1 PHYTOPLANKTON BIOMASS and COMPOSITION in APALACHICOLA BAY, a SUBTROPICAL RIVER DOMINATED ESTUARY in FLORIDA by PAULA A. VIVER

PHYTOPLANKTON BIOMASS AND COMPOSITION IN APALACHICOLA BAY, A SUBTROPICAL RIVER DOMINATED ESTUARY IN FLORIDA

PAULA A. VIVEROS BEDOYA

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2014

To my beloved parents and grandparents who worked really hard so I could become the first doctor in the family. To Alejandro my daily source of motivation and inspiration.

ACKNOWLEDGMENTS

This research was supported by the NSF-SEAGEP fellowships program, a

Graduate Research Fellowship from the Estuarine Reserves Division, Office of Ocean and Coastal Resource Management at the National Oceanic and Atmospheric

Administration (NOAA), a Research Assistantship Match from the University of Florida, a Delores Auzenne Dissertation Award and the Supplemental Retention Award program of the Office of Graduate Minority Programs at the University of Florida.

Invaluable field assistance was generously provided by personnel from the

Apalachicola National Estuarine Research Reserve (ANERR). I would like to thank Nikki

Dix, Loren Mathews, Bailey Trump and Ake Srifa from the Phlips Lab for their help during different stages of this research. Special thanks to Jynessa Dutka-Gianelli for her support with GIS, and Nikolay Bliznyuk for offering statistical support for this research.

I would also like to thank Shirley Baker, Mark Brenner, Lee Edminston and Karl

Havens for their feedback and support during the development of this research. I give special thanks to my advisor Ed Phlips for giving me this opportunity and for his constant guidance and advice.

I want to express my gratitude to my family, specially my parents Tina y Julio, and my brothers Andres y Felipe for always believing in me and offering me their love. I thank my husband Luke for his patience and unconditional support through this journey and my son Alejandro for giving me additional inspiration. Thanks also to my parents-in- law Nancy y Norris for their encouragement and support. Last but not least I would like to thank my wonderful friends for helping me keep a smile and a positive attitude.

Thank you all I would have not done it without you.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 7

LIST OF FIGURES ...... 9

ABSTRACT...... 12

CHAPTER

1 INTRODUCTION ...... 14

2 SPATIAL AND TEMPORAL DYNAMICS OF PHYTOPLANKTON BIOMASS AND RIVER DISCHARGE IN THE APALACHICOLA ESTUARY, FLORIDA, USA ...... 18

Methods ...... 19 Site Description ...... 19 Field Procedures ...... 20 Water Chemistry ...... 21 Statistical Analyses ...... 22 Results ...... 22 Physical- Chemical Variables ...... 22 River discharge...... 22 Salinity patterns ...... 23 Temperature ...... 23 Nutrient patterns ...... 24 Phytoplankton Biomass Patterns ...... 25 Changes in Phytoplankton Biomass...... 27 Discussion ...... 28 Salinity and Nutrient Ecoclines ...... 29 Temporal Variability in Physical and Biological Factors ...... 32

3 SPATIAL AND TEMPORAL PATTERNS OF PHYTOPLANKTON COMPOSITION IN A SUBTROPICAL ESTUARY, APALACHICOLA BAY, FLORIDA, USA ...... 57

Methods ...... 58 Site Description ...... 58 Field Procedures ...... 59 Water Chemistry ...... 59 Nutrient Limitation Bioassays ...... 60 Phytoplankton Analysis ...... 61 Statistical Analyses ...... 62

Results ...... 63 Physical-Chemical Variables ...... 63 River discharge...... 63 Nutrients...... 63 Salinity ...... 64 Temperature ...... 64 Color ...... 65 Secchi depth ...... 65 Nutrient limitation bioassays ...... 66 Cluster analysis ...... 66 Phytoplankton abundance...... 67 Seasonality of Phytoplankton Biovolume ...... 68 Relationships Between Phytoplankton Community Assemblages and Environmental Variables...... 71 Discussion ...... 74

4 SUMMARY ...... 116

LIST OF REFERENCES ...... 119

BIOGRAPHICAL SKETCH...... 123

LIST OF TABLES

Table page

2-1 Summary characteristics of sampling sites for nutrient and chlorophyll a in the Apalachicola NERR SWMP...... 36

2-2 Duncan’s Multiple Range Test for salinity during high discharge and low discharge ...... 36

2-3 Duncan’s Multiple Range Test for temperature during high discharge and low discharge...... 36

2-4 Duncan’s Multiple Range Test for total soluble phosphorus (TSP) during high discharge and low discharge...... 37

2-5 Duncan’s Multiple Range Test for total soluble nitrogen (TSN) during high discharge and low discharge...... 37

2-6 Duncan’s Multiple Range Test for chlorophyll a during high discharge and low discharge...... 38

2-7 Spearman rank correlation coefficients (top) and p-values (bottom) for selected variables at different sites across Apalachicola Bay...... 38

2-8 Results from t-Tests used to compare the significance of mean values for the four variables during high discharge and low discharge...... 39

2-9 Spearman rank correlation coefficients (top) and p-values (bottom) between Chlorophyll a, TSP and TSN at different sites across Apalachicola Bay...... 39

2-10 Summary statistics during high discharge for variables measured monthly at eight selected sites from March 2007- September 2012...... 40

2-11 Summary statistics during low discharge for variables measured monthly at eight selected sites from March 2007- September 2012...... 41

3-1 Duncan tests for total phosphorus (TP) in Apalachicola Bay during low and high river discharge ...... 83

3-2 Duncan tests for total nitrogen (TN) in Apalachicola Bay during low and high river discharge ...... 83

3-3 Duncan tests for silica (Si) in Apalachicola Bay during low and high river discharge ...... 84

3-4 Duncan tests for salinity during low and high discharge. Concentrations expressed as psu. Letters indicate groups based on mean values...... 84

3-5 Summary statistics for physical variables in Apalachicola Bay during high and low river discharge. LD= low discharge (left), HD= high discharge (right)...... 85

3-6 Percent of limitation by different treatments in the six bioassay experiments conducted at six selected sites ...... 85

3-7 Duncan tests for chlorophyll a in Apalachicola Bay during low and high river discharge...... 86

3-8 Duncan tests for phytoplankton biovolume in Apalachicola Bay during low and high river discharge ...... 86

3-9 Summary statistics for variables measured monthly at the twelve sampling sites in Apalachicola Bay during high and low river discharge ...... 87

3-10 Major phytoplankton blooms at the twelve sampling sites in Apalachicola Bay form June 2008 to June 2010...... 90

3-11 List of Species observed in the Apalachicola Estuary, Florida, USA from June 2008 to 2010...... 91

LIST OF FIGURES

Figure page

2-1 Map showing the Apalachicola Bay estuary and the location of the sites of study...... 42

2-2 Average monthly river discharge for the Apalachicola River from January 2000 to December 2012 at the Sumatra gauge...... 43

2-3 Time series plots of salinity (gray) vs river discharge (black line) at six selected sites across Apalachicola Bay...... 44

2-4 Seasonal distribution of salinity (bars) vs river discharge (black line) at six selected sites across Apalachicola Bay ...... 45

2-5 Time series plots showing temperature at three selected sites in Apalachicola Bay...... 46

2-6 Time series plots of TSP (gray) vs river discharge (black line) at eight selected sites across Apalachicola Bay...... 47

2-7 Seasonal distribution of TSP (bars) vs river discharge (black line) at eight selected sites across Apalachicola Bay from March 2007 to September 2012. .. 48

2-8 Time series plots of TSN (gray) vs river discharge (black line) at eight selected sites across Apalachicola Bay...... 49

2-9 Seasonal distribution of TSN (bars) vs river discharge (black line) at eight selected sites across Apalachicola Bay from March 2007 to September 2012. .. 50

2-10 Relationship between salinity and phytoplankton biomass (CHL a [μg L-1]) at six selected sites across Apalachicola Bay...... 51

2-11 Time series plots of chlorophyll a (gray) vs discharge (black line) at six selected sites across Apalachicola Bay...... 52

2-12 Relationship between chlorophyll a and discharge at six different sites in Apalachicola Bay from April 2002 to September 2012...... 53

2-13 Seasonal distribution of chlorophyll a (bars) vs discharge (black line) at eight selected sites across Apalachicola Bay from March 2007 to September 2012 ... 54

2-14 Chlorophyll trends under high and low discharge at four different sites in Apalachicola Bay from April 2002 to September 2012 ...... 55

2-15 Chlorophyll trends under high and low discharge at four different sites in Apalachicola Bay from April 2002 to September 2012 ...... 56

3-1 Average monthly river discharge for the Apalachicola River from June 2008 to June 2010. Line indicates the calculated grand mean (532 m3 s-1)...... 97

3-2 Temperature at three selected sites (231, 161 and 201) from June 2008 to June 2010...... 97

3-3 Cluster analysis grouping of the sampling sites during low discharge based on physical, chemical, and biological characteristics...... 98

3-4 Cluster analysis grouping of the sampling sites during high discharge based on physical, chemical, and biological characteristics...... 99

3-5 Monthly concentrations of total nitrogen (TN, light gray) and dissolved inorganic nitrogen (DIN, dark gray), at six representative sampling sites...... 100

3-6 Temporal variation in monthly concentrations of total phosphorus (TP, light gray) and soluble reactive phosphorus (SRP, dark gray) ...... 101

3-7 Mean total phosphorus (TP), total nitrogen (TN) and Silica (Si) from June 2008 to June 2010 for eight selected sites in Apalachicola Bay ...... 102

3-8 Mean chlorophyll a (Chl a), phytoplankton biovolume (BV) and carbon (C) from June 2008 to June 2010 for eight selected sites in Apalachicola Bay ...... 103

3-9 Time series plots of chlorophyll a (gray) vs discharge (black line) at six selected sites in Apalachicola Bay, from June 2008 to June 2010...... 104

3-10 Phytoplankton biovolume (106 µm3 ml-1) subdivided into major groups at the river, north estuary (site 171), East Bay (site 191), and Mid Bay (site 161) ...... 105

3-11 Phytoplankton biovolume (106 µm3 ml-1) subdivided into major groups at the west (sites 141 and 143) and east (sites 221 and 223)...... 106

3-12 Phytoplankton biovolume (106 µm3 ml-1) subdivided into major groups at the outer estuary (sites 131, 151 and 211) and the gulf region (site 201)...... 107

3-13 Canonical correlation analysis plots of the major phytoplankton groups at four selected sites in Apalachicola Bay ...... 108

3-14 Distribution of total phytoplankton in the Apalachicola estuary with relationship to total phosphorus (TP) and total nitrogen (TN)...... 110

3-15 Distribution of total phytoplankton in the Apalachicola estuary with relationship to salinity and temperature...... 111

3-16 Distribution of small phytoplankton (<20 µm), large phytoplankton (>20 µm) and chain-forming centric diatoms across salinity gradients ...... 112

3-17 Distribution of common bloom forming species in relationship to salinity in the Apalachicola estuary ...... 113

3-18 Distribution of common bloom forming species in relationship to temperature (ºC) in the Apalachicola estuary ...... 114

3-19 Distribution of phycocyanin-rich and phycoerythrin-rich cyanobacteria in the Apalachicola estuary with relationship to salinity ...... 115

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

PHYTOPLANKTON BIOMASS AND COMPOSITION IN APALACHICOLA BAY, A SUBTROPICAL RIVER DOMINATED ESTUARY IN FLORIDA

Paula A. Viveros Bedoya

May 2014

Chair: Edward Phlips Major: Fisheries and Aquatic Sciences

The integrity of estuaries throughout the world is being impacted by human-driven changes in the ecosystems including their watersheds. The Apalachicola River estuary is an example of a major ecosystem in jeopardy of significant change as a consequence of reduced river discharge, which is a key regulator of physical-chemical processes such as salinity, nutrient concentrations, and water residence time. The overall goal of this study was to describe how changes in river discharge influence phytoplankton community structure, biomass, and dynamics. Phytoplankton composition and physical- chemical variables were determined on a monthly basis for two years (June 2008-June

2010) at 12 sampling sites within the bay. Seasonality of phytoplankton biomass was determined using a longer pre-existing data set (2002-2012). Mean salinities in the

Apalachicola Bay varied significantly between the low- and high-discharge periods, which caused salinity to double at some of the sites. Mean total soluble phosphorus

(TSP) concentrations also varied seasonally and spatially due to the influence of both the Apalachicola River and the Gulf of Mexico in providing phosphorus for the estuary.

In terms of nitrogen The Apalachicola River was a major source for the estuary independent of the season. Nutrient limitation bioassays indicated that phosphorus was

the primary limiting nutrient for the bay. Mean chlorophyll a concentrations and mean phytoplankton biovolumes were higher in the low-discharge season than in the high- discharge season at most sampling sites. The phytoplankton community exhibited seasonal changes, with diatoms being the dominant group throughout most of the year, but with some biovolume peaks caused by dinoflagellates. Cyanobacteria were more abundant during periods of low discharge and warmer temperatures. Results indicated that seasonal patterns in river discharge affect salinity and nutrient concentrations, and were correlated to phytoplankton abundance, biovolume, and composition.

CHAPTER 1 INTRODUCTION

Estuaries are biotically rich and highly productive ecosystems along earth’s coastal zones, where there is interaction between the ocean, the land and the atmosphere (Odum, 1971; Day et al., 1989). Estuaries provide more ecosystem services per unit surface area than any other biome on the planet (Costanza et al.,

1997). The integrity of estuaries throughout the world is, however, being compromised by human-driven changes in the ecosystems, including their watersheds. Most estuaries in the US and worldwide currently face challenges associated with increased human development. Nutrient enrichment from agricultural, urban and industrial development can lead to increased harmful algal blooms (HABs). Additionally, changes in temperature, precipitation and hydrology can impact estuarine systems and affect their biota.

One major class of estuaries is those influenced by inputs of large rivers. This study focused on one such ecosystem, Apalachicola Bay, located in the subtropical Gulf of Mexico. Estuaries are subject to a high degree of environmental variability, including daily, seasonal and inter-annual changes in temperature, light, hydrology, and chemistry

(Livingston, 1983). Some examples of the physical/chemical factors that regulate estuaries are river discharge, nutrient inputs (mainly nitrogen, phosphorus, and silica), light availability, temperature, turbidity and seasonality (Eyre et al., 1999). The biotic composition of estuaries is strongly influenced by these regulating factors.

Historically, there have been many ecological studies in temperate estuaries, , whereas subtropical and tropical estuaries have received less attention. Unlike temperate estuaries, tropical and subtropical estuaries are subject to less pronounced

seasonality, highly variable rainfall patterns, larger inter-annual variation in nutrient inputs, higher annual total light availability and higher average temperature (Eyre et al.,

1999). Consequently, the principles governing ecological relationships in temperate estuaries are not necessarily the same as those in subtropical and tropical estuaries, and the biotic communities found in temperate estuaries differ from those observed in tropical and subtropical ones.

Subtropical estuaries, such as Apalachicola Bay are important both ecologically and economically. They serve as a spawning ground and nursery for aquatic wildlife and support shellfish and finfish industries in many regions worldwide. The study of ecological interactions between phytoplankton, water quality variables and environmental factors in subtropical estuaries is particularly relevant today. There is a need to improve the level of knowledge on how the food web, and in particular, primary producers are affected by physical-chemical changes occurring in these estuaries and their watersheds.

Phytoplankton are the major primary producers in most estuarine systems throughout the world (Day et al. 1989). The relative influence of environmental factors on estuarine phytoplankton production varies from one estuary to another. It is therefore important to understand the factors that regulate phytoplankton production and biomass in estuaries (Mortazavi et al. 2000a).

The Apalachicola Bay is noteworthy because of the strong influence of the

Apalachicola River on the ecology of the Bay (Livingston, 1983; Mortazavi et al., 2000;

Mortazavi et al., 2000; Edmiston et al., 2008; Edmiston, 2008; Putland et al., 2013). It is the major source of freshwater for the bay, which is dependent on these hydrologic

inputs to sustain the integrity of the ecosystem, including a large and economically important oyster industry (Wilber, 1992; Edmiston, 2008; Wang et al., 2008).

The inflow of the Apalachicola River has been reduced in recent years, as a consequence of both drought conditions and increased upstream anthropogenic water withdrawal (Livingston 2001). Consequently, the integrity of the estuary is currently endangered, including the structure and function of the main primary producer community in the bay, phytoplankton.

The rapid increase in water withdrawals from the river, to supply increasing human development in the watershed, has raised serious concerns about the potential impacts of changes in flow on phytoplankton and the overall ecology of the bay

(Mortazavi et al., 2000; Huang and Spaulding, 2002; Livingston, 2007; Putland and

Iverson, 2007; Edmiston, 2008; Wang et al., 2008; Huang, 2010; Putland et al., 2013).

Among the major concerns is the relation between river discharge and the phytoplankton community, which represents a large fraction of the base of the bay’s food web.

The specific impacts of water withdrawal are not fully understood. Nevertheless, it can be hypothesized that alteration of salinity regimes and nutrient loads will change the estuary’s biotic community, including the phytoplankton community. Some studies have already shown that continuing reductions in flow from the Apalachicola River will significantly reduce productivity in the Bay, thereby negatively affecting critical fish and oyster populations (Livingston, 1997; Putland 2007a; Wilber 1992). The overall goal of this study was to describe how spatial and temporal patterns in the structure and abundance of estuarine phytoplankton are related to changes in river discharge and

related shifts in key physical and chemical variables such as salinity, nutrient concentrations and water residence times.

CHAPTER 2 SPATIAL AND TEMPORAL DYNAMICS OF PHYTOPLANKTON BIOMASS AND RIVER DISCHARGE IN THE APALACHICOLA ESTUARY, FLORIDA, USA

Phytoplankton composition and biomass in estuaries is affected by numerous physical, chemical, and biological factors. Site-specific mechanistic understanding of phytoplankton dynamics is required to address the driving factors for shifts in composition and biomass (Cloern, 2001, 2010). Historically, temperate estuaries have been the focus of more studies than subtropical and tropical estuaries. Compared to temperate ecosystems, tropical and subtropical environments are typically subject to less pronounced seasonal variability in temperature and solar light flux, but can exhibit wide swings in rainfall associated with tropical storms, which in turn impact nutrient loading (Eyre et al. 1999). Consequently, the ecological principles ruling phytoplankton dynamics in temperate estuaries are not necessarily the same for subtropical and tropical estuaries.

Phytoplankton communities found in tropical and subtropical estuaries can also differ substantially from those in temperate systems (Reynolds 2006). The purpose of this study was to examine phytoplankton dynamics in Apalachicola Bay, a sub-tropical river-dominated estuary that is experiencing major anthropogenically driven changes in hydrology. The overall objective was to examine the relationship between trends in river discharge and phytoplankton biomass. The specific objectives were to identify phytoplankton biomass trends during high and low discharge regimes over a ten-year period, i.e. 2002-2012, and to describe the relationship between river discharge, nutrient concentrations, and chlorophyll a, a proxy for phytoplankton biomass. The objectives were pursued within the context of the following contrasting hypotheses:

1. Periods of below-average river discharge result in higher phytoplankton biomass levels because of the combination of longer water residence time, higher and more stable salinity and warmer temperatures.

2. Periods of above-average river discharge result in lower phytoplankton biomass levels because the combination of shorter water residence time, large swings in salinity and cooler temperatures.

Methods

Site Description

This study was carried out in the Apalachicola Bay National Estuarine Research

Reserve (ANERR), located in the Florida panhandle on the north coast of the Gulf of

Mexico (Figure 2-1). Due to its latitude Apalachicola Bay is considered a subtropical estuary (between 23 º and 30 º north), and is strongly influenced by the currents of the

Gulf of Mexico, which replenish the system with warm waters from the Caribbean Sea.

The Apalachicola River is the main source of fresh water for the Apalachicola estuary, and consists of a tri-river drainage system, including approximately 19,200 square miles (~49,730 km2) in Georgia, Alabama, and Florida. The confluence of the

Chatahoochee and Flint rivers forms the headwaters of the Apalachicola system, which drains about 1,030 square miles (~2670 km2) (Livingston 1983).

ANERR is part of a coordinated national monitoring program called the System-

Wide Monitoring Program (SWMP) that was established in 1995 with the goal of identifying and tracking short-term variability and long-term changes in representative estuarine ecosystems and coastal watersheds. The SWMP was designed to be a phased monitoring approach that focused on three different ecosystem characteristics, abiotic factors, biological monitoring, and watershed and land use classifications

(Edmiston et al., 2008). The main goal of implementing the SWMP for the ANERR was

to examine the relationship among short-term variability, long-term change, and other environmental factors on the productivity of the Apalachicola Bay system.

As part of the SWMP, monthly grab samples are collected at 11 sites located across Apalachicola Bay to monitor spatial and temporal fluctuations in nutrients and chlorophyll a concentrations in diverse areas of the bay. The stations were chosen to help determine the influence of the river, local rainfall, adjacent habitats and man’s impact on these water variables. Sampling sites are located in the lower Apalachicola

River, in the coastal area, offshore of the barrier islands, at the SWMP datalogger locations, and throughout the bay.

Field Procedures

Monthly grab samples were collected at the eleven stations (Table 2-1) within and adjacent to Apalachicola Bay. In most cases all grab samples were collected on the same day, unless it wasn’t possible because of bad weather. Water temperature, salinity, and dissolved oxygen were measured at surface and bottom depths for each station with a YSI 85 handheld meter. Only surface measurements for temperature and salinity were considered in this study.

A horizontal Van Dorn-style sampler was used to collect 2.2 liters of water from a depth of 0.5 meters at all stations not associated with a SWMP datalogger site. At the

Cat Point and Dry Bar SWMP datalogger stations, water samples were collected at depths of approximately 2 and 1.5 meters (0.5 m from the bottom), respectively, depths equivalent to the probes of the dataloggers deployed at these sites. At the East Bay datalogger station, water samples were collected from surface (0.5 meters) and bottom

(1.5 meters) depths, equivalent to the depths of the two dataloggers deployed at this site. Triplicate samples were collected each month at one station, rotating through all

station locations. Replicate samples were collected with separate dips of the horizontal sampler.

Water from the Van Dorn sampler was delivered into a polyethylene graduated cylinder. The water sample was then filtered through glass-fiber filters (0.7-μm pore size) for soluble inorganic nutrients and chlorophyll a (CHL) determination. The glass- fiber filter for chlorophyll a analysis and the water samples were transported on ice to the University of Florida laboratory in Gainesville for subsequent processing.

Water Chemistry

Nitrite (NO2) concentrations were determined by mixing the sample with color reagent (phosphoric acid, sulfanilalimide, and N-1-naphthylethylene diamine dihidrochloride) to form a purple azodye (APHA, 1998). Colorimetric quantification was completed on a Bran + Luebbe Autoanalyzer 3 system. Concentrations of nitrate (NO3) and ammonium (NH4) were first reduced to NO2 and then measured as described above. NO3 was reduced to NO2 through a copperized cadmium redactor (APHA,

1998). NH4 was oxidized to NO2 with hypochlorite in an alkaline medium using potassium bromide as a catalyst (Strickland and Parsons, 1972). Dissolved inorganic nitrogen (DIN) was calculated by summing NH4 + NO3 + NO2. Soluble reactive phosphorus (SRP) concentrations were determined by mixing the sample with color reagent (sulfuric acid, ammonium molybdate, ascorbic acid, and antimony potassium tartrate) to form a blue dye (APHA, 1998). Colorimetric quantification was completed on a Hitachi U-2810 (Tokyo, Japan) dual-beam scanning spectrophotometer. CHL was processed using the Sartory and Grobbelaar (1984) hot ethanol extraction method and concentrations (not corrected for pheophytin) were determined spectrophotometrically according to Standard Methods (APHA, 1998). All processing and analytical methods

conformed to the guidelines of the laboratory’s National Environmental Laboratory

Accreditation Program certification.

Statistical Analyses

The SAS Enterprise Guide statistical package for PCs (Version 4.3) was used to carry out statistical analyses. Distributions of most variables were non-normal

(determined by the Shapiro-Wilk and Kolmogorov-Smirnov goodness-of-fit tests), necessitating the use of non-parametric Spearman rank correlation analysis to explore relationships between them. Duncan’s multiple range tests were used to compare the means of the different variables among sites. T-tests were used to evaluate the significance of the seasonal differences between means in low and high discharge for the physical-chemical variables.

Results

Physical- Chemical Variables

River discharge

A twelve-year (January 2000- December 2012) monthly average river discharge for the Apalachicola River at the Sumatra gauge (about 30 km above the river mouth), was calculated with data from the USGS National Water Information System

(http://waterdata.usgs.gov). During that time span, discharge maxima occurred during late winter and early spring, whereas summer months were characterized by discharge minima (Figure 2-2). The grand mean for the twelve years of river discharge data was

532 m3 s-1. The minimum discharge value was 159 m3 s-1 and the maximum 2358 m3 s-

Salinity patterns

A wide range of salinities was observed across the different sites (Table 2-2).

During low discharge, mean salinities ranged from 0.1 psu at Site 231 in the river to

33.3 psu in the gulf, at Site 201. During high discharge, mean salinities ranged from 0.0 psu at Site 231 in the river to 32.3 psu in the gulf at Site 201 (Table 2-2). The results from the Spearman rank correlation coefficients showed a significant negative correlation between river discharge and mean salinities at most sites, except Site 231 in the river and Site 201 in the gulf (Table2-7).

In general, salinity values exhibited a gradient that increased with distance from the river mouth, highlighting the importance of discharge from the Apalachicola River in controlling this variable throughout the bay. The results from the t-test showed a significant difference in the mean salinities between high and low discharge at most sites, except the river and the gulf, Sites 231 and 201, respectively (Table 2-8).

Salinities at Site 231 in the river and Site 201 in the gulf were steady because of the sampling regime used for these two sites, which consisted of sampling once a salinity reading of a given value was reached (0.1 ppt or less for the river; 32 ppt or more for the gulf).

Temperature

Spearman rank correlation coefficients demonstrate a strong negative correlation between river discharge and temperature (Table 2-7). During low discharge, temperature ranged from 24.4 ˚C at Site 141 in Dry Bar to 25.1 ˚C Site 201 in the gulf.

During high discharge, temperature ranged from 17.2 ˚C at Site 231 in the river to 19.3

˚C at Site 191 in East Bay. (Table 2-3). Time series plots showed that water

temperatures exhibited a seasonal trend, with higher temperatures usually occurring from May to October and lower temperatures occurring in January and February (Figure

2-5).

Nutrient patterns

Nutrient concentrations for the study period were analyzed under both high- discharge and low-discharge periods. Mean TSP concentrations were generally higher during periods of low discharge than periods of high discharge (Table 2-4 and Figures

2-6 and 2-7). The t-test showed that the difference between mean TSP concentrations measured during high and low discharge was not significant at most sites, except Site

231 in the river (Table2-8). During low discharge, mean TSP concentrations ranged from 11 µg P L-1 at Site 171 near the East Bay Bridge to 22 µg P L-1 at Site 201 in the gulf (Table 2-4).

During high discharge, mean TSP concentrations ranged from 12 µg P L-1 at Site

191 in East Bay to 18 µg P L-1 at Site 231 in the river (Table 2-4). Time series plots show an inverse relationship between river discharge and TSP concentrations at most sites (Figure 2-6), with the highest concentration peaks occurring during periods of extended low discharge at most sites, except Site 231 in the river. The Spearman rank correlation coefficients (Table 2-7) showed no significant correlation between TSP and river discharge at most sites, except Site 231 in the river and Site 201 in the gulf. In the low discharge period, TSP concentrations increased with distance from the river to the gulf (Table 2-3), but conversely in the high discharge period TSP concentrations decreased with distance from the river to the gulf.

Mean TSN concentrations were generally higher during periods of high discharge than periods of low discharge at most sites, except Site 201 in the gulf, where mean

TSN concentrations were lower during high discharge (Table 2-5 and Figures 2-8 and 2-

9). During both high and low discharge, mean TSN concentrations followed a gradient that decreased with distance from the river towards Site 151 in Pilots Cove, near the barrier islands (Table 2-5). During low discharge, mean TSN concentrations ranged from 136 µg N L-1 at Site 201 in the gulf to 546 µg N L-1 at Site 231 in the river (Table 2-

5).

During high discharge, mean TSN concentrations ranged from 119 µg N L-1 at

Site 201 in East Bay to 614 µg N L-1 at Site 231 in the river (Table 2-5). Regardless of the season, Site 231 in the river always displayed the highest mean concentrations of

TSN and Site 201 in the gulf the lowest. Time series plots showed a positive relationship between river discharge and TSN concentrations (Figure 2-8). The results of the t-test for TSN showed a significant difference between means during high and low discharge at most sites, except the river and the gulf, Sites 231 and 201, respectively

(Table 2-8). Spearman rank correlation coefficients showed a significant correlation between TSN and river discharge at most sites (Table 2-7), but again sites 231 in the river and 201 in the gulf were exceptions.

Phytoplankton Biomass Patterns

Duncan’s multiple range tests were conducted for chlorophyll a during both high and low discharge periods (Table 2-6). Chlorophyll a concentrations were higher in low discharge than in high discharge at most sites, except at gulf, Site 201, where chlorophyll a concentrations were slightly more elevated during high discharge. During low discharge, chlorophyll a concentrations ranged from 4.9 µg Chl a L -1 at Site 201 in the gulf to 19.1 µg Chl a L -1 at site 191 in East Bay. During high discharge, chlorophyll

a concentrations ranged from 3.8 µg Chl a L -1 at Site 231 in the river to 17.1 µg Chl a L

-1 at site 191 in East Bay.

Although differences in mean chlorophyll a concentrations between high and low discharge were observed, the t-test results showed a lack of significance between the differences in means at most sites (Table 2-8). The differences in mean chlorophyll a concentrations were only significant at three sites, 231 in the River, and those sites located near the oyster bars, Site 141 in Dry Bar and Site 221 in Cat Point (Table 2-8).

Chlorophyll a and river discharge were negatively correlated at site 231 in the river, site 171 in East Bay Bridge, and in the oyster bars, sites 141(Dry Bar) and 221

(Cat Point) (Table 2-7). At the rest of the sites, a lack of correlation between chlorophyll a and river discharge was observed (Table 2-7).

Time series plots showed that there is an inverse relationship between river discharge and chlorophyll a (Figure 2-11). During periods of prolonged high discharge, such as spring 2005 and 2010, chlorophyll a concentrations throughout the bay fell below 5 µg Chl a L -1 at most sites (Figure 2-12). Conversely, during periods of extended low discharge, like summer/fall 2010 and 2011, chlorophyll a levels tended to peak at the different sites, particularly at Site 191 in East Bay, located in the northeast portion of the bay. In general, Site 191 stood out because it exhibited the highest mean phytoplankton biomass during both periods, with concentrations of 17.1 µg Chl a L -1 in high discharge and 19.1 in low discharge.

A closer look at the relationship between discharge and chlorophyll a indicated that most sites exhibited chlorophyll optima at discharges <1000 m3 s-1 but, there were

regional differences in the way discharge affects chlorophyll patterns at the different locations (Figure 2-12).

The relationship between salinity and phytoplankton biomass varied at the different locations in the estuary (Figure 2-10). Site 171 near the East Bay Bridge displayed maximum chlorophyll a concentrations between 10 and 20 psu. Site 191 in

East Bay displayed maximum chlorophyll a concentrations between 3 and 20 psu. In the middle of the bay, Dry Bar (Site 141) and Cat Point (Site 221), which are located close to the main oyster bars, and site 161 in the center of the bay, displayed maximum chlorophyll a concentrations between 10 and 30 psu. At site 151 Pilots Cove, located near the barrier islands, the optimum salinity range for maximum chlorophyll a concentrations varied from about 15 to 35 psu.

Changes in Phytoplankton Biomass

There are empirical indications that changes may have occurred in the chlorophyll a concentrations in Apalachicola Bay in the last ten years (Figures 2-14 and

2-15). Chlorophyll levels showed a long-term increase at all sites in the bay under both high and low discharge regimes (Figures 2-8 and 2-9). Although these relationships were not statistically significant (R2 < 0.25), there were a few sites such as 191 in East

Bay, 141 in Dry Bar, and 221 in Pilots Cove, where the steepness of the slopes indicated increases in chlorophyll a concentrations, especially after 2005. These increases in phytoplankton biomass were more pronounced during low discharge than during high discharge.

Discussion

River dominated estuaries frequently exhibit significant variation in environmental conditions, which affect phytoplankton composition and biomass (Smayda, 1978;

Malone et al., 1988; Eyre and Balls, 1999; Mortazavi et al., 2000; Murrell et al., 2007;

Quinlan and Phlips, 2007; Paerl et al., 2010). The variability can take the form of spatial gradients in key variables such as salinity and nutrient concentrations, sometimes referred to as ecoclines (Attrill and Rundle, 2002; Murrell et al., 2007; Quinlan and

Phlips, 2007). An ecocline represents a gradient of change in key variables (both spatial and ecological) between two systems (e.g. river to ocean); it is a response to gradual differences in at least one major environmental factor. Additional factors can influence the gradient and the character of transitional states (Attrill and Rundle, 2002).

Ecoclines are important features of Apalachicola Bay, and the specific character and dynamics of these ecoclines are strongly influenced by river discharge. The central goal of this study was to determine whether the abundance of phytoplankton in the estuary is linked to discharge from the Apalachicola River. Certain regions of the

Apalachicola Bay estuary manifest links between river discharge, ecoclines in key environmental variables, and phytoplankton biomass, whereas other regions exhibit different or lesser relationships, likely because of mitigating factors such as water residence time, temperature, or grazing pressure.

I examined the major ecolines within the bay with respect to salinity, nutrient level, water residence times) and explored their potential links to variability in phytoplankton biomass. I found that river discharge exerts some control over phytoplankton biomass (Table 2-6 and Figures 2-11, 2-12 and 2-13), however the effects should be considered within the context of related factors that regulate losses

and gains in algal biomass, such as salinity, nutrient concentrations, temperature, zooplankton grazing, water residence time and tidal water exchange with the Gulf of

Mexico.

Salinity and Nutrient Ecoclines

The most obvious ecocline directly associated with river discharge in

Apalachicola Bay is salinity (Livingston, 1984). Salinity values increase with distance from the river mouth, and from west to east, reflecting the predominant flow of gulf water from the eastern inlet of the bay toward the western outlet (Edmiston, 2008). The relative influences of the river and gulf on salinity in the bay depend on the amount of discharge. This is illustrated by differences in mean salinity at Site 141 in the middle of the bay during high and low discharge periods. During high discharge, the mean salinity was 12.8 psu whereas during low discharge it was 24.5 psu, reflecting the importance of higher salinity water coming from the Gulf of Mexico during the latter period.

A previous study of Apalachicola Bay reported that the concentration of chlorophyll a peaked in waters with salinities between 5 and 25 psu (Putland et al.,

2013), however my results indicate that the optimal salinity ranges vary by region in the bay (Figure 2-10). At bay sites close to river inflow (i.e. Sites 171 and 191), salinities commonly fall below 5 psu. Peaks in phytoplankton biomass associated with prolonged periods of low salinity, which are common in this region, are dominated by freshwater species of algae (Chapter 3). Conversely, peaks in phytoplankton biomass associated with prolonged periods of mesohaline (i.e. 5-20 psu) conditions in the same region are dominated by marine phytoplankton taxa (Chapter 3).

In the mid-bay, the highest phytoplankton biomass peaks are associated with mesohaline/polyhaline conditions (i.e. 10-30 psu), which are most commonly related to periods of low-moderate river discharge conditions (Figure 2-10), during which phytoplankton communities are dominated by marine taxa (Chapter 3). Salinities below

10 psu in the mid-bay are generally associated with relatively low phytoplankton biomass (Figure 2-10), reflecting the effect of reduced water residence times and increased osmotic stress experienced during periods when large freshwater inflows from the river result in low and variable salinities. The latter condition is a feature of some river-dominated estuaries in which there is a region of the ecocline at which salinities are frequently high enough to cause mortality of freshwater species, but too low and variable to support high biomass of marine species (Eyre and Balls, 1999;

Murrell et al., 2007).

To evaluate the role of salinity in defining phytoplankton biomass potential, it is worthwhile to keep in mind that salinity is important, but may be associated with a broad range of other variables, such as nutrient availability, water residence time, light transmission through the water column and grazing activity, which in Apalachicola Bay includes both benthic and planktonic grazing.

Nutrient concentration gradients are important ecoclines in Apalachicola Bay.

Previous studies highlighted the importance of the Apalachicola River in adding nutrients to the estuary, which enhances phytoplankton production (Livingston, 1983;

Mortazavi et al., 2000; Mortazavi et al., 2001; Edmiston, 2008). In many river- dominated estuaries, peak phytoplankton biomass is found between the light-limited

oligohaline (i.e. 0.5-5 psu) upper estuary and nutrient-limited mixoeuhaline (i.e. >30 psu), marine-dominated region.

The spatial distribution of phytoplankton maxima in Apalachicola Bay deviates somewhat from this pattern, in part because of barrier islands that affect tidal water exchange, and in part because of the unique character of the Apalachicola River discharge. The impacts of discharge on nutrient concentrations in the bay vary by region and nutrient type. The positive correlation between river discharge and total soluble nitrogen concentration in the river is matched by positive correlations between river discharge and total soluble nitrogen (DIN) at all sites in the bay (Tables 2-5 and 2-

7 and Figures 2-8 and 2-9). By contrast, the positive relationship between discharge and total soluble phosphorus concentrations in the river is not seen in the bay (Figures

2-6 and 2-7), where the correlation is negative (Tables 2-4 and 2-7). These contrasting relationships with respect to river discharge and nutrients (DIN and TSP) indicate that the river is a source of nitrogen enrichment for the bay, but the situation for phosphorus is more complicated, possibly because of nutrient contributions from the Gulf of Mexico and advective and diffusive fluxes of nutrients from bottom sediments in the bay.

In terms of sources of phosphorus to support phytoplankton production in

Apalachicola Bay, it is noteworthy that mean TSP concentrations in the Apalachicola

River are highest during high discharge periods, whereas mean concentrations in the

Gulf of Mexico are comparatively low. Conversely, during the summer, when mean river discharge is low, mean TSP levels are also low in the river, but concentrations in the gulf are high relative to the river and bay. The latter relationships suggest that the gulf may be a source of phosphorus for phytoplankton production in the summer.

Phosphorus budgets for Apalachicola Bay (Mortazavil et al., 2000) estimated that 78% of annual total phosphorus input to the bay comes from the Apalachicola River and 22% from the Gulf of Mexico, of which 41% is in soluble form. The TSP gradients observed in the current study (Table 2-4, Figures 2-6 and 2-7) suggest that the influence of the Gulf of Mexico on phosphorus supply to the bay is strongest during summer. The importance of the latter observation is highlighted by the results of nutrient limitation bioassays, which show a predominance of phosphorus limitation (Chapter 3).

A long-standing paradigm in aquatic science is that primary production in freshwater systems is often limited by phosphorus availability, whereas the primary limiting nutrient in most marine systems is nitrogen (Board, 2000). There are of course many exceptions to this general rule (Howarth, 1988; Myers and Iverson, 1982), and

Apalachicola Bay is a case in point (Mortazavi et al., 2000; Myers and Iverson, 1982).

Results of bioassays in the bay indicate that phosphorus is more often the primary limiting nutrient for phytoplankton production (Viveros et al., 2014), in part a result of the relatively low phosphorus levels in the Apalachicola River, as indicated by the high DIN/SRP ratios (i.e. >80) (Putland et al., 2013). The high nitrogen to phosphorus ratios may reflect the relatively small amount of human disturbance in the watershed in terms of nutrient enrichment (Howarth, 1988; Board, 2000).

Temporal Variability in Physical and Biological Factors

The effects of temporal shifts in salinity and nutrient ecoclines in Apalachicola

Bay on phytoplankton biomass must be viewed within the context of variation in key physical and biological factors, such as temperature, light flux, water residence times, and grazing rates. These factors play a major role in defining the relationships between nutrient load and phytoplankton biomass (Reynolds, 2006). Elevated nitrogen and

phosphorus loads to the bay from the river, particularly during the winter and early spring enhance the potential for increased biomass, but low temperatures, reduced water residence times and high salinity variability have a negative effect on the attainment of that potential (Mortazavi et al., 2000). Similarly, spring peaks in benthic grazer activity can exacerbate the loss of biomass.

Water temperatures in Apalachicola Bay are highly correlated with air temperature because the bay is shallow and the water column is frequently wind-mixed.

Only incipient thermal stratification has been detected because of frequent mixing in the bay (Livingston, 1983). Temperature ranges from 5 to 33 °C within a year, with peak temperatures generally occurring in July and August and lowest temperatures occurring from December through February (Livingston, 1984). During the period this study was conducted mean temperature in the estuary was 18 °C during high discharge and 24.6

°C during low discharge. Spearman rank correlation coefficients demonstrate a strong negative correlation between river discharge and temperature (Table 2-7).

This relationship could be seen as an autocorrelation, given that the high discharge period occurs in the winter and spring months. Therefore, the combination of lower temperature and high discharge may act to control phytoplankton biomass during the high discharge season. Conversely, the combination of higher temperature and low discharge could facilitate phytoplankton growth during the summer and fall.

This study demonstrated that river discharge plays a major role in controlling phytoplankton biomass. Previous studies found that export from Apalachicola Bay provided a significant control on phytoplankton biomass during winter months, but on an annual basis, grazing by zooplankton and oysters accounted for 80% of the chlorophyll

a loss from the estuary (Mortazavi et al., 2000). The present study did not evaluate the role of grazers in controlling phytoplankton biomass, and the limited temporal scope of previous studies made it difficult to assess quantitatively the impact of zooplankton grazing. Although large oyster populations in the bay undoubtedly have an impact on phytoplankton biomass, little research has been done to quantify that impact.

Apalachicola Bay exhibits higher chlorophyll a concentrations during late spring and summer months when river discharge is low and water residence time is high.

Previous studies have found similar patterns in other subtropical estuaries. In Escambia

Bay, near Pensacola, Florida, chlorophyll a concentrations generally were also higher during the summer and lower during the winter and spring, and freshwater flow appears to be an important driver of phytoplankton dynamics (Murrell et al., 2007). In the

Matanzas River Estuary (MRE), near St. Augustine, Florida, phytoplankton biomass in the winter was relatively low mainly because of the combination of low temperature and light availability, consistent tidal water exchange and bivalve grazing throughout the year. Relatively low levels of phytoplankton standing stock and small inter-annual variability within the MRE reflect a balance between gain and loss processes (Dix et al.,

2013). Similar studies in Australian tropical estuaries found suppressed phytoplankton biomass as a consequence of increased turbidity from freshwater floods (Eyre and

Balls, 1999).

Results from this study support the hypothesis that periods of below average river discharge are associated with higher phytoplankton biomass levels. It is possible to hypothesize that this may be a result of longer water residence time, higher and more stable salinity and warmer temperatures. Ecoclines were important features of

Apalachicola Bay, and the specific character and dynamics of these ecoclines is influenced by changes in river discharge. Discharge from the Apalachicola River has an effect on salinity and TSN concentrations.

Table 2-1. Summary characteristics of sampling sites for nutrient and chlorophyll a in the Apalachicola NERR SWMP. Site Water depth average number Site name (m) Bottom habitat 141 Dry Bar 1.7 Oyster bar 151 Pilot's Cove 1.8 Patchy seagrass 161 Mid Bay 2.2 Sandy silt 171 East Bay Bridge 1.6 Silty clay 181 East Bay Surface 1.7 Clayey sand 201 Sikes Cut Offshore 5 Sand 221 Cat Point 1.8 Oyster bar 231 River 3.5 Sandy silt

Table 2-2. Duncan’s Multiple Range Test for salinity during high discharge and low discharge from March 2007 to September 2012. Concentrations expressed as psu. Letters indicate groups based on mean values. Low discharge salinity High discharge salinity Duncan Grouping Mean SITE Duncan Grouping Mean SITE A 33.4 201 A 32.3 201 B 29.2 151 B 17.5 151 C 24.8 221 C 12.8 141 C 24.5 141 C 12.6 221 C 24.2 161 C 10.3 161 D 12.7 171 D 3.5 191 D 11.8 191 D 3.2 171 E 0.1 231 D 0.1 231

Table 2-3. Duncan’s Multiple Range Test for temperature during high discharge and low discharge from March 2007 to September 2012. Concentrations expressed as °C. Letters indicate groups based on mean values. Low discharge temperature High discharge temperature Duncan Duncan Grouping Mean SITE Grouping Mean SITE A 25.1 201 A 19.3 191 A 24.9 231 A 18.4 201 A 24.9 171 A 18.4 171 A 24.7 151 A 17.9 161 A 24.6 191 A 17.8 151 A 24.6 221 A 17.8 221 A 24.5 161 A 17.6 141 A 24.4 141 A 17.2 231

Table 2-4. Duncan’s Multiple Range Test for total soluble phosphorus (TSP) during high discharge and low discharge from March 2007 to September 2012. Concentrations expressed as µg P L-1. Letters indicate groups based on mean values. Low discharge TSP High discharge TSP Duncan Duncan Grouping Mean SITE Grouping Mean SITE A 22 201 A 18 231 B A 17 151 B A 14 171 B A C 16 221 B A 14 221 B A C 16 141 B A 13 161 B C 14 161 B A 13 201 B C 13 231 B 12 141 B C 13 191 B 12 151 C 11 171 B 12 191

Table 2-5. Duncan’s Multiple Range Test for total soluble nitrogen (TSN) during high discharge and low discharge from March 2007 to September 2012. Concentrations expressed as µg N L-1. Letters indicate groups based on mean values. Low discharge TSN High discharge TSN Duncan Duncan Grouping Mean SITE Grouping Mean SITE A 547 231 A 614 231 B 356 171 B A 549 171 B 314 191 B C 506 191 C 260 221 D C 426 161 C 233 161 D 361 221 C 228 141 D E 340 141 D 168 151 E 255 151 D 136 201 F 119 201

Table 2-6. Duncan’s Multiple Range Test for chlorophyll a during high discharge and low discharge from March 2007 to September 2012. Concentrations expressed as µg L-1. Letters indicate groups based on mean values. Low discharge chlorophyll High discharge chlorophyll Duncan Duncan Grouping Mean SITE Grouping Mean SITE A 19.1 191 A 17.1 191 B 10.7 141 B 8.4 161 C B 9.4 221 B 7.7 171 C B 9.1 171 C B 7.0 141 C B 9.0 161 C B 6.6 221 C D 6.9 151 C B 6.6 151 D 5.3 231 C B 5.1 201 D 4.9 201 C 3.8 231

Table 2-7. Spearman rank correlation coefficients (top) and p-values (bottom) for selected variables at different sites across Apalachicola Bay. Data from March 2007 to December 2012. Discharge m3 s-1 141 151 161 171 191 201 221 231 TSP -0.24 -0.09 -0.10 0.05 -0.20 -0.05 -0.04 0.46 0.05 0.52 0.41 0.68 0.10 0.70 0.76 <.0001 TSN 0.48 0.26 0.58 0.66 0.47 -0.12 0.24 0.33 <.0001 0.05 <.0001 <.0001 <.0001 0.40 0.05 0.01 Chlorophyll a -0.30 0.01 -0.16 -0.32 -0.12 0.04 -0.27 -0.39 0.01 0.96 0.20 0.01 0.34 0.79 0.03 0.00 Temperature -0.49 -0.50 -0.48 -0.45 -0.39 -0.53 -0.47 -0.56 <.0001 0.00 <.0001 0.00 0.00 <.0001 <.0001 <.0001 Salinit y -0.67 -0.60 0.71 -0.78 -0.57 -0.36 -0.67 -0.38 <.0001 <.0001 <.0001 <.0001 <.0001 0.01 <.0001 0.00

Table 2-8. Results from t-Tests used to compare the significance of mean values for the four variables during high discharge and low discharge from March 2007 to September 2012. Salinity Site CHLa µg L-1 (psu) TSN µg L-1 TSP µg L-1 141 Dry Bar 0.0476 0.0001 0.0002 0.1478 151 Pilots Cove 0.6967 0.0001 0.0245 0.1559 161 Mid Bay 0.6083 0.0001 0.0001 0.8091 171 East Bay Bridge 0.2995 0.0001 0.0001 0.0511 191 East Bay 0.4097 0.0001 0.0003 0.4651 201 Sike's Cut 0.7985 0.041 0.4357 0.0202 221 Cat Point 0.0049 0.0001 0.0133 0.3537 231 River 0.0414 0.0215 0.1356 0.0001

Table 2-9. Spearman rank correlation coefficients (top) and p-values (bottom) between Chlorophyll a, TSP and TSN at different sites across Apalachicola Bay. Data from March 2007 to December 2012. TSP Site 141 151 161 171 191 201 221 231 Chlorophyll a 0.05465 -0.0524 0.03093 -0.1876 -0.0009 0.19495 -0.027 -0.3119 0.6605 0.7016 0.8052 0.1376 0.9942 0.1538 0.8286 0.0102 TSN ______Chlorophyll a -0.4142 -0.1777 -0.3511 -0.3457 -0.1347 -0.0827 -0.1706 -0.1633 0.0005 0.1902 0.0038 0.0051 0.2773 0.5483 0.1675 0.1866

Table 2-10. Summary statistics during high discharge for variables measured monthly at eight selected sites from March 2007- September 2012. High Discharge Site Variable Mean Median Std Dev Minimum Maximum N 141 TSP µg P L-1 12 10 7 0 27 22 TSN µg N L-1 340 349 129 159 528 22 CHLa µg L-1 7.0 5.6 3.9 2.0 19.2 22 TEMP ° C 17.6 17.5 6.0 5.7 28.0 21 SAL (psu) 12.8 11.8 8.2 2.4 31.9 22 151 TSP µg P L-1 12 10.5 9.4 0.0 47.0 20 TSN µg N L-1 255 208 151 44 581 20 CHLa µg L-1 7 7 3 2 11 19 TEMP ° C 17.8 17.0 5.9 6.2 27.6 19 SAL (psu) 17.5 16.8 9.2 2.5 32.1 20 161 TSP µg P L-1 13 10 11 4 51 22 TSN µg N L-1 426 390 185 226 1053 22 CHLa µg L-1 8.4 6.0 5.7 1.1 24.4 22 TEMP ° C 17.9 18.0 5.6 6.4 27.5 22 SAL (psu) 10.3 10.8 6.8 0.3 23.4 22 171 TSP µg P L-1 14 14 7 2 26 22 TSN µg N L-1 549 554 155 285 893 22 CHLa µg L-1 7.7 5.3 6.2 1.6 23.9 22 TEMP ° C 18.4 18.4 6.1 6.4 28.9 20 SAL (psu) 3.2 2.0 3.9 0.0 16.9 21 191 TSP µg P L-1 12 11 6 3 28 22 TSN µg N L-1 506 484 199 227 954 22 CHLa µg L-1 17.1 17.4 11.0 2.3 48.3 22 TEMP ° C 19.3 18.1 5.5 11.8 29.9 19 SAL (psu) 3.5 2.9 3.3 0.1 11.3 20 201 TSP µg P L-1 13 14 5 3 22 18 TSN µg N L-1 119 95 78 0 300 18 CHLa µg L-1 5.1 3.6 3.6 1.5 15.5 18 TEMP ° C 18.4 16.9 4.9 10.6 27.6 18 SAL (psu) 32.3 32.0 1.5 30.2 35.3 18 221 TSP µg P L-1 14 11 8 5 44 22 TSN µg N L-1 361 371 174 10 746 22 CHLa µg L-1 6.6 6.6 2.9 2.4 14.0 22 TEMP ° C 17.8 17.7 5.4 6.2 27.2 21 SAL (psu) 12.6 12.6 6.7 0.5 24.4 22 231 TSP µg P L-1 18 18 5 11 31 22 TSN µg N L-1 614 626 178 228 953 22 CHLa µg L-1 3.8 3.4 2.6 1.5 13.6 22 TEMP ° C 17.2 16.5 5.6 6.2 26.6 21 SAL (psu) 0.1 0.1 0.0 0.0 0.1 22

Table 2-11. Summary statistics during low discharge for variables measured monthly at eight selected sites from March 2007- September 2012. Low Discharge Site Variable Mean Median Std Dev Minimum Maximum N 141 TSP µg P L-1 16 9 16 2 71 45 TSN µg N L-1 227 220 99 21 500 45 CHLa µg L-1 10.7 9.3 10.8 0.6 76.5 45 TEMP ° C 24.4 27.4 6.5 8.7 31.5 45 SAL (psu) 24.5 24.4 6.6 12.4 35.2 45 151 TSP µg P L-1 17 11 14 0 65 37 TSN µg N L-1 168 177 78 17 346 37 CHLa µg L-1 6.9 6.4 3.5 0.6 17.3 37 TEMP ° C 24.7 27.2 6.5 8.5 31.4 37 SAL (psu) 29.2 29.0 4.7 15.9 35.4 37 161 TSP µg P L-1 14 11 10 3 46 44 TSN µg N L-1 233 242 98 33 416 44 CHLa µg L-1 9.0 8.5 4.5 0.8 21.7 44 TEMP ° C 24.5 27.3 6.6 9.3 32.1 44 SAL (psu) 24.2 24.9 6.1 8.8 35.2 44 171 TSP µg P L-1 11 10 6 2 28 42 TSN µg N L-1 356 352 133 114 614 42 CHLa µg L-1 9.1 9.0 4.4 0.4 26.0 42 TEMP ° C 24.9 28.1 6.8 6.9 33.1 42 SAL (psu) 12.7 12.2 6.4 1.2 30.7 42 191 TSP µg P L-1 13 11 5 5 27 45 TSN µg N L-1 314 298 131 121 707 45 CHLa µg L-1 19.1 18.6 7.8 0.8 36.7 45 TEMP ° C 24.6 26.7 6.9 5.0 33.5 43 SAL (psu) 11.8 10.8 6.9 0.1 29.8 43 201 TSP µg P L-1 22 12 20 1 81 37 TSN µg N L-1 136 128 76 3 315 37 CHLa µg L-1 4.9 4.3 3.5 0.6 16.9 37 TEMP ° C 25.1 28.3 5.9 11.4 30.8 37 SAL (psu) 33.4 33.8 1.9 29.8 37.0 37 221 TSP µg P L-1 16 13 12 3 49 45 TSN µg N L-1 260 228 141 90 731 45 CHLa µg L-1 9.4 8.5 4.9 0.4 21.8 45 TEMP ° C 24.6 26.9 6.5 7.0 31.4 45 SAL (psu) 24.8 24.3 5.5 11.1 35.2 45 231 TSP µg P L-1 13 12 5 5 31 44 TSN µg N L-1 547 522 168 185 1042 45 CHLa µg L-1 5.3 4.9 2.8 0.6 12.6 45 TEMP ° C 24.9 27.3 6.4 9.7 32.3 45 SAL (psu) 0.1 0.1 0.0 0.1 0.1 45

Figure 2-1. Map showing the Apalachicola Bay estuary and the location of the sites of study.

Figure 2-2. Average monthly river discharge for the Apalachicola River from January 2000 to December 2012 at the Sumatra gauge. Line indicates calculated grand mean (532 m3 s-1).

River River Discharge m

psu

Salinity, Salinity,

- 1

Figure 2-3. Time series plots of salinity (gray) vs river discharge (black line) at six selected sites across Apalachicola Bay.

River River Discharge m

Salinity, psu Salinity,

- 1

Figure 2-4. Seasonal distribution of salinity (bars) vs river discharge (black line) at six selected sites across Apalachicola Bay from March 2007 to September 2012.

River MidBay Gulf

30.0

20.0 Temperature ˚ ˚ C Temperature 10.0

0.0

11 11

07 08 09 10 12

07 08 09 10

07 08 09 10 12

- -

- - - - -

------

- - - - -

Jun

Mar

Mar Mar Mar Mar Mar

Jun Jun Jun Jun Jun

Dec Sep

Dec Dec Dec Dec Sep Sep Sep Sep Sep

Figure 2-5. Time series plots showing temperature at three selected sites in Apalachicola Bay.

River Discharge m

TSP µg L µg TSP

- 1

Figure 2-6. Time series plots of TSP (gray) vs river discharge (black line) at eight selected sites across Apalachicola Bay.

River River Discharge m

TSP µg L µg TSP

- 1

Figure 2-7. Seasonal distribution of TSP (bars) vs river discharge (black line) at eight selected sites across Apalachicola Bay from March 2007 to September 2012.

River Discharge m

TSN µg L µg TSN

- 1

Figure 2-8. Time series plots of TSN (gray) vs river discharge (black line) at eight selected sites across Apalachicola Bay.

River River Discharge m

TSN µg L µg TSN

- 1

Figure 2-9. Seasonal distribution of TSN (bars) vs river discharge (black line) at eight selected sites across Apalachicola Bay from March 2007 to September 2012.

171 East Bay Bridge 191 East Bay 30 30 25 25 20 20 15 15 10 10 5 5 0 0 0 10 20 30 40 0 10 20 30 40

141 Dry Bar 221 Cat Point

30 30 1 - 25 25

µgL 20 20 a 15 15

CHL CHL 10 10 5 5 0 0 0 10 20 30 40 0 10 20 30 40

161 Mid Bay 151 Pilots Cove 30 30 25 25 20 20 15 15 10 10 5 5 0 0 0 10 20 30 40 0 10 20 30 40 Salinity (psu)

Figure 2-10. Relationship between salinity and phytoplankton biomass (CHL a [μg L-1]) at six selected sites across Apalachicola Bay.

River River Discharge m

µg L µg

CHL CHL

- 1

Figure 2-11. Time series plots of chlorophyll a (gray) vs discharge (black line) at six selected sites across Apalachicola Bay.

171 East Bay Bridge 191 East Bay 30 30 25 25 20 20 15 15 10 10 5 5 0 0 0 500 1000 1500 2000 2500 0 500 1000 1500 2000 2500

141 Dry Bar 221 Cat Point

30 30 1

- 25 25

20 20 µg L µg

a 15 15

CHL 10 10 5 5 0 0 0 500 1000 1500 2000 2500 0 500 1000 1500 2000 2500

161 Mid Bay 151 Pilots Cove 30 30 25 25 20 20 15 15 10 10 5 5 0 0 0 500 1000 1500 2000 2500 0 500 1000 1500 2000 2500

River Discharge m3 s-1

Figure 2-12. Relationship between chlorophyll a and discharge at six different sites in Apalachicola Bay from April 2002 to September 2012.

171 East Bay Bridge 191 East Bay 25 1000 25 1000 20 800 20 800 15 600 15 600 10 400 10 400 5 200 5 200

0 0 0 0

Jul

Apr Oct

Jan Jun

Mar

Apr Oct

Feb

Aug Sep Nov Dec

Jan Jun

May

Mar

Feb

Nov Aug Sep Dec May 141 Dry Bar 221 Cat Point 25 1000 25 1000 20 800 20 800 15 600 15 600

10 400 10 400 River River Discharge m

5 200 5 200 1

- 0 0 0 0

Jul

Apr Oct

Jan Jun

Mar

Feb

Mar

Sep µgL Aug Nov Dec

Feb

Aug Sep Nov Dec

May

May a

161 Mid Bay 151 Pilots Cove

CHL CHL 3

25 1000 25 1000 s - 20 800 20 800 1 15 600 15 600 10 400 10 400 5 200 5 200

0 0 0 0

Jul

Apr Oct

Jan Jun

Mar

Feb

Aug Sep Nov Dec

May May

201 Sike’s Cut 231 River 25 1000 25 1000 20 800 20 800 15 600 15 600 10 400 10 400 5 200 5 200

0 0 0 0

Jul

Apr Oct

Oct

Apr

Jan Jun

Mar

Feb

Aug Sep Nov Dec

Aug Sep Nov Dec May May

Figure 2-13. Seasonal distribution of chlorophyll a (bars) vs discharge (black line) at eight selected sites across Apalachicola Bay from March 2007 to September 2012.

171 East Bay Bridge HD 171 East Bay Bridge LD 35 35 30 y = 0.0015x - 51.477 30 y = 0.0016x - 56.703 R² = 0.1524 25 R² = 0.1373 25 20 20 15 15 10 10 5 5 0 0

191 East Bay HD 191 East Bay LD

35 y = 0.0026x - 89.75 35 y = 0.0037x - 130.31 30 R² = 0.091 30 R² = 0.243 25 25 20 20 15 15 10 10 5 5 0 0

141 Dry Bay HD 141 Dry Bar LD 35 35 y = 0.0014x - 47.117 30 30 y = 0.0014x - 48.365 R² = 0.1922 25 R² = 0.1356 25 20 20 15 15 10 10 5 5 0 0

221 Cat Point HD 221 Cat Point LD 35 35 y = 0.0012x - 40.545 30 30 y = 0.0025x - 87.937 R² = 0.1318 25 25 R² = 0.243 20 20 15 15 10 10 5 5 0 0

Figure 2-14. Chlorophyll trends under high and low discharge at four different sites in Apalachicola Bay from April 2002 to September 2012 (HD =high discharge; LD= low discharge).

161 Mid Bay HD 161 Mid Bay LD 35 35 y = 0.0015x - 49.954 30 y = 0.0018x - 63.265 30 R² = 0.142 R² = 0.1596 25 25 20 20 15 15 10 10 5 5 0 0

151 Pilots Cove HD 151 Pilots Cove LD 35 35 y = 0.0012x - 41.684 y = 0.0012x - 44.222 30 30 R² = 0.2193 25 R² = 0.1614 25 20 20 15 15 10 10 5 5 0 0

231 River HD 231 River LD 35 35 30 30 y = 0.0006x - 19.461 y = 0.0006x - 20.788 R² = 0.0517 25 R² = 0.0918 25 20 20 15 15 10 10 5 5 0 0

201 Sike’s Cut HD 201 Sike’s Cut LD 35 35 30 y = 0.0013x - 47.396 30 y = 0.0007x - 23.063 R² = 0.1 25 R² = 0.111 25 20 20 15 15 10 10 5 5 0 0

Figure 2-15. Chlorophyll trends under high and low discharge at four different sites in Apalachicola Bay from April 2002 to September 2012 (HD =high discharge; LD= low discharge).

CHAPTER 3 SPATIAL AND TEMPORAL PATTERNS OF PHYTOPLANKTON COMPOSITION IN A SUBTROPICAL ESTUARY, APALACHICOLA BAY, FLORIDA, USA

The combination of land and oceanic influences in estuaries results in high spatial and temporal variability of key environmental factors, such as salinity, nutrient concentrations, light attenuation and water residence time. All of these factors are potentially important in regulating phytoplankton community structure, biomass and function (Eyre et al., 1999). The biotic composition of estuaries is strongly influenced by these regulating factors. The relative influence of environmental factors on the phytoplankton community depends on the specific character and location (e.g. climatic zone) of individual systems. The influences of watersheds associated with estuaries are particularly pronounced in semi-enclosed systems subject to inputs from large rivers. In such systems, changes associated with human development can play major roles in ecosystem structure and function, including phytoplankton communities.

Apalachicola Bay is an example of such a system in the subtropical environment of

Florida’s panhandle, and was the subject of this study.

The Apalachicola River is the main source of fresh water for Apalachicola Bay, and consists of a watershed of approximately 19,200 square miles (~49,730 km2) encompassing parts of Georgia, Alabama and Florida (Edmiston, 2008). The watershed has been subject to rapid human development over the past century, including the growth of Atlanta, the largest metropolitan area in the southeastern United

States. Whereas much of the watershed has escaped extensive industrialization, the burgeoning consumptive water demand has created a serious issue with respect to flows needed to service the needs of the estuary, particularly during drought years

(Huang, 2010; Putland et al., 2013). Significant reductions in freshwater discharge to

the bay during low rainfall years are implicated in mass mortalities of oysters, which are a keystone species and support a large shellfish industry (Livingston, 1983; Mortazavi et al., 2000; Huang and Spaulding, 2002; Edmiston, 2008; Huang, 2010). There are also serious concerns about the consequences of low discharge on the phytoplankton community, which forms a large part of the bay’s primary production and carbon availability for the food web (Putland and Iverson, 2007; Putland et al., 2013). It has recently been hypothesized that prolonged periods of low river discharge result in low levels of primary production and undesirable changes in the structure of phytoplankton communities (Putland et al., 2013).

The specific objectives of this study were (1) to characterize the composition and abundance of the phytoplankton community within different regions of Apalachicola Bay;

(2) to describe temporal trends in composition and abundance; and (3) to compare phytoplankton assemblages under high and low discharge. The results of the study are discussed within the context of the aforementioned hypotheses regarding the impacts of changes in discharge on phytoplankton abundance.

Methods

Site Description

This study was carried out in the Apalachicola estuary, located in the Florida panhandle on the northern coast of the Gulf of Mexico (Figure 2-1). The Apalachicola River is the main source of freshwater for the Apalachicola estuary, and consists of a tri-river drainage system, including approximately 19,200 square miles (~49,730 km2) in

Georgia, Alabama, and Florida. The confluence of the Chatahoochee and Flint rivers forms the headwaters of the Apalachicola system, which drains about 1,030 square miles (~2,670 km2) (Livingston, 1983). The estuary may be divided into four sections

based on both natural bathymetry and artificial structural alterations: East Bay (North

East), St. Vincent Sound (West side), Apalachicola Bay (Middle), and St. George Sound

(East). Apalachicola Bay is behind a well-developed barrier island complex composed of four islands: St. Vincent, Cape St. George, St. George, and Dog Island, lying roughly parallel to the mainland (Edmiston, 2008).

Field Procedures

Twelve sampling sites were selected for this study (Figure 2-1), with the intention of obtaining extensive coverage of the different geographic regions of the estuary.

Collections were made monthly for a two-year period, from June 2008 to June 2010.

The primary phytoplankton samples were collected with a vertically integrating sampling tube (integrated pole), which collects water evenly from the surface to 0.1 m from the bottom. Samples were split into two subsamples, one preserved with Lugol’s and the other with glutaraldehyde in 0.1 M sodium cacodylate buffer. Basic water column characteristics (depth, salinity, and temperature) were obtained in situ in cooperation with the on-going ANERR monitoring program, at the same time that samples were taken for analyses of chemical variables such as chlorophyll a concentrations and nutrient concentrations (total soluble phosphorus, total soluble nitrogen, and soluble reactive phosphorus). Additional analyses were carried out on samples collected under this project: total phosphorus (TP), total nitrogen (TN), silica (Si), total suspended solids

(TSS), turbidity, and color dissolved organic matter (CDOM).

Water Chemistry

Whole water samples were used to determine concentrations of TN, TP, Si, and

Chl a. To determine TN and TP, samples were digested and measured colorometrically on a Bran-Luebbe autoanalyzer (TN) and a dual-beam scanning spectrophotometer

(TP; APHA, 1998). Chlorophyll a was processed from filters using the Sartory and

Grobbelaar (1984) hot ethanol extraction method and concentrations were determined spectrophotometrically according to Standard Methods (APHA, 1998). Si concentrations were corrected for turbidity and measured on a dual-beam scanning spectrophotometer following Standard Methods (APHA, 1998).

For soluble nutrient and CDOM analyses, whole water was filtered through glass-fiber filters (0.7-µm pore size). NH4 and NO3+NO2 concentrations were determined by conversion to NO2 and read colorometrically on a Bran-Luebbe autoanalyzer (Strickland and Parsons, 1972; APHA, 1998). Dissolved inorganic nitrogen (DIN) was calculated by summing NH4+NO3+NO2. SRP concentrations were measured via the ascorbic acid method on a dual-beam scanning spectrophotometer following Standard Methods

(APHA, 1998). Color dissolved organic matter (CDOM) values were measured against a platinum-cobalt standard using a dual-beam scanning spectrophotometer (APHA,

1998).

Nutrient Limitation Bioassays

For each nutrient limitation experiment, 5 L of water were collected using an integrated sampling pole at each of six sites: 231 in the River, 171 near The East Bay

Bridge, 143 in St. Vincent Sound, 223 in St. George Sound, 161 in Mid Bay and 201 in the gulf. Water was transported to the lab in a large, clear carboy covered with a white plastic bag (to best simulate natural light conditions) and closed with a foam stopper (to allow gas exchange). At the lab, water was transferred to a large mixing tank and stirred continuously during experimental set-up. Water from each site was divided into 300-ml aliquots and poured into 15 Erlenmeyer flasks (500-ml) to create five treatment groups

(control, N addition, P addition, N+P addition, and N+P+Si addition) in triplicate.

-1 -1 Nutrients were added to obtain final concentrations of 400 μg NO3-N L , 400 μg Si L ,

-1 and 40 μg PO4-P L . Flasks were incubated in temperature-controlled water baths illuminated from the bottom. Temperatures were set at ambient levels measured on the day of sampling and light flux was fixed at 80 µE m-2 s-1. To correspond with seasonal differences in day length, photoperiods were set at 12 hours light, 12 hours dark in

November and February, and 14 hours light, 10 hours dark in May, June, July and

September. Flasks were swirled and sampled for algal biomass every 24 hours for seven days. Algal biomass was estimated using in vivo fluorescence of Chl a using a

Turner Designs TD-700 Fluorometer (Sunnyvale, CA) at fixed time intervals over a 6–8- day incubation period.

Phytoplankton Analysis

Picoplanktonic cyanobacteria were counted after a subsample was filtered onto 0.2-µm

Nuclepore filters and mounted between microscope slides and coverslips with immersion oil. Samples were kept frozen, and within 48 h of sampling cells were counted with a Nikon research microscope equipped with autofluorescence (green and blue light excitation). Numerical abundances of cyanobacterial cells were determined by counting a minimum of five grids of the ocular micrometer at 1,000X magnification. After five grids, the number of additional grids counted was dependent on cell density, until reaching a count of 100 cells of the dominant taxon (Phlips et al., 1999). Biovolumes were determined using the closest shape method (Smayda, 1978).

General phytoplankton composition was determined for integrated samples from all the sites on a monthly basis using the Utermohl method (Utermohl, 1958). Samples preserved in Lugol’s were settled in 19-mm-diameter cylindrical glass chambers, and counted. Phytoplankton cells were identified and counted at 400X and 100X with a

Nikon phase contrast inverted microscope. At 400X, a minimum of 100 cells of a single taxon and 30 grids were counted. If 100 cells were not counted within 30 grids, up to a maximum of 100 grids were counted, or until 100 cells of a single taxon was reached. At

100X, a total bottom count was completed for taxa >30 mm. Counts for individual taxa were converted to cell volumes using the closest geometric shape method (Smayda,

1978). Mean volume was calculated for each species from specific phytoplankton dimensions measured for a minimum of 30 randomly selected cells or as many as possible for rare species.

Statistical Analyses

The SAS Enterprise Guide statistical package for PCs (Version 4.3) was used to carry out statistical analyses. Distributions of most variables were non-normal (determined by the Shapiro-Wilk and Kolmogorov-Smirnov goodness-of-fit tests), necessitating the use of non-parametric Spearman rank correlation analysis to explore relationships between them. Duncan’s multiple range tests were used to compare the means of the different variables among sites. Canonical Correlation Analyses (CCA) were performed in R, using the CCA package, to evaluate the statistical relationships between phytoplankton composition and abundance and environmental variables (nutrients, chlorophyll a, temperature and salinity). For clustering the sites with respect to discharge and salinity values, Cluster Analysis was performed in R using the principal components method and the hclust function.

Results

Physical-Chemical Variables

River discharge

Discharge trends in the Apalachicola estuary from January 2000 to December

2012 were discussed in Chapter 2 (Figure 2.2). During the two years of this study, four low-discharge periods and three high-discharge periods were indentified (Figure 3-1).

The longer low-discharge periods began in the month of June and extended until

November in 2008 and September in 2009. Additionally, February 2009 and June 2010 were characterized as short-duration, low-discharge periods. The first high-discharge period was short and occurred during December 2008 and January 2009, the second one ran from March through June 2009, and the third one, which was also longest, went from October 2009 through May 2010.

Nutrients

Mean total nitrogen (TN) total phosphorus (TP) and Silica (Si) varied slightly from between low and high discharge periods (Tables 3-1, 3-2, 3-3 and Figure 3-7). Nutrient distribution portrays the influence of both the Apalachicola River and the Gulf of Mexico in nourishing the Bay on a seasonal basis.

During low discharge, mean TP concentrations ranged from 19.4 µg P L-1 at Site

171 near the East Bay Bridge, to 42.5 µg P L-1 at Site 221 in Cat Point (Table 3-1,

Figure 3-7). During high discharge, TP concentrations ranged from 21.1 µg P L-1 at Site

201 in the gulf to 41.4 µg P L-1 at Site 231 in the river (Table 3-1, Figure 3-7).

TN concentrations tended to be higher in sites near the river mouth and lower in sites near outlets to the gulf, during both high and low discharge (Table 3-2, Figure 3-7).

During low discharge, mean TN concentrations ranged from 221.9 µg N L-1 at Site 201

in the gulf to 617.29 µg N L-1 at Site 191 in East Bay. During high discharge, mean TN concentrations ranged from 169.64 µg N L-1 at Site 201 in the gulf to 514.81 µg N L-1 at

East Bay (191).

Silica (Si) concentrations were strongly influenced by river discharge, and were higher in sites adjacent to the Apalachicola River, decreasing with proximity to the barrier islands and the gulf (Table 3-3, Figure 3-7). During low discharge, mean Si concentrations ranged from 1495 µg Si L-1 at Site 221 in Cat Point to 7474 µg Si L-1 at

Site 231 in the river. During high discharge, mean Si concentrations ranged from 819 µg

Si L-1 at Site 201 in the gulf to 6534 µg Si L-1 at Site 231 in the river.

Salinity

Measurements at the 12 sampling sites revealed a salinity gradient that decreases with distance from the river to the gulf (Table 3-4). Salinities at Site 231 in the river and Site 201 in the gulf were constant because of the sampling strategy used for these two sites, which consisted of taking the sample only after a certain salinity value was reached (≤0.1 ppt for the river; ≥32 ppt for the gulf). Mean salinities in

Apalachicola Bay vary significantly between the low- and high-discharge periods (Table

3-4). During low discharge, salinities ranged from 9.9 psu at Site 171 near the East Bay

Bridge to 28.8 psu at Site 131 in West Pass (Tables 3-4 and 3-9). During high discharge, salinities ranged from 1.9 psu at Site 171 in East Bay Bridge to 16.8 psu at

Site 151 in Pilots Cove (Tables 3-4 and 3-9).

Temperature

Temperature variations in the estuary from June 2008 to June 2010 were similar to those described in Chapter 2 for the period March 2007 to September 2012 (Figures

2-4 and 3-2). From June 2008 to June 2010, water temperatures exhibited inter-season

variability, with higher temperatures usually occurring from May to October and lower temperatures occurring from December to February (Table 3-5, Figure 3-2).

Mean water temperatures were higher during low discharge and ranged from

25.7 ˚C at Site 223 in St. Vincent Sound to 27.8˚C at Site 191 in East Bay. Mean water temperatures were lower during high discharge and ranged from 17.7 ˚C at Site 231 in the river to 20.2˚C at Site 191 in East Bay. Site 191 in East Bay displayed the highest mean temperature among all the sites during both low and high discharge (Tables 3-5 and 3-9).

Color

Color dissolved organic matter (CDOM) values reflected proximity to the river inflow. The CDOM pattern was similar during both low and high discharge (Tables 3-5 and 3-9). In general, mean CDOM values were higher at sites 191 in East Bay and 231 in the river and decreased with distance from the river, with the lowest mean values displayed at site 201 in the gulf. During low discharge, CDOM ranged from 6.8 PCU at

Site 201 in the gulf to 47 PCU at Site 191 in East Bay. During high discharge, CDOM values ranged from 6.8 PCU at Site 201 in the gulf to 120 PCU at Site 191 in East Bay, and 132 PCU at Site 143 in St. Vincent Sound. The high mean CDOM concentrations displayed at Site 143 in St. Vincent Sound in April 2009 (1392 PCU) were the result of an exceptionally high discharge event.

Secchi depth

Light transmission through the water column was, on average, slightly higher during low discharge than during high discharge (Table 3-5). Site 191in East Bay displayed the lowest mean Secchi depth values during both seasons, 0.6 m during low

discharge and 0.5 m during high discharge. Site 201 in the gulf displayed the largest mean Secchi depth values during both seasons, 2.0 m during low discharge and 1.8 m during high discharge (Tables 3-5 and 3-9). Mean Secchi depth at site 231 (River) doubled from 0.6 m during high discharge to 1.2 m during low discharge (Table 3-5).

Nutrient limitation bioassays

Bioassays (Table 3-6) showed that phosphorus was the primary limiting nutrient for phytoplankton growth in the river, in the north and in the center of the bay (sites 231,

171 and 161), except when the presence of surplus nutrients was indicated by significant growth in the control groups (i.e., no added nutrients). Even in cases for which the bioassays showed no initial nutrient limitation, the mesocosms eventually shifted to phosphorus limitation or phosphorus-nitrogen co-limitation, as biomass levels peaked in the incubations from these sites (Table 3-6). Conversely, nitrogen or a combination of nitrogen and phosphorus limited phytoplankton growth in the west and east portions of the bay, as well as in the gulf (Table 3-6). Phytoplankton growth was never limited by silica (Si) during any of the incubations.

Cluster analysis

Cluster analyses based on physical-chemical variables (nutrients, chlorophyll a, temperature, and salinity) were performed during low- and high-discharge periods from

March 2007 to September 2012 (Figures 3-3 and 3-4). Cluster analyses illustrated the influence of river discharge in structuring temporal and spatial patterns in physical- chemical variables.

In general, Site 231 in the river and adjacent sites such as 191 in East Bay and

171 near the East Bay Bridge tended to group together, independent of discharge. Site

143 in St. Vincent Sound tended to group with these lower-salinity sites during periods

of low discharge, but during periods of high discharge it grouped with the bay sites, especially with Site 223 in St. George Sound (Figures 3-3 and 3-4).

Sites located within the bay, such as 141 in Dry Bar, 161 in Mid Bay, 221 in Cat

Point, 211 in Nick’s Hole, 223 in St. George Sound, and 131 in West Pass tended to form a large cluster that varied according to the discharge period (Figures 3-3 and 3-4).

Site 151 in Pilots Cove is close to the barrier islands and is greatly influenced by the marine waters of the gulf. This site merged with the bay cluster during high discharge, however during low discharge it grouped with Site 201 in the gulf (Figures 3-3 and 3-4).

Site 201 was in a separate cluster during both high and low discharge.

Phytoplankton abundance

Mean chlorophyll a concentrations were higher at Site 191 in East Bay and lower at Site 231 in the river and at Site 201 in the gulf (Table 3-7, Figure 3-8). During low discharge, mean chlorophyll a concentrations ranged from 5.5 µg L-1 at Site 201 in the gulf to 20.8 µg L-1 at Site 191 in East Bay. During high discharge, mean chlorophyll a concentrations ranged from 4.0 µg L-1 at Site 231 in the river to 14.4 µg L-1 at Site 191 in

East Bay. Mean values were slightly higher in the low-discharge season than in the high-discharge season at the 12 sampling sites. The average mean chlorophyll a concentrations for the bay increased from 7.6 µg L-1 in the high-discharge season to 9.9

µg L-1 in the low-discharge season.

In general, chlorophyll a patterns from June 2008 to June 2010 were similar to those observed for the longer data set discussed in Chapter 2. Time-series plots showed that river discharge and chlorophyll a are inversely related (Figure 3-9). During periods of prolonged high discharge, such as October 2009 through May 2010, chlorophyll a concentrations remained low at most sites (Figure 3-9). Conversely, during

periods of extended low discharge, like June to September 2008, chlorophyll a levels tended to peak at the different sites, particularly at Site 191 in East Bay. During the two years this study was carried out, Site 191 in East Bay displayed the highest mean phytoplankton biomass during both low and high discharge.

Similar to chlorophyll a, phytoplankton biovolumes were higher during low discharge than during high discharge (Table 3-8, Figure 3-8). The average mean phytoplankton biovolume for the entire bay doubled from high discharge (1.6 x 106 µm3 ml-1) to low discharge season (3.3 x 106 µm3 ml-1), however, the spatial and temporal trends observed for phytoplankton biovolume were different from those observed for chlorophyll a (Figures 3-8, 3-10, 3-11 and 3-12). Mean phytoplankton biovolume tends to be higher at Site 201 in the gulf and lower at site 231 in the river independent of the season (Figure 3-8). Spatial trends in the distribution of phytoplankton biovolume were similar to those observed for carbon (Figure 3-8). Sites 191 in East Bay, 151 in Pilots

Cove, and 201 in the gulf displayed the highest values for both phytoplankton biovolume and carbon.

Seasonality of Phytoplankton Biovolume

Phytoplankton blooms for the Apalachicola Bay were established using a 10% exceedance criterion, i.e. the biovolume level that was exceeded on <10% of the sampling events. Bloom conditions within the bay sites (e.g. 141, 151, 161, 171, 221),

East Bay (191), and the gulf (201) corresponded to total individual biovolumes exceeding 3 x 106 µm3 ml-1. At site 231 in the river, phytoplankton blooms under this definition were not observed and phytoplankton biovolumes were low and steady.

Seasonal patterns of phytoplankton biovolume in the estuary varied between the two years of sampling, and there were more phytoplankton blooms during the first low-

discharge season (June-November 2008) than in any other season (Figures 3-10, 3-11 and 3-12). The first low-discharge season included in the study exhibited moderate to high peaks in phytoplankton biovolume, which varied temporally and spatially and were mostly dominated by diatoms. In the northern area of the bay, more specifically at Site

171 near the East Bay Bridge and at Site 191 in East Bay, Leptocylindrus danicus,

Cerataulina pelagica and Thalassionema nitzschioides were the dominant species. L. danicus was particularly important because it formed the greatest bloom observed during the period of study (13.86 x 106 µm3 ml-1) at Site 191 in East Bay. At Site 161 in

Mid Bay, moderate peaks in biovolume of T. nitzschioides, L. danicus, Pseudosolenia calcar-avis, and Fragilaria spp. were observed. In the west, at Site 141 in Dry Bar and

143 in St. Vincent Sound, Thalassiosira spp., L. danicus and P. calcar-avis dominated peaks of phytoplankton biovolume. In the outer portion of estuary, at sites 131 in West

Pass, 151 in Pilots Cove, and 211 in Nick’s Hole, Fragilaria spp., Hemialus hauckii, L.s danicus, T. nitzschioides and Rhabdonema adriaticum were the major bloom formers.

At Site 201 in the gulf, the phytoplankton community was dominated by the diatoms L. danicus, P. calcar-avis, and Fragilaria spp. In the east part of the estuary, at sites 221 in

Cat Point and 223 in St. George Sound, L. danicus and P. calcar-avis were the predominant species. November 2008 was an outstanding month in that blooms of various species occurred at 10 of the 12 sampling sites.

The first high-discharge season occurred in December 2008-January 2009, and coincided with a general pattern of low biovolumes at most sites in the estuary. Three sites, 191 in East Bay, 151 in Pilots Cove, and 223 in St. George Sound were the exception, and displayed moderate to intermediate biovolumes, dominated by diatoms

at Pilots Cove and St. George Sound, and dinoflagellates of the genus Protoperidinium at East Bay.

During the low-discharge period of February 2009, elevated biovolume levels and some exceptional blooms were observed in seven of the twelve sites, including Site 191 in East Bay and other sites located in the middle and outer estuary and the gulf (161,

221, 151, 223, 131 and 201). In this period, the dinoflagellate Prorocentrum minimum was in bloom at East Bay and the diatoms C. pelagica, Skeletonema costatum, R. adriaticum and Fragilaria spp. were the main contributors to the blooms in the middle and outer estuary.

The second high discharge season (March-June 2009) followed the February low-discharge event. During that time, noticeable reductions in phytoplankton biovolume occurred across the estuary, especially in April 2009, when biovolumes throughout the estuary reached dramatic low levels. Site 201 in the gulf was the exception, where a bloom of P. calcar-avis was recorded. Also, during May and June 2009, cyanobacteria reached high biovolume levels at sites 191 in East Bay, 151 in Pilots Cove and 211 in

Nick’s Hole.

Phytoplankton biovolumes remained low in the east and middle estuary during the third low-discharge period (June-September 2009), but was moderate at East Bay

(Site 191), in the outer estuary (Sites 151 in Pilots Cove and 211 in Nick’s Hole), the western part of the estuary (Site 143 in St. Vincent Sound) and the gulf (Site 201). At these sites, a combination of cyanobacteria and diatoms dominated the community.

During the third and longest high-discharge period (October 2009-May 2010) eight of the 12 sites (171, 191, 161, 141, 143, 131, 223, and 201) displayed blooms in

November 2009, which were composed of a mix of dinoflagellates, diatoms, cyanobacteria and other groups. These blooms covered the north, west and east portions of the estuary, and were followed by significant reductions in phytoplankton biovolumes at all sites. Later in the season, diatoms of the genus Fragilaria formed blooms at Site 201 in the gulf (4 x 106 µm3 ml-1) during January 2010 and at Site 131 in

West Pass (16.4 x 106 µm3 ml-1) during Mach 2010. This latter bloom was the third largest one detected in the estuary during the study period. Also, a phytoplankton bloom of the dinoflagellate P. minimum was recorded at Site 161 in Mid Bay in April 2010.

River discharge was low in the last month of sampling for this study (June 2010), however, phytoplankton biovolumes were low at most sites. Sites 161 in Mid Bay and

131 in West Pass were the exception, as a combination of cyanobacteria and diatoms of the genus Fragilaria reached bloom levels in these regions.

Relationships Between Phytoplankton Community Assemblages and Environmental Variables

The major algal groups observed within the Apalachicola estuary during the study period included: diatoms (88 taxa), dinoflagellates (58 taxa), cyanobacteria (9 taxa), green algae (35 taxa), euglenoids (4 taxa), cryptophytes (4 taxa), and prasinophytes (7 taxa) (Table 3-12). The dominant phytoplankton species varied temporally and spatially at the different sites. Bloom-forming species included centric diatoms, pennate diatoms and dinoflagellates.

The relationship between the major phytoplankton groups and environmental variables was explored at four selected sites using Canonical Correlation Analysis

(CCA) (Figure 3-13). In the river (Site 231), cyanobacteria were positively correlated with temperature and diatoms were correlated with chlorophyll a. At Site 161 in Mid Bay,

dinoflagellates were positively correlated with Secchi depth, and again diatoms were correlated with chlorophyll a and cyanobacteria with temperature. At Site 151 in Pilots cove, dinoflagellates were positively correlated with Secchi depth, and cyanobacteria and diatoms correlated with temperature and salinity, respectively. At Site 201 in the gulf, cyanobacteria correlated positively with temperature and diatoms with total phosphorus (TP).

Although no obvious relationship with phosphorus was detected (Figure 3-14) concentrations in the range of 10 to 90 µg L-1 seemed to be optimal for phytoplankton biolvolume peaks. Phytoplankton biovolume showed the highest bloom intensities at concentrations of nitrogen between 200 and 600 µg L-1 (Figure 3-14). Total phytoplankton biovolume optima were observed at salinities between 15 and 25 psu

(Figure 3-15). Total phytoplankton biovolumes were also affected by temperature, with most of the peaks occurring between 15 and 25 º C (Figure 3-15).

Moderate amounts of small phytoplankton (biovolume ≤ 2000 µm3) were observed over a broad spectrum of salinities (0 to 35 psu), however peak concentrations were more common in salinities between 5 and 27 psu (Figure 3-16).

Large phytoplankton (biovolume ≥ 2000 µm3) were also abundant at different salinities, but noticeable peaks in biovolume tended to occur at salinities between 15 and 35 psu

(Figure 3-16). Chain-forming centric diatoms were a conspicuous component of the phytoplankton biovolume, with several species forming medium to large blooms at salinities between 15 and 25 psu (Figure 3-16).

Three species of centric diatoms were abundant throughout the study, L.danicus,

C. pelagica, and P. calcar-avis. These species were present at different salinity ranges,

however C. pelagica reached bloom levels when salinity was ~20 psu (Figure 3-17).

The other two species, L. danicus and P. calcar-avis reached bloom conditions frequently at salinities between 18 and 34 psu.

The most prominent pennate diatom taxa forming blooms were species belonging to genera Fragilaria, Amphiprora and Pleurosigma, as well as T. nitzchioides

(Figure 3.17). The most conspicuous blooms of pennate diatoms were formed by T. nitzchioides at salinities of about 15 to 32 psu, and Fragilaria sp. at salinities between

25 and 35 psu (Figure 3.17). The dinoflagellates, P. minimum, Gyrodinium spirale and

Akashiwo sanguinea were observed over a wide range of salinities. P. minimum showed somewhat different salinity preferences in terms of small-magnitude bloom events (Figure 3-17).

Temperature was another key factor correlated with the distribution of certain species of centric diatoms. L. danicus, C. pelagica, and P. calcar-avis had different preferences in terms of temperature (Figure 3-18). Peaks in biovolume of C. pelagica were observed around 15 ºC, whereas P. calcar-avis tended to prefer temperatures around 20 ºC. In the case of L. danicus, the range in temperature preferences was larger (18 to 32 ºC), and high biovolumes for this species were recorded at 18, 25 and

30-32 ºC (Figure 3-18).

Some pennate diatoms also showed preferred ranges in temperature (Figure 3-

18). Peaks in biovolume of Fragilaria spp. were recorded at a wide range of temperatures from 10 to 32 ºC, whereas species of the genus Pleurosigma were restricted to temperatures between 18 and 25 ºC. T. nitzchioides was observed over a broad spectrum of temperatures, however major blooms of this species tended to be

restricted to temperatures around 30 ºC. The dinoflagellate P. minimum exhibited higher biovolumes between 15 and 25 ºC. A. sanguinea, also a dinoflagellated had higher biovolumes were observed around 20 ºC and 28 ºC (Figure 3-18).

Cyanobacteria also were a major component of the phytoplankton biovolume in the estuary, especially during low discharge (Figures 3-10, 3-11, 3-12 and 3-19).

Phycocyanin-rich cyanobacteria exhibited a wide range of salinity distribution, with biovolume peaks occurring at salinities between 3 and 20 psu (Figure 3-19).

Phycoerythrin-rich cyanobacteria, on the other hand, tended to display peaks in biovolume at salinity ranges between 25 and 35 psu (Figure 3-19).

Discussion

This study revealed spatial differences in phytoplankton composition and biovolume in the Apalachicola estuary. Sampling sites included in the study can be roughly organized into five groups; 1) The river (Site 231) and the site close to the river discharge (Site 171), 2) East Bay in the northeast corner of Apalachicola Bay (Site 191),

3) the central bay and oyster bars (Sites 143, 141, 161, 221, and 223), 4) the outer bay

(Sites 131, 151, and 211), and 5) Site 201 in the Gulf of Mexico.

Sites near the river outflow had the lowest mean phytoplankton biovolume values, in part reflecting the high light attenuation and short water residence times in the river. The latter region also showed strong temporal changes in composition attributable to shifts in salinity from fresh to saline conditions related to levels of river discharge (Figure 3-10).

Site 191 in East Bay stood apart from other regions in the study because it displayed the greatest variability in dominant species. Peaks in phytoplankton biovolume were at different times dominated by diatoms, dinoflagellates, and

picoplanktonic cyanobacteria (Figure 3-10). The site also exhibited high chlorophyll a levels. These observations may be attributable to several unique characteristics of East

Bay, including longer water residence times than the other sites in the bay and influences of allochthonous inputs from small creeks in the region, which discharge colored waters of relatively high nutrient content.

In the central bay, where large oyster bars are located, mixing of river and marine waters provide conditions more conducive to high phytoplankton biovolumes and proliferation of marine phytoplankton species, including increased light availability in the water column and more consistent mid-range salinities (i.e. >5 psu). These findings are in agreement with previous studies in the Apalachicola estuary (Putland and Iverson,

2007; Putland et al., 2013), which point out that during the summer months (defined as

May to October), the highest concentrations of chlorophyll a occur in samples from sites with salinities between 5 and 23 psu.

Similar spatial trends are observed in other estuaries associated with discharges from turbid or highly colored rivers, such as the Amazon River in Brazil (Smith Jr and

Demaster, 1996; Santos et al., 2008), the Pearl (Dai et al., 2006) and Huange ( Turner et al., 1990) Rivers in China, the Mississippi (Liu et al., 2004; Rabalais et al., 2004) and

Suwannee (Bledsoe et al., 2004; Quinlan and Phlips, 2007) Rivers in the Gulf of

Mexico, and the Nile River in Africa (Oczkowski et al., 2009). Phytoplankton maxima in these systems often occur in the nearshore transition zone between light-limited river water and the nutrient-limited offshore water. The location and magnitude of the phytoplankton maxima are dictated by the rates of discharge from the river and its light

attenuation properties, which in turn can influence the productivity of higher trophic levels, as well as the potential for and distribution of hypoxia.

In terms of phytoplankton composition, Sites 161, 141, and 221 provide an example of the patterns in the central bay and oyster bars. Site 161 exhibited higher mean phytoplankton biovolume, 3.74 x 106 µm3 ml-1, during low discharge, and lower phytoplankton biovolume 2.3 x 106 µm3 ml-1 during high discharge. Bloom events were often dominated by one or more species of centric, chain-forming diatoms, including L. danicus, P. calcar-avis, and C. pelagica. Pennate diatom species in the genus Fragilaria were also common bloom formers in the region. Some dinoflagellate blooms were observed at this site, as well as other sites in the mid- and outer bay, most prominently

A. sanguinea and P. minimum. Understanding the factors that regulate blooms of A. sanguinea and P. minimum is important, because of their potential for forming harmful algal blooms (Phlips et al., 2002; Heil et al., 2005; Phlips et al., 2012).

Earlier studies report that among harmful algal bloom species, P. minimum is important for the following reasons: it is widely distributed geographically in temperate and subtropical waters; it is potentially harmful to humans via shellfish poisoning; it has detrimental effects at both the organismal and broader environmental levels; blooms appear to have undergone a geographic expansion over the past several decades; and, a relationship appears to exist between blooms of this species and changing environments in coastal systems (Heil et al., 2005). Although it was beyond the scope this study to determine the particular conditions that may trigger harmful algal blooms

(HABs) of specific taxa, the dinoflagellate P. minimum was found frequently in

temperatures between 15 to 25 ºC and at different salinity regimes (Table 3-10 and

Figure 3-17).

A. sanguinea has also been reported to have the potential to develop blooms under conditions of increased water residence time and high temperature in San

Francisco Bay (Cloern et al., 2005). In Apalachicola Bay, A. sanguinea was often found to form medium-size blooms (Table 3-10), in waters with temperatures around 20 ºC and was tolerant of a wide range of salinities (Figures 3-17 and 3-18). Picoplanktonic cyanobacteria were frequently abundant and dominant in the central bay and oyster bars, especially during periods of low discharge and warm temperatures (Figure 3-10).

These findings highlight the often-overlooked importance of considering the role of these species in the ecology of estuaries (Phlips et al., 1999; Murrell and Lores, 2004;

Putland et al., 2013).

Sites 141 in Dry Bar and 221 in Cat Point are located at major oyster bars

(Figure 3-11). In a similar way to Site 161, bloom events were often dominated by one or more species of centric, chain-forming diatoms, including L.s danicus, P. calcar-avis, and C. pelagica. Pennate diatom species of the genus Fragilaria, however, were not very common at these two sites. Phytoplankton biovolumes at these sites were slightly lower than at site 161, possibly because of constant grazing from the oyster bars.

Although it was beyond the scope of this study to evaluate the impact of grazers on phytoplankton abundance, a previous study reported that up to 80% of phytoplankton biomass is consumed by grazers in the water column ((Mortazavi et al., 2000).

Sites 131 and 151 provide examples of the outer estuary. This region is greatly influenced from the Gulf of Mexico, and with respect to species composition, some of

the dominant species recorded in this region were similar to those found in the central estuary (e.g. L. danicus, P. calcar-avis, and Fragilaria spp.). However, marine species such as R. adriaticum and H. hauckii were also major bloom formers. This could have been the result of increased influence of marine phytoplankton species coming from the

Gulf of Mexico through West Pass.

The fifth region examined in the study was outside the bay in the open waters of the Gulf of Mexico (Site 201). Like much of the mid- and outer bay, biovolume peaks at

Site 201 were dominated by marine diatoms. Although many of the same species observed in the bay were important at Site 201, the cosmopolitan species S. costatum was added to the list of bloom-formers. Mean biovolume levels were relatively high, reflecting the high productivity of the near-shore shelf environment of the northern Gulf of Mexico, which receives high nutrient inputs from numerous large rivers, such as the

Mississippi (Rabalais et al., 2004).

In comparison with the first year of sampling (June 2008-May 2009), the second year (June 2009-June 2010) was characterized by a generalized reduction in phytoplankton biovolumes throughout all regions in the estuary, except for Site 131 in

West Pass, next to the gulf. The phytoplankton community was mostly dominated by diatoms. A previous study conducted in the estuary found similar results (Estabrook,

1973). However, a transition from a mostly diatom-dominated community during the first year to a heterogenous mix, including diatoms, dinoflagellates and cyanobacteria was observed during the second year (Figures 3-10, 3-11, and 3-12). This was possibly the result of interactions among numerous environmental factors, including a longer and

more pronounced high-discharge period from October 2009 to May 2010 (Figure 3-1), which resulted in shorter residence times and highly variable salinities.

One of the central issues facing the ecology of Apalachicola Bay is the impact of anticipated future declines in river discharge (Mortazavi et al., 2001; Huang and

Spaulding, 2002; Livingston, 2007; Edmiston, 2008; Huang, 2010; Putland et al., 2013).

In 2012, widespread mass mortalities of oysters in the bay were attributed to persistent high-salinity conditions caused by drought-induced, low river discharge. The resulting negative effects on the important oyster industry in the bay heightened concerns about the effects of low river discharge on the overall ecology of the bay, including the phytoplankton community, which forms a large portion of primary production in the bay.

Putland et al. (2013) hypothesized that persistent low river discharge will reduce phytoplankton production and biomass because of diminished nutrient loads from the river. They also suggested that the structure of the phytoplankton community will change from one in which smaller phytoplankton species (< 20 µm) are major components to one dominated by larger species (> 20 µm), in particular diatoms.

This study presents a more complex picture of the relationships between discharge and phytoplankton biomass, although there is support for aspects of the aforementioned hypothesis. One of the principal expectations associated with the latter hypothesis is that peak phytoplankton biomass should occur at salinities between 5 and

25 psu, because these areas represent a water column where river inflows have contributed significantly to the surface mixed layer. Phytoplankton biomass peaks in the mesohaline to lower polyhaline (i.e. 5-25 psu) regions of estuaries are common because the area possesses a combination of nutrient-rich river water with relatively

clear marine water, yielding an optimal blend for primary production (Quinlan and

Phlips, 2007). In Apalachicola Bay, many of the highest peaks in phytoplankton biovolume were observed at salinities between 15 and 27 psu, although there were also peaks in the upper polyhaline (27-35 psu) or oligohaline range (0.1-5 psu).

The results of this study indicate somewhat variable trends in the relative importance of small- and large-celled phytoplankton across the salinity gradient in

Apalachicola Bay. Small-celled phytoplankton did not indicate dramatically higher peaks in biovolume in the mesohaline-lower-polyhaline salinity range, except for picoplanktonic cyanobacteria (Figures 3-16 and 3-19). As observed by Putland et al.

(2013), biovolume peaks of phycocyanin-rich picoplanktonic cyanobacteria were abundant in the mesohaline-lower-polyhaline salinity range (Figure 3-19). Large-celled phytoplankton showed a relatively wide range of salinity over which biovolume peaks were observed, i.e. 10-33 psu (Figure 3-16).

The group that showed the most dramatic trend in biovolume peaks in the upper- mesohaline to lower polyhaline range was chain-forming centric diatoms (Figure 3-16).

Most of the highest peaks in biovolume were observed during the late fall or early spring when temperatures were relatively low (near 20 °C) (Figure 3-18). Prominence of chain- forming centric diatoms under the aforementioned conditions is in part attributable to several features common to these species, including their high growth rates (Stolte and

Garcés, 2006) and tolerance of a wide range of salinities and temperatures (Smayda

1980; Reynolds 2006). These traits help explain their cosmopolitan, global distribution

(Smayda 1980; Reynolds 2006).

The larger question of whether low river discharge results in low phytoplankton primary production and biomass is complicated by the need to define the relative role of the Gulf of Mexico as a source of nutrients compared to the role of the Apalachicola

River. During periods of high river discharge (i.e. winter and early spring), phosphorus and nitrogen levels in the river are elevated compared to low-discharge periods.

Whereas the elevated nutrient loads during the latter periods represent a potential for increased phytoplankton biomass, high discharge can also reduce water residence time and increase salinity variability, both of which have a negative effect on biomass.

The other complicating factor is the role of nutrients from the Gulf of Mexico in supporting phytoplankton production. Previous studies (Mortazavi et al., 2000, 2001) estimated that only 23% of annual nutrient load to Apalachicola Bay comes from the gulf, with the rest coming from the river. However, it is possible that this approximation underestimates the gulf contribution, which depends on specific environmental conditions that vary from year to year. The northern Gulf of Mexico is a relatively nutrient-rich region, at least compared to many open-ocean environments, because of the numerous regional inputs from major eutrophic rivers, such as the Mississippi River

(Rabalais et al., 2004) and Suwannee River (Quinlan and Phlips, 2007). These regional river influences are reflected in the fact that average total phosphorus concentrations in the Gulf of Mexico, just outside of Apalachicola Bay, are twice as high as in the

Apalachicola River during the spring-summer low-discharge period (Table 2-3).

Because phosphorus appears to be the dominant limiting nutrient for phytoplankton growth in the bay, the gulf could represent an important nutrient source during the latter period. This possibility needs to be further explored to evaluate more accurately the

overall consequences of future reductions in river discharge on phytoplankton primary production and biomass.

In summary results from this study indicated regional differences in the composition and abundance of phytoplankton in the estuary, which had a strong connection to changes in river discharge and salinity gradients. River discharge alone played an important role in decreasing water residence time and, as a consequence, decreased phytoplankton biomass and biovolume due to osmotic stress and rapid flushing. However phytoplankton blooms are highly dynamic and drawing major conclusions from a two year study would be a precipitate action.

Table 3-1. Duncan tests for total phosphorus (TP) in Apalachicola Bay during low and high river discharge. Concentrations expressed as µg P L-1. Letters indicate groups based on mean values.

Low-discharge TP High-discharge TP Duncan Grouping Mean N SITE Duncan Grouping Mean N SITE A 43 11 221 A 41 14 231 B A 38 11 131 B A 39 14 171 B A 37 11 201 B A 38 14 191 B A C 35 11 191 B A 38 13 131 B A C 34 11 211 B A 37 14 161 B A C 33 11 161 B A 37 12 143 B A C 32 8 223 B A C 34 14 221 B A C 30 11 151 B A C 31 14 141 B A C 30 8 143 B A C 28 13 211 B C 24 11 231 B A C 28 13 223 B C 22 11 141 B C 25 13 151 C 19 11 171 C 21 13 201

Table 3-2. Duncan tests for total nitrogen (TN) in Apalachicola Bay during low and high river discharge. Concentrations expressed as µg N L-1. Letters indicate groups based on mean values.

Low-discharge TN High-discharge TN Duncan Grouping Mean N SITE Duncan Grouping Mean N SITE A 617 10 191 A 515 14 191 A 600 10 231 A 512 14 171 B A 505 10 171 A 472 14 231 B C 402 10 141 B A 427 14 161 B C D 378 10 221 B A 415 14 221 B C D 376 7 143 B A 407 12 143 B C D 373 10 161 B A 392 14 141 C D 343 10 131 B A 377 12 223 C D 310 10 211 B A 370 13 211 C D 299 7 223 B C 299 13 151 C D 285 10 151 B C 285 13 131 D 222 10 201 C 170 10 201

Table 3-3. Duncan tests for silica (Si) in Apalachicola Bay during low and high river discharge. Concentrations expressed as µg Si L-1. Letters indicate groups based on mean values.

Low-discharge Si High-discharge Si Duncan Grouping Mean N SITE Duncan Grouping Mean N SITE A 7474 11 231 A 6535 14 231 B 5424 11 191 B A 6121 14 171 B 4745 11 171 B C 5083 14 191 C 2790 8 143 D C 4531 14 161 C 2572 11 141 D C E 4224 14 221 C 2472 11 161 D C E 4095 14 141 D C 2197 11 131 D E 3591 12 143 D C 2062 8 223 D E 3541 13 211 D C 1828 11 221 D E 3278 13 223 D C 1531 11 151 E 2933 13 151 D C 1495 11 211 E 2849 13 131 D 989 11 201 F 820 13 201

Table 3-4. Duncan tests for salinity during low and high discharge. Concentrations expressed as psu. Letters indicate groups based on mean values.

Low discharge salinity High discharge salinity Duncan Grouping Mean N SITE Duncan Grouping Mean N SITE A 32.9 11 201 A 32.4 13 201 B A 28.8 11 151 B 16.8 13 131 B C 26.2 8 223 B 16.7 13 151 B C D 25.9 11 211 C B 13.5 12 143 B C D 24.8 11 221 C B 13.2 13 223 B C D 24.7 11 131 C B 11.7 13 211 E C D 23.9 11 161 C B 11.6 14 141 E D 21.3 11 141 C B 11.2 14 221 E 19.9 8 143 C D 8.3 14 161 F 10.7 11 191 E D 3.1 12 191 F 9.9 11 171 E 1.8 14 171 G 0.1 11 231 E 0.0 14 231

Table 3-5. Summary statistics for physical variables in Apalachicola Bay during high and low river discharge. LD= low discharge (left), HD= high discharge (right).

SITE Temp LD Temp HD Secchi LD Secchi HD CDOM LD CDOM HD 231 26.3 17.7 1.2 0.6 31.2 66.6 171 27.1 18.8 1.1 0.7 19.8 57.3 191 27.8 20.2 0.6 0.5 47.0 120.6 131 26.8 18.6 1.0 0.9 10.8 21.1 141 26.8 18.2 1.0 0.8 11.6 26.4 143 25.7 17.9 0.9 0.7 14.1 132.1 151 26.7 18.7 1.3 1.0 8.1 20.4 161 26.8 18.3 1.2 1.0 10.8 40.3 211 27.0 18.6 1.3 0.9 10.4 31.3 221 27.0 18.3 0.9 0.9 13.9 42.8 223 25.7 18.9 1.2 1.1 14.6 33.6 201 26.6 19.2 2.0 1.8 6.8 6.8 Average 26.7 18.6 1.1 0.9 16.6 49.9

Table 3-6. Percent of limitation by different treatments in the six bioassay experiments conducted at six selected sites. U= unlimited, P= phosphorus-limited, N= nitrogen-limited, NP= co-limited by nitrogen and phosphorus.

Site Season Initial Primary Low U 100% P 100% 231 (River) High P 50%, U 50% P 100%

Low NP 25%, P 75% NP 25%, P 75% 171 (North) High P 50%, U 50% P 100%

Low P 50%, NP 50% P 50%, NP 50% 161 (Center) High NP 50%, U 50% NP 50%, P 50%

Low N 50%, P 25%, NP 25% N 50%, P 25%, NP 25% 143 (West) High N 50%, U 50% N 50%, P 50 %

Low P 50%, NP 50% P 50%, NP 50% 223 (East) High N 50%, U 50% N 50%, P 50%

Low NP 50 %, N 50% NP 50 %, N 50% 201 (Gulf) High NP 100% NP 100%

Table 3-7. Duncan tests for chlorophyll a in Apalachicola Bay during low and high river discharge. Concentrations expressed as µg L-1. Letters indicate groups based on mean values.

Low-discharge chlorophyll High-discharge chlorophyll Duncan Grouping Mean N SITE Duncan Grouping Mean N SITE A 20.8 11 191 A 14.4 14 191 B 13.0 8 143 B A 11.0 12 143 C B 11.1 11 221 B C 8.0 14 161 C B D 10.0 11 141 B C 7.5 12 223 C B D 9.8 11 161 B C 6.9 13 131 C B D 9.3 11 131 B C 6.9 14 171 C B D 8.8 11 171 B C 6.6 12 151 C B D 8.5 11 211 B C 6.6 14 141 C D 8.4 8 223 B C 6.3 12 211 C D 7.2 11 151 B C 6.1 14 221 C D 6.9 11 231 C 5.8 13 201 D 5.5 11 201 C 4.0 14 231

Table 3-8. Duncan tests for phytoplankton biovolume in Apalachicola Bay during low and high river discharge. Concentrations expressed as 106 µm3 ml-1. Letters indicate groups based on mean values.

Low-discharge biovolume High-discharge biovolume Duncan Grouping Mean N SITE Duncan Grouping Mean N SITE A 4.7 8 143 A 2.8 13 131 A 4.4 11 201 B A 2.5 13 201 A 4.2 11 191 B A 2.3 14 161 A 4.2 7 223 B A C 2.0 13 151 B A 3.7 11 161 B A C 1.9 14 191 B A 3.6 11 131 B A C 1.6 12 143 B A 3.2 11 141 B A C 1.5 13 223 B A 3.1 11 221 B A C 1.3 13 211 B A 3.0 11 151 B A C 1.2 14 141 B A 2.9 11 211 B C 1.0 14 171 B A 2.2 11 171 B C 1.0 14 221 B 0.9 11 231 C 0.4 14 231

Table 3-9. Summary statistics for variables measured monthly at the twelve sampling sites in Apalachicola Bay during high and low river discharge from June 2008 to June 2010. Std Std Site Mean Mean Dev Dev Min. Min. Max. Max. Variable (Low) (High) (Low) (High) (Low) (High) (Low) (High) 231 CHL a 6.9 4.0 3.3 3.2 2.6 1.5 12.6 13.6 TP µg 24 41 6 13 9 23 32 63

TN µg 600 472 218 150 268 263 875 711

Si µg 7474 6535 642 1474 6254 3098 8278 8800

TEMP 26.3 17.7 5.8 6.3 12.3 6.2 30.8 26.6

SAL 0.1 0.1 0.0 0.0 0.1 0.0 0.1 0.1

SECCHI 1.2 0.6 0.3 0.3 0.8 0.3 1.7 1.2

CDOM 31.2 66.6 22.6 39.6 16.1 27.3 96.1 186.1

171 CHL a 8.8 6.9 3.0 5.2 4.3 1.6 13.7 17.7 TP µg 19 39 7 14 10 18 27 66

TN µg 505 512 127 141 319 365 776 793

Si µg 4745 6121 1372 1575 3151 3102 7015 9757

TEMP 27.1 18.8 5.2 6.8 15.2 6.4 31.6 28.9

SAL 9.9 1.9 4.1 2.5 4.4 0.0 16.3 9.1

SECCHI 1.1 0.7 0.4 0.2 0.7 0.4 2.1 0.9

CDOM 19.8 57.3 20.5 45.1 8.9 21.8 81.0 187.7

191 CHL a 20.8 14.4 5.7 9.9 14.6 2.3 32.6 35.1 TP µg 35 38 14 13 16 19 57 64

TN µg 617 515 155 144 431 231 973 799

Si µg 5424 5083 2492 1328 604 2366 8880 7671

TEMP 27.8 20.2 5.1 6.3 16.7 13.0 31.4 29.9

SAL 10.7 3.2 6.5 3.9 1.3 0.1 21.4 11.3

SECCHI 0.6 0.5 0.2 0.2 0.3 0.2 1.0 1.0

CDOM 47.0 120.6 41.3 134.4 16.1 20.3 140.1 496.6

161 CHL a 9.8 8.0 3.6 5.9 5.1 1.1 15.5 24.4 TP µg 33 37 18 22 22 20 85 108

TN µg 373 427 203 172 202 109 918 717

Si µg 2472 4531 1665 2014 801 1864 6247 9335

TEMP 26.8 18.3 5.5 6.7 14.4 6.4 30.5 27.5

SAL 24.0 8.3 4.0 6.8 20.5 0.3 31.5 23.4

SECCHI 1.2 1.0 0.4 0.6 0.4 0.2 1.8 2.6

CDOM 10.8 40.3 6.3 40.1 1.6 9.3 27.4 145.8

141 CHL a 10.0 6.7 3.6 3.1 3.5 2.8 17.9 12.8 TP µg 22 31 11 16 10 10 47 72

TN µg 402 392 138 164 180 210 697 798

Si µg 2572 4095 1392 1645 443 1971 4355 7209

Table 3-9. Continued Std Std Mean Mean Min. Min. Max. Max. Site Variable Dev Dev (Low) (High) (Low) (High) (Low) (High) (Low) (High) TEMP 26.8 18.2 5.3 7.0 14.9 5.7 30.8 28.0

SAL 21.3 11.6 6.4 8.3 12.4 2.4 34.1 27.6

SECCHI 1.0 0.8 0.4 0.4 0.3 0.2 1.8 1.4

CDOM 11.6 26.4 10.3 27.0 2.3 9.4 40.7 98.0

143 CHL a 13.0 11.0 7.8 11.3 3.8 1.7 23.9 39.1 TP µg 30 37 15 35 7 12 54 142

TN µg 376 407 133 243 248 86 598 956

Si µg 2790 3591 1156 1565 1088 929 4584 5875

TEMP 25.7 17.9 5.7 7.1 15.3 5.6 30.3 28.6

SAL 19.9 13.6 5.4 10.9 11.2 0.6 26.6 31.9

SECCHI 0.9 0.7 0.4 0.3 0.3 0.2 1.5 1.2

CDOM 14.1 132.1 8.2 397.2 5.9 4.2 28.9 1393.0

221 CHL a 11.1 6.1 5.6 2.3 2.8 2.4 21.8 11.4 TP µg 43 34 17 12 22 23 83 63

TN µg 378 415 146 195 251 17 740 663

Si µg 1827 4224 1245 1465 134 2130 4851 6320

TEMP 27.0 18.3 5.1 6.3 15.8 6.2 31.4 27.2

SAL 24.7 11.2 5.0 7.2 19.0 0.5 33.2 24.4

SECCHI 0.9 0.9 0.3 0.3 0.4 0.4 1.4 1.7

CDOM 13.9 42.8 11.7 50.0 7.2 10.7 42.5 199.7

223 CHL a 8.4 7.5 4.9 3.7 2.6 2.5 17.2 15.3 TP µg 32 28 14 10 11 17 48 55

TN µg 299 377 61 181 218 63 391 612

Si µg 2062 3278 1478 1648 234 767 3943 5814

TEMP 25.7 18.9 5.8 6.4 15.1 6.6 31.2 26.9

SAL 26.2 13.3 5.2 8.8 18.3 2.2 33.2 32.4

SECCHI 1.2 1.1 0.3 0.8 0.7 0.4 1.5 3.2

CDOM 14.6 33.6 9.6 38.6 5.8 9.6 36.3 153.0

131 CHL a 9.3 7.0 4.6 4.3 4.4 2.6 20.1 18.3 TP µg 38 38 17 21 20 13 79 75

TN µg 343 285 132 169 157 52 590 632

Si µg 2197 2849 1449 1718 380 473 4335 6125

TEMP 26.8 18.6 5.4 6.4 14.7 7.4 30.6 27.3

SAL 24.7 16.8 6.2 11.1 17.2 1.8 34.4 34.9

SECCHI 1.0 0.9 0.6 0.5 0.3 0.3 2.2 2.0

CDOM 10.8 21.1 7.7 21.7 0.0 3.7 31.7 69.8

151 CHL a 7.2 6.7 3.1 3.2 4.0 2.1 13.3 11.2 TP µg 30 25 21 11 6 10 86 42

Table 3-9. Continued Std Std Mean Mean Min. Min. Max. Max. Site Variable Dev Dev (Low) (High) (Low) (High) (Low) (High) (Low) (High) TN µg 285 299 115 154 120 137 569 606

Si µg 1531 2933 916 1825 151 759 2777 6088

TEMP 26.7 18.7 5.2 6.8 15.1 6.2 31.4 27.6

SAL 28.8 16.7 4.3 9.8 20.1 2.5 34.5 32.1

SECCHI 1.3 1.0 0.4 0.4 0.5 0.5 2.3 2.0

CDOM 8.1 20.4 5.9 19.1 0.2 5.0 24.0 63.9

211 CHL a 8.5 6.3 4.1 3.2 3.8 1.9 18.8 13.0 TP µg 34 28 21 14 11 12 83 60

TN µg 310 370 159 172 192 52 743 682

Si µg 1495 3541 1173 1679 268 868 4030 5552

TEMP 27.0 18.6 5.2 6.7 16.3 6.1 31.6 26.8

SAL 25.8 11.8 4.0 7.8 20.4 1.3 32.1 24.0

SECCHI 1.3 0.9 0.4 0.5 0.7 0.4 2.2 2.4

CDOM 10.4 31.3 5.7 39.7 4.3 6.4 26.1 153.6

201 CHL a 5.5 5.9 3.4 4.0 1.7 1.5 12.9 15.5 TP µg 37 21 22 10 8 3 73 35

TN µg 222 170 119 67 81 71 453 256

Si µg 988 820 781 738 259 0 2945 2635

TEMP 26.6 19.2 5.0 5.4 14.8 10.6 30.4 27.6

SAL 32.8 32.4 1.8 1.4 30.5 30.2 35.8 35.0

SECCHI 2.0 1.8 1.2 1.2 0.5 0.5 4.0 4.0

CDOM 6.8 6.8 4.5 3.1 0.8 1.9 15.9 14.6

Table 3-10. Major phytoplankton blooms at the twelve sampling sites in Apalachicola Bay form June 2008 to June 2010.\ Maximum Number of Bloom Species biovolume observations frequency

Prorocentrum minimum 10,117,318.19 105 8

Protoperidinium spp 4,989,537.00 69 1

Gymnodinium c.f. 1,660,554.98 9 1

Akashiwo sanguineum >45µ<65µ 1,588,204.71 64 4

Gyrodinium spirale c.f. <80µ 1,131,743.34 83 1

Cryptophyte (>5µ<15µ) 1,368,241.20 282 1

Fragilaria sp. 17,896,284.00 30 15

Leptocylindrus danicus 13,861,936.41 78 14

Cerataulina pelagica 8,988,936.98 51 8

Pseudosolenia calcar-avis 5,026,694.40 111 3

Rhabdonema adriaticum 4,800,000.00 7 4

Thalassionema nitzschioides (>30µ apical) 4,212,512.04 112 6

Pleurosigma/Gyrosigma (40µ transapical) 3,404,153.30 16 1

Navicula 25µ 3,198,427.64 24 1

Coscinodiscus 2,997,085.00 13 1

Skeletonema cf. costatum 2,612,179.20 58 1

Thalassiosira 10µ cell 2,608,600.50 55 1

Pleurosigma/Gyrosigma (10-20µ transapical) 2,507,400.00 250 1

Aulacoseira sp. 2,100,189.00 35 1

Cerataulina pelagica 1,713,123.38 51 8

Hemialus hauckii 1,706,964.40 25 2

Centric diatom 10µ 1,470,302.10 246 1

Dactyliosolen fragilissimus (>10µ < 20µ transapical) 1,257,217.07 9 1

Synechococcus (phycocyanin) 1,623,424.11 183 3

Phycocyanin (spherical picoplankton) 1,536,614.40 281 8

Table 3-11. List of Species observed in the Apalachicola Estuary, Florida, USA from June 2008 to 2010. Phytoplankton Species Sites 171 191 141 221 161 151 201 231 Green algae - Chlorophyceae

Actinastrum hantzschii 1 ...... Ankistrodesmus convolutus 4 6 2 2 3 2 . 5 Ankistrodesmus falcatus 4 4 . 4 2 2 . 11 Ankistrodesmus nannosolene . 1 . . 1 . . 1 Chlamydomonas 8 8 7 8 8 6 3 7 Closteriopsis sp...... 1 Closterium (c shaped) 2 1 . . 1 . . . Closterium (straight) 1 1 . 3 2 . . . Coelastrum cambricum ...... 1 1 Coelastrum sp. . . 1 . . . . 2 Crucigenia quadrata 1 1 2 1 1 . . 3 Crucigenia rectangularis . . . . . 1 . . Crucigenia sp. . 1 . . 1 . . 1 Crucigenia tetrapedia . 1 . . 1 . . 2 Dictospherium puchellum . . . 2 . . . 2 Eudorina 2 ...... 1 Kirchneriella contorta . . . 1 . . . 1 Micractinium pusillum . 1 . . . . . 2 Oocystis 5 1 1 . . . . 4 Pandorina sp...... 1 Pediastrum duplex 1 1 . . . . . 6 Pediastrum simplex . 1 1 . . . . 5 Pleodorina . 1 . . . . . 1 Scenedesmaus bijuga 3 2 . 2 1 2 . 6 Scenedesmus acutiformis . . . . 1 . . . Scenedesmus denticulatus ...... 1 Scenedesmus dimorphus . 1 ...... Scenedesmus quadricauda 11 7 3 4 5 . . 14 Scenedesmus sp. 6 1 1 2 4 . . 8 Staurastrum sp...... 2 Tetraedron minimum ...... Tetraedron regulare var incus ...... 1 Tetrastrum . . . 1 . . . . Tetrastrum hetero ...... 1 Treubaria . 1 ...... Pseudobodo tremulans 2 3 . 1 . 2 1 1

Table 3-11. continued Phytoplankton Species Sites 171 191 141 221 161 151 201 231 Eutreptia cf. . . . 1 . 1 1 2 Eutreptia globulifera . . 1 1 1 . . . Trachlemonas cf . 1 ...... Dinoflagellates - Dinophyceae Akashiwo sanguineum 7 15 5 6 7 7 5 . Amphidinium crassum ...... 1 . Amphidinium sp. . 1 . 1 2 . . . Ceratium fusus (Steidenger) . . . 2 1 . 6 . Ceratium hircus (Steidenger) . . . . . 1 2 . Ceratium lineatum . 1 2 2 2 6 9 . Ceratium sp. . . 2 . 1 3 1 . Cochlodinium polykrikodos . 1 . 1 1 1 1 . Cocholidium citron . . . 1 . . . . Dinophysis caudata 2 . 2 5 1 3 . . Diplosoid . . . 1 . . 1 . Gonyaulax polygramma (Steidenger) . . 1 . 1 . 2 . Gonyaulax sp...... Gymnodinium sp A <15µ 18 13 19 15 19 20 18 6 Gymnodinium sp B >15 <50 µ 6 4 3 4 4 3 5 1 Gyrodinium >5 <25 µ 3 2 5 2 3 2 8 . Gyrodinium instriatum 3 2 1 . . . . . Gyrodinium spirale 5 7 4 9 8 13 17 1 Hermesinum adriaticum (Sornia) 1 1 . . 1 . . . Heterocapsa sp. 2 4 3 5 6 2 5 . Karenia longicanalis . . . . 1 1 . . Karlodinium veneficum 5 8 6 6 11 8 9 2 Katodinium >25<50 ...... Katodinium glaucum 1 1 2 . 1 . 5 . Katodinium rotundatum 1 1 3 3 1 2 1 . Kryptoperidinium foliaceum cf . 1 ...... Oxyphysis oxytoxides 3 2 2 4 3 5 2 . Oxyrrhis marina ...... Polykrikos hartmanni 2 2 1 1 1 1 . . Polykrikos schwartzi (Steidenger) . . . . 1 . 1 1 Prorocentrum scutellum ...... Prorocentrum balticum . . . . 1 . . . Prorocentrum compressum ...... Prorocentrum gracile ...... 2 . Prorocentrum mexicanum 1 . . . . . 2 .

Table 3-11. Continued Phytoplankton Species Sites 171 191 141 221 161 151 201 231 Prorocentrum micans 3 6 9 9 13 14 7 1 Prorocentrum minimum 11 10 10 8 13 9 9 1 Prorocentrum rathymum . . . . . 1 2 . Prorocentrum sp...... 1 2 . Protoperidinium conicum . . . 2 1 2 . . Protoperidinium crassipes ...... Protoperidinium divergens ...... 1 . Protoperidinium excentricum . . . . . 1 1 . Protoperidinium leonis cf . . . 1 . . . . Protoperidinium oblongum ...... Protoperidinium pallidum ...... 1 . Protoperidinium pellucidium . 1 . . 1 . . . Protoperidinium quinquecorne ...... 1 Protoperidinium sp. 5 10 4 8 4 7 10 1 Pyrodinium bahamense var. bahamense . 2 . . . . 1 . Pyrophacus horologium <50u 1 1 2 1 1 1 5 . Pyrophacus sp. 1 2 2 1 2 3 5 . Scrippsiella sp. 5 3 5 6 4 6 4 1 Takayama pulchella . . . 1 . . . . Takayama sp 2 4 3 . 5 2 2 . Takayama tasmanica . . . . . 1 . . Torodinium terado ...... 1 . Warnowia sp . . . . 1 . 1 . Cryptophytes- Cryptophyceae Cryptomonas <30µ 3 4 1 2 . 2 1 3 Cryptophytes 25 26 27 26 26 25 23 29 Hillea . . 1 2 2 . . . Rhodomonas 1 2 1 2 1 . . 1 Diatoms- Bacyllariophyceae Amphiprora A 5 <50 µ 2 2 6 10 6 4 1 Amphiprora B >50 <100 µ . 3 . 3 . . . . Asterionella glacialis (Hasle) 7 4 15 12 15 17 19 1 Asterolampra (30µ) . . . . . 1 . . Aulocoseira sp. 2 2 1 2 2 1 . 2 Bacillaria paradoxa 2 . 5 3 1 3 7 1 Bacteriastrum furcatum . . 1 . . . . . Bacteriastrum hylinum . . 1 . . . 2 . Bacteriastrum sp...... 1 5 . Bidduphila alternans . . . . . 5 7 .

Table 3-11. Continued Phytoplankton Species Sites 171 191 141 221 161 151 201 231 Bidduphila sp. 1 . 2 . 2 2 4 . Centric chain >5 <30µ 19 4 6 9 9 12 13 26 Centric diatom A >5 < 25µ 60 56 66 73 66 55 55 44 Centric diatom B >30 <60µ 25 19 48 41 42 40 43 5 Centric diatom C >70 <100µ 4 4 6 4 3 7 11 2 Centric diatom D >100 <150µ 0 0 1 0 2 1 0 0 Cerataulina 1 . . . . . 2 . Cerataulina pelagica 4 2 8 4 5 6 7 . Chaetoceros aequatorialis . . . 2 2 2 1 . Chaetoceros affins ...... Chaetoceros cf. costatus . . . . 1 1 1 . Chaetoceros diadema . . . 1 . . 1 . Chaetoceros simplex (Hasle) 1 1 2 . 1 2 1 . Chaetoceros sp. >5 <30 µ 3 4 9 5 4 12 16 0 Chaetoceros subtillis (Hasle) . 1 1 1 . 1 . . Chaetoceros tenuissimus . 1 1 . . . . . Corethron sp...... Coscinodiscus sp. 2 2 2 2 2 3 . . Cylindrotheca sp 9 11 7 12 10 10 15 4 Dactyliosolen fragilissimus 3 . 7 3 2 6 9 . Diploneis sp. A >50<100 ...... 1 . Diploneis sp. B >25<50 1 . 1 3 1 . 1 . Diploneis sp. C >5<25 1 1 . 1 . 1 1 . Epithemia sp. 1 . . . . 1 . . Eucampia sp . . . 1 . . 3 . Fragilaria gramatophora . . . 1 . 2 3 1 Fragilaria sp. . . 1 1 3 2 9 . Guinardia delicatata ...... 3 . Guinardia flaccida 3 2 4 4 7 9 17 . Guinardia striata 2 1 5 10 7 13 17 . Hemiaulus hauckii 2 2 1 1 3 4 8 . Hemiaulus sinensis ...... Leptocylindrus danicus 2 3 6 6 7 10 14 . Leptocylindrus minimus 9 6 8 8 8 11 13 2 Licmophora abbreviata ...... Licomophora sp . . 2 1 . . 2 . Melosira granulata 3 1 . . . . 1 6 Melosira sp. 7 4 2 3 2 . 1 9 Navicula plagiotropis ...... 1 .

Table 3-11. Continued Phytoplankton Species Sites 171 191 141 221 161 151 201 231 Navicula sp. A >75 < 150 µ 1 1 1 2 1 Navicula sp. B >5 <75µ 0 0 5 4 3 4 6 0 Nitchia cf. delicate 12 13 17 16 14 20 19 4 Nitzschia sp. 3 4 5 8 6 2 3 1 Odontella aurita ...... 2 . Odontella moblilienis (Hasle) . . 3 1 1 6 9 . Odontella regia . . 1 . . 2 2 . Odontella sp 10-15µ diameter ...... Paralia sulcata (Ricard) 3 2 5 4 6 11 15 . Pennate diatom sp. A <5 transapical >5 <50 apical 43 40 51 56 40 49 57 24 Pennate diatom sp. B <5 transapical >50 <100 apical 10 2 10 7 4 8 17 4 Pennate diatom sp. C <5 transapical >100 <250 apical 5 5 3 5 9 8 2 15 Pennate diatom sp. D <5 transapical >250 <450 apical 0 0 0 1 2 1 6 0 Pennate diatom sp. E 6-15 transapical >5 <50 apical 3 2 4 4 8 7 8 0 Pennate diatom sp. F 6- 15 transapical >50 <100 apical 7 5 2 6 3 9 8 2 Pennate diatom sp. G 6-15 transapical >100 <250 apical 9 6 5 11 4 6 3 7 Pennate diatomsp. H 6-15 transapical >250 <600 apical 1 0 1 0 2 4 2 0 Pennate diatom sp. I 30 transapical >10 <100 apical 0 4 1 2 1 4 1 0 Pennate diatom sp. J 30 transapical >100 <200 apical 0 1 0 1 0 0 2 1 Plagiogramma vanheurckii . . 1 . . . 1 . Pleurosigma/Gyrosigma A >5 < 20µ transapical) 10 24 20 32 20 34 41 9 Pleurosigma/Gyrosigma B >30 < 40 µ transapical) 6 2 2 3 3 6 7 0 Pseudo nitzchia sp. 6 5 8 8 8 13 20 1 Pseudo nitzchia turgidula c.f. . . 1 . 1 1 2 . Pseudo-nitzschia calliantha . . 1 . 1 1 2 . Pseudosolenia calcar-avis 6 8 5 31 9 4 11 0 Rhabdonema adriaticum 1 . . . . 4 . . Rhizosolenia setigera 0 0 0 4 1 1 5 0 Rhizosolenia styliformis . . . . . 1 2 . Skeletonema costatum 3 3 5 4 8 7 7 . Surirella sp. A >5 <50 µ . 2 ...... Surirella sp. B >50 < 100 µ 1 3 . 1 . . . 2 Thalassionema bacillare . . 2 1 . 3 2 . Thalassionema frauenfeldii ...... 1 Thalassionema javanicum 2 2 1 . 2 1 . 1 Thalassionema nitzschioides 18 9 23 22 20 25 25 1 Thalassiosira chain >5 <30µ 6 5 8 6 6 5 5 0 Triceratium . . . 1 . . . . Tropidoneis lepidoptere . . . . . 1 . .

Table 3-11. Continued Phytoplankton Species Sites 171 191 141 221 161 151 201 231 Cyanobacteria – Cyanophyceae Anabaena sp. . 1 . 1 . . 1 2 Chrococcus 4µ . . . . . 1 . . Chrococcus minutus 1 . 2 1 2 . . . Merismopedia sp. 5 2 . 1 1 2 . 9 Merismopedia tennuissima . 1 ...... Microcystis sp...... 1 Oscillatoria sp...... 1 . 3 Spherical picoplankton 47 42 48 48 46 49 49 36 Synechococcus 27 26 27 22 28 30 24 21 Raphydophyceae Chatonella 1 1 ...... Generic raphidiophyceae 1 2 ...... Prasinophyceae Chrysochromulina sp. . . 1 . 1 1 . . Flagellate ovoid <15 µ 11 12 4 7 4 5 3 7 Flagellate ovoid >15 µ 1 1 1 2 . 1 1 4 Flagellate spherical 10-15 µ 11 14 10 14 8 11 11 11 Micromonas cf. 9 5 7 3 8 4 3 4 Pyramimonas sp. 2 2 3 2 3 2 2 0 Unidentified haptophyte 1 1 1 2 . 2 . .

2500

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Nov Nov May May Figure 3-1. Average monthly river discharge for the Apalachicola River from June 2008 to June 2010. Line indicates the calculated grand mean (532 m3 s-1).

River MidBay Gulf

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Figure 3-2. Temperature at three selected sites (231, 161 and 201) from June 2008 to June 2010.

Figure 3-3. Cluster analysis grouping of the sampling sites during low discharge based on physical, chemical, and biological characteristics.

Figure 3-4. Cluster analysis grouping of the sampling sites during high discharge based on physical, chemical, and biological characteristics.

1200 231 River 1200 171 East Bay Bridge 1000 1000 800 800 600 600 400 400 200 200

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Figure 3-5. Monthly concentrations of total nitrogen (TN, light gray) and dissolved inorganic nitrogen (DIN, dark gray), at six representative sampling sites in Apalachicola Bay. Concentrations expressed as µg N L-1.

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120 231 River 120 171 East Bay Bridge 100 100 80 80 60 60 40 40 20 20

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Figure 3-6. Temporal variation in monthly concentrations of total phosphorus (TP, light gray) and soluble reactive phosphorus (SRP, dark gray), at six representative sampling sites in Apalachicola Bay. Concentrations expressed as µg P L-1.

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Figure 3-7. Mean total phosphorus (TP), total nitrogen (TN) and Silica (Si) from June 2008 to June 2010 for eight selected sites in Apalachicola Bay. Low= Low discharge, High= High discharge.

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Figure 3-8. Mean chlorophyll a (Chl a), phytoplankton biovolume (BV) and carbon (C) from June 2008 to June 2010 for eight selected sites in Apalachicola Bay. Low= Low discharge, High= High discharge.

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River River Discharge m

µgL

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Figure 3-9. Time series plots of chlorophyll a (gray) vs discharge (black line) at six selected sites in Apalachicola Bay, from June 2008 to June 2010.

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River Region River

Relative Biovolume

East Bay East Central Bay Central

Figure 3-10. Phytoplankton biovolume (106 µm3 ml-1) subdivided into major groups at the river, north estuary (site 171), East Bay (site 191), and Mid Bay (site 161). Dominant species during blooms: Diatoms 1.Leptocylindrus danicus, 2.Pseudosolenia calcar-avis, 4.Cerataulina pelagica, 5.Fragilaria spp., 9.Coscinodiscus, 11.Thalassionema nitzschioides, 12.Skeletonema costatum. Dinoflagellates: 14.Akashiwo sanguinea, 15.Prorocentrum minimum, 16.Protoperidinium spp. Cyanobacteria: 19.Picocyanobacterium. Gray boxes indicate periods of high discharge.

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Relative Biovolume Central Bay Central

Figure 3-11. Phytoplankton biovolume (106 µm3 ml-1) subdivided into major groups at the west (sites 141 and 143) and east (sites 221 and 223) portions of the estuary. Dominant species during blooms: Diatoms 1.Leptocylindrus danicus, 2.Pseudosolenia calcar-avis, 4.Cerataulina pelagica, 6. Thalassiosira spp., 7.Pleurosigma spp., 8.Navicula spp., 9.Coscinodiscus spp. Dinoflagellates: 14.Akashiwo sanguinea, 15.Prorocentrum minimum, Cyanobacteria: 19.Picocyanobacterium. Gray boxes indicate periods of high discharge.

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Bay

Outer Outer

Relative Biovolume Gulf

Figure 3-12. Phytoplankton biovolume (106 µm3 ml-1) subdivided into major groups at the outer estuary (sites 131, 151 and 211) and the gulf region (site 201). Dominant species during blooms: Diatoms 1.Leptocylindrus danicus, 2.Pseudosolenia calcar-avis, 4.Cerataulina pelagica, 5.Fragilaria spp., 10.Rhabdonema adriaticum, 11.Thalassionema nitzschioides, 13.Hemialus hauckii. Cyanobacteria: 19.Picocyanobacterium. * Combination of different diatoms. Gray boxes indicate periods of high discharge.

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231 River

161 Mid Bay

Figure 3-13. Canonical correlation analysis plots of the major phytoplankton groups at four selected sites in Apalachicola Bay based on physical, chemical, and biological characteristics.

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151 Pilots Cove

201 Sike’s Cut

Figure 3-13. Continued

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Total Phytoplankton 25

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µm 15 6

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0 0 200 400 600 800 1000 TN, µg L-1

Figure 3-14. Distribution of total phytoplankton in the Apalachicola estuary with relationship to total phosphorus (TP) and total nitrogen (TN). Observations are expressed as biovolume and where taken from June 2008 to June 2010.

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Total Phytoplankton 25

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Figure 3-15. Distribution of total phytoplankton in the Apalachicola estuary with relationship to salinity and temperature. Observations are expressed as biovolume and where taken from June 2008 to June 2010.

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All 20 Sites Small Phytoplankton

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Figure 3-16. Distribution of small phytoplankton (<20 µm), large phytoplankton (>20 µm) and chain-forming centric diatoms across salinity gradients in the Apalachicola estuary. Observations are expressed as biovolume and where taken from June 2008 to June 2010.

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Figure 3-17. Distribution of common bloom forming species in relationship to salinity in the Apalachicola estuary. Observations are expressed as biovolume and where taken from June 2008 to June 2010.

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Figure 3-18. Distribution of common bloom forming species in relationship to temperature (ºC) in the Apalachicola estuary. Observations are expressed as biovolume and where taken from June 2008 to June 2010.

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Phycocyanin-rich Picocyanobacteria 3.0

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Figure 3-19. Distribution of phycocyanin-rich and phycoerythrin-rich cyanobacteria in the Apalachicola estuary with relationship to salinity. Observations are expressed as biovolume and where taken from June 2008 to June 2010.

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CHAPTER 4 SUMMARY

Apalachicola Bay is a sub-tropical river-dominated estuary which is experiencing major anthropogenically-driven changes in hydrology. This study was conducted in

Apalachicola Bay with the overall goal of describing how spatial and temporal patterns in the structure and abundance of phytoplankton are related to changes in river discharge, and shifts in key physical and chemical parameters, such as salinity, nutrient concentrations and water residence times. This initiative was undertaken in two different approaches under the premise that river discharge is a crucial element driving key physical-chemical parameters and ultimately phytoplankton abundance and composition.

The first approach consisted of identifying phytoplankton biomass trends during periods of below and above average discharge over a ten year period (i.e. 2002-2012) and exploring the relationship between river discharge, nutrient concentrations, and chlorophyll a. River discharge exerted significant control on phytoplankton biomass in most regions of the estuary and, in combination with nutrient gradients and salinity, acted as ecoclines for phytoplankton biomass. Ecoclines were important features of

Apalachicola Bay, and the specific character and dynamics of these ecoclines was strongly influenced by changes in river discharge. Discharge from the Apalachicola

River had a strong effect on salinity and TSN, and was auto-correlated with temperature. It also appeared to regulate TSP concentrations in regions adjacent to the river, but had little effect on TSP concentrations in regions close to the Gulf of Mexico.

In Apalachicola Bay lower mean chlorophyll a concentrations were detected during high discharge, when the combination of lower temperature and high residence time limited

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phytoplankton growth. Conversely, the combination of higher temperature and low discharge, and therefore high residence time could have facilitated phytoplankton biomass to reaching maximum levels during the summer and fall.

The second approach attempted to characterize the composition and abundance of the phytoplankton community within different regions of Apalachicola Bay, considering temporal trends and including comparisons of the phytoplankton assemblage under different discharge regimes. Numerous species of diatoms were the main component of the community, especially during the first year of sampling. The second year was characterized by community transitions from a diatom-dominated system to a more heterogeneous one, displaying a mix of diatoms, dinoflagellates, and cyanobacteria. In comparison with the first year of sampling, the second year was characterized by a generalize reduction in phytoplankton. This was possibly the result of interactions among numerous environmental factors, including a longer and more pronounced high discharge period from October 2009 to May 2010, which resulted in shorter residence times and highly variable salinities.

With respect to phytoplankton size distribution, biovolume of small-celled phytoplankton did not show dramatically higher peaks in biovolume in the mesohaline- lower polyhaline salinity range, except for picoplanktonic cyanobacteria peaks, which were abundant in mesohaline-lower polyhaline salinity range. Large-celled phytoplankton showed a relatively wide range of salinity over which biovolume peaks were observed, i.e. 10-33 psu. The group showing the most dramatic trend in biovolume peaks in the upper-mesohaline to lower polyhaline range was chain-forming centric diatoms. In general, diatoms were the major dominant group, however

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dinoflagellates were important, including a few species with potential to develop harmful algal blooms (HABs) such as Prorocentrum minimum.

In summary, results from this study indicated regional differences in the composition and abundance of phytoplankton in the estuary, which had a strong connection to changes in river discharge and salinity gradients. River discharge alone played an important role in decreasing water residence time and, as a consequence, decreased phytoplankton biomass and biovolume due to osmotic stress and rapid flushing. However phytoplankton blooms are highly dynamic and drawing major conclusions from a two year study would be a premature action.

The task of defining the factors that drive phytoplankton community assembly and succession remains a central challenge in aquatic ecology (Hutchinson, 1961,

Cloern and Dufford, 2005). As Hutchinson (1961) pointed out, it is hard to understand how, in turbulent open water, many physical opportunities for niche diversification can exist, and how only a few organisms can be favored by peculiar chemical conditions at the surface. Although our results highlight the importance of factors such as salinity and river discharge in shaping the phytoplankton community in Apalachicola Bay, several additional factors (e.g. residence time, loss due to grazing by zooplankton and oysters, and nutrient gradients) remain to be considered in order to explain, in a more holistic fashion, the dynamics of the phytoplankton community in this estuary.

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BIOGRAPHICAL SKETCH

Paula Viveros grew up in the city of Armenia, Colombia. She graduated from

Universidad del Quindío in July of 2001 with a B.S. in biology. Her undergraduate research focused on orchid distribution and taxonomic composition in two natural reserves of Quindío, Colombia. In May 2007 she graduated from Universidad del

Quindío with her M.S. in plant biology, concentrating her studies on plant taxonomy and diversity. Her thesis examined distribution and taxonomic composition of

Pleurothallidinae (Orchidaceae) in the Quindío region of Colombia. In 2008 Paula began her doctoral studies on estuarine and phytoplankton ecology at the University of Florida in Fisheries and Aquatic Sciences.

Her research focused on factors controlling phytoplankton abundance, species composition, and seasonality in Apalachicola Bay, a subtropical estuary in Florida.

Her research and studies were funded by NOAA's National Estuarine Research

Reserve Graduate Research Fellowship, South East Alliance for Graduate Education and Professoriate fellowships and awards, and the Delores Auzenne Dissertation

Award. She received her Ph.D. from the University of Florida in the spring of 2014.

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