Abiotic Influences on and Community Dynamics of Benthic Dinoflagellate Species in the Florida Keys

ABIOTIC INFLUENCES ON AND COMMUNITY DYNAMICS OF BENTHIC DINOFLAGELLATE SPECIES IN THE FLORIDA KEYS

A Thesis

Presented to

The Faculty of the College of Arts and Sciences

Florida Gulf Coast University

In Partial Fulfillment

Of the Requirement for the Degree of

Master of Science

Jessica Elizabeth Schroeder

2020

Florida Gulf Coast University Thesis

APPROVAL SHEET

This thesis is submitted in partial fulfillment of the

requirements for the degree of

Master of Science

Jessica E. Schroeder

Approved:

______Michael L. Parsons, Ph.D. Committee Chair/Advisor

Hidetoshi Urakawa, Ph.D.

Serge Thomas, Ph.D.

The final copy of this thesis has been examined by the signatories, and we find that both the content and the form meet acceptable presentation standards of scholarly work in the above mentioned discipline.

Acknowledgments

I would like to thank all of the people who helped and contributed to this thesis research. My committee members, Drs. Michael Parsons, Hidetoshi Urakawa, and Serge

Thomas, thank you all for your help, not only on this project, but in my education here at

FGCU since 2012. I would also like to thank the Coastal Watershed Institute and

CiguaHAB teams, for your contributions to the project as a whole and for the grant funding that allowed me to work at FGCU and paid for my schooling. And a special thanks to my family and friends for constantly helping me through the stressful times and always pushing me to be the best. Also, a special thanks to my boyfriend, Andrew for helping with my chlorophyll measurements and always motivating me to perfect my thesis.

Table of Contents Abstract ...... 1 Chapter 1: Background ...... 2 Ciguatera Fish Poisoning & Toxins ...... 2 Symptoms & Occurrence of CFP ...... 3 Gambierdiscus Discovery ...... 4 Habitat ...... 5 Chapter 2: Introduction ...... 7 Objectives ...... 10 Chapter 3: Material and Methods ...... 11 Study Sites ...... 11 Figure 1...... 11 Sample Collection...... 12 Sample Analysis ...... 13 Figure 2...... 14 Figure 3...... 14 Data Analysis ...... 15 Chapter 4: Results ...... 17 Abiotic Parameters ...... 17 Figure 4...... 17 Table 1...... 18 Relationships ...... 18 Table 2. AIC results based on the parameters from the DistLM...... 19 Figure 5...... 20 Figure 6...... 20 Figure 7...... 21 Figure 8...... 21 Figure 9...... 22 Figure 10...... 22 Relationships: Site, Season, and Location ...... 23 Correlations Between Environmental Parameters and Cell Densities ...... 23 Table 3...... 24 Niche Optima and Tolerance ...... 24 Figure 11...... 25 Figure 12...... 26 Figure 13...... 26 Figure 14...... 27 Figure 15...... 27 Figure 16...... 28 Figure 17...... 28 Figure 18...... 29 Site Relationships ...... 29

iii

Figure 19...... 30 Figure 20...... 30 Figure 21...... 31 Figure 22...... 31 Season and Location Relationships ...... 32 Figure 23...... 32 Figure 24...... 33 Chapter 5: Discussion ...... 34 5.1 Dinoflagellate Species Individually: Autecology ...... 34 Table 4...... 35 5.2 Dinoflagellate Species Assemblages: Synecology ...... 36 5.3 Discussion ...... 37 5.4 Significance and Future Implications ...... 37 References ...... 40

Abstract

Gambierdiscus is an epibenthic dinoflagellate genus containing some species that produce a toxin (ciguatoxin), which causes Ciguatera Fish Poisoning (CFP). These dinoflagellates live on macrophytes that are consumed by herbivorous fish and invertebrates, thereby introducing ciguatoxin into the reef food web through bioaccumulation and biomagnification processes. Such grazers are later predated on by larger fish, which when consumed by people, can lead to CFP. Other dinoflagellates coexist with Gambierdiscus spp., including potentially toxigenic Prorocentrum and

Ostreopsis spp. The research presented here focuses on the community ecology of

Gambierdiscus spp. and these other dinoflagellates, and the abiotic factors that affect their distribution, abundance, and compositions. New findings in this project have provided information about community dynamics and the influence of abiotic factors. The data suggest that physical and chemical parameters influence dinoflagellate densities and the differences in site community composition. These epibenthic dinoflagellates thrive in high temperatures but may compete in other environmental conditions. The three dinoflagellates were more abundant in the Florida Bay than Atlantic Ocean sites. The three dinoflagellates coexist, but there were no differences in abundances seasonally and between sites, suggesting subtle niche partitioning may be taking place.

Chapter 1: Background

Ciguatera Fish Poisoning & Toxins

Ciguatera Fish Poisoning (CFP) is a circumtropical illness caused by the consumption of contaminated reef fishes. Reef fishes known to cause CFP include grouper

(Epinephelinae family), snapper (Lutjanidae family), barracuda (Sphyraena family), hogfish (Lachnolaimus maximus ) and other resident and migrating fishes. CFP is the most common form of non-bacterial seafood poisoning which is linked to harmful algal blooms

(HABs). CFP is associated with fish caught in circumtropical coastal waters between 35°N and 35°S latitudes (Parsons et al. 2017). Gambierdiscus is an epibenthic dinoflagellate, which is found attached to a macroalgal host that is then consumed by herbivorous fishes.

Upon consumption by these herbivorous fishes, toxin pre-cursors produced by toxigenic

Gambierdiscus species are metabolized and assimilated into the fish tissue as ciguatoxins.

Ciguatoxins are heat-stable and lipid soluble, and resistant to cooking and freezing, such that impacted fishes cannot be detoxified before consumption (Withers 1982). After these toxic dinoflagellates are consumed and bioaccumulated by herbivores, the toxins are subsequently biomagnified through the food chain.

More than 400 fishes have been identified as vectors of CFP (Helfrich & Banner

1968; Hirama et al. 2001). There are multiple congeners of ciguatoxin (CTX) based on where they originated (e.g., Pacific Ocean versus Caribbean Sea) and how they were oxidized by fish enzymatic pathways; some compounds are more toxigenic than others

(Gopalakrishnakone et al. 2016). All of the CTX congeners are ladderlike, cyclic polyethers which bind to and activate the voltage-sensitive sodium channels (VSSC;

Gawley et al. 1992).

The genus Gambierdiscus , is comprised of 16 known species of armored dinoflagellates which prefer warm waters (Hoppenrath et al. 2019; Rains & Parsons 2015).

The genus is morphologically distinguished by its plate tablature, which follows a specific pattern on the Kofoidian plates; Po, 3’, 7”, 6C, 6 or 7S, 5’”, 1P, and 2”” (Litaker et al.

2009). Gambierdiscus species are known to adhere to macroalgal hosts and other substrates by polysaccharide mucus onto which they are solidly tethered even when subjected to high wave events, although they prefer calmer waters with less wave action

(Adachi & Fukuyo 1979). The macroalgal host provides surface area for multiple species of dinoflagellates to adhere to, but calcareous hosts, such as the macroalga Halimeda species, have more mass than the softer structures (Lobel et al. 1988). Gambierdiscus growth is influenced primarily by temperature, although other anthropogenically and naturally occurring environmental changes may be important as well (i.e., coral reef damage; Parsons et al. 2012 and references therein). Many studies have shown that the growth preferences of Gambierdiscus vary by locale, thus resulting in a broad range of potential habitats where cells can grow and thrive. Generally, Gambierdiscus prefers shallow water habitats and an annual temperature range between 21 and 31 °C (Parsons et al. 2012). High Gambierdiscus densities are most commonly found in a high and stable salinity habitat with low light levels (below 10% of incident irradiance) and ample substrate

(macrophyte hosts) to settle upon (Parsons et al. 2012).

Symptoms & Occurrence of CFP

CFP is most common in the tropics but is found worldwide because of the export of warm water fish to markets and the expansion of habitat for Gambierdiscus species in

response to a warming climate (Parsons et al. 2010). Over 50,000 people suffer from ciguatera fish poisoning annually, with gastrointestinal, neurological, and cardiovascular symptoms possibly resulting in paralysis, coma, or death (Gopalakrishnakone et al. 2016).

Although fatal incidences are low, the occurrence of CFP in the tropics remains high. The illness is often misdiagnosed by medical doctors unfamiliar with CFP and its symptoms

(Ting & Brown 2001). Symptoms typically last several weeks but the people affected can be sensitized and may experience recurring symptoms for years (Falconer 2012).

Ciguatera can render many marine fish potentially toxic, causing a hardship on those stakeholders that rely on these fish for their main source of protein (Bomber 1985).

Fisheries are an important resource to many, especially in the tropics, and CFP represents a health threat for the inhabitants in the form of gastrointestinal, neurological, and cardiovascular distress (Lewis 1992). CFP can also sensitize the consumer to hot and cold sensations, with a low fatality rate, the long-term health effects could be severe (Chateau-

Degat et al. 2005). The public health risk is not localized, however, as areas prone to CFP also rely heavily on tourism to support their economy in many cases (Lewis 1992). In the atoll island communities of the Pacific, the risk of CFP is compounded by the possible loss of fish as a protein source for these communities, as they avoid eating local fishes

(Gopalakrishnakone et al. 2016).

Gambierdiscus Discovery

Ciguatoxin was discovered by Dr. Scheuer, who was a professor at the University of Hawaii, in specimens of the moray eel and was determined to be a lipid containing quaternary nitrogen, hydroxyl, and carbonyl function (Scheuer 1969). Ciguatoxin was

found in samples collected from the Gambier Islands, an archipelago in French Polynesia, in detritus that had settled on the dead coral in the area (Yasumoto et al. 1977). Two representative species, parrotfish and surgeonfish, were examined and the fat-soluble toxin was found in these species. The detrital mass (and fish gut contents) revealed the presence of dinoflagellates that were further examined and initially identified as Diplopsalis sp., later described as Gambierdiscus toxicus (Adachi and Fukuto 1979). This finding was a major break-through in ciguatera research, shifting the focus from fish to algae .

Habitat

The Gambierdiscus community has been known to vary from site-to-site and season-to-season, as well as exhibit preferences of substrate (Parsons et al. 2017; Parsons

& Preskitt 2007). Surface area is key to the abundance of the epiphytic dinoflagellates, although calcareous algae (e.g. Halimeda ) have more mass than non-calcareous species

(e.g. Dictyota ) meaning more delicate species generally provide more of an area to settle upon (Lobel et al. 1988). Gambierdiscus cells tend to attach to the algal surface, utilizing the three-dimensional structure to minimize light exposure in the shallow locations and avoid turbulent wave action, allowing them to inhabit areas that would otherwise be high light intensity environments (Rains & Parsons 2015; Parsons et al. 2012). Gambierdiscus are known to coexist with many other toxigenic benthic dinoflagellates including

Amphidinum, Coolia, Prorocentrum, and Ostreopsis species, all influenced by various environmental factors, both natural and anthropogenic, that can impact the microbial communities (Parsons & Preskitt 2007; Parsons et al. 2012). These dinoflagellates also compete for space, light, and nutrient availability (Tindall & Morton 1998). They are

influenced by other factors such as water advection or turbulence and temperature. For example, epiphytic dinoflagellate densities are higher in areas like lagoons that are protected (Richlen & Lobel 2011).

Nutrient concentrations correlate with dinoflagellate densities on their macroalgal hosts in some – but not all – cases (Parsons & Preskitt 2007; Anderson et al. 2008). In some cases, Gambierdiscus are most abundant with the highest nutrient concentrations; for example in Puako, Hawaii, which is prone to ciguatera, higher cell densities were observed nearshore where the nutrient concentrations were higher (Parsons et al. 2012).

Gambierdiscus and Ostreopsis species are known to be stimulated in growth by the anthropogenic and naturally occurring environmental changes, such as global warming and nutrient run off (Parsons et al. 2012). In a study in the Florida Keys, Gambierdiscus cell densities were higher at higher temperatures, greater than 26ºC, while other dinoflagellates, including Ostreopsis , were more abundant in temperatures less than 26ºC (Bomber 1985).

Dinoflagellates are known to inhabit the intertidal and sub-tidal zones inhabited by macroalgae (Bomber 1985). Habitat expansion for Gambierdiscus and other dinoflagellates is occurring due to climate change because of the increase in temperature, heating the waters to the optimal temperature ranges for toxic dinoflagellates blooms, which in turn increases the risk of ciguatera poisoning and other toxins in endemic regions as well as nonendemic regions (Gopalakrishnakone et al. 2016).

Chapter 2: Introduction

Ciguatera Fish Poisoning (CFP) is an illness that is caused by the consumption of contaminated reef fish (Parsons et al. 2017). The consumption of the benthic dinoflagellate, Gambierdiscus, which inhabits macrophytes such as Halimeda, is consumed by herbivorous fishes and in turn consumed by predatory reef fishes. These reef fish that are known to be vectors include grouper (Epinephelinae family), snapper

(Lutjanidae family), barracuda ( Sphyraena family), hogfish (Lachnolaimus maximus) and other resident and migrating fishes (Parsons et al. 2017). The toxin is fat soluble and metabolizes into the fish tissue, being inconspicuous and causing symptoms in human consumers, while also being unable to be cooked or frozen out (Withers 1982).

Ciguatoxins (CTXs) activate the sodium channels resulting in the depolarization of the cell, causing symptoms in humans including gastrointestinal, neurological and cardiovascular problems (Antilla et al. 2015).

Gambierdiscus is a benthic dinoflagellate, that has been identified as the source of ciguatoxins that cause CFP, is a genus of photosynthetic, epibenthic, armored dinoflagellates, containing currently at least 16 species (Yasumoto et al. 1977; Hoppenrath et al. 2019; Litaker et al. 2009; Kretzschmar et al. 2019). These toxigenic dinoflagellates prefer a sheltered habitat, like lagoons and mangrove forests, to protect them from wave activity, wind, and land water runoff (Hoppenrath et al. 2014). Gambierdiscus species increase in abundance as temperature and changing storm patterns increase, which is important as the Earth is experiencing a potential warming period that may cause abnormal storm patterns such as hurricanes and increases in water temperatures (Chateau-Degat et al. 2005). With the expansion of warmer waters, the possibility of more blooms and/or

more toxigenic species abundance could spread, causing a potential impact on humans.

Gambierdiscus are known to coexist with many other toxigenic benthic dinoflagellates including Prorocentrum and Ostreopsis. These dinoflagellates all prefer low energy environments and require algae host substrates with high surface area to settle upon. They also compete for space, light, and nutrient availability (Tindall & Morton 1998).

Prorocentrum species are potentially toxigenic and known to produce okadaic acid and its analogs (Kumagai et al. 1986), the causative toxin in diarrheic shellfish poisoning

(Faust 1999; Faust 1993). Prorocentrum lima, for example, has been found to contribute to diarrheic shellfish toxins in coastal areas with high turbulence, which resuspends the cells in the water column, making them more available for the filter-feeders

(Gopalakrishnakone et al. 2016). Toxigenic Prorocentrum species may play a role in ciguatera fish poisoning and have been observed to have dominated in the reef communities

(Bomber 1985). In a study, P. lima species was studied in reef and algal environments and was found to be present in the highest abundances on dead corals than on red macroalgae

(Hoppenrath et al. 2014). The specific species that were observed in these samples were

P. belizianum, P. carribaeum, P. compressum, P. concavum, P. elegans, P. emarginatum,

P. hoffmanium, P. lima, and P. tropicalis.

Ostreopsis species, such as Ostreopsis ovata , are bloom-formers that can produce toxins including palytoxin analogs and ovatoxins (Ciminiello et al. 2008; Faust et al. 1996;

Hoppenrath et al. 2014). O. ovata is known to increase in biomass and cell growth rate while maintaining low toxicity in high water temperatures versus higher toxicity and less biomass in lower temperatures, becoming stressed by the lower temperatures and causing them to expend energy on defense rather than growth or biomass (Antilla et al. 2015;

Hoppenrath et al. 2014). O. ovata was also observed to be higher in abundance when nutrients, temperatures, salinity, and wave action were all high, allowing this species to expand in biomass and abundance under these environmental conditions (Antilla et al.

2015). Ostreopsis heptagona is known to be toxic and produce large filamentous strands of mucus when they bloom (Hoppenrath et al. 2014).

Abiotic factors, including wave height, temperature, photosynthetically active radiation (PAR), salinity, and nutrients can influence the abundance of benthic dinoflagellates. In a study at the Johnston Atoll in the Pacific Ocean, water motion was studied to determine its influence on the dinoflagellates (Richlen & Lobel 2011). The study found that sites with a lot of water motion supported less dinoflagellates than the lagoons.

Low energy habitats such as mangrove forests and lagoons are known to be a more habitable environment than reefs for these dinoflagellate species (Tindall & Morton 1998).

Research has been done to determine the optimal salinity levels for these species to thrive, resulting in a range of 30-39 ppt to have better growth and abundance (Tindall & Morton

1998). With climate change occurring, ciguatera fish poisoning is known as an environmental indicator for the warming of the waters; but there appears to be a delay between temperature spikes and the onset of CFP (e.g., 13-17 months in French Polynesia;

Chateau-Degat et al. 2005). Dinoflagellate growth is positively correlated to the seawater temperature, making climate change very influential to the dinoflagellate communities

(Chateau-Degat et al. 2005).

The species that cohabit the same macroalgal hosts likely influence each other via carrying capacity and/or symbiotic factors. The carrying capacity may increase due to symbiotic relationships that mutually benefit the species that inhabit the host algae.

Alternatively, competition (and/or allelopathy) among dinoflagellate species for space and resources may result in competitive exclusion. Because benthic dinoflagellates are typically found on the same host algae, they likely encounter similar exogenous conditions such as abiotic factors (light, temperature and salinity) and available nutrients, leading to a possible Paradox of the Plankton situation. The goal of this study, therefore, was to determine how the three most common genera of epibenthic dinoflagellates

(Gambierdiscus, Prorocentrum, and Ostreopsis ) were influenced by changing environmental conditions in the Florida Keys.

Objectives

This research builds upon previous research done in the Florida Keys on Ciguatera

Fish Poisoning, which focused primarily on the autecology of Gambierdiscus (Parsons et al. 2017; Rains & Parsons 2015) or on nearshore, shallow water locations (Bomber 1985).

This research includes other epiphytic dinoflagellates (i.e., Prorocentrum and Ostreopsis ) and includes sampling sites further offshore than previous studies. The purpose of this thesis was to study the ecology of benthic dinoflagellates found in the Florida Keys and the abiotic factors controlling them. The first objective was to determine the influence of abiotic factors (PAR, waves, temperature, salinity, ortho-phosphate, nitrate, nitrite, and ammonium) on Gambierdiscus, Prorocentrum and Ostreopsis species that cohabit within the same environment, such as the macroalgal species, Halimeda. This objective uses an autecological approach (i.e., focusing on individual species). The second objective was to explore the role of abiotic factors that affect the dinoflagellate species assemblages in the

Florida Keys (i.e., a synecological approach).

Chapter 3: Material and Methods

Study Sites

Four sites around Long Key and Lower Matecumbe Key in the Florida Keys were examined for this study (Parsons et al. 2017). The sites were Heine Grass Bed (HGB),

Tomato Patch Hardbottom (TPH), Long Key Hardbottom (LKH), and Tennessee Reef

Lighthouse (TRL) (Figure 1). These sites are named based on certain descriptive factors:

HGB is a bayside seagrass bed environment on the bayside of Lower Matecumbe; TPH is a bayside hardbottom environment about 50 meters from Highway 1; LKH is a hardbottom, reef environment on the ocean side of Long Key near channel marker 44; TRL is a reef site near the Tennessee Reef lighthouse (Parsons et al. 2017).

Figure 1. Map of sample sites: 1) Heine Grass Bed (HGB), 2) Tomato Patch Hardbottom (TPH), 3) Long Key Hardbottom (LKH), 4) Tennessee Reef Lighthouse (TRL) (adapted from Parsons et al. 2017).

Sample Collection

The samples were previously collected during trips to the Florida Keys over a period of three years from 2013 to 2015. Macrophyte samples, water samples, and fish species were taken on these research trips, collected along transect lines that were 20 meters long and separated by five meters (Parsons et al. 2017). In this research, Halimeda incrassata was the only macroalgal species studied because it harbors large numbers of epiphytic dinoflagellates (Parsons et al. 2017) and was present at all four sampling sites.

The Halimeda samples were collected via SCUBA diving, where thalli were clipped at their base and inserted in 50 mL screw-capped polypropylene centrifuge tubes, and placed in a cooler with ambient seawater for transport back to shore (Parsons et al. 2017). The samples were processed back onshore by shaking followed by filtering samples with ambient seawater five times using 200 and 20-micrometer sieves (PVC and mesh; 6.3 cm diameter) to separate and concentrate the dinoflagellates from the other material on the macrophytes (Richlen & Lobel 2011; Parsons et al. 2017). Samples were preserved with

1% glutaraldehyde (by volume) and placed in a refrigerator at 4 °C until counting (Parsons et al. 2017). The Halimeda thalli were then patted dry and weighed for the wet weight on a Mettler Toledo AL204 balance. Temperature and light data were collected in the field using Onset  HOBOs  (data loggers; model number UA-002-64) that were positioned at the end of one of the transect pins at each site, recording data every 15 minutes. The loggers were exchanged on a monthly basis and the data were downloaded and analyzed thereafter.

Salinity (surface and bottom) was measured using a YSI PRODSS multisonde. Nutrient samples were collected in triplicate at each site each month in bottom waters (<0.3 m from the bottom) in acid-washed 250 mL amber glass bottles, filtered onshore and frozen until

processing. Nutrient samples were analyzed using a Bran + Luebbe Seal AutoAnalyzer 3 for determining nutrient levels in the water column (nitrate, nitrite, ortho-phosphate, silicate and ammonium) (Parsons et al. 2017). Simulated wave data were obtained from

Wind Guru ( https://www.windguru.cz/ ; GFS 27 km daily archive; United States-

Islamorada) and using wind data retrieved from the National Climatic Data Center

(http://www.ncdc.noaa.gov ; Marathon Airport, FL; Daily Summaries data set), corrected for fetch. Wind corrections were applied as weights multiplied by the wave data as outlined in Stanca & Parsons (2017).

Sample Analysis

An Olympus IX71 epifluorescence inverted microscope (DAPI filter and transmitted light) was used to identify and enumerate the armored dinoflagellates in the samples. Three 3 mL triplicate subsamples were originally examined for Gambierdiscus cells using three wells of a 6 well tissue culture well plate (Parsons et al. 2017). Additional counts were conducted to enumerate Prorocentrum and Ostreopsis cells using the remaining sample left after the initial Gambierdiscus counts (6 mL left). For these counts, each well of a four-well slide was filled with 0.5 mL from the original sample, a drop of

Uvitex  was added to stain the armored, cellulosic plates covering the cells of the armored dinoflagellates (Parsons et al. 2017) and samples were examined. Each sample was counted until ten cells of both Ostreopsis and Prorocentrum were identified and enumerated. All cell counts are reported as cells per gram wet weight Halimeda.

In vivo chlorophyll-a (a proxy for total epiphytic autotrophic biomass) was also determined for each sample by analyzing 1 mL of each sample in a Turner Designs Trilogy

fluorometer fitted with an in vivo chlorophyll-a module. The cell density and in vivo chlorophyll-a data were entered into Excel and the monthly means per site were calculated.

Figure 2. Ostreopsis heptagona from a sample at TRL in January of 2014. Uvitex stained the cellulosic plates, which fluoresces under UV light (DAPI filter).

Figure 3. Prorocentrum emarginatum from a sample at TRL in January of 2014. P. emarginatum cells are identified by the deep and narrow V-shaped periflagellar area.

Data Analysis

All data analyses took place using the following statistical programs; Primer 7.0,

IBM SPSS Statistics, and C2 Statistics. The C2 program was used to determine the optimum value and tolerance ranges for each environmental parameter on the three dinoflagellate genera, using the transfer function and weighted average selection. Prior to analysis in the Primer 7.0 and SPSS programs, the cell density data were LOG(X+1) transformed. The environmental data were also normalized in the Primer 7.0 workspace to give the data similar magnitudes and ranges. Pearson correlations among the variables were determined in SPCC using the bivariate correlation function. In Primer 7.0 workspace, the transformed data were used to perform a SIMPROF analysis, which will output the statistically significant similarities between biotic data. A resemblance analysis was performed on both the environmental and biotic data sets, to pair the samples together to be analyzed. A BEST analysis was used to find similarities between the environmental parameters and the dinoflagellate assemblages to test for relationships between data sets.

Distance-based linear models (DistLM) were performed in the PERMANOVA+ workspace to identify particular predictor variables that can be used to describe the dissimilarity or similarity among samples and test for variability. The samples were organized into groups (factors): sites (LKH, TRL, HGB, TPH), seasons (winter: December through February; spring: March through May; summer: June through August; and fall:

September through November), and locations (LKH, TRL are Ocean; HGB, TPH are Bay).

These factors were applied for both dinoflagellate abundance and environmental data comparisons, to examine how much variation in the dinoflagellate abundance data could be explained by each factor (BEST and DistLM). The environmental data were also

grouped into indicator categories (physical: waves, temperature, PAR, salinity; and chemical: nutrients) for the environmental data for similar explanations of variation (but by parameters rather than samples as factor is used for). Using the normalized environmental data, a draftsman plot was created along with the -1:1 correlation to show us the interrelations between variables in the multivariate environmental data. This would also indicate collinearities and the need to remove collinear parameters to prevent redundancy (and the potential over-influence of collinear parameters).

Chapter 4: Results

Abiotic Parameters

The correlations between the environmental parameters showed that there may be a common driver (i.e., seasonal changes, weather, etc.) influencing the environmental parameters and the dinoflagellates (Figure 4). There is no evidence of collinearity and the parameters are independent of one another with the only waves versus temperature showing a linear or negative relationship (r = -0.75). High waves and low temperatures may not be the driver, but instead reflect seasonal conditions, such as winter. Therefore, both parameters were left in the analysis to reflect this potential seasonal influence. The ranges, averages, and standard deviations of the abiotic data are displayed in Table 1.

Figure 4. The Draftsman Plot shows the normalized abiotic data is appropriate and that the parameters respond to one another.

Table 1. The ranges, averages, and standard deviations of environmental parameters throughout the three years (2013 to 2015).

Parameter Range Average Standard Deviation Waves 0.105-4.36 0.885 0.477 Temperature 18.4-39.2 27.5 3.01 (°C) PAR (µM/m 2/sec) 6.19-133 38.6 15.4 Salinity (ppt) 30-42 36.8 1.85 - NO 3 (µM) 0.012-5.85 0.417 0.867 - NO 2 (µM) 0.002-0.324 0.0258 0.0533 Ortho-P (µM) 0.010-0.566 0.0744 0.0682 + NH 4 (µM) 0.09-4.60 1.39 0.845

Relationships

The samples could not be separated into distinctive groupings (clusters) using the dinoflagellate assemblage data with (p=0.445) and without (p=0.663) the chlorophyll data in the SIMPROF analysis. Similarly, the DistLM results suggest that assemblages are responding similarly across sites, seasons, and locations, corroborating the lack of distinction (Table 2). The analysis was done using the Akaike’s Information Criterion

(AIC), which comes from the likelihood theory and smaller values of AIC will represent a better model. In this case PAR had the smallest AIC value making it the best fitting model parameter. The dinoflagellate assemblages in the samples appeared to be more influenced by lower light, temperature, and salinity levels as well as higher wave action, although there was little separation by location, season or site in this regard (Figures 5, 7, & 9).

These conditions are typical of winter months and may indicate that the dinoflagellates are responding more to changing environmental conditions in the winter versus summer. The three overall most influential parameters (ammonium, salinity and PAR) also displayed

similar negative relationships; the dinoflagellates were more influenced by lower levels of these three parameters (Figures 6, 8, & 10). Each figure (5-10) was fitted to the axes based on the distance based linear redundancy analysis (dbRDA), but the low amount of variation explained by each axis (<4% in all cases) indicates that overall, the environmental parameters used in the analysis had little statistical influence on the dinoflagellate assemblages. Therefore, other analyses need to be done to explain variation in the species assemblages.

Table 2. AIC results based on the parameters from the DistLM.

PARAMETERS AIC

Waves 669.61

Temperature 669.14

PAR 668.8

Salinity 669.41

- NO 3 670.95

Ortho-P 672.83

- NO 2 674.81

+ NH 4 677.02

Figure 5. The DistLM and distance based linear redundancy analysis (dbRDA) of the top four physical parameters versus samples (distinguished as ocean and bay sites).

Figure 6. The DistLM and distance based linear redundancy analysis (dbRDA) of the top environmental parameters overall versus samples (distinguished as ocean and bay sites).

Figure 7. The DistLM and distance based linear redundancy analysis (dbRDA) of the top four physical parameters versus samples (distinguished by season).

Figure 8. The DistLM and distance based linear redundancy analysis (dbRDA) of the top environmental parameters overall versus samples (distinguished by season).

Figure 9. The DistLM and distance based linear redundancy analysis (dbRDA) of the top four physical parameters versus samples (distinguished by site).

Figure 10. The DistLM and distance based linear redundancy analysis (dbRDA) of the top environmental parameters overall versus samples (distinguished by site).

Relationships: Site, Season, and Location

BEST analysis was performed on the dinoflagellate abundance data to determine the best possible environmental parameter combinations that correspond with site, season, and location variability. None of the relationships, however, were statistically significant, based on the Rho values (all >0.05). The BEST analysis alone, therefore, does not explain the differences in dinoflagellate assemblages.

Correlations Between Environmental Parameters and Cell Densities

Using the transformed cell density data and the normalized environmental data in the SPSS statistics program, bivariate correlation was performed. The correlations between genera and abiotic factors are displayed in Table 5. The highlighted cells are the significant correlations, at either 0.05 (*) or 0.01 (**). The only correlation between genera and abiotic factors was Ostreopsis species and PAR at a 0.05 significance level. Interestingly, the three species assemblages all positively correlated with one another at a 0.01 significance level.

Table 3. Correlations between abiotic factors, cell densities, and chlorophyll. The highlighted cells represent the significant values; 0.05 (*) or 0.01 (**). P (cells/gram) O (cells/gram) G (cells/gram) Chl

Waves -0.153 -0.102 0.050 -0.079

Temperature 0.126 0.146 -0.025 0.153

PAR 0.134 .205 * -0.043 0.075

Salinity -0.052 -0.026 -0.148 -0.011

NO3 0.156 0.072 0.024 0.024

Ortho-P 0.024 0.038 -0.069 -0.022

NO2 0.096 0.068 0.025 0.031

NH4 0.137 0.087 -0.101 0.150

P (cells/gram) 1 .808 ** .284 ** 0.141

O (cells/gram) .808 ** 1 .284 ** 0.056

G (cells/gram) .284 ** .284 ** 1 -0.059

Chl 0.141 0.056 -0.059 1

Niche Optima and Tolerance

The optima and tolerances of the niche that the three dinoflagellate genera thrive in were evaluated based in the C2 statistics program. Figures 10-17 depict the optimum (bars) and the tolerance (error bars) that each genus may occupy as a niche. The optimum wave heights for Prorocentrum and Ostreopsis were similar while Gambierdiscus had a higher optimum wave activity (Figure 11). The optimum temperature and salinity were similar between all three genera, about 23°C. The tolerances showed the temperature and salinity ranges were constant among the three dinoflagellates (Figures 12 & 13). PAR was similar amongst Prorocentrum and Ostreopsis , about 39 to 40 µM/m 2/sec, while Gambierdiscus

had a lower optimum, at about 33 µM/m 2/sec. The tolerance for Prorocentrum and

Ostreopsis were a range for 22 to 55 µM/m 2/sec (Figure 14). The optimum level of nitrate was similar amongst all three genera at about 0.5 µM, although tolerance levels were wide ranging (Figure 15). The optimum level of nitrite was similar for Prorocentrum and

Ostreopsis at about 0.03 µM , and lowest for Gambierdiscus at about 0.01 µM. The tolerance for Prorocentrum and Ostreopsis was about -0.02 to 0.08 µM, while

Gambierdiscus’ tolerance was about -0.03 to 0.07 (Figure 16). The optimum level of ortho- phosphate was similar between Prorocentrum and Ostreopsis , about 0.07 µM, while the

Gambierdiscus optimum was higher, about 0.09 µM. The tolerances for Ostreopsis and

Prorocentrum were in a range of 0.01 to 0.13 µM and Gambierdiscus was in a range of

0.03 to 0.15 µM (Figure 17). The optimum level of ammonium was highest for

Prorocentrum at 1.5 µM, with a tolerance range of 0.75 to 2.25 µM , lower for Ostreopsis, and lowest for Gambierdiscus . The lowest level of ammonium mimicked the optimum with

Gambierdiscus with the lowest tolerance level (Figure 18).

1.4

1.2

0.8

0.6

Average Average Waves 0.4

0.2

0 P cells/g O cells/g G cells/g Species

Figure 11. Optimum and highest tolerance level of wave activity that the three dinoflagellate genera experience.

10 Temperature Temperature (C) 5

0 P cells/g O cells/g G cells/g Species

Figure 12. Optimum and lowest tolerance level of temperature for the three dinoflagellate genera.

40 39 38 37 36 35 Salinity (ppt) 34 33 32 P cells/g O cells/g G cells/g Species

Figure 13. Optimum and lowest tolerance of salinity for the three dinoflagellate genera.

40 /sec) 2 30

20 PAR (µM/m 10

0 P cells/g O cells/g G cells/g Species

Figure 14. Optimum and lowest tolerance of PAR for the three dinoflagellate genera.

1.5

0.5

Nitrite (uM) 0 P cells/g O cells/g G cells/g -0.5

-1 Species

Figure 15. Optimum and highest tolerance of nitrate for the three dinoflagellate genera.

0.1

0.08

0.06

0.04

0.02 Nitrate (uM) 0 P cells/g O cells/g G cells/g -0.02

-0.04 Species

Figure 16. Shows the optimum and highest tolerance of nitrite for the three dinoflagellate species.

2.5

1.5

1 Ammonium (uM)

0.5

0 P cells/g O cells/g G cells/g Species

Figure 17. Optimum and lowest tolerance of ammonium for the three dinoflagellate genera.

0.16

0.14

0.12

0.1

0.08

0.06

Ortho-phosphate (uM) 0.04

0.02

0 P cells/g O cells/g G cells/g Species

Figure 18. Optimum and lowest tolerance of ortho-phosphate for the three dinoflagellate genera.

Site Relationships

Cell densities per site were similar amongst the three dinoflagellates, although

Gambierdiscus was as abundant as the other two genera at the bay sites and background offshore. There appears to be significant spikes in abundances in the three years of abundance data (Figures 19-22). The spikes that occurred in 2013 were: HGB in February,

TRL in September where Gambierdiscus was the lowest, and LKH in October where

Ostreopsis was the highest. The spikes that occurred in 2014 were in January for both

LKH and TRL where Gambierdiscus was the lowest. The spikes that occurred in 2015 were: HGB in August and October where Gambierdiscus remained very low, LKH in

October where Gambierdiscus remained very low, and TRL in March where

Gambierdiscus was highest, and in August Ostreopsis was highest and Gambierdiscus was very low.

LKH Abundances of Benthic Dinoflagellates per Month 800 700 600 500 400 300

Cells/gram 200 100 0 Jul-13 Jul-14 Jul-15 Jan-13 Jan-14 Jan-15 Sep-13 Sep-14 Sep-15 Nov-13 Nov-14 Nov-15 Mar-13 Mar-14 Mar-15 May-13 May-14 May-15 Date

P cells/g O cells/g G cells/g

Figure 19. LKH abundances per month from 2013 through 2015.

TRL Abundances of Benthic Dinoflagellates per Month 1200 1000 800 600 400 Cells/gram 200 0 Jul-13 Jul-14 Jul-15 Jan-13 Jan-14 Jan-15 Sep-13 Sep-14 Sep-15 Nov-13 Nov-14 Nov-15 Mar-13 Mar-14 Mar-15 May-13 May-14 May-15 Date

P cells/g O cells/g G cells/g

Figure 20. TRL abundances per month from 2013 through 2015.

HGB Abundances of Benthic Dinoflagellates per Month 2500

2000

1500

1000 Cells/gram

500

0 Jul-13 Jul-14 Jul-15 Jan-13 Jan-14 Jan-15 Sep-13 Sep-14 Sep-15 Nov-13 Nov-14 Nov-15 Mar-13 Mar-14 Mar-15 May-13 May-14 May-15 Date

P cells/g O cells/g G cells/g

Figure 21. HGB abundances per month from 2013 through 2015.

TPH Abundances of Benthic Dinoflagellates per Month 2500 2000 1500 1000

Cells/gram 500 0 Jul-13 Jul-14 Jul-15 Jan-14 Jan-15 Jan-13 Sep-13 Sep-14 Sep-15 Nov-13 Nov-14 Nov-15 Mar-14 Mar-15 Mar-13 May-14 May-15 May-13 Date

P cells/g O cells/g G cells/g

Figure 22. TPH abundances per month from 2013 through 2015.

Season and Location Relationships

The abundances were affected by seasons: winter was December through February, spring was March through May, summer was June through August, and fall was September through November (Figure 23). Prorocentrum was most abundant across all seasons. The abundances were affected by the locations, bay and ocean (Figure 24). The three genera were more abundant at the bay sites versus the ocean sites.

Abundance affected by Seasons 900 800 700 600 500 400 Cells/g 300 200 100 0 Winter Spring Summer Fall Season

P O G

Figure 23. The abundances of the three species affected by season. Prorocentrum was the most abundant species during all of the seasons.

Location affecting the Abundance 1600

1400

1200

1000

800

600 Cells/g 400

200

0 Bay Ocean -200 Location

P O G

Figure 24. The abundances affected by location; bay and ocean.

Chapter 5: Discussion

5.1 Dinoflagellate Species Individually: Autecology

The C2 analysis, which displayed the optima and tolerances of the dinoflagellate genera with the environmental parameters that were present in their habitat, showed that there was a slight difference in optimums and tolerances. The niche all three genera prefer would have higher temperatures (around 23°C), but they differ in preferences for other environmental parameters (Table 4; Bomber 1985). There were multiple spikes in abundances but no specific pattern, while all three genera abundances are more abundant in the bay than the ocean (Figures 19-22).

The seasonal differences show that there were no specific patterns, Gambierdiscus was more abundant in the winter while Prorocentrum was more abundant in spring, summer, and fall (Figure 23). Gambierdiscus appears to cohabit closely with other dinoflagellates, such as Prorocentrum and Ostreopsis , all requiring similar levels of the available nutrients, light, and temperature, although slightly different because of basic necessities of each genera. The dinoflagellate assemblages in the samples appeared to be more influenced by lower light, temperature, and salinity levels as well as higher wave action, although there was little separation by location, season, or site in this regard

(Figures 5, 7, & 9). These conditions are typical of winter months and may indicate that the dinoflagellates are responding more to changing environmental conditions in the winter versus summer.

Table 4. The lows, mediums, and highs of environmental parameters based on the genera. Tem Salinity + Waves PAR Nitrate Nitrite Ortho-P NH 4 p (C) (ppt)

Gambierdiscus High High High Low Low Low High Low

Ostreopsis Medium High Medium High Low High Medium Medium

Prorocentrum Medium High Medium Medium Medium High Medium High

5.2 Dinoflagellate Species Assemblages: Synecology

The SIMPROF did not show any indication of significant groupings while the

DistLM suggests there are negative relationships. There was no indication that there were influences by sites, seasons, and locations. The results of the dbRDA were less than 4% which indicated that the environmental parameters could explain little of the assemblage variability (Figures 5-8). There was some evidence of environmental influence, however.

The dinoflagellate assemblages appeared to be more influenced by lower light, temperature, and salinity coupled with higher wave action – conditions typical of winter months – which may indicate that the dinoflagellates are responding more to changing environmental conditions in the winter versus summer. Morton et al. (1992) also noted that benthic dinoflagellate assemblages tended in warmer versus cooler temperature conditions.

The dinoflagellate densities were highest at the bay sites while the ocean site densities remained low for all three genera (Figure 24). This observation may reflect the lower water motion at the bay sites, in a similar manner to Richlen and Lobel (2011) who found that dinoflagellate abundance was negatively correlated with water motion. The only significant relationship between dinoflagellate genera and environmental parameters was the positive correlation between Ostreopsis and PAR. In high light intensity environments, Ostreopsis are known to produce a mucus and settle (Parsons et al. 2017).

Interestingly, the three dinoflagellate genera were positively correlated with one another, indicating that they all appear to be responding similarly to environmental dynamics.

Parsons & Preskitt (2007) experienced similar abundances between multiple dinoflagellate

species but on different substrates and environmental parameters, concluding that substrates and environmental parameters could be species specific.

5.3 Discussion

In conclusion, the three dinoflagellate genera coexist but there are differences in abundances both seasonally and between locations. Relationships are not easily explained by the environmental data (DistLM, BEST, and correlations). There could be some evidence of subtle niche partitioning when looking at the optima data from the C2 analysis, or the environmental factors that are driving the partitioning were missed when sampling.

It could be that the true driver parameter was not measured, like pH or trace metals, or that conditions were a function of rapid change (minutes to hours) that we did not pick up during the routine monthly sampling. Other factors that could be important in studying would be grazing on the host algae or metals (Bomber et al. 1989). Additionally, the spatial distribution of dinoflagellates may be a factor; i.e., they spread out on the thallus or be right next to one another, two scenarios that would not have been accounted for using the shaking and sieving method employed in this study. Lastly, the analyses done here were genera- level; species-specific variation was not examined and the apparent subtleties in environmental preferences may be at this lower taxonomic scale.

5.4 Significance and Future Implications

The research done on this project can influence the future public knowledge of

Ciguatera Fish Poisoning (CFP) and therefore reduce outbreaks. To better understand how

CFP initiates, the community dynamics of Gambierdiscus needs to be studied. These cells

cohabitate with many organisms which can affect their abundance, which in turn may influence toxin loading on the reef and subsequent outbreaks of CFP. In the future, this research will give way to a better understanding of how Gambierdiscus species survive, how other species compete for the available resources, and how abiotic factors influence

Gambierdiscus densities.

Future research could be done to obtain a better understanding of the individual

Gambierdiscus species and the community in which they live. There is little known about the other host algae community dynamics and how other dinoflagellates affect the abundance of Gambierdiscus , as well as the extreme conditions which could influence abundance. Another experiment that could examine the age of the macroalgae and how that effects the dinoflagellate community that lives upon it. Studying the extent of the macroalgal community and how species are cohabitating upon them would give answers of why the species are not showing signs of competition and instead are coexisting. An experiment on the thallisphere could be done to examine the environment surrounding the macroalgal species and from these results, a growth curve for the environmental parameters could be done. More studies should be done on the grazing pressures of the algal species that are present in these sites, based on the Coral Point Count (CPC) data that identified the different macroalgal species and surfaces that dinoflagellates can settle on. My concluding questions, based on my research are: were they present after grazing or before the algae was grazed upon? If this is before the algae was grazed upon, could that mean that the grazers will consume all of the cells present on the macroalgae? If this was after grazing, are the fish selecting the areas that are not covered in epiphytic algae? This could

influence the abundance of the species and the toxicity of fish species in the sites being studied.

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