The photosynthetic resilience of Porites furcata to salinity disturbance
Derek Manzello ()) 
Diego Lirman
Rosenstiel School of Marine and Atmospheric Science
University of Miami
4600 Rickenbacker Cswy.
Miami, FL 33149
E-mail: dmanzello@rsmas.miami.edu
Tel.:  305-361-4389  
Fax:  305-361-4292
Keywords	 Porites furcata - Biscayne Bay – Salinity – Photosynthesis
Introduction
Benthic habitats of Biscayne Bay, a shallow lagoon adjacent to the city of Miami, Florida, contain a limited number of coral species that represent a subset of the species found at nearby reefs of the Florida Reef Tract.  The physical characteristics of this basin (i.e., variable salinity and temperature, high sedimentation) make it a marginal environment for coral growth (Lirman et al. 2003).  Salinity fields of Biscayne Bay are influenced by freshwater inputs from canal, surface, and groundwater sources that create a near-shore environment with low mean salinity and high salinity fluctuation.  For example, salinity fell to levels below 25 ppt in this area for 188 days in 1998 and 156 days in 1999 (Lirman et al. 2003).  Siderastrea radians and Porites furcata are the most abundant coral species found along this near-shore environment (Lirman et al. 2003), suggesting their high tolerance to low and variable salinity.  In this study, we test the hypothesis that a high physiological tolerance for salinity fluctuations enables P. furcata to occupy these marginal environments.      
	Osmotic stress has long been recognized as a limiting physical parameter for marine organisms, as emphasized by the negative correlation observed between marine species and salinity levels in estuarine environments (Wells 1961).  Changes in salinity are known to pose a significant metabolic drain on marine organisms, and disrupt normal cellular electrochemical processes, enzyme kinetics, and nerve conduction (Vernberg and Vernberg 1972).  In corals, sudden changes in salinity are known to negatively impact reproduction (Richmond 1993), photosynthesis and respiration (Muthiga and Szmant 1987; Moberg et al. 1997; Nystrom et al 1997; Porter et al. 1999), and survivorship (Marcus and Thourhaug 1981; Coles 1992; Krupp et al. 1998).  Corals are considered to be stenohaline with a limited ability to adapt to or survive salinity changes (Wells 1957; reviewed by Coles and Jokiel 1992).  Mortality in corals is predicted to occur at salinities lower than 25 ppt (Edmondson 1928) or higher than 45 ppt (Jokiel et al. 1974).  However, other studies suggest that corals can tolerate both low (17.5-28 ppt) (Vaughn 1919; Edmondson 1928) and high salinities (38.5-52.5 ppt) (Edmondson 1928; Wells 1957).  In this study, we evaluate the photosynthetic response of P. furcata to sub-optimal salinity (20-45 ppt) levels in a series of exposure experiments of different duration (2 to 24 hr). 
Methods
Colonies of Porites furcata were collected from Biscayne Bay, FL in May 2001 and transferred to outdoor mesocosms.  The colonies collected (n = 2, 25 cm in diameter) were taken from adjacent clumps.  Considering the high degree of fragmentation and asexual recruitment in this branching species (Soong 1991) it was assumed that these colonies were ramets from a common genet.  Colonies were divided into small, cylindrical pieces (2-3 cm in height) and allowed to acclimate to ambient conditions for at least one week prior to the exposure experiments.  Ambient conditions in the outdoor flow-through mesocosms was characterized by normal oceanic salinities (35 ppt) with a natural light environment shaded with UV filters to reduce the potential for photodamage in collected corals.  Only healthy coral fragments with dark pigmentation and fully extended polyps were used and fragments were assigned blindly to each treatment.
Fragments of P. furcata were exposed to salinities of 20, 25, 35 (controls, ambient
salinity), and 45 ppt for intervals of 2, 4, 6, and 24 hours within aerated 20-liter
containers placed within a water table to control temperature.  Elevated salinities were
achieved by the addition of Instant Ocean salts to seawater whereas low salinities were
created through the addition of freshwater to seawater.  Salinity was checked at regular intervals (i.e., every 2 hours) except for the 24 hr exposures where salinity was
checked upon leaving the lab at ~ 1700 hrs and returning the next day at 0700 hrs. 
Salinity did not change overnight in the aerated 20-liter buckets.  All exposure experiments were carried out under ambient light inside an outdoor greenhouse.   Coral fragments (n=5 per treatment) were placed in translucent and opaque 1-liter containers with both experimental and control salinities, and then sealed airtight to prevent gas diffusion.  Concurrently, control light and dark containers with both experimental and control salinities were set up without coral fragments to account for photosynthesis and respiration due to the plankton and bacterial components of seawater.  Corals (n=5) and water controls (n=4) were kept within the sealed containers for 2 hours, after which dissolved oxygen was measured with a YSI-3000 oxygen electrode.  The surface area of each fragment was calculated assuming a cylindrical shape, and rates of photosynthesis and respiration were normalized to the area of tissue (McCloskey et al. 1978).  To reduce the amount of error due to the daily variation in photosynthesis, as well as the variable light intensity during incubation times, the parameters obtained during each experiment were compared only to those obtained for control corals that were run simultaneously during each exposure.  Net photosynthetic rates were expressed as changes in mg O2 cm-2 hr-1.  Relative photosynthesis was expressed as the percent deviation from the mean value obtained for the corresponding coral controls.
To evaluate recovery, coral fragments were returned to ambient salinity following exposure to sub-optimal salinity.  During this recovery phase, corals were placed in flow-through, aerated tanks.  Recovery periods were 24 hr for corals exposed to salinity treatments for 2, 4, and 6 hrs, whereas corals exposed for 24 hr were left to recover for both 24 hr and 7 days.  Photosynthesis and respiration rates were measured for these corals under ambient salinity conditions using the methods previously described.
Results and Discussion
Figure 1
Changes in salinity were found to influence photosynthesis of Porites furcata based on salinity values and exposure length.   The initial response (i.e., after 2 hours) of corals exposed to all sub-optimal salinity treatments was a significant drop in relative photosynthetic rates (ANOVA, P < 0.05, Tukey a posteriori comparisons) (Fig. 1).  After sudden exposure, P. furcata fragments displayed a full retraction of polyps and a copious production of mucus qualitatively indicating colony stress.  
Table 1
Significant reductions in net photosynthesis were also documented for colonies exposed to 20 ppt and 25 ppt from 2-24 hr, and for colonies exposed to 45 ppt after 2 and 6 hr of exposure (t-tests, P < 0.05) (Table 1).  While reductions in mean net photosynthesis compared to controls were measured for corals exposed to 45 ppt for 4 and 24 hrs, these differences were not significant (Table 1).  It is important to note that no tissue mortality was observed under any of the treatments used in this study despite the documented decreases in relative and net photosynthesis.  The reported tolerance of P. furcata to salinity fluctuations is similar to that described for its congeneric P. porites, which can withstand salinity deviations from 20 to 40 ppt (Marcus and Thouraug 1981).
The magnitude of the initial (i.e., after 2 hours) as well as the final (i.e., after 24 hr) decrease in relative photosynthesis was influenced by salinity level.  The biggest decrease was documented for corals exposed to 20 ppt and the smallest for corals exposed to 45 ppt (Fig. 1).  Following the initial significant decrease, fragments of P. furcata displayed short-term recovery responses.  For example, corals exposed to 20 and 45 ppt exhibited an increase in relative photosynthesis after 4 hr (Fig. 1).  A similar recovery response was observed between 6 and 24 hr of exposure for colonies exposed to 20 and 45 ppt.  Coral colonies exposed to 25 ppt showed an increase in relative photosynthesis from 4 to 6 hr of exposure and no change between 6 and 24 hr (Fig. 1).  For corals exposed to low salinities, these recovery responses were short-lived, and relative photosynthesis after 24 hr remained below that recorded after the initial decrease.  However, the short-term recovery response of corals exposed to high salinity resulted in higher values of relative photosynthesis at 4 and 24 hr compared to those recorded after 2 hr (Fig. 1).
	Respiration rates of experimental corals did not differ significantly from controls and followed no apparent trends. A Pg/R (gross photosynthesis/respiration) ratio > 1.0 indicates that photosynthetically fixed carbon exceeds basal metabolic demands, whereas a Pg/R ratio < 1.0 implies that the coral is utilizing more carbon than what is fixed through photosynthesis (McCloskey et al. 1978).  In this study, Pg/R ratios of experimental corals never fell below one, indicating the maintenance of autotrophy while subjected to salinity stress during daylight hours (Table 1) (McCloskey et al. 1978).  
Figure 2
Photosynthetic recovery was rapid for P. furcata fragments exposed to sub-optimal salinity for 2, 4, and 6 hr.  Following 24 hr of recovery, photosynthetic rates increased for all treatments (except those exposed to 45 ppt for 24 hr), highlighting the metabolic resilience (i.e., rate of return to a non-disturbed state) of P. furcata.  The lower mean relative photosynthetic rate for corals exposed to 45 ppt for 24 hrs following a day of recovery is likely due to the high variability in net photosynthesis for this treatment (Fig. 2) given there was no significant difference in net photosynthesis between experimental and control corals initially after the exposure period (Table 1).  Recovery was slower for corals exposed for prolonged periods of time (24 hr), but all treatments recovered completely after one week indicating that P. furcata can withstand acute and prolonged salinity stress without lasting physiological impairment.  
It has been proposed that the ability of hermatypic corals to tolerate salinity stress is due to a behavioral response of the coral to retract its polyps under osmotic stress, thus decreasing the amount of tissue surface area in contact with the surrounding seawater (Muthiga and Szmant 1987). The largest percent decrease in photosynthesis seen in the first two hours may be explained by the initial retraction of the polyps documented here, which may cause a decrease in the intensity and amount of photosynthetically active radiation (PAR) reaching the zooxanthellae.
The subsequent short-term recovery or acclimation response documented here for fragments of P. furcata may represent a limited capacity for osmoregulation.  The possibility of osmoregulation in corals has been dismissed as corals have been historically considered osmoconformers (Wells 1957; reviewed by Coles and Jokiel 1992).  However, Vernberg and Vernberg (1972) note that an organism can be both an osmoconformer and euryhaline through the toleration of tissue dilution and the likely dependence on more cellular osmoregulatory abilities.   Notably, a freshwater Cnidarian in the genus Hydra was shown to have the ability to osmoregulate (Lilly 1955).  The results reported here warrant further study into the possible cellular mechanism(s) responsible for the documented physiological acclimation and tolerance response.  
The ability of corals found in marginal habitats to survive both short as well as longer-term salinity fluctuations is essential for their persistence in such environments.  While sub-optimal salinity patterns can persist in the near-shore environment of coastal lagoons like Biscayne Bay for several days, short-term (< 24 hrs) changes in salinity are not uncommon as a result of canal runoff or storm events.  In this study, we document the following aspects of the response of P.  furcata to salinity stress that allow this species to persist in such habitats:  1) the capability of surviving sudden changes in salinity without tissue mortality, 2) Pg/R ratios never falling below one, indicating the maintenance of autotrophy while subjected to salinity stress, 3) acclimation to short-term changes in salinity, and 4) rapid recovery of photosynthetic rates after a return to ambient salinity.  Finally, the documented resistance and resilience of this species to salinity stress correlates well with its reported distribution within near-shore environments of Biscayne Bay where it can reach densities of up to 5 colonies m-2 (Lirman et al. 2003).  
	In sum, we report a high tolerance to salinity fluctuation in P. furcata, which may play a critical role in the ability of this coral to inhabit marginal reef-environments that limit the distribution of other hermatypic coral species.  Similarly, the physiological tolerance of P. furcata documented here likely acts as a determining factor in the population structure of coral communities exposed to frequent salinity changes.

Acknowledgements
Financial support was provided by NOAA Coastal Ocean Program (Award #NA67RJ0149) and EPA STAR Program (#R-827453-01-0).  Richard Curry, Science Director of Biscayne National Park, provided field support for this project.  This manuscript was greatly improved by comments from D. DiResta, P. W. Glynn, P. Fong and two anonymous reviewers.  Field and laboratory assistance was kindly provided by L. Kaufman, B. Orlando, and P. Biber.  I would like to express my gratitude to the guest editors of this issue, C. Perry and P. Larcombe for their help and support.  This work was partly carried out under the requirements of a thesis submitted to the University of Miami for the distinction of Summa cum laude graduation honors. 

References
Coles SL (1992) Experimental comparison of salinity tolerances of reef corals from the 
Arabian Gulf and Hawaii. Proc 7th Int Coral Reef Symp 1: 227-234
Coles SL, Jokiel PL (1992) Effects of salinity on corals. In: Connell DW, Hawker DW 
(eds) Pollution in tropical aquatic systems. CRC Press, Boca Raton, FL, pp 147-166 
Edmondson CH (1928) The ecology of a Hawaiian coral reef. Bull Bernice P Bishop Mus 
45: 1-64
Jokiel PL, Coles SL, Guither EB, Key GS, Smith SV, Townley SJ (1974) Final Report, 
EPA Project No. 18050 DDN
Krupp DA, Hunter CL, Jokiel PL, Neves E, Gilette PA (1998) The Phoenix Effect: 
regeneration of cryptic residual tissue in Hawaiian corals subjected to osmotic 
stress. Am Zool 37(5): 56A
Lilly SJ (1955) Osmoregulation and ionic regulation in Hydra. J Exp Biol 23: 423-439
Lirman D, Orlando B, Macia S, Manzello D, Kaufman L, Biber P, Jones T (2003) 
Coral communities of Biscayne Bay, Florida and adjacent offshore areas: Diversity, abundance, distribution, and environmental correlates. Aquatic Conserv: Mar Freshw Ecosyst 13: 121-135.
Marcus J, Thouraug A (1981) Pacific versus Atlantic responses of the subtropical 
hermatypic coral Porites spp to temperature and salinity effects. Proc 4th Int Coral Reef Symp 2: 15-20
McCloskey LR, Wethey DS, Porter JW (1978) Measurement and interpretation of 
photosynthesis and respiration in corals. In: Coral Reefs: Research Methods. UNESCO Monographs on Oceanographic Methodology, No. 5. UNESCO, pp 379-396
Moberg F, Nystrom M, Kautsky N, Tedengren M, Jarayabhand P (1997) Effects of 
reduced salinity on the rates of photosynthesis and respiration in the hermatypic corals Porites lutea and Pocillopora damicornis. Mar Ecol Prog Ser 157: 53-59
Muthiga NA, Szmant AM (1987) The effects of salinity stress on the rates of aerobic 
respiration and photosynthesis in the hermatypic coral Siderastrea siderea. Biol Bull 173: 539-551
Nystrom M, Moberg F, Tedengren M (1997) Natural and anthropogenic disturbance on 
reef corals in the inner Gulf of Thailand: physiological effects of reduced salinity, copper, and siltation. Proc 8th Int Coral Reef Symp 2: 1893-1898
Porter JW, Lewis SK, Porter KG (1999) The effect of multiple stressors on the Florida 
Keys coral reef ecosystem: A landscape hypothesis and a physiological test. Limnol Oceanogr 44: 941-949
Richmond RH (1993) Effects of coastal runoff on coral reproduction. In: Ginsburg RN 
(ed) Proc Colloquium on Global Aspects of Coral Reefs: Health, Hazards, and History, Rosenstiel School of Marine and Atmospheric Science, Miami, pp 360-364 
Soong K (1991) Sexual reproductive patterns of shallow-water reef corals in Panama. 
Bull Mar Sci 49: 832-846.
Vaughn TW (1919) Corals and the formation of coral reefs. Ann Rep Smithsonian Inst 
17: 189-238
Vernberg FJ, Vernberg WB (1972) Environmental physiology of marine animals. 
Springer-Verlag, New York, NY
Wells JW (1957) Corals. Geol Soc Am Mem 67: 1087
Wells JW (1961) The fauna of oyster beds, with special reference to the salinity factor. 
Ecol Monographs 31: 239-266

Figure Legend

Figure 1.  Relative photosynthesis of experimental colonies of Porites furcata subjected to 20, 25, and 45 ppt for 2, 4, 6, and 24 hr.  Relative photosynthesis was expressed as the percent deviation from the mean value obtained for the corresponding coral controls. 
20 ppt. One-factor ANOVA, DF=4, F=21.1, P < 0.0001, Tukey a-posteriori comparisons
25 ppt. One-factor ANOVA, DF=4, F=20.1, P < 0.0001, Tukey a-posteriori comparisons
45 ppt. One-factor ANOVA, DF=4, F=3.7, P = 0.02, Tukey a-posteriori comparisons
Figure 2. Comparison of relative photosynthesis of Porites furcata after initial exposure and 24 hrs and 7 days of recovery for all treatments. Relative photosynthesis was expressed as the percent deviation from the mean value obtained for the corresponding coral controls.

Table 1. Net photosynthesis and Pg/R ratios of experimental and control colonies of Porites furcata for each salinity exposure.  Control corals exposed to ambient salinity of 35 ppt.  Signficant differences between control and experimental corals were determined using Student’s t-test.  Values represent means (+/- sem).
Salinity 	Duration	Net Photosynthesis	
(mg O2 cm-2 h-1)	 	Gross Photosynthesis/Respiration	
(ppt)	 of	 	 		Pg/R	 	
 	Exposure (hr)	Experimental	Control	p value	Experimental	Control	
20	2	2.77 (0.41)	10.2 (2.1)	0.011	1.48	3.57	
20	4	3.76 (0.60)	11.9 (1.0)	0.0017	2.32	4.40	
20	6	1.75 (0.55)	16.3 (3.0)	0.0036	1.31	5.32	
20	24	2.62 (0.43)	10.3 (1.4)	0.0019	1.52	3.24	
25	2	5.35 (0.53)	10.8 (2.3)	0.04	3.01	3.22	
25	4	3.98 (0.85)	13.5 (1.7)	0.0012	2.10	3.68	
25	6	4.38 (0.38)	 11.2 (2.5)	0.025	2.24	2.84	
25	24	5.70 (1.3)	14.8 (1.8)	0.0021	1.42	4.80	
45	2	3.64 (0.73)	6.28 (0.67)	0.014	1.88	2.69	
45	4	3.11 (0.86)	4.25 (1.72)	ns	1.80	2.57	
45	6	3.51 (0.71)	8.00 (0.39)	< 0.001	1.74	2.81	
45	24	3.51 (0.53)	5.24 (1.56)	ns	2.13	2.51