M .R. Lombardi et al. J. Exp. Mar. Biol. Ecol. 252 2000 75 –84
77
FRR fluorescence methods Kolber et al., 1998. New instruments have been developed that incorporate protocols to measure the multiple photochemical turnover
PAM, and single photochemical turnover of PSII FRR in the laboratory and in the field Schreiber et al., 1986; Gorbunov et al., 2000. In particular, PAM measurements of
fluorescence in corals have become widespread Brown et al., 1999; Ralph et al., 1999; Warner et al., 1996, 1999. Previous studies using PAM fluorescence protocols have
indicated that corals exposed to bleaching conditions, such as elevated seawater
9
temperature, yield progressively lower DF 9 F of PSII fluorescence values before they
m
bleach Warner et al., 1999. Similar results have been obtained with FRR fluorometers Lesser and Gorbunov, in press.
Since the quantum yield of PSII fluorescence is an important indicator of functional PSII units we examined the association of color variability corresponding with
9
zooxanthellae loss, or decrease of photosynthetic pigment, with changes in the DF 9 F
m
of PSII fluorescence. In this paper we describe the use of a newly described in situ FRR fluorometer Gorbunov et al., 2000, to examine the fluorescence yields of zooxanthellae
in hospite on corals recovering from a bleaching event due to elevated seawater temperatures.
2. Materials and methods
This study was conducted at the Caribbean Marine Research Center on Lee Stocking Island LSI, Bahamas during June, 1999. Preliminary dives were made on a number of
reef sites to determine the most appropriate methodology to conduct the work described below. Montastraea faveolata and Diploria labyrinthiformes were selected as the coral
species to be studied due to the variability in bleaching observed within their respective populations. Horseshoe Reef depth 10–12 m and Rainbow Gardens depth 3–4 m
were chosen as research sites, as they hosted populations of both coral species. Both of these sites were affected by a bleaching event that occurred in May 1998 by the
intrusion of warm maximum temperature 34–358C Bahama Bank water onto the Exuma island chain Lesser, personal observation. Both of these coral species bleached
around LSI and at the two study sites in June of 1998. At that time the pattern of bleaching observed was that whole colonies were bleaching with very little hetero-
geneity in color. That is, colonies largely conformed to the light coloration pattern described below Lesser, personal observation.
At each of the two sites, five colonies of Montastraea faveolata and three colonies of Diploria labyrinthiformes were identified and non-destructively tagged. These colonies
contained regions of color that were representative of the remaining visual bleaching damage across the entire population of these two species when this study was conducted.
Corals with dead regions were not used during this study. A 25325 cm quadrat was placed on each colony and photographed with a Nikonos V underwater camera 15 mm
lens. Three FRR fluorescence measurements were then taken within each quadrat. The location of the fluorescence measurements within a quadrat was carefully recorded to
correlate the respective fluorescence measurement with the visual color variability of the coral colony. Color variability reflected a range of bleaching from highly bleached to no
78 M
.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
m
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
9
protocol while illuminated is defined by F 9 steady state and F maximum
m
components of the yield Kolber et al., 1998. From these values the quantum yield of
9
PSII fluorescence DF 9 F can be calculated Gorbunov et al., 2000. The instrument
m
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
9
differences in DF 9 F at the 5 level. When significant treatment effects occurred, a
m
post hoc test Student–Newman–Keuls [SNK] was used to determine differences in
9
DF9 F between the three shades of coral cover. A two-way ANOVA was run for each
m
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.
9
Both DF 9 F and percent cover were arcsine transformed before analysis.
m
3. Results
