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The effect of salinity on growth rate and branch morphology in tank cultivated Grateloupia filicina (rhodophyta) in Hawaii
Aquatic Botany, 27 {1987) 187-193
187
Elsevier Science Publishers B.V., Amsterdam-- Printed in The Netherlands
Short Communication T H E E F F E C T O F S A L I N I T Y ON G R O W T H R A T E A N D B R A N C H MORPHOLOGY IN TANK CULTIVATED GRATELOUPIA FILICINA (RHODOPHYTA) IN HAWAII
EARL ZABLACKIS'
Department o[ Botany, University of Hawaii, Honolulu, HI 96822 ( U.S.A.) (Accepted for publication 17 July 1986)
ABSTRACT Zablackis,E., 1987. The effect of salinity on growthrate and branch morphologyin tank cultivated Grateloupia filicina ( Rhodophyta ) in Hawaii.Aquat. Bot., 27: 187-193. The red algal species Grateloupia filicina {Lamour.) C. Agardh was grown in tank culture at fivesalinities.There was no statistically significantdifferencebetweengrowthrate at the different salinities tested. However,reduced salinity initiated production of significantlygreater numbers of branches in three branch orders in this species,thus demonstrating environmentalcontrol over branching due to salinity levels.
INTRODUCTION The red algal genus Grateloupia C. Agardh is of interest ecologically, chemically and commercially because it is common worldwide, often a conspicuous member of intertidal and nearshore algal communities and contains appreciable quantities of carrageenan (Zablackis, 1986) in its cell walls. In Hawaii, Grateloupia filicina (Lamour.) C. Agardh is almost always found in an area with some source of freshwater nearby and therefore is naturally subject to various salinity regimes. Salinity is an i m p o r t a n t environmental parameter {Gessner and Schramm, 1971 ) affecting distribution, growth, morphology and chemical composition (Haug and Larson, 1958; Kim, 1970) of algae. There are relatively few studies on the effect of salinity on the morphology and a n a t o m y of algae. One theory ( Levring, 1969 ) is t h a t lowered salinities cause reductions in form, i.e. smaller thalli. On the other hand, J o r d a n a n d Vadas (1972) demonstrated in Fucus vesiculosus L. t h a t branching and vesiculation increased with decreasing salinity. Other studies have shown t h a t salinity affects cell size. For instance, ~Presentaddress: Botany Department, Universityof California,Santa Barbara, CA 93106 (U.S.A.)
0304-3770/87/$03.50
© 1987 Elsevier Science Publishers B.V.
188 Ogata and Schramm (1971) found that cells of Porphyra umbilicalis (L.) J. Ag. grown at reduced salinities were smaller and lighter in color than cells grown at higher salinities, while DeSouza (1981) could find no relationship in Gracilaria verrucosa (Huds.) Papenf. between salinity and cell size. The current interest in farming economically useful algae has led to a demand for knowledge of the optimal growth conditions for such algae. During the course of a study on the effect of salinity on carrageenan composition and quality in Grateloupia filicina, a correlation was found between salinity and branch morphology. MATERIALSAND METHODS The experiments were carried out at Anuenue Fisheries Research Station at Sand Island, Oahu Island, Hawaii. Five identical fiberglass tanks (117 × 117 × 60 cm, approximately 820 1), one for each salinity to be tested, were aligned in series. Uniform water motion was provided by a paddle wheel system driven by a 1725 rpm motor modified with reduction gearing to turn the paddles at 26 rpm. The tanks were maintained outdoors and subject to ambient temperatures (20-25°C), no shade was provided. Salinity levels (15, 20, 25, 30 and 35%o ) were obtained by dilution of the Anuenue well-water (seawater, 35%o S) with fresh tap water. The seawater was first aged two weeks in an "aging" tank and then p u m p e d through a swimming pool filter to the growth tanks. The water was changed every two days and the tanks were scrubbed weekly to remove epiphytes. NO3-N was kept at 1.1 mg 1-1. To effectively eliminate genetic variability, thallus fragments of Grateloupia filicina were taken from a long running batch culture ( 35%o S) started from 2 germlings from the same parent that were allowed to fragment and grow vegetatively for several months. Thalli weighing 385 g (wet) were placed in each tank. The thalli were weighed weekly for 5 weeks. All thalli were removed from their respective tanks, rinsed with running seawater for 10-15 min and then, in a standard manner, shaken vigorously to remove excess water before weighing. A change in weight was recorded and a daily growth rate calculated using the following formula: [ ( W T f / W T i ) (l/x)-1] ×100 where WTf=final weight in grams, WTi=initial weight in grams and I= interval in days. The data were ranked and then analyzed by Friedman's method ( Sokal and Rohlf, 1969) for random blocks. Twenty segments at least 10 cm in length were selected at random from each salinity treatment. Each branch was assigned a number with respect to its position relative to the main axis. Branches originating on the main axis were assigned a first order ranking, branches originating on first order branches
189 6
~2
15
20 25 30 SALINITY Ippt]
35
Fig. 1. M e a n growth rates of Grateloupia filicina grown in five salinities from 21 April 1984 to 26 May 1984. T h e vertical lines in the bars indicate the s t a n d a r d error of the mean ( n = 5 ).
were assigned a second order ranking and so on. The number of first, second and third order branches within 5 cm of the apex of each thallus was recorded. All first order branches longer than I m m were measured and an average length of first order branches for each thallus was calculated. The data were analyzed with ANOVA. Significantly different means were contrasted using Scheff~'s multiple comparison test. RESULTS
The growth rates for Grateloupia filicina grown in the five salinity treatments decreased weekly. Overall growth rates (Fig. 1 ) ranged from 4.46% per day for thalli grown in 15%o S to 3.68% per day for thalli grown in 30%c salinity. There was no significant difference based on Friedman's method ( Friedman, 1937) for random blocks in growth rates between salinities tested. Figure 2 illustrates the morphological differences in thalli grown under the five different salinity treatments. The n u m b e r of first, second and third order branches (Figs. 2 and 3, Table I) increased with decreasing salinity. Analysis of variance (Table II) and Scheff~'s test (Scheff~, 1953) showed the number of first, second and third order branches to be significantly different ( a = 0.05), while first order branch length was not significantly different in the five salinities tested. There were three significantly different groups of salinities with respect to the number of first and second order branches (1) 15 %c, (2) 20-25 %c and (3) 30-35%c, and two groups of salinities for third order branches (1) 15%o and (2) 20-35%0. DISCUSSION
Grateloupia filicina can be maintained for m a n y m o n t h s in tank culture with growth rates of 6-12% per day (Zablackis, 1986) if the seawater is continu-
190
A
D
\
c
Fig. 2. Grateloupia filicina thalli grown at five salinities: (A) 15%o; (B) 20%o; (C) 25%o; (D) 30%o; and (E) 35%o. Scale=5 ram.
ously flowing and the tank population is thinned weekly to reduce overcrowding, mutual shading and nutrient depletion. The lower and decreasing weekly growth rates obtained here are possibly due to the lack of periodic reductions in biomass and lower nutrient and water turnover rates imposed in the experiment to maintain constant salinities.
191 400-
E:~ 1st r ~ 2nd I~ 3rd
¢n uJ 3ooz<
~2oo-
o
15
20
25
SAUNITY
30
35
[ppti
Fig. 3. Distributions of mean number of first, second and third order branches on 5-cm lengths of Grateloupiafilicina thalli grown in five salinities. The vertical lines in the bars indicate the standard error of the mean ( n = 20). No significant difference between growth rates and salinity was found. Howe v e r , m o r p h o l o g i c a l c h a n g e s o c c u r r e d i n t h a l l i ( F i g s . 2 a n d 3 ) g r o w i n g i n differing salinities. T h e n u m b e r of b r a n c h e s of each order i n c r e a s e d w i t h TABLE I Mean number of first (1), second (2) and third (3) order branches and length (L) of first order branches from 5-cm-long Grateloupia filicina thallus tips grown in five salinities ( S ), n = 20 S (%o)
1
2
3
L (mm)
15 20 25 30 35
83.8 67.6 59.9 37.7 35.5
352.4 181.7 151.2 15.1 12.9
51 19.9 11.8 0 0
6.96 7.69 12.4 10.9 13.4
TABLE II Analysis of variance and significant differences between salinity levels based on Scheff~'s test for the number of first, second and third order branches and the length of first order branches of
Grateloupia filicina Salinity (%0) 1
Analysis of variance Dependent variable
df
F
15
20
25
30
35
No. 1st order branches No. 2nd order branches No. 3rd order branches Length of 1st order branches
4 4 4 4
48.1 39.2 15.36 2.71
A A A A
B B B A
B B B A
C C B A
C C B A
1Salinities with the same letter are not significantly different, n ~- 20
192
decreasing salinity. Reduced salinities produced significantly greater numbers of first, second and third order branches. The results indicate that the overall thallus appearance of Grateloupia filicina is highly dependent on the salinity of the surrounding seawater, and further indicate that this species can indeed tolerate a fairly wide salinity range for extended periods of time, as suspected previously on field collections, without significantly affecting the growth rate. The increased branching described here is similar to the increased vesiculation and branching reported by Jordan and Vadas (1972) in Fucus vesiculosus, and may be a common response to reduced salinities. Recently Sideman and Mathieson (1985) showed that the major differences in morphology in Fucus distichus L. were genetically determined, but also that there was some morphological variation within populations which was environmentally controlled. These experiments show that the salinity level is one of the environmental parameters that can affect the morphology of Grateloupia
filicina. ACKNOWLEDGMENTS
I would like to thank the staff at Anuenue Fisheries Research Station, Honolulu, Hawaii for their assistance and use of their facilities. Many thanks to Karla McDermid for help in photography, typing and review of the manuscript. Finally I would like to thank Dr. M.S. Doty and Dr. I.A. Abbott for their guidance, many helpful discussions and their critical reviews of this work.
REFERENCES De Souza, Y.G., 1981. Relationship of salinity to morphological and physiological variation in estuarine populations ofGracilaria verrucosa. Masters Thesis, Biology Department, San Francisco State University, San Francisco, CA, 83 pp. (unpublished). Friedman, M., 1937. The use of ranks to avoid the assumption of normality implicitin the analysis of variance. J. Am. Stat. Assoc., 32: 675-701. Gessner, R. and Schramm, W., 1971. Salinity-Plants. In: O. Kinne (Editor), Marine Ecology, Environmental Factors. Part 2. Wiley, N e w York, pp. 705-820. Hang, A. and Larson, B., 1958. Influence of habitat on the chemical composition of Ascophyllum nodosum. Nature (London), 181: 1224. Jordan, A.J. and Vadas, R.L., 1972. Influence of environmental parameters on intraspecificvariation in Fucus vesiculosus. Mar. Biol., 14: 248-252. Kim, D.H., 1970. Economically important seaweeds in Chile-I.Gracilaria. Bot. Mar., 13: 140-162. Levring, T., 1969. The vegetation in the sea. In: T. Levring, H.A. Hoppe and O.J. Schmid (Editors),Marine Algae. A Survey of Research and Utilization.Botanica Marina Handbooks, Vol. 1, Cramer de Gruytar and Co., N e w York, pp. 1-46. Ogata, E. and Schramm, W., 1971. Some observations on the influence of salinityon growth and photosynthesis in Porphyra umbilicali,s. Mar. Biol., 10: 70-76.
193 Scheff$, H.A., 1953. A method for judging all contrasts in the analysis of variance. Biometrika, 40: 87-104. Sideman, E.J. and Mathieson, A.C., 1985. Morphological variation within and between natural populations of non-tide pool Fucus distichus (Phaeophyta) in New England. J. Phycol., 21: 250-257. Sokal, R.R. and Rohlf, F.J., 1969. Biometry. W.H. Freeman, San Francisco, 776 pp. Zablackis, E., 1986. Responces to salinity in the red algae Gracilaria and Grateloupia. M.Sc. Thesis, University of Hawaii, Honolulu, HI, 73 pp. (unpublished).
187
Elsevier Science Publishers B.V., Amsterdam-- Printed in The Netherlands
Short Communication T H E E F F E C T O F S A L I N I T Y ON G R O W T H R A T E A N D B R A N C H MORPHOLOGY IN TANK CULTIVATED GRATELOUPIA FILICINA (RHODOPHYTA) IN HAWAII
EARL ZABLACKIS'
Department o[ Botany, University of Hawaii, Honolulu, HI 96822 ( U.S.A.) (Accepted for publication 17 July 1986)
ABSTRACT Zablackis,E., 1987. The effect of salinity on growthrate and branch morphologyin tank cultivated Grateloupia filicina ( Rhodophyta ) in Hawaii.Aquat. Bot., 27: 187-193. The red algal species Grateloupia filicina {Lamour.) C. Agardh was grown in tank culture at fivesalinities.There was no statistically significantdifferencebetweengrowthrate at the different salinities tested. However,reduced salinity initiated production of significantlygreater numbers of branches in three branch orders in this species,thus demonstrating environmentalcontrol over branching due to salinity levels.
INTRODUCTION The red algal genus Grateloupia C. Agardh is of interest ecologically, chemically and commercially because it is common worldwide, often a conspicuous member of intertidal and nearshore algal communities and contains appreciable quantities of carrageenan (Zablackis, 1986) in its cell walls. In Hawaii, Grateloupia filicina (Lamour.) C. Agardh is almost always found in an area with some source of freshwater nearby and therefore is naturally subject to various salinity regimes. Salinity is an i m p o r t a n t environmental parameter {Gessner and Schramm, 1971 ) affecting distribution, growth, morphology and chemical composition (Haug and Larson, 1958; Kim, 1970) of algae. There are relatively few studies on the effect of salinity on the morphology and a n a t o m y of algae. One theory ( Levring, 1969 ) is t h a t lowered salinities cause reductions in form, i.e. smaller thalli. On the other hand, J o r d a n a n d Vadas (1972) demonstrated in Fucus vesiculosus L. t h a t branching and vesiculation increased with decreasing salinity. Other studies have shown t h a t salinity affects cell size. For instance, ~Presentaddress: Botany Department, Universityof California,Santa Barbara, CA 93106 (U.S.A.)
0304-3770/87/$03.50
© 1987 Elsevier Science Publishers B.V.
188 Ogata and Schramm (1971) found that cells of Porphyra umbilicalis (L.) J. Ag. grown at reduced salinities were smaller and lighter in color than cells grown at higher salinities, while DeSouza (1981) could find no relationship in Gracilaria verrucosa (Huds.) Papenf. between salinity and cell size. The current interest in farming economically useful algae has led to a demand for knowledge of the optimal growth conditions for such algae. During the course of a study on the effect of salinity on carrageenan composition and quality in Grateloupia filicina, a correlation was found between salinity and branch morphology. MATERIALSAND METHODS The experiments were carried out at Anuenue Fisheries Research Station at Sand Island, Oahu Island, Hawaii. Five identical fiberglass tanks (117 × 117 × 60 cm, approximately 820 1), one for each salinity to be tested, were aligned in series. Uniform water motion was provided by a paddle wheel system driven by a 1725 rpm motor modified with reduction gearing to turn the paddles at 26 rpm. The tanks were maintained outdoors and subject to ambient temperatures (20-25°C), no shade was provided. Salinity levels (15, 20, 25, 30 and 35%o ) were obtained by dilution of the Anuenue well-water (seawater, 35%o S) with fresh tap water. The seawater was first aged two weeks in an "aging" tank and then p u m p e d through a swimming pool filter to the growth tanks. The water was changed every two days and the tanks were scrubbed weekly to remove epiphytes. NO3-N was kept at 1.1 mg 1-1. To effectively eliminate genetic variability, thallus fragments of Grateloupia filicina were taken from a long running batch culture ( 35%o S) started from 2 germlings from the same parent that were allowed to fragment and grow vegetatively for several months. Thalli weighing 385 g (wet) were placed in each tank. The thalli were weighed weekly for 5 weeks. All thalli were removed from their respective tanks, rinsed with running seawater for 10-15 min and then, in a standard manner, shaken vigorously to remove excess water before weighing. A change in weight was recorded and a daily growth rate calculated using the following formula: [ ( W T f / W T i ) (l/x)-1] ×100 where WTf=final weight in grams, WTi=initial weight in grams and I= interval in days. The data were ranked and then analyzed by Friedman's method ( Sokal and Rohlf, 1969) for random blocks. Twenty segments at least 10 cm in length were selected at random from each salinity treatment. Each branch was assigned a number with respect to its position relative to the main axis. Branches originating on the main axis were assigned a first order ranking, branches originating on first order branches
189 6
~2
15
20 25 30 SALINITY Ippt]
35
Fig. 1. M e a n growth rates of Grateloupia filicina grown in five salinities from 21 April 1984 to 26 May 1984. T h e vertical lines in the bars indicate the s t a n d a r d error of the mean ( n = 5 ).
were assigned a second order ranking and so on. The number of first, second and third order branches within 5 cm of the apex of each thallus was recorded. All first order branches longer than I m m were measured and an average length of first order branches for each thallus was calculated. The data were analyzed with ANOVA. Significantly different means were contrasted using Scheff~'s multiple comparison test. RESULTS
The growth rates for Grateloupia filicina grown in the five salinity treatments decreased weekly. Overall growth rates (Fig. 1 ) ranged from 4.46% per day for thalli grown in 15%o S to 3.68% per day for thalli grown in 30%c salinity. There was no significant difference based on Friedman's method ( Friedman, 1937) for random blocks in growth rates between salinities tested. Figure 2 illustrates the morphological differences in thalli grown under the five different salinity treatments. The n u m b e r of first, second and third order branches (Figs. 2 and 3, Table I) increased with decreasing salinity. Analysis of variance (Table II) and Scheff~'s test (Scheff~, 1953) showed the number of first, second and third order branches to be significantly different ( a = 0.05), while first order branch length was not significantly different in the five salinities tested. There were three significantly different groups of salinities with respect to the number of first and second order branches (1) 15 %c, (2) 20-25 %c and (3) 30-35%c, and two groups of salinities for third order branches (1) 15%o and (2) 20-35%0. DISCUSSION
Grateloupia filicina can be maintained for m a n y m o n t h s in tank culture with growth rates of 6-12% per day (Zablackis, 1986) if the seawater is continu-
190
A
D
\
c
Fig. 2. Grateloupia filicina thalli grown at five salinities: (A) 15%o; (B) 20%o; (C) 25%o; (D) 30%o; and (E) 35%o. Scale=5 ram.
ously flowing and the tank population is thinned weekly to reduce overcrowding, mutual shading and nutrient depletion. The lower and decreasing weekly growth rates obtained here are possibly due to the lack of periodic reductions in biomass and lower nutrient and water turnover rates imposed in the experiment to maintain constant salinities.
191 400-
E:~ 1st r ~ 2nd I~ 3rd
¢n uJ 3ooz<
~2oo-
o
15
20
25
SAUNITY
30
35
[ppti
Fig. 3. Distributions of mean number of first, second and third order branches on 5-cm lengths of Grateloupiafilicina thalli grown in five salinities. The vertical lines in the bars indicate the standard error of the mean ( n = 20). No significant difference between growth rates and salinity was found. Howe v e r , m o r p h o l o g i c a l c h a n g e s o c c u r r e d i n t h a l l i ( F i g s . 2 a n d 3 ) g r o w i n g i n differing salinities. T h e n u m b e r of b r a n c h e s of each order i n c r e a s e d w i t h TABLE I Mean number of first (1), second (2) and third (3) order branches and length (L) of first order branches from 5-cm-long Grateloupia filicina thallus tips grown in five salinities ( S ), n = 20 S (%o)
1
2
3
L (mm)
15 20 25 30 35
83.8 67.6 59.9 37.7 35.5
352.4 181.7 151.2 15.1 12.9
51 19.9 11.8 0 0
6.96 7.69 12.4 10.9 13.4
TABLE II Analysis of variance and significant differences between salinity levels based on Scheff~'s test for the number of first, second and third order branches and the length of first order branches of
Grateloupia filicina Salinity (%0) 1
Analysis of variance Dependent variable
df
F
15
20
25
30
35
No. 1st order branches No. 2nd order branches No. 3rd order branches Length of 1st order branches
4 4 4 4
48.1 39.2 15.36 2.71
A A A A
B B B A
B B B A
C C B A
C C B A
1Salinities with the same letter are not significantly different, n ~- 20
192
decreasing salinity. Reduced salinities produced significantly greater numbers of first, second and third order branches. The results indicate that the overall thallus appearance of Grateloupia filicina is highly dependent on the salinity of the surrounding seawater, and further indicate that this species can indeed tolerate a fairly wide salinity range for extended periods of time, as suspected previously on field collections, without significantly affecting the growth rate. The increased branching described here is similar to the increased vesiculation and branching reported by Jordan and Vadas (1972) in Fucus vesiculosus, and may be a common response to reduced salinities. Recently Sideman and Mathieson (1985) showed that the major differences in morphology in Fucus distichus L. were genetically determined, but also that there was some morphological variation within populations which was environmentally controlled. These experiments show that the salinity level is one of the environmental parameters that can affect the morphology of Grateloupia
filicina. ACKNOWLEDGMENTS
I would like to thank the staff at Anuenue Fisheries Research Station, Honolulu, Hawaii for their assistance and use of their facilities. Many thanks to Karla McDermid for help in photography, typing and review of the manuscript. Finally I would like to thank Dr. M.S. Doty and Dr. I.A. Abbott for their guidance, many helpful discussions and their critical reviews of this work.
REFERENCES De Souza, Y.G., 1981. Relationship of salinity to morphological and physiological variation in estuarine populations ofGracilaria verrucosa. Masters Thesis, Biology Department, San Francisco State University, San Francisco, CA, 83 pp. (unpublished). Friedman, M., 1937. The use of ranks to avoid the assumption of normality implicitin the analysis of variance. J. Am. Stat. Assoc., 32: 675-701. Gessner, R. and Schramm, W., 1971. Salinity-Plants. In: O. Kinne (Editor), Marine Ecology, Environmental Factors. Part 2. Wiley, N e w York, pp. 705-820. Hang, A. and Larson, B., 1958. Influence of habitat on the chemical composition of Ascophyllum nodosum. Nature (London), 181: 1224. Jordan, A.J. and Vadas, R.L., 1972. Influence of environmental parameters on intraspecificvariation in Fucus vesiculosus. Mar. Biol., 14: 248-252. Kim, D.H., 1970. Economically important seaweeds in Chile-I.Gracilaria. Bot. Mar., 13: 140-162. Levring, T., 1969. The vegetation in the sea. In: T. Levring, H.A. Hoppe and O.J. Schmid (Editors),Marine Algae. A Survey of Research and Utilization.Botanica Marina Handbooks, Vol. 1, Cramer de Gruytar and Co., N e w York, pp. 1-46. Ogata, E. and Schramm, W., 1971. Some observations on the influence of salinityon growth and photosynthesis in Porphyra umbilicali,s. Mar. Biol., 10: 70-76.
193 Scheff$, H.A., 1953. A method for judging all contrasts in the analysis of variance. Biometrika, 40: 87-104. Sideman, E.J. and Mathieson, A.C., 1985. Morphological variation within and between natural populations of non-tide pool Fucus distichus (Phaeophyta) in New England. J. Phycol., 21: 250-257. Sokal, R.R. and Rohlf, F.J., 1969. Biometry. W.H. Freeman, San Francisco, 776 pp. Zablackis, E., 1986. Responces to salinity in the red algae Gracilaria and Grateloupia. M.Sc. Thesis, University of Hawaii, Honolulu, HI, 73 pp. (unpublished).