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.R. Lombardi et al. J. Exp. Mar. Biol. Ecol. 252 2000 75 –84
bleaching and was categorized into three groups, light, intermediate, and dark colored regions. This simple breakdown encompassed a much more variable color palette,
however three regions allowed for an easier analysis of the relationship between the variability of color and fluorescence yields within coral colonies. All measurements were
9
taken between 9 and 10 AM EST to avoid the mid-day depression in DF 9 F
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measured in these corals using FRR fluorescence protocols Lesser and Gorbunov, in press.
The FRRF exposes the coral to a sequence of flashlets that gradually close PSII reaction centers, resulting in an increase in chlorophyll fluorescence Kolber and
Falkowski, 1992. The fluorescence yields during the time of an FRRF excitation
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protocol while illuminated is defined by F 9 steady state and F maximum
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components of the yield Kolber et al., 1998. From these values the quantum yield of
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PSII fluorescence DF 9 F can be calculated Gorbunov et al., 2000. The instrument
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enables the diver to monitor and focus the target organism with a Marshall Electronics black and white video camera. A pair of IR laser diodes is incorporated into the LCD
display to aid in focussing and controlling distance to target. A piezo key trigger allows the diver to ‘‘shoot’’ the target. Upon shooting, an image is captured and stored Fig. 1,
as well as the associated fluorescence data. This instrument is described in greater detail by Gorbunov et al. 2000.
Each quadrat photograph was scanned using a Nikon slidescanner at 300-dpi resolution. The images’ gamma curves were adjusted using the visually segregated
regions of light, intermediate, and dark color variability within the colony. The same procedure was performed on all images. Using the PVC quadrat as a reference, each
image was cropped to the equivalent of 25325 cm. These cropped images were then resized to 4003400 pixels in Adobe Photoshop 5.0. The images were then highlighted
using Jasc Paint Shop Pro. The best representation of light, intermediate, and dark areas were isolated and highlighted to yield an image containing four colors including black
backdrop representing non-coral cover. Next, all highlighted images were adjusted to grayscale and saved Fig. 1. Finally, the three areas representing the different shades of
coral cover were quantified as percent cover using NIH Image v1.61.
A three-way ANOVA using species, site, and color as treatments was run to examine
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differences in DF 9 F at the 5 level. When significant treatment effects occurred, a
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post hoc test Student–Newman–Keuls [SNK] was used to determine differences in
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DF9 F between the three shades of coral cover. A two-way ANOVA was run for each
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of the color regions to examine the effects of site and species on the percent cover of each color region light, intermediate, and dark occupied on the coral colony surface.
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Both DF 9 F and percent cover were arcsine transformed before analysis.
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3. Results
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The three way ANOVA of log transformed fluorescence data DF 9 F revealed
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9
significant ANOVA: P 50.0004 treatment effects of color on DF 9 F Fig. 2. A post
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hoc SNK analysis of color revealed significant SNK: P ,0.05 differences between all treatment groups light, intermediate, and dark. These significant differences show that
M .R. Lombardi et al. J. Exp. Mar. Biol. Ecol. 252 2000 75 –84
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Fig. 1. Top: a Cropped 4003400 pixels, grayscale quadrat photo of Montastraea faveolata at the Horseshoe Reef site. b Highlighted light, intermediate, and dark regions of M
. faveolata. Bottom: Grayscale images captured from CX-100-30 frame grabber Imagination Vision Systems Specialists CCD camera portion
of the FRR fluorometer. c Montastraea faveolata and d Diploria labyrinthiformes.
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light regions contain zooxanthellae with lower DF 9 F of PSII fluorescence
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0.33760.014, mean6S.E., followed by intermediate 0.38260.012, and dark 0.43860.019 regions Fig. 2.
There was also a significant difference ANOVA: P 50.04 between sites all samples
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pooled, where DF 9 F of the shallower site Rainbow Gardens 0.37260.014 were
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significantly less than that of the deeper site Horseshoe Reef 0.40060.015 Fig. 3.
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There was no significant difference in DF 9 F of PSII fluorescence between species
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ANOVA: P .0.05 when all samples are pooled. Additionally, there were no significant interactions species3sites ANOVA: P .0.05.
80 M
.R. Lombardi et al. J. Exp. Mar. Biol. Ecol. 252 2000 75 –84
9 Fig. 2. Mean 6S.D. quantum yield of PSII fluorescence DF 9 F values for light N 516, 0.33760.014,
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intermediate N 516, 0.38260.012, and dark N 516, 0.43860.019 colored regions. Treatment groups with similar superscripts are not significantly different from one another.
A two-way ANOVA of arcsine transformed percent cover data did not reveal significant differences between species ANOVA: P .0.05 or sites ANOVA: P .0.05
for any of the color regions. However, there was a significant treatment interaction between species and site for the light region P 50.037. For the dark color region there
was also a significant treatment interaction between species and site P 50.021. Colonies of Montastraea faveolata were dominated mean6S.D., 68.567.00 by
9 Fig. 3. Mean 6S.D. DF 9 F
values for each site. Horseshoe Reef HR yields significantly higher quantum
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yield of PSII fluorescence N 524, 0.40060.015 values than the shallower site Rainbow Gardens RG N 524, 0.37260.013.
M .R. Lombardi et al. J. Exp. Mar. Biol. Ecol. 252 2000 75 –84
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Fig. 4. Percent cover Mean6S.D. of light, intermediate, and dark regions at for Montastraea faveolata and Diploria labyrinthiformes at each site. Dark regions are significantly greater in percent cover suggesting that
the coral colonies are in a state of recovery despite having variable quantum yields from bleached to unbleached regions.
darker, ‘‘healthier’’, color at Horseshoe Reef compared with light 13.764.33 and intermediate 17.865.95 regions Fig. 4. Although the dark color region also covers
a higher percentage of corals at Rainbow Gardens 49.367.47, there is an increased percentage of light 17.464.60 and intermediate 33.367.81 regions of color
compared to Horseshoe Reef. Diploria labyrinthiformes was also dominated by the darker color regions at both sites, however, the distribution of dark color at Rainbow
Gardens 73.4616.37 was greater than at Horseshoe Reef 45.761.63 showing a different pattern compared to that observed for M
. faveolata Fig. 4.
4. Discussion
