PHOSPHORUS LIMITATION IN REEF MACROALGAE OF SOUTH FLORIDA
by
Courtney Kehler
A Thesis Submitted to the Faculty of
The Charles E. Schmidt College of Science
In Partial Fulfillment of the Requirements for the Degree of
Master of Science
Florida Atlantic University
Boca Raton, Florida
December 2012
ii ACKNOWLEDGEMENTS
The author would like to thank the staff and other graduate students at HBOI for their immense help in preparing this thesis and navigating the graduate requirements. I want to thank my advisor, Dr. Brian Lapointe, for his guidance, help in the field and use of his lab and data. A special thanks to Laura Herren, without her assistance my thesis would have never been completed. Finally, thanks to my parents for their endless support and love.
iii ABSTRACT
Author: Courtney Kehler
Title: Phosphorus Limitation in Reef Macroalgae of South Florida
Institution: Florida Atlantic University
Thesis Advisor: Dr. Brian Lapointe
Degree: Master of Science
Year: 2012
Nitrogen (N) has traditionally been regarded as the primary limiting nutrient to algal growth in marine coastal waters, but recent studies suggest that phosphorus (P) can be limiting in carbonate-rich environments. To better understand the importance of P, alkaline phosphatase activity (APA) was measured in reef macroalgae in seven counties of south Florida; several significant trends emerged: 1) APA decreased geographically from the highest values in Dade > Monroe > Palm Beach > St. Lucie > Broward > Martin
> Lee counties 2) APA varied temporally with increasing nutrient-rich runoff in the wet season 3) APA varied due to taxonomic division Phaeophyta > Rhodophyta >
Chlorophyta 4) Nutrient enrichment experiments demonstrated that increased N- enrichment enhanced P-limitation while increased P decreased P-limitation. These results suggest that high APA observed in carbonate-rich waters of Dade County and low APA in Broward County resulted from high nutrient inputs associated with anthropogenic nutrient pollution.
iv PHOSPHORUS LIMITATION IN REEF MACROALGAE OF SOUTH FLORIDA
List of Figures………………………………………………………………………...... vii
List of Tables……………………………………………………………………………..ix
Introduction…………………………………………………………………………….….1
Hypotheses………………………………………………………………………...6
Methods………………………………………………………………………………...... 7
Study Areas………………………………………………………………………..7
Measurement of Alkaline Phosphatase Activity…..…………………………...... 9
Nutrient Enrichment Experiment...... 11
Results………………………………………………………………………………...... 13
Spatial Variation………………………………………………………………....13
Temporal Variation...….…...... 14
Taxonomic Variation...... 16
Nutrient Enrichment Experiment…………………………………………...... 17
Discussion………………………………………………………………………………..20
Spatial Variation…………………………………………………………………21
Temporal Variation………………………………………………………...... 26
Taxonomic Variation…………………………………………………………….27
Nutrient Enrichment Experiment...……………………….……………………...29
v Conclusions………………………………………………………………………...... 31
Appendix…………………………………………………………………………………33
References………………………………………………………………………………..49
vi FIGURES
Figure 1. Macroalgae sampling location in south Florida……………………………….33
Figure 2. Spatial variation in mean APA……….……………………….……………….34
Figure 3. Taxonomic variation in mean APA……….………………………….………..34
Figure 4. Temporal variation in mean APA at Looe Key, Monroe County.…………….35
Figure 5. Temporal variation in mean APA at Pepper Park, St. Lucie County……….....35
Figure 6. APA of Cladophora catenata collected at Looe Key, Monroe County, in
response to different levels of DIN and SRP enrichment .…………………..……....36
Figure 7. APA of Taonia sp. collected at Looe Key, Monroe County, in response to
different levels of DIN and SRP enrichment ……………………….….……………36
Figure 8. APA of Laurencia poiteaui collected at Looe Key, Monroe County, in
response to different levels of DIN and SRP enrichment ……………………..…....37
Figure 9. APA of Spatoglossum schroedi collected at Pepper Park, St. Lucie County,
in response to different levels of DIN and SRP enrichment……………...……….....37
Figure 10. APA of Gracilaria tikvahiae collected at Pepper Park, St. Lucie County,
in response to different levels of DIN and SRP enrichment ………..………..….....38
Figure 11. APA of Cladophora prolifera collected at Pepper Park, St. Lucie County,
in response to different levels of DIN and SRP enrichment .……...………………..38
Figure 12. Taxonomic and spatial variation in mean APA in south Florida……..……...39
vii Figure 13. Mean APA versus water column TDN:TDP (a) and tissue N:P (b) by
county in south Florida………………………………….……………………..……40
viii TABLES
Table 1. South Florida macroalgal collection sites…………………...... …...…………...41
Table 2. Summary of Two-Way ANOVA of APA as a function of phylum, season
and the phylum*season interaction in Pepper Park, St. Lucie County and
Looe Key, Monroe County…………………..…………………………..…...... 42
Table 3. Summary of Two-Way ANOVA of APA as a function of dissolved
inorganic nitrogen (DIN), soluble reactive phosphorus (SRP), and the
DIN*SRP interaction for species collected at Pepper Park, St. Lucie County……...43
Table 4. Summary of Two-Way ANOVA of APA as a function of dissolved
inorganic nitrogen (DIN), soluble reactive phosphorus (SRP), and the
DIN*SRP interaction for species collected at Looe Key, Monroe County……….....44
Table 5. Historical APA at Looe Key and Pine Channel, Monroe County…..……….....45
Table 6. Comprehensive list of sampled macroalgae from the seven counties………….46
ix INTRODUCTION
Coral reefs are complex, biologically diverse ecosystems adapted to oligotrophic conditions. Anthropogenic activities inevitably add nitrogen (N) and phosphorus (P) to coastal waters resulting in eutrophication. Eutrophication is the increase in the rate of supply of organic matter to an ecosystem (Nixon 1995). With increasing eutrophication, coral reef ecosystems can undergo major ecological changes including increased coral disease and die-off, and most notably, the replacement of corals by benthic macroalgae
(Lapointe 1997, NRC 2000, Lapointe et al. 2004, 2005 a, b). Macroalgal blooms are considered to be a major factor in coral reef decline around the world, including the Great
Barrier Reef (Bell 1992, Schaffelke 2001), the Bahamas (Barile and Lapointe 2005), southeast Florida (Lapointe 1997, Lapointe et al. 2005 a, b, Lapointe 2007), Hawaii
(Smith 1983), Jamaica (Lapointe et al. 2011) and the Florida Keys (Lapointe et al. 1994,
Lapointe et al. 2004). Escalating anthropogenic influences alter the ecosystems ability to cope with disturbances and effect functional diversity (Nystrom et al. 2000).
While N is the primary limiting nutrient to macroalgal growth in temperate siliciclastic environments, P-limitation has been documented in a variety of carbonate- rich environments, such as Bermuda, Jamaica, the Bahamas, and the Florida Keys
(Lapointe 1989, Lapointe et al. 1992). P-limitation is attributed to the geochemical process of phosphate (PO₄³⁻) binding to calcium carbonate (CaCO₃²⁺), which reduces the amount of soluble reactive phosphorus (SRP) in groundwater, sediments and
1 subsequently, the water column (Lapointe et al. 1992, Teichberg 2007). The rock unit,
Miami Limestone, which is composed of CaCO₃2+, extends up to Palm Beach County where it intercepts the coquina shell composed Anastasia Formation (Hine 2009).
Therefore, counties sampled in the Miami Limestone will have mostly carbonate-rich sediments (Monroe, Dade and Broward counties), while counties above this formation will be composed of mostly siliciclastic-rich sediments (Palm Beach, Martin, St. Lucie and Lee counties, Scott 1997).
Measurements of alkaline phosphatase activity (APA) have been used as a proxy of P-limitation in both freshwater and marine environments. Phosphatase is a class of enzymes that promotes the degradation of biologically unavailable complex organic phosphorus compounds into useable orthophosphates (Kuenzler and Perras 1965). Most macroalgae utilize inorganic P, known as soluble reactive phosphorus (SRP), yet most P is generally present in the ocean in the form of dissolved organic phosphorus (DOP; Lee
1999). Hence, marine macroalgae use alkaline phosphatase to cleave the needed SRP from the DOP pools (Hernandez et al. 1999, Hoppe 2003). APA of macroalgae in a wide variety of environments, including the carbonate-rich waters of Jamaica, the Bahamas,
Belize, Bermuda, and the Florida Keys have high APA; whereas macroalgae in siliciclastic-rich waters such as Woods Hole, Massachusetts have generally low APA
(Lapointe et al. 1992). This confirms strong P-limitation in carbonate-rich areas as well the use of APA as a proxy for estimating the degree of P-limitation (Lapointe and
O’Connell 1989, Lapointe and Clark 1992, Lapointe et al. 1992, Lapointe et al. 2004).
APA can be used as an accurate indicator of P-limitation in marine waters because a
2 significant linear relationship between APA and N:P ratios have been established
(Lapointe et al. 1990, Urnezis 1995, Lapointe and Bedford 2010).
Another method to assess P-limitation is through the use of the Redfield ratio.
Redfield (1934) found that the ratio of carbon:nitrogen:phosphorus (C:N:P) for phytoplankton in marine waters averaged 106:16:1. Atkinson and Smith (1983) expanded this concept in their investigation of C:N:P of benthic marine plants and reported a mean
C:N:P tissue ratio of 700:35:1, concluding that N:P ratios of > 35:1 were evidence of P- limitation and N:P ratios of < 10:1 were evidence of N-limitation in benthic marine plants. Lapointe et al. (1992) found that macroalgae in siliciclastic-rich areas had lower
N:P ratios (< 10:1) compared to carbonate-rich environments that had N:P ratios of >
35:1. In a regional assessment, elevated N:P and C:P ratios indicated primary P-limitation of growth in the carbonate-rich environments of Bermuda, the Florida Keys, the
Bahamas, Belize, and Jamaica (Lapointe et al. 1992).
Increases in the amount of nutrient-rich runoff to surface water and groundwater flows, especially during the wet season, alter tissue and water column N:P ratios and potentially APA in coastal waters. Research has found macroalgae tissue N:P ratios correlate significantly and positively with APA in carbonate-rich areas (Urnezis 1995).
Therefore, in carbonate-rich areas, where P is chemically scavenged, increasing N inputs associated with the wet season increase the N:P ratio of macroalgae forcing them into P- limitation; Gracilaria tikvahiae collected from the Florida Keys increased its N:P ratio from 8:1 in the dry season to 30:1 in the wet season (Lapointe 1987). In contrast, in siliciclastic-rich areas where P is not actively scavenged, the N:P ratio and APA decrease.
For example, in Lee County N:P ratios decreased significantly from August (27.4 ± 11.4 3 µmol PO₄-3 g dry weight-1 h-1) to October (17.51 ± 6.74 µmol PO₄-3 g dry weight-1 h-1,
Lapointe and Bedford 2007).
Taxonomy also plays an important role in the C:N:P composition and abundance of macroalgae in coral reef environments. Significant differences have been identified among the three phyla of macroalgae (Rhodophyta, Chlorophyta and Heterokontophyta, here after referred to as Phaeophyta). The morphology and physiology of algae play a critical role in nutrient requirements, uptake kinetics, and storage capacities. For example, in southeast Florida, Phlyum Phaeophyta had elevated C:P and N:P ratios when compared to the other two phyla, suggesting that phaeophytes are naturally adapted to P- limited conditions (Lapointe et al. 2005). Taxonomic shifts can be used as indicators of nutrient pollution on coral reefs since the appearance of bio-indicator species often signals an advance stage of eutrophication (Lapointe et al. 2004). This was first documented in the 1970s when the chlorophyte, Dictyospheria cavernosa increased its biomass in response to point source introduced anthropogenic nutrients, overgrowing reef corals in Kaneohe Bay, Hawaii (Smith 1984). Further, studies in the siliciclastic Waquoit
Bay, Massachusetts, found that the chlorophyte, Cladophora vagabunda and the rhodophyte, Gracilaria tikvahiae, significantly increased their biomass with increasing
N-enrichment. In addition, C. vagabunda increased its biomass three fold over G. tikvahiae, suggesting phlya differences exist between rhodophytes and chlorophytes (Fox et al. 2008). Finally, increasing anthropogenic nutrients to coral reef in O’ahu, Hawaii shifted the dominating macroalgae division. When nutrient concentrations were low, phaeophytes dominated but with increasing nutrient concentrations dominance shifted to non-native rhodophytes (Lapointe and Bedford 2011). 4 Experimental nutrient enrichment has long been documented to cause shifts in
N:P and APA. Lapointe’s (1981) experimental nutrient enrichment study of Gracilaria tikvahiae collected in the Florida Keys, showed proportional increases in N:P with increasing N-enrichment. In addition, N:P was inversely proportionate with increasing P- enrichment. Laboratory experiments conducted on Cladophora prolifera reflected this pattern with N-enrichment increasing N:P and APA, and P-enrichment decreasing N:P and APA (Lapointe and O’Connell 1989).
This study addressed gaps in knowledge of P-limitation in reef macroalgae of south Florida by assessing how environmental and taxonomic factors cause variability of
APA both in the field and laboratory. This study builds on previous ecological studies of reef macroalgae by measuring APA, and comparing those data to previous studies that measured C:N:P ratios, total N (TN), and total P (TP) as indicators of P-limitation and N- enrichment (Lapointe et al. 1992, Lapointe 1997, Lapointe et al. 2005 a, b, Lapointe and
Bedford 2007). Measurements of APA provide new insights and understanding to how south Florida’s coral reefs are being impacted by the increase in land-based nutrient loading to the ecosystem.
Hypotheses
This study involved both field and laboratory research. In the field study, macroalgae were sampled spatially within seven counties and temporally within two counties. In addition, ancillary laboratory experiments were conducted to substantiate
APA trends found in the field regarding the importance of the N:P ratio. The following specific hypotheses were addressed: 5 H1: P-limitation will vary in south Florida macroalgae as a result of sediment type with higher P-limitation occurring in carbonate-rich waters of Monroe, Dade, and
Broward counties and lower P-limitation in siliciclastic-rich waters of Palm Beach,
Martin, St. Lucie and Lee counties.
H2: Macroalgal APA will increase temporally in carbonate-rich areas and decrease in siliciclastic-rich areas with increasing nutrient-rich runoff.
H3: APA will vary due to taxonomic phyla with the highest values occurring in
Phaeophyta > Rhodophyta > Chlorophyta.
H4: In our ancillary laboratory experiment, APA will increase as N-enrichment increases and decrease with increases in P-enrichment.
6 METHODS
Study Area
The persistently pleasant climate of south Florida combined with abundant arable land has resulted in an excellent habitation area for humans. Human population in Florida has increased from 530,000 in 1900 to over 18 million in 2010 with an additional 87.3 million visitors per year (US Census 2010). Population increases have resulted in major changes to the natural landscape and hydrology of Florida. Initially, extensive drainage of wetlands and clearing of uplands were done to create agricultural areas severing the hydrological connection between Lake Okeechobee and the Everglades (Light and
Dineen 1994). Water from Lake Okeechobee that once flowed into the Everglades is now shunted in an unnatural direction, east through the St. Lucie Canal and west through the
Calooshatchee River (Light and Dineen 1994). Flooding in the late 1940s spurred the creation of the 550,000 acre Everglades Agricultural Area by draining the remaining northern Everglades. In addition, the middle of the Everglades was converted to Water
Conservation Areas surrounded by leeves, connected by pumps and gates, highly regulating water flow through the system (Bancroft 1996). Finally, land east of the Water
Conservation Areas historically had surface flow that travelled west through the
Everglades and into the Gulf of Mexico. Rain that falls on the highly developed east coast of Florida now drains east into the Atlantic Ocean. Highly controlled hydrological flow
7 of water with the addition of local nutrients due to rapid population growth has caused significant declines in water quality due to delivery of anthropogenic nutrients.
In this study, reef macroalgae were sampled among seven counties in south
Florida, including Monroe (Florida Keys), Dade, Broward, Palm Beach, Martin, St. Lucie and Lee counties. The geology of south Florida can influence the degree of P-limitation in coastal waters (Lapointe et al. 1992); hence an understanding of different geological substrata is needed. The Florida peninsula lies on the Florida Platform that formed during the Cretaceous period. The Florida Platform is composed of three layers of rock: 1) a basement layer of igneous and metasedimentary, 2) a 2-6 km thick carbonate (limestone and dolomite) layer, 3) a 1-150 m veneer of siliciclastic sands (Hine 2009). In the middle of the Oligocene period, extreme sea level drop filled seaways with siliciclastic sediment and transported it across the Florida Platform. Later in the Pleistocene period, an elevated shallow water shelf formed that produced the well-known rock units, the Miami
Limestone, the Key Largo Limestone and the Anastasia Formation (Hine 2009). The
Miami Limestone boundary lies between Broward and Palm Beach counties where it intercepts the coquina shell composed Anastasia Formation (Hine 2009). Therefore, counties sampled in the Miami Limestone formation will have mostly carbonate-rich sediments (Monroe, Dade and Broward counties), while counties above this formation will be composed of mostly siliciclastic-rich sediments (Palm Beach, Martin, St. Lucie and Lee counties).
8 Measurement of Alkaline Phosphatase Activity (APA) in Reef Macroalgae
Samples of macroalgae were collected via SCUBA or snorkeling by Dr. Brian
Lapointe and research assistants. Collections were made from different reef locations that are part of the Harbor Branch Oceanographic Institute (HBOI) at Florida Atlantic
University (FAU) South Florida Harmful Algal Bloom (HAB) monitoring network, including Monroe, Broward, Dade, Palm Beach, Martin, St. Lucie and Lee counties
(Figure 1, Table 1). Samples were collected from all counties in the dry season
(December to May, 2011), and from Monroe and St. Lucie counties in the wet season
(August to November, 2011). Once collected, the samples were placed in coolers or 10 L buckets (with lids to maintain a dark environment) and held in ambient seawater. Two portable battery-powered BigBubbles® were attached to the container and used to maintain adequate dissolved oxygen levels (> 5 mg/L) until samples were analyzed. All
APA analyses took place within 24 hours in the HBOI HAB Lab in Fort Pierce, Florida.
Prior to analysis, macroalgae were separated, identified, and hand-cleaned of epiphytes and sediment. Algae were identified to the lowest taxon possible using Littler and Littler
(2000).
The protocol for measuring APA was modified from Kuenzler and Perras (1965).
The assay bottles (250 mL HDPE) were acid washed with a 10% HCl solution and then triple rinsed with deionized water (DIW). The assay media was made according to
Kuenzler and Perras (1965) as modified for marine algae by Lapointe et al. (1992). The assay solution included: 1) 500 mL NPP stock (1.0 g nitrophenol phosphate substrate plus
25 g magnesium sulfate dissolved in 500 mL DIW), 2) 1 L TRIS buffer (121.14 g of tris(hydroxymethyl)aminomethane buffer dissolved in 1000 mL DIW), and 3) 8.5 L of 9 ambient seawater filtered through a 10 µm filter. A total of 200 mL of assay solution was added to each bottle and a portion of the thallus (~ 1-2 g wet weight) was added. Four replicates were ran and these were incubated for one hour under ambient irradiance (100-
300 µmol photons cm-2 s-1) and temperature (25 °C). Every 15 minutes the bottles were inverted to ensure mixing of the NPP solution and macroalgae. Two blank bottles, one containing assay solution with no macroalgae and one containing DIW, were also incubated to correct for possible microbial (viruses, bacteria and phytoplankton < 10 µm) activity. After one hour of incubation a sample of the assay solution was drawn from each bottle with a 10 mL automatic pipette. To prevent contamination, a clean pipette tip was used for blanks and assay media. One test tube filled with DIW was used to zero the spectrophotometer. Absorbance was determined using a HACH DR/2000 spectrophotometer. A standard curve slope (0.0167) was used together with absorption of the para-nitrophenol concentration read at 410 nm (Lapointe and O’Connell 1989).
Directly following the spectrophotometer analysis, the macroalgae tissue was dried at 65 °C for 24-48 hours in a Fisher Isotemp oven. The dried macroalgae were weighed using a Denver Instrument MXX 123 balance to 0.001 g. The resulting APA was determined in micromoles of phosphorus released per gram dry weight per hour
(µmol PO₄-3 g dry weight-1 h-1, Equation 1).
Equation 1. APA = ((Absorbance of sample/h –mean Absorbance of blanks/h )/0.0167) (200 mL/L) grams dry weight of test macroalgae
10 Nutrient Enrichment Experiment
Nutrient enrichment assays were performed on three species of macroalgae collected at two reef sites: 1) shallow (< 10 fsw) rubble zone on the western back reef of
Looe Key in Monroe County, which is hypothesized to be P-limited 2) shallow reef (< 10 fsw) at Pepper Park in St. Lucie County hypothesized to be N-limited. Species collected at Looe Key included Cladophora catenata, Taonia sp., and Laurencia poiteaui. Species collected at Pepper Park included Cladophora prolifera, Spatoglossum schroedi, and
Gracilaria tikvahiae. The experiment was a factorial design, in which four replicate plants of target species were weighed (~ 1-2 g wet weight per replicate). The enrichment protocol entailed exposing experimental macroalgae to varying nitrogen (as NaNOз and
NH4Cl in equal parts) and phosphorus (as NaH2PO4 * 2 H2O) in 250 mL Erlenmeyer
- flasks with corresponding treatments: 1 µM and 15 µM of DIN (NOз⁻+ NO2 ), 0.1 µM and 1 µM of SRP (PO₄³-), 1 µM DIN + 0.1 µM SRP, 1 µM DIN + 1 µM SRP, 15 µM
DIN + 0.1 µM SRP, 15 µM DIN + 1 µM SRP, and a control that received only seawater.
Treatments were maintained in the Percival incubators for 6 days. All treatments were placed under controlled light (200 µmol photons m-2 s-1) and temperature (25 °C) in
Percival incubators at the HBOI HAB Lab. The seawater used in the experiment was created from 10 L of water and 650 mL of Instant Ocean®. Salinities of 36.4‰ and 34‰ were used to mimic field conditions at Looe Key and Pepper Park, respectively. APA was measured at the end of the experiment on day 6. Macroalgae were agitated on day 2, 3, and 5 and was pulsed with 10 mL of their corresponding treatment. On day 4, a complete water change was performed.
11 Statistical Analysis
Statistical differences in APA were measured using One-Way and Two-Way
ANOVAs in Statistical Analysis System (SAS) version 9.2 and 9.3. A General Linear
Model (GLM) was used to determine significance. For all analyses, differences were considered significant at p < 0.05. Data were appropriately transformed to reach normality based on the p-value of residuals presented in the Shapiro-Wilks test. A priori contrasts were written to prevent Type 1 errors due to alpha inflation for spatial, temporal and taxonomic among county ANOVAs. A posteriori tests ran included a Tukey-Kramer and a least square means for effects for spatial, temporal and taxonomic within county
ANOVAs. For One-Way ANOVAs, Levene’s tests for homogeneity of variance among groups were ran. One-Way ANOVAs were used to assess the effects of spatial, temporal, and taxonomic variations among counties in APA. Since APA for overall taxonomic and spatial variations’ APA were averaged across all counties; data were broken down and ran per county. Two-Way ANOVAs were used to assess the main effects of phylum, season and the phylum*season interaction on macroalgal APA in Pepper Park, St. Lucie
County and Looe Key, Monroe County. Two-Way ANOVAs were also used to assess main effects of DIN and SRP enrichment and the DIN*SRP interaction on APA for each species tested in the laboratory nutrient enrichment experiment.
12 RESULTS
Spatial Variation Among Counties
A total of 816 assays were performed and the overall mean APA (in µmol PO₄-3 g dry weight-1 h-1 ± S.E.) in macroalgae varied significantly among south Florida counties
(ANOVA F = 53.7, p < 0.001; Figure 2). When individual counties were analyzed, macroalgae collected in carbonate-rich Monroe (23.3 ± 2.70) and Dade (26.0 ± 3.77) counties had significantly higher APA (p < 0.001 for all counties) than those collected in
Palm Beach (14.8 ± 2.43), Broward (6.43 ± 1.09), Martin (5.04 ± 1.20), St. Lucie (7.45 ±
1.42) and Lee (4.98 ± 0.807) counties. The APA recorded for macroalgae collected in
Palm Beach County were significantly higher than the APA for macroalgae collected in
Broward (p < 0.001), Martin (p < 0.001), St. Lucie (p = 0.042), and Lee counties
(p = 0.017).
Spatial Variation Within Counties
Our study sampled several sites within Dade (3), Monroe (3), Palm Beach (4),
Broward (2) and Martin (2) counties (Table 1). Macroalgae collected within Dade and
Monroe counties produced statistically significant APA between sample sites.
Conversely, macroalgae collected within Palm Beach, Broward and Martin counties displayed statistically similar APA between sample sites.
APA in macroalgae collected at Dade County produced marginally significant
13 (F = 3.13, p = 0.051) differences between Cape Florida Lighthouse and Rickenbacker sample sites. APA for macroalgae collected in Monore County was significantly
(F = 5.01, p = 0.008) different between the Pine Channel and Looe Key sample sites. The individual macroalgae species Penicillus dumesotus sampled at both these sites, had APA that also showed significant difference (p = 0.041). In Palm Beach County, APA did not vary significantly (F = 1.41, p = 0.842) among the four sample sites, Princess Anne,
North Colonel Ledge, Jupiter’s Ledge and Timmy’s Deep. Similar to Palm Beach,
Broward County macroalgae APA did not display significant (F = 3.55, p = 0.065) differences between the sample sites, John U. Lloyd State Park and Ft. Lauderdale Anglin
Pier. Finally, APA for macroalgae collected in Martin County at the Three Holes and
Nine Steps sites, were statistically (F = 0.110, p = 0.740) similar.
Temporal Variation Among Counties
Variations in mean APA (in µmol PO₄-3 g dry weight-1 h-1 ± S.E.) between the dry and wet season in macroalgae from Looe Key and Pepper Park showed similar trends
(Figure 4 and 5). During the wet season both counties had chlorophytes decrease their
APA, while APA in rhodophytes and phaeophytes increased. At Looe Key, rhodophytes were the only taxonomic phyla that had significantly higher APA in the wet (36.6 ± 5.15) than in the dry season (18.4 ± 3.10, Figure 4). At Pepper Park, chlorophytes had significantly lower APA in the wet season (1.11 ± 0.360) compared to the dry season
(2.68 ± 0.625). In addition, phaeophytes had significantly higher APA in the wet season
(11.4 ± 3.93) versus the dry season (6.49 ± 3.93, Figure 5).
14 Temporal Variation Within Counties
Mean APA (in µmol PO₄-3 g dry weight-1 h-1 ± S.E.) in macroalgae collected in
Monroe County varied significantly for taxonomic phlya (F = 82.7, p < 0.001), season (F
= 7.16, p = 0.008) and the interaction between phylum*season (F = 10.4, p < 0.001, Table
2). Though mean APA in phaeophytes increased from the dry (32.1 ± 6.05) to wet season
(37.9 ± 4.68), the increase was not significant (p = 0.192); with the exception of Dictyota cervicornis, which increased APA from 52.3 ± 7.31 in the dry season to 89.2 ± 5.32 in the wet season (p = 0.005). Chlorophytes APA decreased from the dry (8.87 ± 1.56) to the wet (7.29 ± 0.930) season, but this decrease was not significant (p = 0.192). Mean
APA for rhodophytes increased significantly (p < 0.001) between the dry (18.4 ± 3.10) and the wet (36.6 ± 5.15) season. Individual species collaborated this finding as APA in
Chondria capillaris increased significantly from the dry (26.4 ± 6.15) to the wet (63.3 ±
9.74; p = 0.019) season, as did Heterosiphonia gibbessii, (dry 25.9 ± 6.02, wet 47.3 ±
2.01, p = 0.011).
APA in macroalgae sampled in St. Lucie County differed significantly between taxonomic phyla (F = 34.0, p < 0.001), season (F = 5.31, p = 0.026) and the interaction between phylum*season (F = 15.6, p < 0.001, Table 2). Phaeophytes significantly
(p < 0.001) increased their APA between the dry (6.50 ± 1.56) and wet (11.4 ± 3.93) season. Rhodophytes increased their APA between the dry (11.5 ± 2.80) and wet (12.3 ±
2.15) season though this increase was non-significant (p = 0.184). Unlike rhodophytes and phaeophytes, chlorophytes APA were actually significantly lower (p = 0.008) in the wet season (1.01 ± 0.360) compared to the dry season (2.69 ± 0.630).
15 Taxonomic Variation Among Counties
Overall, mean APA (in µmol PO₄-3 g dry weight-1 h-1 ± S.E.) varied significantly
(F = 9.85, p < 0.001) between taxonomic phyla (Figure 3). Mean APA in phaeophytes
(14.2 ± 2.24, n = 50) were significantly higher than rhodophytes (11.8 ± 2.03, p = 0.004, n = 82) and chlorophytes (9.74 ± 1.64, p < 0.001, n = 72). Though rhodophytes had higher mean APA than chlorophytes, these differences were not significant (p = 0.344).
Taxonomic Variation Within Counties
In Dade County, significant differences in APA were observed for the taxonomic phyla (F = 4.47, p = 0.014). APA for chlorophytes (33.9 ± 4.88) were significantly higher
(p = 0.011) than phaeophytes (16.8 ± 2.62), but were statistically similar
(p = 0.267) to rhodophytes (23.9 ± 2.90). Likewise, APA in rhodophytes did not vary significantly (p = 0.187) from phaeophytes. In Monroe County, APA was significantly
(F = 13.9, p < 0.001) different between all three macroalgae taxonomic phyla. The APA of phaeophytes (33.9 ± 5.31) was significantly higher than that of chlorophytes (9.87 ±
1.56, p < 0.001) and rhodophytes (26.6 ± 3.80, p = 0.001); though rhodophytes had significantly (p = 0.003) higher APA than chlorophytes. Similar to Monroe County, taxonomic phyla of macroalgae in Broward County had significantly (F = 7.38, p = 0.002) different APA. APA for phaeophytes (8.40 ± 1.62) were significantly higher than chlorophytes (3.91 ± 1.08, p = 0.005) and rhodophytes (9.74 ± 0.790, p = 0.008).
Though rhodophytes had significantly higher APA than chlorophytes (p = 0.719).
APA from macroalgae collected in Palm Beach County varied significantly (F =
30.4, p < 0.001) by taxonomic phyla. Phaeophytes (17.4 ± 1.37) had significantly higher 16 APA than both chlorophytes (8.41 ± 1.83, p < 0.001) and rhodophytes (7.71 ± 1.57, p <
0.001). Although chlorophytes and rhodophytes had statistically similar (p = 0.958) APA.
In St. Lucie County, APA for macroalgae were significantly (F = 24.8, p < 0.001) different between all three taxonomic phlya. Chlorophytes (2.16 ± 0.439) showed significantly lower APA than rhodophytes (10.5 ± 2.76, p = 0.001) and phaeophytes (7.35
± 2.09, p = 0.038). In addition, rhodophytes produced significantly (p < 0.001) higher
APA than phaeophytes.
Unlike all other counties sampled, APA for macroalgae taxonomic phyla collected at Martin County were not significantly (F = 1.35, p = 0.264) different.
Although chlorophytes (3.68 ± 0.916) had lower APA than phaeophytes (6.16 ± 1.98) and rhodophytes (7.18 ± 2.27), these variations were not significant (p = 0.420, p =
0.318, respectively). APA produced in phaeophytes and rhodophytes were also statistically similar (p = 0.993). Finally, macroalgae collected at Charlie’s Cut in Lee
County showed significant (F = 40.9, p < 0.001) differences in APA amongst the phyla.
Rhodophytes (1.44 ± 0.202) had significantly lower APA than chlorophytes (6.08 ±
0.794, p < 0.001) and phaeophytes (9.86 ± 0.709, p < 0.001). Chlorophytes also had significantly lower APA than phaeophytes (p = 0.003).
Nutrient Enrichment Experiment
A total of 216 assays were ran and overall mean APA (in µmol PO₄-3 g dry weight-1 h-1 ± S.E.) suggested nutrient limitation for all macroalgae species sampled. In
Looe Key species sampled included Cladophora catenata, Laurencia poiteaui and
Taonia sp. In Pepper Park species sampled included Spatoglossum schroedi, Cladophora 17 prolifera, and Gracilaria tikvahiae. In all species, APA proportionally increased with increasing N-enrichment and decreased with increasing P-enrichment (Table 3 and 4).
Overall the ANOVA model statement for Cladophora catenata showed significant (F = 3.19, p = 0.011) effects on APA due to nutrient enrichment. Both DIN and SRP had significant effects on APA (p = 0.004 and p = 0.008, respectively), but the interaction between DIN*SRP was not significant (p = 0.979, Figure 6). For Tanoia sp., the overall model statement showed that the nutrient treatments did not significantly
(F = 0.620, p = 0.749) alter APA. DIN, SRP and the DIN*SRP interaction had no statistical significant effect (p = 0.283, p = 0.409, p = 0.971, respectively; Figure 7).
Laurencia poiteaui’s overall ANOVA model statement displayed significant (F = 2.96, p
= 0.016) variation in APA due to experimental nutrient treatments. DIN did not alter
APA significantly (p = 0.097), while SRP did significantly alter APA (p = 0.002). The
DIN*SRP interaction was also not significant (p = 0.599). SRP control of APA was supported by individual treatment findings. The 1 µM SRP (13.7 ± 1.81) treatment had significantly lower APA than the 0 µM DIN + 0µM SRP (control, 22.7 ± 1.21), 1 µM
DIN (25.5 ± 1.59), 15 µM DIN (26.3 ± 4.83), and the 1 µM DIN + 0.1 µM SRP (23.7 ±
0.77) treatments (p < 0.001, p = 0.006, p = 0.007, and p = 0.018, respectively, Figure 8).
The overall ANOVA model statement showed that nutrient treatments in
Spatoglossum schroedi had significant (F = 1.66, p = 0.015) effects on APA. Unlike all other species sampled, only DIN had a significant effect on APA (p = 0.021). SRP and the DIN*SRP interaction did not significantly effect APA (p = 0.676 and p = 0.487, respectively). Support for N-enrichment importance to APA is supported by individual treatments. 0 µM SRP + 0 µM DIN (control, 21.6 ± 4.98) treatment was significantly 18 similar to the 0.1 µM SRP (21.5 ± 7.24) and 1 µM SRP (21.0 ± 13.4) treatments (p =
1.000, p = 0.200, respectively). Overall, samples of this species collected in the field had significantly (F = 12.6, p = 0.001) lower APA than recorded in the laboratory experiment.
Field samples APA (3.01 ± 0.390) was significantly lower than the 15 µM DIN, 15 µM
DIN + 1 µM SRP and 15 µM DIN + 0.1 µM SRP treatments (p = 0.025, p < 0.001, and p
= 0.050, respectively, Figure 9), further supporting the importance of N-enrichment to
APA.
The overall ANOVA model statement for Gracilaria tikvahiae showed that nutrient enrichment treatments had significant effects (F = 7.34, p < 0.001) on variations in APA. Both DIN and SRP significantly altered APA (p = 0.004 and p < 0.001, respectively) APA in the 15 µM DIN (11.4 ± 1.78) treatment was significantly higher than the 0.1 µM SRP (4.09 ± 0.73), 1 µM SRP (2.85 ± 0.320), 1 µM DIN + 1 µM SRP
(2.80 ± 0.331), 1 µM DIN + 0.1 µM SRP (5.60 ± 0.800), and the 15 µM DIN + 1 µM
SRP (3.84 ± 1.33) treatments (p < 0.001, p < 0.001, p < 0.001, p = 0.01, and p < 0.001, respectively; Figure 10). In general, Cladophora prolifera exposure to nutrient enrichment treatments had significant effects (F = 4.62, p = 0.001) on APA. Unlike the other two species collected at Pepper Park, DIN did not have a significant effect on APA
(p = 0.178). SRP did have a significant effect on APA (p < 0.001), but the DIN*SRP interaction did not (p = 0.932). Individual treatments supported the importance of P- enrichment as the 1 µM SRP (2.60 ± 0.431), 1 µM DIN + 1 µM SRP (2.54 ± 0.440), and the 15 µM DIN + 1 µM SRP (3.28 ± 0.930) treatments APA were lower than all other treatments (Figure 11).
19 DISCUSSION
Atkinson (1987) was the first to suggest that measuring APA provided a direct determination of P-cycling and P-availability to macroalgae on coral reefs. Since then
APA has been used to measure the degree of P-limitation. For example, in carbonate-rich
Bermuda the marine chlorophyte Cladophora prolifera had high N:P (62.6 ± 33.7) and
APA (48.0 ±1.00 µmol PO₄-3 g dry weight-1 h-1) indicating P-limitation (Lapointe and
O’Connell 1989). Extensive regional studies were conducted that discovered elevated seawater DIN:SRP, high APA, and greater macroalgae C:P and N:P ratios in carbonate- rich tropical environments that suggested constant P-limitation of benthic primary producers (Lapointe et al. 1992)
Field and laboratory work suggest patterns in mean APA (in µmol PO₄-3 g dry weight-1 h-1 ± S.E.) due to spatial, temporal, taxonomic, and N and/or P-enrichment. Our study supported the effect of Florida’s geology in regulating biogeographic patterns on
APA of benthic macroalgae. APA was greater in the carbonate-rich waters of Monroe and
Dade counties during both the dry and the wet season. In comparison, APA in siliciclastic-rich waters of Palm Beach, Martin, St. Lucie, and Lee counties were lower in both dry and wet season. Broward County was the only county that did not support our spatial hypothesis, and is discussed in more detail below. Our laboratory experiment supported trends displayed in the field. APA proportionally increased with increasing N- enrichment and decreased with P-enrichment.
20 Spatial Variation in Alkaline Phosphatase Activity
APA for macroalgae collected in Broward County did not support our hypothesis of P-limitation. APA was lower than expected, even though the area is carbonate-rich and historically supported coral reef growth. This appears to result from high seawater concentrations of dissolved P in Broward County, which reduces the need for APA in reef macroalgae. Lapointe (2007) showed relatively high concentrations of SRP and TDP (0.2
µM and 0.5 µM) in Broward County, which would result from intense human activities that includes nutrient-rich effluent from two oceanic outfalls (North Broward County and
Hollywood), tidal discharges from two inlets, and numerous septic tanks. δ15N analyses of macroalgae collected from these reefs indicated a significant contribution of sewage N
+ to TDN values as well as matched values for NH4 - rich effluent from the Broward
County sewage outfalls (Hoch et al. 1995, Lapointe 2007, Risk et al. 2009). Broward
County has been experiencing blooms of the cyanobacterium Lyngba sp. (Lapointe and
Clark 1992). Because cyanobacteria fix N, it is likely that nutrients other than N are contributing to these blooms. In Moreton Bay, Queensland, elevated water column P concentrations were shown to maximize the growth of large blooms of Lyngba sp.
(Elmetri and Bell 2004). Therefore, it appears that anthropogenic influences have cause substantial variations in SRP concentrations that have lowered the N:P ratio and APA, shifting Broward County macroalgae away from strong P-limitation towards N- limitation.
APA in Dade County not only supported our spatial hypothesis of P-limitation in carbonate-rich waters but also reflected an imbalanced stoichiometry due to high N- enrichment, increasing the N:P from human activities on the watershed. The values were 21 higher than expected, indicating severe P-limitation, especially in Biscayne Bay. This is attributed to the calcareous sediment coupled with N-rich runoff from pollution. As of
1998, Dade County had approximately 116,300 septic tanks (USGS 1998). These tanks deliver high levels of N, 1.5 billion L N/day or ~6000 metric tons N/year, to adjacent surface waters (Hazen and Sawyer 1994). Sewage, in combination with fertilizers from agricultural runoff in south Dade County, result in very high DIN concentrations in canals that function as point source discharges, such as Mowry (C-103), Turkey Point, C-
111 and the Black Point (C-1) canals (Figure 1). For example, significant nutrient inputs into the Mowry Canal have produced unnaturally high DIN:SRP (~2500:1), reflecting N contamination by upland sources (Lapointe 1999). Macroalgae sampled in Biscayne Bay mirror this with consistently high C:P and N:P ratios, as well as low C:N (Collado-Vides et al. 2011). For example, Anaydomene stellata had a tissue N:P of 143:1; while on average macroalgae tissue N:P was > 70:1 (Collado-Vides et al. 2011, Lapointe 1997).
High levels of land-based N inputs contributed to Dade County’s coastal water column
TDN of > 10 µM, higher than the other six counties sampled during this study (Lapointe
2007). Water quality has declined as a function of increasing N concentrations and simultaneous decreasing P, raising the N:P ratio and therefore increasing the degree of P- limitation (Abbott et al. 2005, Carey et al. 2011). Our results, combined with these data, suggest increasing severity of P-limitation throughout Dade County due to anthropogenically caused eutrophication (Figure 13).
APA in Monroe County supported the hypothesis of high APA in carbonate-rich environments. Natural P-limitation is supported by the lowest water column TDP from the seven counties sampled, as well as the highest water column N:P (B. Lapointe 22 unpublished data; Figure 13). Shifts in N:P and APA in the Keys have been documented in the last three decades due to changes in the quantity of regional-scale N-rich runoff. In the 1980s the N:P ratio and APA indicated P-limitation, 41:1 and 61:1, respectively, due to rapid urbanization of central and south Florida coupled with little regulation of nutrients entering waterways (Lapointe et al. 1992). In the early 1990s, the N:P and APA increased to 86:1 and 63:1, respectively, in nearshore waters (Urnezis 1995). These increases in the N:P and APA were a result of large amounts of N-rich, P-depleted, water being discharged via the Shark River Slough from agricultural areas in the northern
Everglades between 1991 and 1995, increasing N:P ratios and P-limitation downstream in
Florida Bay and the Florida Keys (Lapointe et al. 1993). Water managers have since decreased discharges from the Everglades, lowering the N:P and APA as seen in this study (Table 5); however, plumes of turbid, nutrient-rich, high chlorophyll water from
Florida Bay are still transported out to the offshore reefs of the Florida Keys (Lee and
Smith 2002), including Looe Key (Lapointe et al. 2004).
Increases in the N:P ratios in the nearshore waters of the Florida Keys are also due to increasing localized urban development where cesspits and septic tanks enrich groundwaters with high concentrations of N relative to P (Lapointe et al. 1990). The chemical scavenging of P relative to N has resulted in N-rich waters discharging into confined and nearshore waters that create a gradient of decreasing APA and N:P from nearshore to offshore waters (Urnezis 1995). Pine Channel is a confined inshore water with poor flushing and is surrounded by developed areas with septic tanks that leach, causing high N:P. On the contrary, Looe Key is 9.26 km offshore and is not as directly impacted by anthropogenic influences, resulting in lower N:P ratios more typical of 23 oceanic waters. Indeed, Urnezis (1995) found higher mean APA and N:P in nearshore
Pine Channel (63.5 ± 25.0 and 97.0 ± 25.0 µmol PO₄-3 g dry weight-1 h-1, respectively) versus offshore Looe Key (34.9 ± 31.3 and 45.0 ± 8.00 µmol PO₄-3 g dry weight-1 h-1, respectively). Our study substantiated these finding with higher APA at Pine Channel,
40.3 ± 3.76 µmol PO₄-3 g dry weight-1 h-1 versus Looe Key 19.9 ± 3.77 µmol PO₄-3 g dry weight-1 h-1. Further, individual species sampled, such as Pencillus dumetosus, reflected this trend with lower APA at Looe Key (8.17 ± 2.12 µmol PO₄-3 g dry weight-1 h-1) and higher APA at Pine Channel (19.2 ± 3.67 µmol PO₄-3 g dry weight-1 h-1).
Reduced APA in reef macroalgae from Palm Beach County supported our hypothesis, indicating moderate P-limitation. Lower levels of P-limitation in Palm Beach
County are due to Palm Beach’s classification as an ecotone. An ecotone is a transition area between biomes. Palm Beach separates the carbonate-rich areas to the south,
Broward, Dade and Monroe counties, from the siliciclastic-rich areas to the north, Martin,
St. Lucie and Lee counties. Therefore, Palm Beach County is a combination of minor carbonate and major siliciclastic sediment (Scott 1997). Sediment content coupled with two inlets, a sewage outfall in Boca Raton, a highly transmissive geological zone that allows sewage enriched submarine groundwater discharges (SGD), and effluent from multiple deep injection wells, makes Palm Beach County susceptible to eutrophication
(Lapointe 1997, Lapointe et al. 2005). Groundwater delivers 5727 metric ton/yr of N and
414 metric tons/yr of P via SGD to the coastal waters of Palm Beach (Finkl and Charlier
2003). This is exemplified in findings of surface water DIN enrichment at the North
Colonels Ledge (NCL) and Princess Anne (PA) sample sites where blooms of the
24 chlorophyte Codium isthmocladum developed in the early 1990s (Lapointe 1997). These sites also exhibited a water column N:P ratios of 37:1 and 36:1, respectively, supporting moderate P-limitation (Lapointe 1997, Figure 13). DIN enrichment was linked to wasterwater discharges through elevated δ15N values in Codium isthmocladum and
Caulerpa brachypus (Lapointe et al. 1997, Lapointe and Bedford 2010). Furthermore, the
South Florida Water Management District diverts excess stormwater runoff from agricultural and urban areas to coastal waters via the West Palm Beach Canal (C-51,
Figure 1). Elevated TP levels in canal water was due to high levels of dissolved P, indicative of fertilizer runoff (Boyer et al. 2011), but chemical scavenging of P and reduced runoff in the dry season resulted in macroalgae tissue N:P of 48:1, indicating P- limitation (Lapointe 2007, Figure 13).
Low APA of reef macroalgae in St. Lucie and Martin counties supported our hypothesis, of low P-limitation due to siliciclastic sediment not chemically interacting with P. This is reflected in the tissue and water column N:P of 31:1 and 26:1, respectively, in St. Lucie County and a TDN:TDP ratio of < 30:1 in Martin County (Lapointe 2007,
Figure 13). Low APA and N:P could be intensified by nutrient-rich fertilizer runoff from agricultural practices; between 2004 and 2006, 52-58% of land use was dedicated to agriculture in the Lake Okeechobee and St. Lucie River watersheds, respectively
(Lapointe et al. 2012). These watersheds carry nutrient-rich runoff, especially during times of excessive freshwater release from Lake Okeechobee associated with stormwater, with DIN:SRP as low as 7.7 ± 3.7 to the St. Lucie Estuary and the Indian River Lagoon that interact with coastal waters through the St. Lucie Inlet (Lapointe et al. 2012).
Evidence of localized nutrient pollution from septic tanks has also been reported in the 25 St. Lucie Estuary and Loxahatchee River Estuary, both which drain into Martin County’s coastal waters (Lapointe et al. 2012).
The lowest APA was in macroalgae collected from Lee County, further supporting our hypothesis. Low macroalgae tissue and water column N:P ratios, 20:1 and 25:1, respectively, suggest the highest amount of P present in the water column compared to all other counties sampled (Lapointe and Bedford 2007, Figure 13). Low N:P ratios from widespread P-enrichment are due to increases in nutrient loading associated with expanding urbanization of the watershed and nutrient-rich runoff from the Peace and
Caloosahatchee Rivers (Figure 1). Rivers carry nutrients from agricultural areas where crops are cultivated in sandy soils with low water-holding capacity, allowing free flow of excess fertilizer. In addition, rivers carry runoff from phosphorite deposits, which has been exacerbated by phosphate mining and are persistent sources of P-enrichment (Odum
1953). Therefore, nutrient-rich runoff can be significant, especially during the wet season, where DIN:SRP ratios have been seen to be as low as 4.96 ± 0.99. Since rhodophytes have lower N:P due to degraded phycobilisomes in N-limited Lee County, high nutrient-rich ruoff can cause extensive rhodophyte blooms as seen in 2003 and 2004
(Lapointe and Bedford 2007).
Temporal Variation in Alkaline Phosphatase Activity
Overall, macroalgae from Looe Key and Pepper Park had similar APA responses and supported our hypotheses. In both counties during the wet season, APA of chlorophytes was significantly lower than rhodophytes and phaeophytes. In addition, chlorophytes decreased their APA, while APA in rhodophytes and phaeophytes 26 increased. In carbonate-rich Looe Key, our temporal hypothesis was supported as rhodophytes and phaeophytes increased their APA in the wet season. Chlorophytes, on the other hand, decreased their APA, but this decrease was not significant. In siliciclastic- rich Pepper Park, our temporal hypothesis was generally supported. Chlorophytes significantly decreased their APA; while rhodophytes APA remained relatively constant, and phaeophytes significantly increased their APA against hypothesis. It is theorized that variations in APA at Pepper Park are due to annual state wide droughts beginning in 2006
(NOAA 2012). In 2011, Pepper Park experienced a yearly rainfall deficient of 12ʺ- 16ʺ decreasing nutrient-rich runoff usually associated with the Florida wet season (NOAA
2012). Consequently, chlorophytes with high surface area:volume (SA:V) depleted the water column of nutrients, forcing phaeophytes with lower SA:V to increase their APA to continue balanced growth.
Taxonomic Variation in Alkaline Phosphatase Activity
Results of this study supported the hypothesized taxonomic variations in APA by macroalgal phyla with values significantly decreasing from Phaeophyta to Rhodophyta.
APA did decrease from Rhodophyta to Chlorophyta but, the decrease was not significant.
We hypothesize that the APA decrease was not significant due to artificially high APA in chlorophytes, because of wound healing in coenocytic green algae, including, Halimeda spp., Caulerpa spp. and Codium spp. Each macroalgal phylum has evolved specific biochemical strategies to succeed as macroalgae do not alter their elemental composition in response to nutrient enrichment in the same way, even under similar N:P ratios (Sterner and Elser 2002). Phaeophytes are considered to be stress tolerators that grow in areas of 27 chronically low availability of resources, such as the relatively oligotrophic waters of the
Florida Keys (Sterner and Elser 2002). Rhodophytes are considered ruderal strategists that are adapted to habitats with high levels of resources but frequent disturbances, such as periods of drought or episodic nutrient-rich stormwater runoff as seen in Lee County
(Sterner and Elser 2002). Chlorophytes are considered competitive strategists that are adapted to relatively fertile conditions and are characterized by high growth rates, which is why they are ubiquitously found in areas of eutrophication (Sterner and Elser 2002).
As competition increased for nutrients, some algae evolved multicellularity. As the size of the cell grew, volume increased, expanding the need for cell walls. Because cell walls are
C-rich and mostly N and P poor, algae with larger cells experience higher C:N and C:P ratios, such as paranchymous phaeophytes (Sterner and Elser 2002). Since multicelluarity increases the volume of the cell, it also decreases the SA:V ratio. The SA:V ratio represents the distance of the algal cells from the edge of the thallus, therefore dictating speed of nutrient uptake (Lobban and Harrison 1985). Species with high SA:V ratios assimilate nutrients quicker and have more rapid growth potential, as seen in the competitive strategists chlorophytes, such as Ulva (Lobban and Harrison 1985). While multicellular phaeophytes and rhodophytes have lower SA:V ratios (Lobban and Harrison
1985). Rhodophytes have compensated for the increased cell size by storing excess nutrients within their cells, referred to as luxury consumption and storage (Lee 1999). In addition, rhodophytes evolved the light harvesting antennae known as phycobilisomes.
Phycobilisomes are composed of phycobiliproteins that can be degraded and used as N sources in times of N-deprivation (Lapointe 1981). Therefore, rhodophytes tend to have
28 higher N:P ratios compared to the other algal divisions, when not in N-limitation (Lee
1999).
Nutrient Enrichment Experiment
In this study, it was hypothesized that APA would proportionally increase with increasing N-enrichment (increasing N:P) and decrease with increasing P-enrichment
(decreasing N:P). Results for all six species in the controlled lab enrichment experiment supported this hypothesis and suggested that field APA variations are caused by N and P enrichment and changes in the N:P ratio. Varying APA in response to the main effects of
N and/or P-enrichment demonstrated that patterns of P-limitation can result from availability of either N or P. For example, all species experienced the highest APA at 15
µM DIN followed by 1 µM DIN and the 15 µM DIN + 0.1 µM SRP (N:P of 150:1), illustrating the importance of N-enrichment to P-limitation. The lack of a significant interaction between DIN and SRP for all species indicates that P and N-enrichment affect
APA independently of one another. We hypothesized that this is because maximum growth rate of macroalgae was supported in treatments with concentrations above 1 µM
DIN and 0.1 µM SRP, and therefore the N:P was not as important as total concentrations of nutrients present. This is consistent with experimental results found in Lapointe’s
(1981) study. In all sampled macroalgae, with the exception of Gracilaria tikvahiae, control treatment APA was extremely similar to APA from the 1 µM DIN + 0.1 µM SRP treatment (N:P 10:1), which supports the suggested nutrient threshold concentrations of 1
µM DIN and 0.1 µM SRP for macroalgal blooms on coral reefs (Lapointe et al.1992,
Lapointe 1997). Concentrations above this cause an increase in opportunistic macroalgae
29 that overgrow slower growing seagrasses and corals. For example, 1 µM DIN sustains the maximum growth rate of highly opportunistic rhodophytes (D’Elia and DeBoer 1978) and chlorophytes (Lapointe 1981, Lapointe 1997).
The chlorophytes Cladophora catenata and Cladophora prolifera had low APA compared to other species sampled which supports our field findings demonstrating dominance of opportunistic chlorophytes in eutrophic areas. The lack of significant effects of P-enrichment on APA in phaeophytes, Taonia sp. and Spatoglossum schroedi, supports our field findings of dominance of phaeophytes in oligotrophic areas. While
SRP enrichment affected APA in both rhodophytes, Gracilaria tikvahiae and Laurencia poiteaui, DIN enrichment only significantly affected APA in Gracilaria tikvahiae. This supports field findings of P-limitation in Monroe County and a tendency towards N- limitation in St. Lucie County.
In Spatoglossum schroedi, higher APA in the 15 µM DIN + 1 µM SRP treatment versus the 15 µM DIN + 0.1 µM SRP treatment, was due to human error during pulsing.
One of the samples missed pulsing during day 5, and it appears to have been from the 15
µM DIN + 1 µM SRP treatment; correct pulsing would have yielded results similar to all other species of increasing APA with increasing N-enrichment.
30 CONCLUSIONS
Results of field and laboratory work showed significant patterns in APA due to spatial, temporal, and taxonomic factors, as well as experimental N and P-enrichment.
Greater APA and P-limitation occurred in carbonate-rich areas, due in part to the build-up of N in the water column associated with anthropogenic eutrophication that increased the
N:P ratio and thus the degree of P-limitation (e.g. Dade and Monroe counties).
Conversely, low APA was associated with the less P-limited waters of Martin, St. Lucie and Lee counties, where N:P ratios are low and N-limitation is common. Broward
County, though carbonate-rich, displayed low APA suggesting that the buffer capacity for carbonate P-adsorption may be diminishing as the limestone becomes supersaturated with P from continued nutrient loading.
Over the last several decades, blooms of macroalgae have been increasing along south Florida’s coastline, contributing to decline and die-off of coral reefs (Lapointe
1997, Lapointe et al. 2005 a, b, Lapointe and Bedford 2007, Lapointe and Bedford 2010).
This is attributed to increasing anthropogenic activities associated with Florida’s expanding population, which is leading to eutrophication of coral reefs. Anthropogenic activities have greatly accelerated eutrophication by increasing transport of nutrients from inland watersheds to coastal water bodies (NRC 2000). Growth rates of macroalgae under high light intensity and warm temperatures common on coral reefs are highly dependent on the concentration of the growth limiting nutrient (NRC 2000). Therefore, even slight
31 increases in ambient dissolved nutrient concentrations can lead to expansion and blooms of macroalgae, as exemplified in Broward, Dade, Lee and Palm Beach counties (Lapointe
2007).
Although humans have greatly altered both the global N and P cycle, more N than
P is delivered to coastal waters, increasing the N:P ratio and therefore APA and the degree of P-limitation. For decades, governments have made simplified assumptions to whether N or P was the limiting nutrient, and therefore relied on P or N controls alone to solve coastal eutrophication (Howarth and Paerl 2008). For example, due to public outcries about fouling in Lake Okeechobee, the Comprehensive Everglades Restoration
Program (CERP) has dedicated over $15 billion dollars in taxpayer money to decrease the amount of P being released, while completely neglecting N-release (CERP 1999).
Unfortunately, this will cause increases in eutrophication to areas of south Florida where
N-limitation is common (e.g. Lee, Martin and St. Lucie counties). Our results support the strong consensus of both estuarine and coastal scientists that there is a need to control both N and P in synchrony, not independently.
32 APPENDIX
Figure 1. Macroalgae sampling locations in south Florida
33
35
)
1 a
- h
30 1 - a 25
20 b
g dry weight dry g
3 - 15 c c 10 c
c µmol PO₄ µmol 5
APA ( APA 0
Dade Monroe Palm Beach St. Lucie Broward Martin Lee
Figure 2. Spatial variation in mean APA (in µmol PO₄-3 g dry weight-1 h-1 ± S.E., n = 204). Dade County (n = 21), Monroe County (n = 62), Palm Beach County (n = 43), St. Lucie County (n = 26), Broward County (n = 18), Martin County (n = 29) and Lee County (n = 5) Letters denote significant differences in mean APA values.
18 a
)
1 16
- h
b 1 - 14 b 12
10
g dry weight dry g
3
- 8 6
4 µmol PO₄ µmol
2 APA ( APA 0 Phaeophytes Rhodophytes Chlorophytes
Figure 3. Taxonomic variation in mean APA (in µmol PO₄-3 g dry weight-1 h-1 ± S.E., n = 204). Chlorophyta (n = 72), Rhodophyta (n = 82) and Phaeophyta (n = 50) Letters denote significant differences in mean APA values.
34 45
c c
) 40
1
- h
c 1
- 35 30 25 b
g dry weight dry g 20
3 - 15 a a 10
µmol PO₄ µmol 5
0 APA ( APA Dry Wet Dry Wet Dry Wet Chlorophytes Rhodophytes Phaeophytes
Figure 4. Temporal variation in mean APA (in µmol PO₄-3 g dry weight-1 h-1 ± S.E., n = 51) at Looe Key, Monroe County. Letters denote significant differences in mean APA values.
16
c
) 14 1
- c c
h
1
- 12 10
8 d g dry weight dry g
6 3 - a 4 b
2 µmol PO₄ µmol 0
APA ( APA Dry Wet Dry Wet Dry Wet
Chlorophytes Rhodophytes Phaeophytes
Figure 5. Temporal variation in mean APA (in µmol PO₄-3 g dry weight-1 h-1 ± S.E., n = 20) at Pepper Park, St. Lucie County. Letters denote significant differences in mean APA values.
35
Figure 6. APA (in µmol PO₄-3 g dry weight-1 h-1) of Cladophora catenata collected at Looe Key, Monroe County, in response to different levels of DIN and SRP enrichment (n = 36).
Figure 7. APA (in µmol PO₄-3 g dry weight-1 h-1) of Taonia sp. collected at Looe Key, Monroe County, in response to different levels of DIN and SRP enrichment (n = 36).
36
Figure 8. APA (in µmol PO₄-3 g dry weight-1 h-1) of Laurencia poiteaui collected at Looe Key, Monroe County, in response to different levels of DIN and SRP enrichment (n = 36).
Figure 9. APA (in µmol PO₄-3 g dry weight-1 h-1) of Spatoglossum schroedi collected at Pepper Park, St. Lucie County, in response to different levels of DIN and SRP enrichment (n = 36).
37
Figure 10. APA (in µmol PO₄-3 g dry weight-1 h-1) of Gracilaria tikvahiae collected at Pepper Park, St. Lucie County, in response to different levels of DIN and SRP enrichment (n = 36).
Figure 11. APA (in µmol PO₄-3 g dry weight-1 h-1) of Cladophora prolifera collected at Pepper Park, St. Lucie County, in response to different levels of DIN and SRP enrichment (n = 36).
38 Rhodophytes Chlorophytes Phaeophytes
40
) 1
- 35
h
1 - 30 25
20
g dry weight dry g
3 - 15 10
µmol PO₄ µmol 5
0 APA ( APA Monroe Dade Broward St Lucie Palm Beach Martin Lee
Figure 12. Taxonomic and spatial variation in mean APA (in µmol PO₄-3 g dry weight-1 h-1 ± S.E., n = 204) values for Chlorophyta (n = 72), Rhodophyta (n = 82) and Phaeophyta (n = 50).
39
Figure 13. Macroalgae tissue and water column N:P versus mean APA (in µmol PO₄-3 g dry weight-1 h-1 ± S.E.) per county sampled. L = Lee County, SL = St. Lucie County, MA = Martin County, BR = Broward County, M = Monroe County, PB = Palm Beach County, and D = Dade County.
40 Sample Sites
Table 1. South Florida macroalgae collection sites
County Map ID Site Latitude Longitude
Monroe 1 Pine Channel 24.694 N 81.402 W
2 Patch Reef 24.614 N 81.409 W
3 Looe Key 24.5454 N 81.407 W
Dade 4 Rickenbacker 25.745 N 80.189 W
5 Cape Florida Lighthouse 25.666 N 80.156 W
6 Black Pointe Marina 25.540 N 80.330 W
Broward 7 John U. Lloyd State Park 26.091 N 80.104 W
8 Ft. Lauderdale Anglin Pier 26.183 N 80.073 W
Palm Beach 9 Princess Anne 26.789 N 80.000 W
10 North Colonel Ledge 26.864 N 80.020 W
11 Jupiter’s Ledge 26.941 N 80.048 W
12 Timmy’s Deep 26.865 N 79.996 W
Martin 13 Three Holes 27.006 N 80.068 W
14 Nine Steps 27.061 N 80.048 W
St. Lucie 15 Pepper Park 27.504 N 80.295 W
Lee 16 Charlie’s Cut 26.569 N 82.207 W
41 Two-Way ANOVA Results
Table 2. Summary of Two-Way ANOVA of APA as a function of phylum, season and the phylum*season interaction in Pepper Park, St. Lucie County and Looe Key, Monroe County.
Site Factor SS d.f. F-value P-value Pepper Model 5.36 5 20.9 <0.001 Park phlyum 3.49 2 34.0 <0.001 season 0.271 1 5.31 0.028 phlyum*season 1.59 2 15.6 <0.001 Error 2.35 46
Looe Key Model 17.4 5 38.7 <0.001 phylum 14.7 2 82.7 <0.001 season 0.643 1 7.16 0.008 phlyum*season 1.87 2 10.4 <0.001 Error 15.7 175
42 Table 3. Summary of Two-Way ANOVA of APA as a function of dissolved inorganic nitrogen (DIN), soluble reactive phosphorus (SRP), and the DIN*SRP interaction for species collected at Pepper Park, St. Lucie County.
Species Factor SS d.f. F-value P-value Cladophora prolifera Model 1.14 8 4.63 0.001 DIN 0.113 2 1.84 0.178 SRP 0.999 2 16.3 <0.001 DIN*SRP 0.025 4 0.212 0.932 Error 0.827 27
Gracilaria tikvahiae Model 239 8 7.34 <0.001 DIN 54.8 2 6.72 0.004 SRP 164 2 20.2 <0.001 DIN*SRP 19.9 4 1.22 0.324 Error 110.1 27
Spatoglossum Model 5780 8 1.66 0.155 schroedi DIN 3890 2 4.47 0.021 SRP 346 2 0.401 0.676 DIN*SRP 1540 4 0.881 0.487 Error 11700 27
43
Table 4. Summary of Two-Way ANOVA of APA as a function of dissolved inorganic nitrogen (DIN), soluble reactive phosphorus (SRP), and the DIN*SRP interaction for species collected at Looe Key, Monroe County.
Species Factor SS d.f. F-value P-value Cladophora Model 0.635 8 3.19 0.011 catenata DIN 0.341 2 6.83 0.004 SRP 0.284 2 5.71 0.008 DIN*SRP 0.011 4 0.111 0.979 Error 0.673 27
Laurencia poiteaui Model 506 8 2.96 0.016 DIN 108 2 2.54 0.097 SRP 337 2 0.002 0.002 DIN*SRP 59.9 4 0.701 0.599 Error 578 27
Taonia sp. Model 32.9 8 0.621 0.749 DIN 17.4 2 1.32 0.283 SRP 12.1 2 0.922 0.409 DIN*SRP 3.34 4 0.133 0.971 Error 177 27
44
Table 5. Historical APA values for Looe Key and Pine Channel sites in Monroe County for 1992, 1996 and 2011.
Historical Current Source Site Species APA APA Lapointe et al. 1992 Florida Keys Laurencia poiteaui 101 10.7 Laurencia intricata 52.8 21.9 Sargassum polyceratium 27.8 24.5 Average 60.9 24.7
Urzenis 1995 Pine Channel Laurencia intricata 38.2 32.9 Dictyota cervicornis 84.7 42.5 Average 63.5 40.3 Looe Key Laurencia poiteaui 21.1 10.7 Average 34.9 19.9
45 Table 6. Comprehensive list of sampled macroalgae from the seven counties (n = 104). M = Monroe County, D = Dade County, BR = Broward County, PB = Palm Beach County, MA = Martin County, SL = St. Lucie County, L = Lee County. Numbers indicate the number of times species was collected within each county.
46 Table 6. Comprehensive list of sampled macroalgae from the seven counties (n = 104). M = Monroe County, D = Dade County, BR = Broward County, PB = Palm Beach County, MA = Martin County, SL = St. Lucie County, L = Lee County. Numbers indicate the number of times species was collected within each county.
47 Table 6. Comprehensive list of sampled macroalgae from the seven counties (n = 104). M = Monroe County, D = Dade County, BR = Broward County, PB = Palm Beach County, MA = Martin County, SL = St. Lucie County, L = Lee County. Numbers indicate the number of times species was collected within each county.
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