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. Matsunaga et al. J. Exp. Mar. Biol. Ecol. 241 1999 193 –205
to those in the Pacific Ocean coast Suzuki et al., 1995 although coastal waters in both coasts had similar major nutrient e.g. phosphate, nitrate concentrations; 2 the
coralline algae grew well at low iron levels found in Japan Sea coast Suzuki et al., 1995; 3 iron bioavailability under sufficient major nutrient e.g. phosphate, nitrate
condition plays an important role to promote oogonium formation, growth rate and pigment synthesis of the kelp Suzuki et al., 1994; 4 organically-complexed iron in
forested stream and river water promoted far more microalgal growth than iron oxides which are predominantly found in the Japan Sea coastal water Matsunaga et al., 1998;
5 iron enrichment by placing iron frames in crust-dominated community stimulated macroalgal growth Matsunaga et al., 1994. Interestingly, however, three years after the
placement of iron frames, coralline algal growth recovered and replaced macroalgal growth on the frames unpublished observation. This suggested that reduced iron
bio-availability may not be the only factor involved in the selection of crustose coralline alga over kelp growth.
We hypothesized that, besides decreasing the potential for organic iron complexation, the reduction of humic substances associated with deforestation had another direct effect
leading to the selection of crustose coralline alga over kelp communities. That is, forest- or river-derived humic compounds may act as inhibitors of crustose coralline algal spore
germination. In this paper, the effects of humic substances on the tetraspore growth of crustose coralline algae and macroalga were investigated.
2. Materials and methods
2.1. Effect of humic substances on the tetraspores growth of crustose coralline algae Lithophyllum spp.
Humic soils was extracted with 0.1 N NaOH, centrifuged, and pH adjusted to 2. After centrifugation, pH of the supernatant was adjusted to 7 with 0.1 N NaOH, and the
supernatant was filtered through a 0.45-mm HA filter membrane. Filtrate and precipitated humic acid after dissolving in NaOH and neutralizing were used for the culture
experiments. Triplicate tissue culture flasks containing 30 ml of filtered seawater 34 p.s.u. were prepared for the experiment. Tetraspores were obtained from mature
coralline alga, Lithophyllum spp. collected at the coastal region in the northern Japan Sea, in August and September, 1993. Tetraspores were adhered onto a glass plate
2.5 3 2.5 cm in filtered seawater 0.45 mm. Number of tetraspores on the plate was counted by a light microscopy initial number: N ¯ 100. The tetraspores on the plate
were immediately transferred to 30 ml of medium. The test media were prepared by the addition of 100 ml of each fulvic and humic acid to make a final concentration of 2 and 1
21
mg l , respectively, to filtered seawater containing nitrate 500 mM and phosphate5
mM. The culture experiment of tetraspores was carried out under the following
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conditions; 80 mE cm s
, 12-h light dark cycle and 108C. After 10 days, the number of germinate alga grown with doubling cell was counted survival number: N . The
t
experiment was repeated five times. The survival ratio and the normality were defined by
K . Matsunaga et al. J. Exp. Mar. Biol. Ecol. 241 1999 193 –205
199
the following equations: R 5 N N 3 100 and Normality 5each survival
5 t
ratio mean survival ratio3100. Error bar shows S.D. n 55.
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The culture experiment of the spores with and without 5 mg l of fulvic acid or
humic acid was also carried out. After 3 or 10 days, the photographs of the spores in both media were taken by a light microscope. The range of humic or fulvic acid used for
this experiment was decided by their ambient concentration measured in the study sites. The concentration of aggregated humic substances in surface seawater at the four river
21
mouths 10–300 m off the coast was found to be a range between 1–10 mg C l by a
fluorescence spectrophotometric method mentioned below. The fluorescence spectrum of humic substances used for these experiments matched the fluorescence spectrum of
organic substances from Pacific Ocean and Japan Sea coasts with riverine influence.
2.2. Effect of different iron species on oogonium formation of Laminaria religiosa Zoospores were obtained from mature sporophytes of Laminaria religiosa which were
harvested at a coastal region in the northern Japan Sea, Hokkaido, in November 1996. The zoospores were adhered onto glass plates 2.532.5 cm in 30 ml of the sterilized
filtrate 0.45 mm sea water. The number of zoospores male and female on each plate was about 150 cells. The iron enrichment media were prepared by the addition of
21
amorphous Fe, fulvic acid–Fe the concentration was adjusted to 1 mg C l , and
nitrate and phosphate. The final concentrations of iron, nitrate and phosphate were 0.2 mM, 500 and 30 mM in all media, respectively. Filtered 0.45 mm, autoclaved natural
sea water ,10 nM Fe, 500 mM nitrate, and 30 mM phosphate was used for the control.
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After 10 days cultivation under the following conditions 108C; 40 mE m s
; 12-h light dark cycle, the number of female gametophytes of L
. religiosa on the glass plates was counted by an optical microscope. The percentages of oogonium formation after 25,
30 and 35 culture days were calculated by the following equation: Oogonium formation rate
5 Number of oogoniums at 25, 30 and 35 days Number of female gametophytes at 10 days 3 100
2.3. Organic substances flux measurement at the Japan Sea and the Pacific Ocean
13
coast and d C in the organic substances
Organic substances were captured with a sediment trap 8Ø350 cm which was settled at 2-m depth from the sea surface for 2–4 h at the four stations A–D in Fig. 1 at
the Japan Sea side and two locations E and F in Fig. 1 at the Pacific Ocean coast during summer 1996–1998, water depth was 5 m, 50–100 m away from the land. Water
in the sediment trap was filtered through a 0.45-mm GF F filter to collect organic matter. Collected organic matter was dissolved in 10 ml of 0.1 N NaOH for 5 h, followed by
neutralization with HCl total organic carbon was analyzed by a total organic carbon analyzer. The fluorescence spectrum of isolated organic matter was analyzed by a
fluorescence spectrophotometer.
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. Matsunaga et al. J. Exp. Mar. Biol. Ecol. 241 1999 193 –205
Isotope ratios in the organic matter collected in 1998 were also measured by a Finnigan Mat Delta plus mass spectrometer, and expressed in per millilitre deviations
from a standard as defined by the following equation:
13
d C 5 R R
2 1 3 1000
sample standard
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where R means C C and Peedee belemnite PDB was used as a standard. The
standard deviation was 60.2‰. 3. Results and discussion
As Berner et al. 1978 showed that humic substances can inhibit inorganic precipitation of calcium carbonate, we demonstrated that fulvic and humic acids isolated
from forest soils inhibited germination of crustose coralline algal spores Fig. 4. Fulvic
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acid completely destroyed tetraspores of Lithophyllum spp. at 5 mg C l after 3 days
incubation Fig. 5. This concentration is similar to ambient levels measured in the Pacific Ocean coast of Hokkaido Island. In contrast, the forest-derived fulvic acid–iron
complex found in the Pacific Ocean coast promoted the highest formation rate 79 of macroalgal oogonium compared to other forms of iron 41 e.g. less bioavailable
ferric oxides that are predominantly found in the Japan Sea coast Fig. 6.
Comparisons of ambient dissolved organic substance composition on two sides of the
Fig. 4. Effect of humic substances on the tetraspores growth of crustose coralline algae Lithophyllum spp.. FA, fulvic acid; HA, humic acid.
K . Matsunaga et al. J. Exp. Mar. Biol. Ecol. 241 1999 193 –205
201
Fig. 5. Effect of humic substances on Lithophyllum spp. Tetraspores of Lithophyllum spp. were destroyed at 5
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mg C l of isolated humic substances and fulvic acid 3 days. A Humic substances after 3 days, B fulvic
acid after 3 days, and C control 10 days. After 3 days, the color of spores turned from pink to black and the spores started to be destroyed. At 10 days, all spores were destroyed completely.
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Fig. 6. Effect of different iron species on oogonium formation of Laminaria religiosa. Fulvic–iron complex promoted the best oogonium formation rate 79 of L
. religiosa at 35 days compared to amorphous Fe 41 and control 3 n 52.
southern Hokkaido Island further supported the effect of terrestrial humic substance on coastal ecosystem. Field surveys indicate that the organic substance flux in coastal water
with riverine influence on both sides of Hokkaido Island is much higher 25–83 mg C
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m h
than that in the crust-dominated Japan Sea coast 1 mg C m h
Table 1. Also, organic substances collected from Pacific Ocean and Japan Sea coasts with
riverine influence were found to have originated from forest-derived humic substances, and the fluorescence spectrum of organic substances in these waters matched the pattern
of fulvic acids from terrestrial origin Fig. 7. In contrast, the fluorescence spectrum of organic substances from the crust-dominated Japan Sea resembled that from phyto-
plankton exudates, and not fulvic acid Fig. 7.
The isotope ratio collected in C, D and F in 1998 was 226.160.4‰ n 53 which
corresponds to the ratio in the aggregated organic matter suspended matter in the river
Table 1 Organic substances flux measurement at the Japan Sea coast and the Pacific Ocean coast n 51
Japan Sea coast Pacific Ocean coast
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Location mg C m
h Location
mg C m h
A 1
E 29
B 83
F 42
C 25
D 38
K . Matsunaga et al. J. Exp. Mar. Biol. Ecol. 241 1999 193 –205
203
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Fig. 7. Fluorescence spectra of organic substances 2 mg l as carbon collected by the sediment traps at
various sampling locations The emission wavelength is 460 nm. A Pattern of organic substances collected at stations B–F in Fig. 1 kelp forest community matched the patterns of fulvic acid and humic acid. B
Pattern of organic substances collected at station A in Fig. 1 crust-dominated community matched the patterns of phytoplankton, Chaetoceros spp. and kelp, Laminaria spp. Both dashed lines show the trap
samples.
mouths at about 10 p.s.u. salinity 226.060.3‰, n 59. The ratio in mountain soil was 226.460.4 n56. The ratios collected in E and A were 225.260.6 n53 and
221.060.5 n52, respectively. The former means that the organic matter collected in the traps contained small amounts of phytoplankton. The latter was close to the ratio
220.860.5, n 510 in phytoplankton collected in both the Japan Sea and Pacific Ocean sides. These compositional difference are consistent with the predominant influence of
terrestrially-derived humic substances in the Pacific Ocean coastal waters, but a
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negligible influence in the crust-dominated community in the southern Hokkaido of Japan Sea waters. Forested watershed changes associated with coastal development are
reported to significantly alter the quality, quantity, and timing of organic substances in streams draining into coastal water by increasing impervious surfaces Wahl et al.,
1996. Therefore, evidence is strong that selection for crustose coralline algae is related to the reduction in the supply of forest derived humic substances. It is apparent that the
replacement of kelp community by crustose coralline algal communities in the southern Hokkaido Island in the Japan Sea is linked to deforestation associated suburban coastal
development.
4. Conclusion
