Intraspecific variations in colour morphs of Hypnea musciformis ( Rhodophyta ) in relation to nitrogen availability

(Intraspecific variations in colour morphs of Hypnea musciformis (Rhodophyta) in relation to nitrogen availability). Effects of nitrate availability in two culture media (von Stosch (VSES), and artificial ASP 12-NTA), and nitrogen sources (seawater enriched with nitrate, ammonium or urea in concentrations ranging from zero to 30 mM) were evaluated in two brown morphs (BR-1, BR-2), one light-green morph (LG) and one dark-green morph (DG) of Hypnea musciformis (Wulfen) J.V. Lamour. Higher growth rates of the four morphs were observed in VSES medium. However, artificial ASP12-NTA medium induced tetrasporangium development in DG morph. Growth rates of the four colour morphs followed kinetic of saturation-type nutrient uptake in treatments with urea. In contrast, growth rates of BR-1, BR-2 and LG morphs were inversely proportional to ammonium concentrations, and those higher than 15 mM were lethal. Growth responses of colour morphs of H. musciformis showed intraspecific variations, and they could be used as bioindicators of nitrogen pollution in marine environment by their low tolerance to ammonium.


Intraspecific variations in colour morphs of Hypnea musciformis (Rhodophyta) in relation to nitrogen availability Introduction
The genus Hypnea (Gigartinales, Rhodophyta) includes 50 species distributed in warm waters (Masuda et al. 1997), much of them with economic importance in several countries (Critchley & Ohno 1998).In Brazil, Hypnea musciformis (Wulfen) J.V. Lamour.has a wide geographical distribution, and is the main raw material for carrageenan production (Berchez et al. 1993).
Along the Brazilian coast, populations of Hypnea musciformis usually have a brown phenotype.However, one population composed by brown and dark-green specimens was found in Espírito Santo State.Besides, a brown plant with only one green branch was collected, and a phycoerythrin-deficient morph (light-green phenotype) was originated from this green branch.In laboratory, these specimens were isolated, and cultured in different environmental conditions as irradiance levels, photoperiod, temperature and salinity in order to evaluate if the thallus colour could be an acclimatation to environmental variations.The colour of these H. musciformis morphs is a stable characteristic, and is not a result of photoacclimation processes (Yokoya et al. 2003, Martins et al. 2008).The occurrence of colour variants in Brazilian red algae was reported to some Gracilaria species, as G. birdiae Plastino & E.C. Oliveira, G. cornea J. Agardh, and G. domingensis (Kützing) Sonder ex Dickie (Plastino 2003).The first report on colour variants of Hypnea musciformis was described by Yokoya et al. (2003), and the light-green morph was characterized by having lower phycoerythrin concentration, and higher phycocyanin and allophycocyanin concentrations than the brown morph.
The major nitrogen storage pools in macroalgae are amino acids and proteins, and in nitrogen-limited condition, phycoerythrin is an important source of nitrogen (Bird et al. 1982).Then, the colour morphs of H. musciformis could be a good experimental systems to study different processes of nitrogen metabolism.
The objectives of our study were to determine the effects of nitrate concentration in different culture media as well as to evaluate different nitrogen sources and concentrations on growth of colour morphs of Hypnea musciformis.

Materials and methods
Unialgal cultures -Four colour tetrasporophytes of Hypnea musciformis (Wulfen) J.V. Lamour.were selected: two brown morphs (BR-1 and BR-2), one light-green morph (LG) and one dark-green morph (DG).The LG morph was a phycoerythrin-deficient mutant, and was originated from a green branch, which had arisen probably as a spontaneous mutation in a brown plant (BR-2) collected from Ponta da Baleia, Espírito Santo, Brazil; both morphs were originated by vegetative propagation in laboratory.BR-1 and DG morphs were originated from carpospores liberated, respectively, by brown and dark-green plants collected from Praia de Formosa, Espírito Santo, Brazil.Voucher specimens were deposited in SP herbarium, with the following accession numbers: SP 365645 (LG morph), SP 365646 (BR-2 morph), SP 365671 (BR-1 morph) and SP 365672 (DG morph).
Nitrogen experiments -Effects of nitrate availability were tested in different culture media: sterilized seawater enriched with von Stosch's solution (VSES), and artificial ASP 12-NTA medium, following Iwasaki (1961), and modified by Yokoya (2000), with different nitrate concentrations (125, 250 and 500 µM) for 28 days.Both culture media presented salinity of 30 psu and pH of 8.0.
Effects of nitrogen sources and concentrations: two inorganic nitrogen sources (sodium nitrate and ammonium chloride) and one organic source (urea) were added to sterilized seawater in concentrations of zero, 5, 10, 15, 20, 25 and 30 µM.Low concentrations of ammonium (zero to 5 µM) were tested in a second experiment to determine the optimal concentrations for growth, as colour morphs did not tolerate higher concentrations of ammonium.Phosphorus was added (Na 2 HPO 4 .12H 2 O) in concentrations to maintain the N:P ratio of 4:1.All treatments were prepared with sterilized seawater provided by only one collection, and experimental period was 21 days.
Other experimental conditions were the same as described for unialgal cultures.Each treatment was tested with three replicates, with four apical segments of 10 mm, cultured in 250 mL Erlenmeyer flasks with 150 mL of medium.Fresh biomass was recorded weekly in the same intervals of medium renewal.
Growth rates were calculated as [ln (B f .B o -1 ).t -1 ], where B o is the initial fresh biomass, B f is the fresh biomass after t days, and t corresponds to the experimental period (Yokoya et al. 2003).
Data analysis -Data were analyzed by one-way and two-way analyses of variance (ANOVA) and, when normality and equal variance tests did not pass, the data were analyzed by ANOVA on Ranks.Student-Newman-Keuls post hoc test was conducted to distinguish significantly different results (p < 0.05) following the ANOVA tests.Statistical tests were performed by SigmaStat software (version 1.0).

Results
Nitrate availability in VSES and ASP 12-NTA media -The highest growth rates of the four morphs were observed in VSES medium.However, differences among treatments (nitrate concentrations ranging from 125 to 500 mM) were not significant, except for BR-2 morph, which showed the highest growth rate at low nitrate concentration (figure 1).In ASP 12-NTA medium, growth rates of the four morphs were lower than those observed in VSES medium, and the highest growth rates of the BR-1 and DG morphs were observed in low nitrate concentration (125 mM) (figure 1).A comparison among the four morphs showed intraspecific variations to the response to nitrate availability (table 1), and DG and LG morphs showed, respectively, the lowest and the highest growth rates in both culture media (figure 2).
The ASP 12-NTA medium stimulated the development of tetrasporangia in DG morph (figure 3A), and specimens cultured under 250 and 125 mM of nitrate became fertile after four and seven weeks, respectively.Some tetraspores germinated "in situ" and tetrasporelings grew on the stichidium (figure 3B), or tetraspores, which were settled onto coverslips placed on the bottom of culture flasks, germinated and gave rise to several tetrasporelings (figure 3C-G).
Effects of nitrogen sources and concentrations -Growth rates of BR-1, BR-2 and LG morphs of Hypnea musciformis cultured with different concentrations of N-nitrate did not vary significantly when compared to the control (figure 4).However, growth rates of DG morph increased significantly under treatments with N-nitrate concentrations from 5 to 30 mM (figure 4).
Variations of growth rates on the studied morphs with the addition of zero to 30 mM of N-ammonium were significantly different (figure 5), except for the DG morph.BR-1, BR-2 and LG morphs showed inverse relationship between growth rates and N-ammonium concentrations, and concentrations equal and higher than 15 mM were lethal for LG and BR-2 morphs, respectively.
The experiment with low concentrations of N-ammonium (from zero to 5 mM) showed that only green morphs presented significant differences on growth rates when compared to the control (figure 6).Optimum concentration for growth of the DG morph was 1,0 mM N-ammonium while LG morph showed higher growth rates at 1.0 and 4.0 mM N-ammonium (figure 6).
Growth rates of the four colour morphs in relation to the increase of N-urea concentration followed kinetic of saturation-type nutrient uptake (figure 7).Growth rates of BR-1 and DG morphs increased at concentrations higher than 5 mM N-urea, and the optimal concentrations for growth were 10 and 5mM N-urea, respectively (figure 7).
A comparison among the four morphs indicated that responses to different nitrogen concentrations (nitrate, ammonium, and urea) as well as nitrogen sources (ammonium and urea) showed intraspecific variations (table 2).Besides, the interaction between nitrogen concentrations and sources had significant effects on colour morphs of H. musciformis (table 3).

Discussion
Seaweeds are able to use different forms of nitrogen, and each species could have ability to use specific nitrogen sources (Hanisak 1983, Lobban & Harrison 1994).Considering seaweed growth, three patterns could be observed: 1. higher growth with nitrate, 2. higher growth with ammonium, and 3. similar growth with nitrate and ammonium (Hanisak 1990).Besides these patterns, some species showed higher growth with addition of organic nitrogen (urea) instead of inorganic nitrogen sources, as observed in the red algae Pterocladiella capillacea (S.G.Gmel.)Santel.& Hommers.(cited as Pterocladia capillacea, Nasr et al. 1968) and the green algae Ulva fasciata Delile (Mohsen et al. 1974).The red alga Gracilaria cornea did not have preference between organic and inorganic nitrogen sources (Navarro-Angulo & Robledo 1999).
The four colour morphs of Hypnea musciformis showed higher growth rates with addition of nitrate and urea.However, ammonium inhibited the growth of BR-1, BR-2 and LG morphs, and concentrations higher than 15 mM were lethal to LG and BR-2 morphs.At high concentrations, ammonium could be toxic for some seaweeds (Waite & Mitchell 1972, Prince 1974), and the four morphs of H. musciformis showed similar responses.These results could be explained by the coupling of nitrogen metabolism and photosynthetic pathways (Turpin 1991), and the increase in intracellular ammonium concentration needs a higher carbon skeleton availability for nitrogen assimilation.Probably, intracellular ammonium could not be assimilated by H. musciformis colour morphs, and its accumulation into the cytoplasm decreased the internal pH, what was prejudicial to the cells (Turpin 1991).In contrast, different results were observed by Haines (1976) in a cultivation system, where H. musciformis showed higher growth in effluent enriched with ammonium than with nitrate, and ammonium uptake was faster than nitrate uptake (Haines & Wheeler 1978).
A comparison among the four morphs showed intraspecific variations in relation to nitrate availability in VSES and ASP 12-NTA media; DG and LG morphs showed, respectively, the lowest and the highest growth rates in both culture media.Then, DG morph showed lower growth rates than the BR-1 morph, and the best performance of wild specimens was also observed in the red specimens of Gracilaria birdiae (cited as Gracilaria sp. by Ursi & Plastino 2001), and in brown specimens of Kappaphycus alvarezii (Doty) Doty ex Silva (Paula et al. 1999).However, different results were observed in BR-2 and LG morphs, since the LG morph (phycoerythrindeficient mutant) had higher growth rate than the BR-2 morph (wild specimen).Similar results were observed for a green variant of Kappaphycus striatum (Schmitz) Doty (Gerung & Ohno 1997), and for yellowish-brown mutant of Porphyra yezoensis Ueda, which had higher growth than the wild specimen and other colour mutants (Yan et al. 2000).
In the four colour morphs of Hypnea musciformis, relationship between growth rates and urea concentrations corresponds to a saturation curve.Similar results were also observed for the red algae Neoagardhiella bailey (Harvey ex Kützing) Wynne & Taylor and Gracilaria foliifera (Forsskal) Bfrgesen, with half saturation constants (Ks) values ranging from 0.2 to 0.4 mM of different nitrogen sources, and maximum values of growth rates at nitrogen concentrations up to 1.0 mM (DeBoer et al. 1978).However, higher values were observed to young specimens of the brown algae Macrocystis pyrifera (L.) Agardh (from 6 to 15 mM) and the green algae Cladophora aff.albida (Hudson) Kützing (30 mM) (Gordon et al. 1981).In the present study, optimum concentration was 5 mM N-nitrate for DG morph of H. musciformis.Similar results were observed to Laminaria saccharina (L.) J.V. Lamour., which growth rate reached the maximum value at nitrogen concentrations of 5 and 10 mM N-nitrate (Wheeler & Weidner 1983).With addition of urea, the optimum concentration was 10 µM for BR-1 and DG morphs.Urea is considered a good nitrogen source for many seaweed species, and, generally its assimilation follows saturation kinetics (Thomas & Harrison 1985).
In conclusion, responses of the four colour morphs showed intraspecific variations in relation to nitrogen sources and concentrations.Moreover, colour morphs of H. musciformis could be used as bioindicators of nitrogen pollution in the marine environment by their low tolerance to ammonium.

Figure 1 .
Figure 1.Growth rates of brown morphs (BR-1 and BR-2), dark-green morph (DG) and light-green morph (LG) of Hypnea musciformis cultured at different concentrations of N-nitrate in Von Stosch and ASP 12-NTA media.Each data point represents the mean (n = 3, ± SD).Treatments marked by different letters are significantly different according to ANOVA and Student-Newman-Keuls comparison test (p < 0.05).To BR-2 and LG morphs the data were analyzed by ANOVA on Ranks.

Figure 2 .
Figure 2. Growth rates of the four morphs of H. musciformis (brown morphs, BR-1, BR-2, dark-green morph, DG, and light-green morph, LG) cultured at different concentrations of N-nitrate in Von Stosch and ASP 12-NTA media.Each data point represents the mean (n = 3, ± SD).Treatments marked by different letters are significantly different according to ANOVA and Student-Newman-Keuls comparison test (p < 0.05).

Figure 4 .
Figure 4. Growth rates of brown morphs (BR-1 and BR-2), dark-green morph (DG) and light-green morph (LG) of H. musciformis cultured at seven different concentrations of N-nitrate.Each data point represents the mean (n = 3, ± SD).Treatments marked by different letters are significantly different according to ANOVA and Student-Newman-Keuls comparison test (p < 0.05).

Figure 5 .
Figure 5. Growth rates of brown morphs (BR-1 and BR-2), dark-green morph (DG) and light-green morph (LG) of H. musciformis cultured at seven different concentrations of N-ammonium.Each data point represents the mean (n = 3, ± SD).Treatments marked by different letters are significantly different according to the ANOVA and Student-Newman-Keuls comparison test (p < 0.05).To BR-1 morph the data were analyzed by ANOVA on Ranks.

Figure 6 .
Figure 6.Growth rates of brown morphs (BR-1 and BR-2), dark-green morph (DG) and light-green morph (LG) of H. musciformis cultured at different concentrations of N-ammonium.Each data point represents the mean (n = 3, ± SD).Treatments marked by the different letter are significantly different according to ANOVA and Student-Newman-Keuls comparison test (p < 0.05).To DG morph the data were analyzed by ANOVA on Ranks.

Figure 7 .
Figure 7. Growth rates of brown morphs (BR-1 and BR-2), dark-green morph (DG) and light-green morph (LG) of H. musciformis cultured at different concentrations of N-urea.Each data point represents the mean (n = 3, ± SD).Treatments marked by different letters are significantly different according to the ANOVA and Student-Newman-Keuls comparison test (p < 0.05).

Table 1 .
Bifactorial analysis based on growth rates of colour morphs (BR-1, BR-2, DG, and LG) of Hypnea musciformis cultured in von Stosch and ASP 12-NTA media with different concentrations of N-nitrate.Variables: N-nitrate concentrations (A), and morphs (B).Bold letters/numbers indicate significant effects.

Table 3
. Bifactorial analysis based on growth rates of colour morphs (BR-1, BR-2, DG, and LG) of Hypnea musciformis cultured in sterilized seawater with addition of different concentrations of N-nitrate, N-ammonium and N-urea.Variables: Nitrogen concentrations (A), nitrogen sources (B).Bold letters/numbers indicate significant effects.

Table 2 .
Bifactorial analysis based on growth rates of colour morphs (BR-1, BR-2, DG, and LG) of Hypnea musciformis cultured in sterilized seawater with addition of different concentrations of N-nitrate, N-ammonium and N-urea.Variables: Nitrogen concentrations (A), morphs (B).Bold letters/numbers indicate significant effects.