear inversely related. Previous studies (Fig. 1). This result is in agreement with previous studies in red seaweeds maintained under laboratory conditions have reporting that the biosynthesis of MAAs is influenced by the shown that the biosynthesis of carbohydrates and nitrogenous irradiance and spectral range of solar radiation, as well as by compounds are differentially regulated by environmental fac- the availability of nutrients required for the biosynthesis of tors depending on physiological requirements of individuals these nitrogen-containing metabolites (Table 7). (Macler 1986;Bird1988; Chopin and Wagey 1999; Collén et al. 2004). Thus, the present study shows that this inverse trend Geographic variation in ecophysiological traits between amounts of cell wall polysaccharides and nitrogenous after cultivation metabolites also occurs in natural populations of red sea- weeds, possibly hinting at the metabolic costs for producing In C. chamissoi, variation among populations in their photo- either of these compounds. synthetic characteristics, levels of photosynthetic pigments, Finally, the data obtained from seaweeds acclimated under antioxidant capacity, and MAA contents were observed after common-garden conditions suggest ecotypic differentiation in acclimation under identical abiotic conditions. Furthermore, C. chamissoi, with the need of a phylogeographic study, the amounts of MAAs and antioxidant capacity, which can whereas G. lingulatum seems to have a more generalist strat- also be considered as important functional traits in red sea- egy. However, further laboratory experiments about the eco- weeds, showed differences among the northernmost and physiological responses of the population of both species to southernmost populations of C. chamissoi. This result may different levels of environmental factors are required. indicate that the intraspecific variation in MAAs has a genetic base in C. chamissoi. However, more experiments under con- Acknowledgements We would like to thank the Centro de Investigación trolled conditions of UV radiation are needed to corroborate y Desarrollo Tecnológico en Algas (CIDTA-UCN) for providing labora- tory facilities. We are grateful to Samanta García and David Yañez for this hypothesis. At present, the genetic or plastic base behind their collaboration in laboratory activities, as well as to David Jofré MAA biosynthesis in algae has been principally examined in Madariaga, Oscar Pino, and Felipe Saéz for their help in the collection green microalgae (Kitzing et al. 2014; Kitzing and Karsten of field samples. 2015) and cyanobacteria (Pattanaik et al. 2008), reporting ge- netic differences among the populations or strains of the same Funding information Financial support for this study was provided by Ph.D Grant CONICYT-Chile 21130402 and FIAC2-UCN1104 to K.V., species. Fondecyt 1131082 to M.T., and FIC-R BIP 30137720-0 to N.C.; C.L. In the case of G. lingulatum, differences in the levels of acknowledges support from the Millennium Nucleus Center for the Study MAAs, antioxidant capacity, photosynthetic pigments (Chl a of Multiple Drivers on Marine Socio-Ecological Systems (MUSELS) and carotenoids) and some photosynthetic characteristics funded by MINECON NC120086.

(ETRmax and Ek) observed in the field samples did not persist after cultivation, suggesting a more generalist strategy (phe- References notypic plasticity) in this species. On the other hand, G. lingulatum seems to be well connected by long-distance dis- persal events along the Chilean coast (López et al. 2017), Barufi BJ, Korbee N, Oliveira MC, Figueroa FL (2011) Effects of N supply on the accumulation of photosynthetic pigments and which could reduce the probability of ecotypic differentiation photoprotectors in Gracilaria tenuistipitata (Rhodophyta) cultured (reviewed in Sanford and Kelly 2011). under UV radiation. J Appl Phycol 23:457–466 Beer S, Eshel A (1985) Determining phycoerythrin and phycocyanin concentrations in aqueous crude extracts of . Aust J Mar Conclusion Freshw Res 36:785–792 Bird KT (1988) Agar production and quality from Gracilaria sp. strain G- 16: effects of environmental factors. Bot Mar 31:33–39 Along the same latitudinal gradient, the intraspecific variation Bischof K, Gómez I, Molis M, Hanelt D, Karsten U, Lüder U, Roleda in phenotypic traits of red seaweeds is strongly dependent on MY, Zacher K, Wiencke C (2006) Ultraviolet radiation shapes sea- life cycle phase and species. Furthermore, sulfated polysac- weed communities. In: Amils R, Ellis-Evans C, Hinghofer-Szalkay H (eds) Life in extreme environments. Springer, Dordrecht, pp 187– charides and MAAs are differentially correlated with the en- 212 vironmental conditions at the site of seaweed origin. For in- Bischof K, Steinhoff FS (2012) Impacts of ozone stratospheric depletion stance, whereas the amounts of MAAs were positively corre- and solar UVB radiation on seaweeds. In: Wiencke C, Bischof K lated to the geographic variation of PAR and Chl a (consid- (eds) Seaweed biology novel insights into ecophysiology, ecology and utilization. Springer, Berlin, pp 433–467 ered as an indirect indicator of nutrient supply) in both species, Boedeker C, Karsten U (2005) The occurrence of mycosporine-like ami- the polysaccharide contents of C. chamissoi were inversely no acids in the gametophytic and sporophytic stages of Bangia correlated to these environmental conditions. In addition, in (Bangiales, Rhodophyta). Phycologia 44:403–408 680 J Appl Phycol (2019) 31:665–682

Bouarab K, Potin P, Correa J, Kloareg B (1999) Sulfated oligosaccharides Franklin LA, Kräbs G, Kuhlenkamp R (2001) Blue light and UV radia- mediate the interaction between a marine red alga and its green algal tion control the synthesis of mycosporine-like amino acids in pathogenic endophyte. Plant Cell 11:1635–1650 (Florideophyceae). J Phycol 37:257–270 Boyer JN, Kelble CR, Ortner PB, Rudnick DT (2009) Phytoplankton Freile-Pelegrín Y, Robledo D, Armisén R, García-Reina G (1996) bloom status: chlorophyll a biomass as an indicator of water quality Seasonal changes in agar characteristics of two populations of condition in the southern estuaries of Florida, USA. Ecol Appl 95: Pterocladia capillacea in Gran Canaria, Spain. J Appl Phycol 8: 556–567 239–246 Brand-Williams W, Cuvelier M, Berset C (1995) Use of a free radical Gerard VA (1988) Ecotypic differentiation in light-related traits of the method to evaluate antioxidant activity. Food Sci Biotechnol 28:25– kelp Laminaria saccharina. Mar Biol 97:25–36 30 Goulard F, Diouris M, Quere G, Deslandes E, Floc'h JY (2001) Salinity Bulboa C, Macchiavello J, Oliveira E, Véliz K (2008) Growth rate dif- effects on NDP-sugar, floridoside, starch and carrageenan yield, and ferences between four Chilean populations of edible seaweed UDP-glucose-pyrophosphorylase and epimerase activities of culti- Chondracanthus chamissoi (Rhodophyta, Gigartinales). Aquac vated Solieria chordalis. J Plant Physiol 158:1387–1394 Res 39:1550–1555 Hayashi L, Bulboa C, Kradolfer P, Soriano G, Robledo D (2014) Buschmann AH, Correa JA, Westermeier R, Paredes MA, Aedo D, Potin Cultivation of red seaweeds: a Latin American perspective. J Appl P, Aroca G, Beltrán J, Hernández-González MC (2001) Cultivation Phycol 26:719–727 of Gigartina skottsbergii (Gigartinales, Rhodophyta): recent ad- Henley WJ, Dunton KH (1995) A seasonal comparison of carbon, nitro- – vances and challenges for the future. J Appl Phycol 13:255 266 gen, and pigment content in Laminaria solidungula and L. Calderón M, Ramírez ME, Bustamante D (2010) Notas sobre tres saccharina (Phaeophyta) in the Alaskan Arctic. J Phycol 31:325– especies de Gigartinaceae (Rhodophyta) del litoral peruano. Rev 321 – Peru Biol 17:115 121 Hernández K, Yannicelli B, Montecinos A, Ramos M, González HE, Carreto JI, Carignan MO (2011) Mycosporine-like amino acids: relevant Daneri G (2012) Temporal variability of incidental solar radiation secondary metabolites. Chemical and ecological aspects. Mar Drugs and modulating factors in a coastal upwelling area (36°S). Prog – 9:387 446 Oceanogr 92-95:18–32 Carrington E, Grace SP, Chopin T (2001) Life history phases and the Hoffmann A, Santelices B (1997) Flora marina de Chile central. biomechanical properties of the red alga Chondrus crispus Ediciones Universidad Católica del Chile, Santiago, Chile. 434 pp (Rhodophyta). J Phycol 37:699–704 Hoyer K, Karsten U, Sawall T, Wiencke C (2001) Photoprotective sub- Chang L, Sui Z, Feng F, Zhou W, Wang J, Kang KH, Zhang S, Ma J stances in Antarctic macroalgae and their variation with respect to (2014) Relationship between gene expression of UDP-glucose depth distribution, different tissues and development stages. Mar pyrophosphorylase and agar yield in Gracilariopsis lemaneiformis Ecol Prog Ser 211:117–129 (Rhodophyta). J Appl Phycol 26:2435–2441 Hoyer K, Karsten U, Wiencke C (2002) Induction of sunscreen com- Chopin T, Kerin BF, Mazerolle R (1999) Phycocolloid chemistry as a pounds in Antarctic macroalgae by different radiation conditions. taxonomic indicator of phylogeny in the Gigartinales, Mar Biol 141:619–627 Rhodophyceae: a review and current developments using Fourier Huovinen P, Gómez I, Figueroa FL, Ulloa N, Morales V, Lovengreen C transform infrared diffuse reflectance spectroscopy. Phycol Res 47: (2004) Ultraviolet-absorbing mycosporine-like amino acids in red 167–188 macroalgae from Chile. Bot Mar 47:21–29 Chopin T, Wagey BT (1999) Factorial study of the effects of phosphorus and nitrogen enrichments on nutrient and carrageenan content in Huovinen P, Matos J, Pinto IS, Figueroa FL (2006) The role of ammoni- Chondrus crispus (Rhodophyceae) and on residual nutrient concen- um in photoprotection against high irradiance in the red alga – tration in seawater. Bot Mar 42:23–31 Grateloupia lanceola. Aquat Bot 84:308 316 Collén PN, Camitz A, Hancock RD, Viola R, Pedersén M (2004) Effect Hurtado MA, Manzano-Sarabia M, Hernández-Garibay E, Pacheco-Ruíz of nutrient deprivation and resupply on metabolites and enzyme I, Zertuche-González JA (2011) Latitudinal variations of the yield related to carbon allocation in Gracilaria tenuistipitata and quality of agar from Gelidium robustum (Gelidiales, (Rhodophyta). J Phycol 40:305–314 Rhodophyta) from the main commercial harvest beds along the Craigie JS (1990) Cell walls. In: Cole KM, Sheath RG (eds) Biology of western coast of the Baja California peninsula, Mexico. J Appl – the red algae. Cambridge University Press, Cambridge, pp 221–257 Phycol 23:727 734 Dodgson K, Price R (1962) A note on the determination of the ester Inskeep WP, Bloom PR (1985) Extinction coefficients of chlorophyll a sulphate content of sulphated polysaccharides. Biochem J 84:106– and b in N, N-dimethylformamide and 80% acetone. Plant Physiol – 110 77:483 485 Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Jouanneau D, Boulenguer P, Mazoyer J, Hebert W (2011) Hybridity of Colorimetric method for determination of sugars and related sub- carrageenans water- and alkali-extracted from Chondracanthus stances. Anal Chem 28:350–356 chamissoi, Mazzaella laminarioides, Sarcothalia crispata,and Enríquez S, Borowitzka MA (2010) The use of the fluorescence signal in Sarcothalia radula. J Appl Phycol 23:105–114 studies of sea grasses and macroalgae. In: Suggett DJ, Borowitzka Karsten U, Sawall T, Wiencke C (1998a) A survey of the distribution of MA, Prásil O (eds) Chlorophyll a fluorescence in aquatic sciences: UV-absorbing substances in tropical macroalgae. Phycol Res 46: methods and applications. Springer, Dordrecht, pp 187–208 271–279 Eswaran K, Suba Rao PV, Mairh OP (2001) Impact of ultraviolet-B Karsten U, Sawall T, Hanelt D, Bischof K, Figueroa FL, Flores-Moya A, radiation on a marine red alga Kappaphycus alvarezii. Indian J Wiencke U (1998b) An inventory of UV-absorbing mycosporine- Mar Sci 30:105–107 like amino acids in macroalgae from polar to warm-temperate re- Ficko-Blean E, Hervé C, Michel G (2015) Sweet and sour sugars from the gions. Bot Mar 41:443–453 sea: the biosynthesis and remodeling of sulfated cell wall polysac- Karsten U, Wiencke U (1999) Factors controlling the formation of UV- charides from marine macroalgae. Perspect Phycology 2:51–64 absorbing mycosporine-like amino acids in the marine red alga Fournet I, Zinoun M, Deslandes E, Diouris M, Floc’h JY (1999) Palmaria palmata from Spitsbergen (Norway). J Plant Physiol Floridean starch and carrageenan contents as responses of the red 155:407–415 alga Solieria chordalis to culture conditions. Eur J Phycol 34:125– Karsten U, Friedl T, Schumann R, Hoyer K, Lembcke S (2005) 130 Mycosporine-like amino acids and phylogenies in green algae: J Appl Phycol (2019) 31:665–682 681

Prasiola and its relatives from the Trebouxiophyceae (Chlorophyta). Memorandum - SeaWIFS Postlaunch. Technical Report Series, pp JPhycol41:557–566 1–49 Karsten U (2008) Defenses strategies of algae and cyanobacteria against Pattanaik B, Roleda MY, Schumann R, Karsten U (2008) Isolate-specific solar UVR. In: Amsler CD (ed) Algal chemical ecology. Springer, effects of ultraviolet radiation on photosynthesis, growth and Berlin, pp 273–296 mycosporine-like amino acids in the microbial mat-forming cyano- Kitzing C, Pröschold T, Karsten U (2014) UV-induced effects on growth, bacterium Microcoleus chthonoplastes. Planta 227:907–916 photosynthetic performance and sunscreen contents in different pop- Pereira L, Mesquita JF (2004) Population studies and carrageenan prop- ulations of the green alga Klebsormidium fluitans (Streptophyta) erties of Chondracanthus teedei var. lusitanicus (Gigartinaceae, from alpine soil crusts. Microb Ecol 67:327–340 Rhodophyta). J Appl Phycol 16:369–383 Kitzing C, Karsten U (2015) Effects of UV radiation on optimum quan- Pereira L, Critchley AT, Amado AM, Ribeiro-Claro P (2009) A compar- tum yield and sunscreen contents in members of the genera ative analysis of phycocolloids produced by underutilized versus Interfilum, Klebsormidium, Hormidiella and Entransia industrially utilized carrageenophytes (Gigartinales, Rhodophyta). – (Klebsormidiophyceae, Streptophyta). Eur J Phycol 50:279 287 JApplPhycol21:599–605 Korbee N, Huovinen P, Figueroa FL, Aguilera J, Karsten U (2005a) Pereira L, van de Velde F (2011) Portuguese carrageenophytes: carra- Availability of ammonium influences photosynthesis and the accu- geenan composition and geographic distribution of eight species mulation of mycosporine-like amino acids in two Porphyra species (Gigartinales, Rhodophyta). Carbohyd Polym 84:614–623 (Bangiales, Rhodophyta). Mar Biol 146:654–654 Platt T, Gallegos CL, Harrison WG (1980) Photoinhibition of photosyn- Korbee N, Figueroa FL, Aguilera J (2005b) Effect of light quality on the thesis in natural assemblages of marine phytoplankton. J Mar Res accumulation of photosynthetic pigments, proteins and 38:687–701 mycosporine-like amino acids in the red alga Porphyra leucosticta (Bangiales, Rhodophyta). J Photochem Photobiol B 80:71–78 Ramírez ME, Santelices B (1991) Catálogo de las algas marinas Lahaye M (2001) Developments on gelling algal galactans, their structure bentónicas de la Costa del Pacífico Temperado de Sudamérica. and physico-chemistry. J Appl Phycol 13:173–184 Monografías Biológicas 5. Pontificia Universidad Católica de Chile, Santiago Lee WK, Lim YY, Leow ATC, Namasivayam P, Abdullah JO, Ho CL (2017a) Biosynthesis of agar in red seaweeds: a review. Carbohyd Ramos-Rodríguez A, Lluch-Cota DB, Lluch-Cota SE, Trasviña-Castro A Polym 164:23–30 (2012) Sea surface temperature anomalies, seasonal cycle and trend – Lee WK, Lim YY, Leow ATC, Namasivayam P, Abdullah JO, Ho CL regimes in the eastern Pacific coast. Ocean Sci 8:81 90 (2017b) Factors affecting yield and gelling properties of agar. J Appl Reis RP, Yoneshigue-Valentin Y, dos Santos CP (2008) Spatial and tem- Phycol 29:1527–1540 poral variation of Hypnea musciformis carrageenan (Rhodophyta, López BA, Tellier F, Retamal-Alarcón JC, Pérez-Araneda K, Fierro AO, Gigartinales) from natural beds in Rio de Janeiro state, Brazil. J Macaya EC, Tala F, Thiel M (2017) Phylogeography of two inter- Appl Phycol 20:1–8 tidal seaweeds, Gelidium lingulatum and G. rex (Rhodophyta: Sanford E, Kelly MW (2011) Local adaptation in marine invertebrates. Gelidiales), along the south East Pacific: patterns explained by Annu Rev Mar Sci 3:509–535 rafting dispersal? Mar Biol. https://doi.org/10.1007/s00227-017- Schreiber U, Neubauer C (1990) O2-dependent electron flow, membrane 3219-5 energization and mechanism of non-photochemical quenching of Macler BA (1986) Regulation of carbon flow by nitrogen and light in the chlorophyll fluorescence. Photosynth Res 25:279–293 red alga Gelidium coulteri. Plant Physiol 82:136–141 Tala F, Velásquez M, Mansilla A, Macaya EC, Thiel M (2016) Latitudinal Macler BA (1988) Salinity effects on photosynthesis, carbon allocation, and seasonal effects on short-term acclimation of floating kelp spe- and nitrogen assimilation in the red alga Gelidium coulteri.Plant cies from the South-East Pacific. J Exp Mar Biol Ecol 483:31–41 Physiol 88:690–694 Tapia FJ, Largier JL, Castillo M, Wieters EA, Navarrete SA (2014) Mariani P, Tolomio C, Baldan B, Braghetta P (1990) Cell wall ultrastruc- Latitudinal discontinuity in thermal conditions along the nearshore ture and cation localization in some benthic marine algae. of Central-Northern Chile. PLoS One 9(10):e110841 – Phycologia 29:253 262 Tasende MG, Cid M, Fraga MI (2012) Spatial and temporal variations of Matsuhiro B, Urzúa CC (1991) Agars from Chilean Gelidiaceae. Chondrus crispus (Gigartinaceae, Rhodophyta) carrageenan content – Hydrobiologia 221:149 156 in natural populations from Galicia (NW Spain). J Appl Phycol 24: Matsuhiro B, Conte AF, Damonte EB, Kolender AA, Matulewicz MC, 941–951 Mejías EG, Pujol CA, Zúñiga EA (2005) Structural analysis and Tasende MG, Cid M, Fraga MI (2013) Qualitative and quantitative anal- antiviral activity of a sulfated galactan from the red seaweed ysis of carrageenan content in gametophytes of Mastocarpus Schizymenia binderi (Gigartinales, Rhodophyta). Carbohydr Res stellatus (Stackhouse) Guiry along Galician coast (NW Spain). J – 340:2392 2402 Appl Phycol 25:587–596 Mercado JM, Jiménez C, Niell FX, Figueroa FL (1996) Comparison of Torres PB, Chow F, Ferreira MJ, dos Santos DYAC (2016) Mycosporine- methods for measuring light absorption by algae and their applica- like amino acids from Gracilariopsis tenuifrons (Gracilariales, tion to the estimation of the package effect. Sci Mar 60:39–45 Rhodophyta) and its variation under high light. J Appl Phycol 28: Molyneux P (2004) The use of the stable free radical 2035–2040 diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity. van de Velde F (2008) Structure and function of hybrid carrageenans. J Sci Technol 26:2119–21 Food Hydrocoll 22:727–734 Navarro NP, Figueroa FL, Korbee N (2017) Mycosporine-like amino acids vs carrageenan yield in Mazzaella laminarioides Véliz K, Chandía N, Rivadeneira M, Thiel M (2017) Seasonal variation (Gigartinales; Rhodophyta) under high and low UV solar irradiance. of carrageenans from Chondracanthus chamissoi with a review of Phycologia 56:570–578 variation in the carrageenan contents produced by Gigartinales. J – O'Reilly JE, Maritorena S, O'Brien MC, Siegel DA, Toole D, Menzies D, Appl Phycol 29:3139 3150 Smith RC, Mueller JL, Mitchell BG, Kahru M, Chavez FP, Strutton Vernet M, Diaz S, Fuenzalida H, Camilion C, Booth CR, Cabrera S, P, Cota GF, Hooker SB, McClain CR, Carder KL, Muller-Karger F, Casiccia C, Deferrari G, Lovengreen C, Paladini A, Pedroni J, Harding L, Magnuson A, Phinney D, Moore GF, Aiken J, Arrigo Rosales A, Zagarese H (2009) Quality of UVR exposure for differ- KR, Letelier R, Culver M (2000) SeaWiFS Postlaunch Calibration ent biological systems along a latitudinal gradient. Photochem – and Validation Analyses, Part 3, vol. 11, NASA Technical Photobiol Sci 8:1329 1345 682 J Appl Phycol (2019) 31:665–682

Wang P, Zhao X, Lv Y, Li M, Liu X, Li G, Yu G (2012) Structural and Yaphe W, Arsenault GP (1965) Improved resorcinol reagent for the de- compositional characteristics of hybrid carrageenans from red algae termination of fructose and 3,6-anhydrogalactose in polysaccha- Chondracanthus chamissoi. Carbohyd Polym 89:914–919 rides. Anal Biochem 13:143–148 Yang MY,Macaya EC, Kim MS (2015) Molecular evidence for verifying the distribution of Chondracanthus chamissoi and C. teedei (Gigartinaceae, Rhodophyta). Bot Mar 58:103–113