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Seasonal variations in environmental factors (irradiance, seawater concentration of NO,, NOpand NH4+, temperature) and in physiological variables of Porphyra umbiljcalis (L.) Kiitzing (intracellular NOn-, NO2and NH; concentrations, nitrate reductase activity, amino acids, soluble proteins, total C, total N, C:N:P ratio) were determined during winter 1991-92 at a site in Lagos (Malaga, southern coast of Spam). In the western Mediterranean Sea, the leafy thallus of P. urnbd~cahs occurs only during winter (mid-December to mid-March). Maximal % cover (80 %) was reached in the first part of February. Conditions of low temperature (14.7 _+ 0.6OC), short days (10 h daylight) and low global solar irradiance (2.5 to 3 4 kWh m-2) prevailed during the period of increase in ?/o cover. The period of decrease in % cover was characterized by long days (12 h) , higher irradiance (3.4 to 5 kWh m-2), and slightly higher water temperature (15.5 f 1.34 "C). NO, concentration in seawater ([NO,],) at the study site ranged from 1 to 6.7 FM, [NOz-], was always lower than l pM, and [NH,'],, the largest source of inorganic nitrogen, ranged from 6.8 to 17.6 pM. Intracellular concentration of NO, ([NO,],) ranged from 2.8 to 51.3 mm01 1-' cell water (11.7 to 210.6 ymol g' dry wt), which represents a concentration factor of up to 4.2 X 104 with respect to the seawater. [NO,-], ranged from 0.2 to 0.7 mm01 1-' cell water and [NH;], from 0.016 to 2.2 mm01 1-' cell water. Nitrate reductase (NR) activity varied widely during the winter cycle (between 1.2 and 9.4 pm01 NOpg-' fresh wt h-'). It was found to be a very sensit.ive parameter for indicating when NOT is being used as the N source for growth. Therefore, low NR activity levels coincided with the highest [NO<ji and were more or less independent of [NOy], when the alga was using NH,' as N source. A large increase in NR activity was detected when [NO.;], decreased sharply. Cysteine was the predominant amino acid, amounting to between 83.5 and 97.2''1, of total amino acid content (5 to 28.3 ymol g-' fresh wt). No correlation was found between total amino acid and soluble protein content. Soluble protein content showed the same seasonal variation as total N and total C. and varied inversely to the C:N ratio. Winter changes in the Redfield ratio, from 258.20:l (24 December 1991) to 495:38:1 (16 March 1992) (in atoms), suggest that primary production of P. umbilicalis is Lunlted by P rather than N on thls coast. INTRODUCTION (Drew 1949). This study focuses on the gametophyte, the leafy thallus. On the southern coast of Spain the Porphyra umbilicalis (Rhodophyta) occurs In 2 very gametophytic form of P. umbilicalis is usually present easily distinguished phases, the leafy, haploid thallus, from December to March. In Porphyra tenera, the conwhich is present in winter, and the so-called conchocelis stage develops conchosporangia under condichocelis stage, a filarnentous form, present in summer tions of short daylength (Dring 1967), and in Porphyra miniata conchospores are released under low-temperature conditions (Chen et al. 1970). The germination of 'Addressee for correspondence these conchospores produces the leafy thallus in the 0 Inter-Research 1993 302 Mar. Ecol. Prog. SE cold season. However, less information exists on the environmental factors determining the disappearance of the gametophytic phase at the end of winter. There is also little information on the circumstances under which the thallus produces monospores, which once released and fixed develop new leafy thalli, or cuspores and p-spores (carpospores), which are involved in the generation of the conchocelis stage. Besides identifying which environmental factors control the transition between both phases, it is also of interest to identify the environmental factor that controls Porphyra primary production. Nitrogen and phosphorus have been reported to limit macroalgal primary production (Hanisak 1983, Lapointe & O'Connell 1989). Hence we focused our attention on both P and N metabolism. In this paper we concentrate on several relevant variables related to N metabolism; P metabolism is treated elsewhere (Hernandez et al. 1993). Here we measure a number of environmental and physiological parameters for Porphyra umbilicalis: external and intracellular concentration of NOT, NO2 and NH4+; nitrate reductase activity; protein and amino acid content; and C:N:P ratio. Our purposes were to: (1) study the annual cycle of P. umbiLicalls in the Mediterranean Sea (south of Spain) from the standpoint of N metabolism; and (2) identify the relative importance of N and P in promoting seasonal growth of the leafy thallus, and ultimately investigate the environmental or biochemical factor controlling the seasonal disappearance of P, um bilicalis. MATERIALS AND METHODS Study site and sampling methods. Porphyra umbilicalls was studied and collected for experiments from a flat rock (upper intertidal zone) in Lagos (Malaga, south of Spain) between December 1991 and March 1992. Plants (ca 3 g fresh weight) were always collected at the same time (11:30 to 12:00 h, solar time) from the same area (1.5 X 0.5 m'), selected at the beginning of the study. Immediately after collection, samples of fresh tissue were wrapped in aluminium foil and immersed in liquid nitrogen. Samples of seawater received the same treatment. Once in the laboratory all samples were maintained at -20°C until analysis. The percentage of the sampling area covered by P. umbilicalis was used to estimate its abundance during the cycle. Percentage cover (% cover) was determined by a modification of the linear transect method: a ruler of 30 cm was thrown 10 times on the selected area, and the mean ratio between the length of the ruler on P. umbilicalis and the length without P. umbilicalis was considered a good estimation of percentage cover. Sea water and rock temperatures were measured with a liquid-in-glass thermometer. Data presented in this study are the means of 2 to 3 replicates (see figure legends). Random plant samples were obtained from the entire selected area and therefore included many different individuals. Hence error bars represent the addition of 2 components, the variance due to the methodological procedure (chemical and biochemical analysis) plus the intraspecific variability of P. umbilicalis in the selected area. Since the selected area was small, it can be assumed that environmental factors were spatially homogeneous. Determination of external and intracellular NO<. NO? and NH4+ concentrations. Nitrate (Wood et al. 1967), NO1 (Shinn 1941) and NH,' (Slawyk & MacIsaac 1972) concentrations were determined in seawater samples by means of a continuous flow analytical system (BRAN+LUEBBE TRAACS 800). Intracellular NO?, NO1 and NH; concentrations were determined as follows. Samples of frozen tissue were dried (7O0C, 24 h) and ground to a homogeneous powder, and 0.035 g of the dry powder was dissolved in 40 m1 of deionized water. After 1 h of continuous shaking, the mixture was filtered (Whatman GF-C), and NOT, NO, and NH4+ determined in the filtrate by the same procedure referred to above (Corzo & Niell 1992a). Amino acids. Preparation o f samples: Samples of frozen tissue were homogenized in methanol (80 %). Precipitated proteins were removed by centrifugation (19 000 X g, 15 min). The pellet containing the proteins was resuspended in 80 % methanol to wash away possible remaining amino acid and centrifuged in the same conditions. To eliminate pigments, supernatants were passed through a solid phase extraction column (C18; Supelco, Bellefonte, USA). Finally, the extract was concentrated to 0.1 m1 under reduced pressure (Speedvac concentrator, Salvant Instruments, USA). Derivatization procedure and HPLC: To obtain the dansyl derivatives of the amino acid, 200 p1 of 40 mM C03Li and 200 p1 of 20 mM dansyl chloride were added to 0.1 m1 of each extract and, after 1 h at room temperature (23 "C), the mixture was filtered (0.22 pm, Millex-GU13, Millipore) (modified from Marquez et al. 1986). A Hewlett-Packard (HP-1090) HPLC equipped with a Rheodyne injection valve, a 20 p1 sample loop and a Supelcosil LC-l8 column (4.6 X 15 cm) (Supelco, Bellafonte, USA) was used for the analysis. Derivatives were separated using the following mixture: 0.008 % triethylamine + 0.6% acetic acid (polar phase) and methanol (nonpolar phase). Derivatives were measured at 254 nm. Individual amino acids were identified by comparison of elution time with an external standard (S~gma) and concentrations were calculated from the peak area. Two analyses were carried out for every sample. Hernandez et al.: Seasonal cycle of Porphyra u~nbll~calis 303 Soluble proteins, pigments and C:N:P ratio. Soluble proteins and phycobiliproteins were extracted by grinding the samples of fresh tissue (frozen in liquid nitrogen; 0.15 g) in 5 m1 of 0.1 M Na+-phosphate, 4 mM EDTA, pH 6.5. Debris was removed by centrifugation (10000 X g, 20 min, 4°C). Phycoerythrin and phycocyanin were determined spectrophotometrically (Beckman DU-7) in the supernatant (Beer & Eshel 1985). Soluble proteins were determined in an aliquot of the same supernatant, as follows: 3 m1 Coomassie Blue was added to 0.1 rnl of sample, and extinction at 595 nm was measured (Beckman DU-7) after 5 min of incubation at room temperature (Bradford 1976). Concentrations were calculated by means of standards made with bovine serum albumin (Sigma). Total C and total N were determined in triplicate on samples of dry ground tissue using a Perkin Elmer 240 CNH elemental analyser. Total phosphorus was determined by acid digestion in triplicate samples of dry ground tissue (Sommer & Nelson 1972). Nitrate reductase activity. Determination of nitrate reductase (NR) activity requires a number of technical procedures difficult to implement in the field. Therefore the specimens have to be brought to the laboratory while ensuring that NR activity is affected as little as possible. In

Seasonal cycle of the gametophytic form of Porphyra umbilicalis: nitrogen and carbon