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ECOLOGY AND DISTRIBUTION OF THE FLORIDA SEA CUCUMBER, HOLOTHURIA FLORIDANA, IN SEAGRASS AND HARD-BOTTOM COMMUNITIES OF THE FLORIDA KEYS

NATHAN PATRICK BERKEBILE

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2017

To everyone in my family, especially my mother and father

ACKNOWLEDGMENTS

I thank my mother and father for creating a hospitable and productive environment during my childhood, which directly resulted in the person I am today. They have supported me throughout my life and have continued doing so throughout my duration, here at University of

Florida. I want to acknowledge Dr. Donald Behringer for being my advisor and all his help guiding me through my project. He has provided me with a wealth of knowledge on experimental design and experimentation. I would also like to acknowledge my committee members; Dr. Shirley Baker and Dr. J. Antonio Baeza, for helping me strengthen and centralize my project and providing me with intellectual input. I am also grateful for Karen Bray and

Cynthia Hight, who provided me with important deadlines throughout my time here. Without their help, in addition to Dr. Behringer’s, my plan of study would not have been kept up to date and I would not have taken the most influential classes that I did. I would also like to thank Dr.

Ed Phlips, Leslie Landauer, and Dr. Patrick Baker for their involvement in processing my chlorophyll a and grain size sediment samples. I could not have asked for better mentors for understanding the methodology. Furthermore, in analyzing and interpreting my data, I want to give an incredible thanks to James Colee, as he explained statistical tests to me in the most basic of terms, allowing me to easily manipulate my data. Finally, I would like to acknowledge every friend and family member that has helped me conduct field survey in the Florida Keys, as they were the backbone to my project. Without all of you, conducting each study would have been nightmarish.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 7

LIST OF FIGURES ...... 8

LIST OF ABBREVIATIONS ...... 10

ABSTRACT ...... 11

CHAPTER

1 INTRODUCTION ...... 13

Sea Cucumbers and their Ecological and Distributional Patterns ...... 13 Species Assessment ...... 13 Effects of Holothurian Foraging on Sediment Characteristics ...... 14 Environmental Variables that Correlate with Holothurian Abundance and Distribution ...... 15 Marine Ornamental Fisheries ...... 17

2 TROPHIC IMPORTANCE OF HOLOTHURIA FLORIDANA IN HARD-BOTTOM AND SEAGRASS HABITATS ...... 20

Trophic Importance of Holothuria floridana ...... 20 Methods ...... 21 Objective 1: Ossicle and Reproductive Comparison between Hard-bottom and Seagrass Morphologies of Holothuria floridana ...... 21 Objective 2: Relative Predation on H. floridana ...... 22 Objective 3: Effect of H. floridana Foraging on Sediment Characteristics ...... 23 Total organic content ...... 25 Chlorophyll a and pheopigments ...... 26 Grain size distribution ...... 27 Results...... 28 Objective 1: Ossicle and Reproductive Comparison between Hard-bottom and Seagrass Morphologies of Holothuria floridana ...... 28 Objective 2: Relative Predation on H. floridana ...... 28 Objective 3: Effect of H. floridana Foraging on Sediment Characteristics ...... 29 Discussion ...... 41 Relative Predation on H. floridana ...... 41 Effect of H. floridana Foraging on Sediment Characteristics ...... 42 Trophic Importance ...... 43

3 ENVIRONMENTAL CORRELATES WITH THE DISTRIBUTION AND ABUNDANCE OF HOLOTHURIA FLORIDANA ...... 45

Distribution of Holothuria floridana ...... 45 Methods ...... 47 Analysis ...... 48 Results...... 48 Discussion ...... 57

4 CONCLUSIONS ...... 60

APPENDIX: SUPPLEMENTARY MATERIALS...... 62

LIST OF REFERENCES ...... 66

BIOGRAPHICAL SKETCH ...... 74

LIST OF TABLES

Table page

2-1 Results of a repeated measures MANCOVA analysis summarize the effect of H. floridana on the chlorophyll a and total organic content within the sediment. Grain size distribution was used as a covariate. Significant values (α < 0.05) are in bold...... 30

3-1 Results of the full multiple linear regression model of environmental variables that correlate with H. floridana distribution. Contribution strength of each variable is denoted in standardized coefficients beta column, which determines if the respective ....49

LIST OF FIGURES

Figure page

1-1 The study area in the Florida Keys is interspersed with seagrass and hard-bottom habitats throughout the Florida Bay and westerly towards the GOM. Black ovals denote all study sites per each objective. An imaginary line of demarcation is seen s. ....19

2-1 Hard-bottom and seagrass sites where holothurians were tethered in...... 32

2-2 The percent mortality of larger and smaller H. floridana was captured within and between seagrass and hard-bottom habitats, in the lower and middle Keys...... 33

2-3 The mean total organic content (%) at Sawyer Key and Channel Key, before and after the 1-month trial...... 34

2-4 The mean total organic content (%) sampled at Sawyer Key, before and after the 1- month trial...... 35

2-5 The mean total organic content (%) sampled at Channel Key, before and after the 1- month trial...... 35

2-6 The mean change in chlorophyll a (ug/mL) at Sawyer Key and Channel Key, before and after the 1-month trial. Error bars represent ± 1 SE...... 36

2-7 The mean chlorophyll a (ug/mL) at Sawyer Key, before and after the 1-month trial...... 37

2-8 The mean chlorophyll a (ug/mL) at Channel Key, before and after the 1-month trial...... 37

2-9 Grain size of the sediment at Sawyer Key...... 38

2-10 Grain size of the sediment at Channel Key...... 39

2-11 Percent change in each grain size category at Sawyer Key over 1-month trial...... 40

2-12 Percent change in each grain size category at Channel Key over 1-month trial...... 40

3-1 Survey sites in the...... 50

3-2 The total number of H. floridana counted at each site within the lower and middle Florida Keys...... 51

3-3 The mean number of H. floridana counted along 50m x 1 m belt transect at sites in the lower and middle Keys...... 52

3-4 The mean sediment depth (m) at lower and middle Keys survey sites...... 52

3-5 The mean chlorophyll a (ug/mL) in lower and middle Keys survey sites ...... 53

3-6 The percent cover of Thalassia testudinum in the ...... 54

3-7 The percent cover of total macroalgae in the ...... 55

3-8 The mean total organic content (%) at lower and middle Keys survey sites...... 56

3-9 The weighted mean of grain size distribution (mm) at the lower and middle Keys survey sites...... 56

A-1 A portion of the body wall from H. floridana was obtained for extraction of ossicles .....62

A-2 A dissection of H. floridana showing reproductively mature gonads. The dorsal body wall was cut, posterior to anterior, to keep mesentery in tact...... 63

A-3 Enclosure used for foraging study. Foam rubber was used to prevent holothurians from escaping or entering, while allowing light penetration...... 64

A-4 Gut of a necropsied Loggerhead sea turtle Caretta caretta, which appears to contain many H. floridana...... 64

A-5 Light microscopy images of the ossicles extracted from the body wall of H. floridana ...65

LIST OF ABBREVIATIONS

CaCO3 calcium carbonate

CHLA chlorophyll a

CO2 carbon dioxide

COI cytochrome oxidase I

DNA deoxyribonucleic acid

ENP Everglades National Park

EtOH ethanol

FAO Food and Agricultural Organization

FWC Florida Fish and Wildlife Conservation Commission

GOM Gulf of Mexico

GPS global positioning system

HB hard-bottom

HCl hydrochloric acid

MANCOVA Multivariate Analysis of Covariance

NOAA National Oceanic and Atmospheric Administration

PCA Principle Component Analysis pH potential of hydrogen

PVC polyvinyl chloride

SG seagrass

TOC total organic content

UF University of Florida

USA United States of America

Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

ECOLOGY AND DISTRIBUTION OF THE FLORIDA SEA CUCUMBER, HOLOTHURIA FLORIDANA, IN SEAGRASS AND HARD-BOTTOM COMMUNITIES OF THE FLORIDA KEYS

Nathan Patrick Berkebile

August 2017

Chair: Donald Behringer Major: Interdisciplinary Ecology

Holothuria floridana is found throughout the Florida Keys, USA with different distributional patterns occurring between large and small phenotypes. H. floridana may be fulfilling a specific niche as a prey item, where predation pressure may have led to adaptations in defensive mechanisms, such as cryptic behavioral tendencies and unpalatabilty. Their abundances are high in seagrass beds, where they can significantly reduce benthic microalgae.

Environmental variables and sediment characteristics also correlate with their distribution and abundances in these areas.

To understand their morphological adaptations, ossicles and reproductive organs were compared. Large and small H. floridana of the lower and middle Keys appear to be the same species. A tethering study was conducted to interpret their relative susceptibility to predation.

Predation occurred most often in the lower Keys, in hard-bottom habitats, and on small H. floridana. To assess foraging impacts on sediment characteristics, an enclosure study was conducted in which H. floridana densities were manipulated. Addition of H. floridana reduced sediment chlorophyll a concentrations, but not total organic content. Vegetative cover- abundance, sediment depth, canopy height and sediment characteristics were measured to

understand correlates with H. floridana distribution. Sediment depth, percent cover-abundances of Thalassia testudinum, total macroalgae and sediment chlorophyll a were significant drivers of their distribution.

In sum, this knowledge will help build a foundation for conservation and informed management. Furthermore, potential food sources for higher trophic predators, such as sea turtles, and seagrass health, which has already been declining in the Florida Bay, will be negatively impacted with overharvesting.

CHAPTER 1 INTRODUCTION

Sea Cucumbers and their Ecological and Distributional Patterns

Species Assessment

Sea cucumbers are pentaradial organisms within the phylum Echinodermata and class

Holothuroidea, characterized by an endoskeleton of calcareous ossicles and a large coelome

(Conand 2004). Each species has specialized shapes and sizes to their ossicles, which are used in taxonomic determination at the species-level and to identify juveniles with phenotypic traits that differ from their adult counterparts (Massin 2000; Michonneau et al. 2015). Previous assessments have relied on ossicle examination as the primary means of species verification and differentiation. However, DNA (e.g. barcoding of cytochrome oxidase I (COI)) sequences have shown that intra-specific variation may warrant a need for taxonomic revisions (Michonneau et al. 2015). Regardless, many species within the Indo-Pacific show different color patterns yet have little to no change in their COI sequences (Michonneau et al. 2015). Furthermore, holothurians show morphological adaptations and live in a variety of habitats including tropical intertidal zones, hard-bottom, seagrass beds, and deep-sea bathyal continental slopes (Hamel and

Mercier 2008; Toral-Grande 2008; Toral-Grande et al. 2008; Michonneau 2015; Barry et al.

2016). Thus, ossicle and genetic examination are critical in determining species relationships.

Few studies have focused on trophic position in holothurians. Although, predator-prey relationships were examined in literature review by Francour (1997) which documented 76 predators on all holothurians including 26 species of fish, 19 species of sea stars, and 17 species of crustaceans as the most important. Many studies have set out to determine predatory species, but a few have shown that it is difficult to discern if the predation events are scavenging, or induced through starvation (Bourjon and Vasquez 2016). Seastars represent the main predators

of holothurians, followed by several fish species. In response to predation, holothurians have adapted defensive mechanisms which include toxicity (Bakus 1974; Bingham and Braithwait

1986; Castillo 2006; Van Dyck et al. 2011), unpalatability (De Vore and Brodie 1982; Bingham and Braithwait 1986; Castillo 2006), evisceration of Cuvierian tubules (Hamel and Mercier

2000; Becker and Flammang 2010; Demeuldre et al. 2014), nocturnal movement behavior

(Bakus 1968), and cryptic behavior (Bakus 1974; Shiell and Knott 2008; So et al. 2010). Juvenile holothurians have also been documented to remain cryptic in their behavior, possibly as a predator avoidance mechanism (Slater et al. 2010). A study by So et al. (2010) documented adaptive responses to predation pressure in Cucumaria frondosa, where fewer small individuals were found on bare sandy habitats, as opposed to hard rocky habitats, where dense populations of larger individuals occurred. This high predation pressure, plus high fishing pressure may have resulted in a behavioral split in the species. A similar combination of predation and fishing pressure is seen in Parastichopus californicus, which are fished commercially and preyed upon by the sea otter, Enhydra lutris. Declines of 100% were seen in P. californicus as a result of otter predation (Larson et al. 2013). These studies show the effect predators can have on distributional patterns and behavioral adaptations. However, holothurians also have impacts when they are feeding.

Effects of Holothurian Foraging on Sediment Characteristics

Holothuria atra and Stichopus chloronotus, both commercially important tropical holothurians, forage on sediment in the Great Barrier Reef of Australia (Uthicke 1999). Their bioturbation disturbs the sediment, which promotes aeration and stratification of the interstitial dissolved organic matter (Massin 1982; Uthicke 1999). As the sediment passes through the gut passage nutrients are regenerated. Ultimately, H. atra and S. chloronotus were found to excrete ammonium and phosphorous, which are important in promoting high productivity in coral reef

ecosystems (Uthicke 2001b). Alternatively, H. atra and S. chloronotus, as well as other holothurians, have been noted to feed on the microalgae and organic matter of dead plant and animal origin, or may be omnivorous (Massin 1982; Uthicke 2001b). Moriarty et al. (1985) found that bacterial and microalgal production was decreased through holothurian feeding.

Similarly, a study by Michio et al. (2003) provided evidence of algal inhibition and a decrease of organic matter within the sediment from S. japonicus feeding. Alternatively, an increase in total alkalinity can occur as the holothurian digests the organic matter within the sediment, which ultimately dissolves CaCO3 and can buffer changes in seawater pH (Schneider et al. 2011).

Similar feeding strategies have been observed in holothurians inhabiting seagrass beds

(Wolkenhauer et al. 2010).

Tropical holothurians use seagrass beds for foraging and can provide direct nutrient release to neighboring communities as they feed on bacteria, microalgae, and organic detritus

(Moriarty et al. 1985; Wiedemeyer 1992; Uthicke 2001b; Grall and Chauvaud 2002;

Wolkenhauer et al. 2010). One such tropical holothurian, H. scabra, relies heavily on seagrass beds for foraging (Wolkenhauer et al. 2010). When H. scabra were excluded from treatment cages, seagrass growth was negatively impacted due to an increase in the organic matter and benthic microalgal biomass within the sediment. Algae is superior in nutrient uptake to seagrass and can pose a threat by intercepting light and blocking diffusion of carbon and oxygen through the seagrass blades (Sand-Jensen 1977; Sand-Jensen et al. 1985; Duarte 1995; Valiela et al.

1997; Hughes et al. 2004; Ralph et al. 2007). Microalgal biomass can be reduced by C. frondosa, as was shown when algal supplements were introduced in an experimental diet (So et al. 2010).

Environmental Variables that Correlate with Holothurian Abundance and Distribution

Holothurians are important in multiple habitats, but it is critical to understand what factors influence their population abundance and distribution. Benthic sediment characteristics

and vegetative cover have been recognized as essential to the distributional patterns of holothurians (Slater and Jeffs 2010; Slater et al. 2010; Dissanayake and Stefansson 2012).

Holothurians in Banco Chinchorro, Mexico prefer waters with low flow and a high organic sediment accumulation, and were consistently found in seagrass habitats (Fuente-Betancourt et al. 2001). A similar pattern is documented in H. atra, where they inhabit low energy seagrass habitats and are distributed relative to mean grain size, organic content (%), gravel, and depth.

Alternatively, H. edulis prefer rocky habitats interspersed with algae and seagrass, where organic content (%) was not significant in their distribution (Dissanayake and Stefansson 2012). These two commercially valuable holothurians are within the same genus and play similar roles in nutrient recycling, but are found in different habitats where the organic content varies. These distributional patterns have also been documented in different lifestages within a single species.

Juvenile Australostichopus mollis are found in extreme abundances within sediments characterized as having high nitrogen content, a high pheopigment:chlorophyll a ratio and small grain size, as opposed to their adult counterpart that inhabit sites with higher chlorophyll a content, coarser grain sizes and lower total organic content (Slater et al. 2010; Slater and Jeffs

2010).

Holothurians are found throughout Florida, USA, but there is little information on their ecological or distributional patterns. Florida is a peninsula, with the most southern tip consisting of a tropical chain of islands, the Florida Keys. This chain of islands is surrounded by the

Atlantic Ocean on the southern side, and the Gulf of Mexico and Florida Bay on the northern side (Figure 1-1). The upper and middle Keys are comprised of Key Largo limestone, whereas the lower Keys are separated with narrow channels and finger banks, characterized by oolitic

Miami limestone (Hoffmeister 1974).

The north of the Florida Keys consists of a mix of hard-bottom and seagrass habitats

(Figure 1-1). The hard-bottom habitats are comprised of sponges, octocorals and scleractinian corals with red, green and brown macroalgae interspersed within, housing organisms such as spiny lobsters, stone crabs, turban snails and holothurians (Behringer and Butler 2006).

Alternatively, the seagrass habitats are characterized by deep sediments and rooted vascular plants such as Thalassia testudinum, Halodule wrightii, and Syringodium filiforme (Zieman et al.

1989; Zieman et al. 1999; Behringer and Butler 2006). Multiple species of holothurians span the east and west coasts of Florida, but here I focus on Holothuria floridana, which is found within seagrass and hard-bottom habitats, in the middle and lower Florida Keys.

Marine Ornamental Fisheries

The global trade of marine ornamental species, including reef fish and invertebrates, has been documented since the 1930’s and has only been expanding in value and demand (Rhyne et al. 2009). Currently, more than 45 countries supply reef fish and invertebrates for the aquarium trade, which includes over 1400 species globally (Wood 2001; Rhyne et al. 2009). A shift to miniature reef ecosystem tanks has increased the need for multiple niche-satisfying invertebrates such as shrimp, crabs, starfish, snails and holothurians. It has been estimated that about 600,000 households in the USA alone have marine aquaria and about 1.5-2 m households, worldwide

(Wabnitz et al. 2003). Invertebrates are not only suffering an increase in demand in the ornamental aquaria trade, but many are simultaneously fished for food, as are holothurians

(Rhyne et al. 2009).

Holothurians feed upon detritus and algae within home aquaria, but are also highly sought after within the Asian food industry where the dried body wall is a high protein delicacy, know as bêche-de-mer (Conand 2004; Wolkenhauer et al. 2010). Malaysian processing plants also commercially produce gamat oil from sea cucumber by-products, thought to be a home

remedy for ailments (Baine and Choo Poh Sze 1999; Conand 2004). Increasing market demands, paired with unregulated fisheries, increases the possibility of fishery collapse (Purcell 2010). An increase in exploited holothurian species has persistently occurred, where 40 species were under harvest in 2006 (Conand 2006), 47 species two years later (Toral-Granda et al. 2008), and finally

66 species four years later (Purcell et al. 2012).

The Western Central Pacific has been experiencing high rates of holothurian overexploitation in boom-and-bust cycles since the 1980’s, but export prices and demand from

Asian countries have steadily increased (Kinch et al. 2008). Recently, overharvesting has reached the Caribbean where not much is known about the biology or ecology of the species in the trade (Toral-Grande 2008). In some countries, economically important fisheries such as the queen conch Strombus gigas and the spiny lobster Panulirus argus, have been overexploited, pushing interest toward holothurians as an economic resource (Fuente-Betancourt et al. 2001). In

Florida, landings quadrupled from 2011 to 2014 (FWC 2014). In response, the Florida Fish and

Wildlife Conservation Commission set a commercial harvest limit of 200 holothurians per day and now require identification of landed species. This multi-specific holothurian fishery is mainly in the Florida Keys, with few landings in other areas of the state. However, Holothuria floridana is the most abundant and targeted more often than other species. Compared to other holothurians, not much is known about this species, other than a few studies pertaining to their growth and reproductive cycles (Edwards 1908; Edwards 1909; Engstrom 1980). There is a fundamental lack of ecological knowledge about how they affect the areas they forage in, their trophic importance, and what drives their distribution. As sustainable management tends to be a reactionary discipline this knowledge will provide critical proactive information.

Figure 1-1. The study area in the Florida Keys is interspersed with seagrass and hard-bottom habitats throughout the Florida Bay and westerly towards the GOM. Black ovals denote all study sites per each objective. An imaginary line of demarcation is seen between the ovals. East of the line can be categorized as the middle and upper Keys, whereas, west of the line can be categorized as the lower Keys.

CHAPTER 2 TROPHIC IMPORTANCE OF HOLOTHURIA FLORIDANA IN HARD-BOTTOM AND SEAGRASS HABITATS

Trophic Importance of Holothuria floridana

Holothurian fisheries have been overexploited throughout the greater Caribbean.

Holothuria floridana, most often landed species in Florida is fished for both the aquarium trade and food industry (Conand 2004; Rhyne et al. 2009; Wolkenhauer et al. 2010). H. floridana is dispersed throughout the middle and lower Florida Keys, in both seagrass and hard-bottom habitats. Sponges, octocorals, scleractinian corals, and patches of red, green, and brown macroalgae are the sessile fauna overlaying the limestone bedrock, that create the 3-dimensional structure of hard-bottom habitats (Hoffmeister 1974; Behringer and Butler 2006). Small H. floridana have a mottled coloration and show cryptic behavior by hiding diurnally under structure in hard-bottom habitats, whereas large H. floridana lie exposed in seagrass beds diurnally and are found in high abundances. The latter do not show different color morphologies or cryptic behavioral traits. While the former are fished for the aquarium trade, the latter are fished for food. The cryptic hiding behavior of the small morph has been seen in other species of holothurians (Hamel and Mercier 1996; Mercier et al. 1999; Mercier et al. 2000; Hamel et al.

2001; Dance et al. 2003; Slater et al. 2010), however understanding the role predation has is unknown. Predation pressure may drive the morphology and behavior of H. floridana, so it is important to understand their relative susceptibility to predation. Differing responses to predation pressure have been documented in C. frondosa, where the smaller individuals are found in sandy habitats and larger in hard rocky habitats (So et al. 2010).

Seagrass beds and hard-bottom habitats intersperse throughout Florida Bay and westerly towards the GOM; seagrass beds are characterized as having deep sediments and rooted vascular plants that contribute a substantial portion of organic carbon to the trophic web (Behringer and

Butler 2006). Seagrass beds are also crucial in providing a habitat for juvenile fish, a foraging- ground for many large fish and invertebrates, protect coastlines from erosion, and are important in nutrient recycling (Grech et al. 2012). Prior work has shown that H. scabra promotes seagrass growth in subtropical Australia through feeding. They reduce the amount of benthic microalgae and total organic content within the sediment (Wolkenhauer et al. 2010). This is particularly valuable to note as seagrass beds in Florida Bay have been slowly degrading since at least 1987

(Hall et al. 1999; Zieman et al. 1999). H. floridana inhabit similar foraging habitats as H. scabra, where they can reach high abundances in seagrass beds within the lower Keys. Their relative vulnerability as prey is important, but it is also vital to understand the effects of their foraging on seagrass beds.

The objective of this chapter was to understand the trophic importance of H. floridana, and whether the morphologies observed in hard-bottom and seagrass beds are different species.

To answer these questions I address the following objectives.

Methods

Objective 1: Ossicle and Reproductive Comparison between Hard-bottom and Seagrass Morphologies of Holothuria floridana

The ossicles of holothurians are unique to each species (Massin et al. 2000). However,

COI barcoding, a method used to determine intra-specific variation where extraction of ossicles may not, can provide additional insight about species status (Michonneau et al. 2015).

Interestingly, COI sequences from H. floridana did not differ from sequences of H. mexicana, a congener (Michonneau pers. comm.). A similar pattern was found in Holothuria edulis and

Holothuria nigralutea in the Pacific, and extracting the ossicles was necessary to determine differences between the two (Michonneau pers. comm.). To that extent, ossicle determination of

small and large H. floridana, in hard-bottom and seagrass habitats, respectively, must initially be examined and compared to fully understand their ecological impact and distributional patterns.

To determine whether small and large holothurians found in the middle and lower Keys, respectively, were both Holothuria floridana, ossicles were extracted from the body wall of 12 individuals (6 large and 6 small) and examined (Hendler et al. 1995; Massin et al. 2000). A sliver of the body wall was cut using a scalpel and placed on a slide with 10% diluted bleach (Figure

A-1). The sample soaked for approximately 5 min, while the ossicles would expel from the body wall. The slide was then examined using a Leica S8APO stereo Microscope. Rosettes and tables are believed to be species-specific and were to be examined to species status.

To simultaneously determine whether small and large H. floridana may be juveniles and adults, respectively, it was necessary to look at the gonads to determine if they were reproductively mature (Engstrom 1980; Conand 1993). Thicker gonads correspond to ovaries or testes that are ready for spawning. Mature ovaries are pink, orange, or red and testes are a cream color. Thinner gonads indicate an organism that is not ready to spawn, possibly juvenile, and gonads of both male and female appear cream in color (Engstrom 1980). Color, length (cm) and weight (g) measurements were taken and compared to values found in Engstrom (1980).

Dissection methods followed Lambert (1985) from posterior to anterior of the dorsal body wall to reduce engagement with the organs and to leave the dorsal mesentery in tact (Figure A-2).

Objective 2: Relative Predation on H. floridana

To determine the relative rate of predation on H. floridana in the wild, a series of tethering experiments were conducted in a completely crossed design to compare location, habitat, and life stage morphology against survival. Tethering has been shown to be a reliable means of measuring relative, not absolute predation. Larger, more abundant H. floridana inhabit seagrasses in the lower Keys, whereas smaller H. floridana are cryptic, hiding under structures in

hard-bottom areas in the middle Keys. Small H. floridana have a mottled yellow and brown color, whereas large H. floridana are dark blue/black in appearance. Large and small H. floridana were tethered within hard-bottom and seagrass habitats within the lower and middle

Keys to determine if their morphology and behavior may be linked to predation pressure.

H. floridana were tethered with 20 cm of 10 lb test monofilament at 2 m intervals along a

50 m transect tape. Stakes were used in soft sediment seagrass habitats, whereas, bricks were used in hard-bottom habitats. Due to the flexible morphology of H. floridana, it was found that piercing the sea cucumber near the anus, and threading monofilament back through the body wall, is the most secure method. Tethers were strung through H. floridana the night before deployment, which allowed assessment of recovery and survival (Da Silva et al. 1986; Cieciel et al. 2009). Sea cucumbers were given the 20 cm tether to allow movement but not entanglement.

Each sea cucumber was measured volumetrically (mL) and placed out in the afternoon and checked every 24 h. Tethers were checked for 3 d and at each check all predation events or missing holothurians were recorded. A missing holothurian was considered eaten if the tether had been severed or there was tissue remaining on the tether. Each holothurian was tethered near shelter (sponge, coral head, etc.) along the transect line. Half (n=10) of the tethered sea cucumbers were of the larger found in the lower Keys and the other half (n=10) were the smaller found in the middle Keys. This process was repeated at two hard-bottom and two seagrass sites within the lower and middle Keys to ensure that any differences between predator assemblages among sites were captured (Figure 2-1).

Objective 3: Effect of H. floridana Foraging on Sediment Characteristics

To determine how H. floridana movement and foraging affects sediment characteristics, including microalgal content, a series of field enclosure experiments were performed in seagrass habitats. Larger H. floridana were used because they are most commonly found there. Two

seagrass sites, Sawyer Key in the lower Keys, and Channel Key in the middle Keys, were chosen based on their similarity. However, where Sawyer Key is replete with H. floridana, Channel Key is largely devoid of H. floridana.

At each site a 50 m transect tape was laid out and the vegetative cover was measured using the point-intercept method. Ten adjacent pairs of 1.0 m2 enclosures were laid on the benthos at each of the two sites. Each pair had similar vegetative cover and each enclosure was delineated with a 20 cm tall strip of 1 cm vexar mesh that was driven down through the sediment to the bedrock below and staked at the corners using galvanized rebar. The enclosures were open top to allow sunlight to penetrate as normal, and foam rubber was wrapped around the top of the enclosures to prevent holothurians from using their tube feet to crawl over (Figure A-3). All large, macro-invertebrates within each enclosure were carefully removed to minimize disturbance to the sediment. Sediment depth was measured using a meter stick in each plot.

Sediment cores were then taken to a depth of 5 cm from three standard locations within each enclosure. Each core was placed into an individual ziplock bag and iced for later analysis. The cores were used to measure total organic carbon, chlorophyll a and grain size distribution. Six similarly sized H. floridana were then placed in one of each of the paired enclosures. The other enclosure remained devoid of H. floridana to serve as a control. Enclosures were checked weekly for one month to ensure the holothurians remained in or out. Missing holothurians were replaced and the loss recorded. At the end of the 1-month experiment the holothurians were released and the plot was re-cored five times, however the new cores were taken 45 to the right of each initial core to ensure that the same location had not been cored again. The sediment cores were then analyzed for total organic content, chlorophyll a, grain size distribution.

A repeated measures MANCOVA was used to determine if total organic content, chlorophyll a and grain size distribution or their interactions were significantly affected by the sea cucumber additions. The model design was a 2 (two levels of site; Channel Key and Sawyer

Key) X 2 (two levels of TRT; addition and exclusion) factorial repeated measures MANCOVA.

Chlorophyll a (CHLA) and total organic content (TOC) were the multivariate response variables, site and treatment were predictor variables, and grain size distribution was used as a covariate.

The data violated Mauchly’s Test of Sphericity (p-value = 0.0001), so univariate tests were run using the same model.

Total organic content

Sediments contain a large variety of organic materials that can interact with clay particles and bind them, absorbing and releasing nutrients, and holding water in the soil (Schumacher

2002). Many holothurians are documented deposit-feeders, but the trophic position of this is H. floridana is unknown. However, if true, it may influence the total organic matter within the seagrass environment through bioturbation (Terrados et al. 1999) or alteration of nutrients and organic matter composition.

Total organic carbon (TOC) was analyzed gravimetrically following the loss-on-ignition method and test-methods A and C from Schumacher (2002) and ASTM (2000), respectively.

Each sediment sample was placed in an aluminum foil mold and any aggregates separated using a spoon or spatula. The sample was weighed and dried at 105° C for 16 h to correct for moisture content (%). The sample was then re-weighed to determine percent moisture content using Eq. 2-

Initial Sample Weight - Final Sample Weight ( ) * 100 (2-1) Initial Sample Weight

The sample was then transferred to a muffle furnace and combusted, uncovered, at 440°

C until completely ashed (approximately 4 h) (Rosenmeier 2005). It was then re-covered and re- weighed. Organic matter (%) was calculated from Eq. 2-2:

(Ash (g) * 100) Ash Content (%) = (2-2) Oven Dried Sample (g) and subsequently Eq. 2-3:

Organic Matter (%) = 100.00 - Ash Content (%) (2-3)

Chlorophyll a and pheopigments

Benthic algae play an important role in primary production and can bind with nutrients from the sediment (Sin et al. 2009). H. floridana may be attracted to habitats with a greater benthic microalgae content if it constitutes a portion of their diet.

Benthic microalgal biomass was estimated by measuring chlorophyll a per unit sediment following McCarthy et al. (2011). To measure the absorbance of the chlorophyll a pigment using a spectrophotometer, EPA Method 150.1 was used.

This following process proceeded in the dark to minimize degradation of the chlorophyll a pigment (McCarthy et al. 2011). Each sediment sample was dried, weighed, and homogenized.

Two standard subsamples (1 cm X 1 cm depth) were taken from each whole sample and weighed. Each subsample was then placed in a 125 ml flask with a 1:10 ratio of sediment to 90%

EtOH, followed by heating in a water bath at 79.1° C for 5 min. This extracted the chlorophyll a from the sediment and dissolved it in solution. Samples sat in the dark for 24 h to fully extract, after which, 8 mL of solution was pippetted and placed in a microcentrifuge tube and centrifuged at 2,000 RPM for 20 min. 3.2 mL of the supernatant was extracted and placed in a scintillation vial and analyzed using a Hitachi spectrophotometer. The samples were analyzed with and without 0.1 mL 1.66% HCl at 664 nm and 665 nm, respectively, to determine the chlorophyll

a:pheopigment ratio (grazing effect). Grazing organisms can convert chlorophyll a to pheopigments, released as feces, and the higher the ratio of pheopigments against the sum total, the higher the grazing effect (Sin et al. 2009). However, other processes are also involved in degrading the chlorophyll (Takamiya et al. 2000; Tanaka and Tanaka 2006), so the ratio was only used in calculating a corrected chlorophyll a value. Corrected chlorophyll a (ug/mL sediment) was calculated from Eq. 2-4, Eq. 2-5 and Eq. 2-6:

0.04 ug 27931 * ( ) * (Abs Init-Abs Acid) = Chl a ( ⁄ ) (2-4) (1*light path (cm)) L

ug 50 mL ug Chl a ( ⁄ )* ( ) = Chl a ( ⁄ ) (2-5) L 1000 mL mL

ug Subsample Core Weight ug Chl a ( ⁄ ) * ( ) = Corrected Chl a ( ⁄ ) (2-6) mL Total Core Weight mL

Where Abs Init = Absorance of chlorophyll a at 664 nm prior to addition of HCl and,

Abs Acid = Absorbance of pheopigments at 665 nm with addition of HCl.

Grain size distribution

Grain size largely depends on the energy of the local environment. Coarse sediments can be found in high energy environments, whereas finer sediments are found in lower energy environments (Folk 1974). Anecdotally, H. floridana appear to prefer low flow environments where water movement is minimal. This preference has also been documented in other holothurians (Fuente-Betancourt et al 2001; Slater and Jeffs 2010; Dissanayake and Stefansson

2012). Grain size measurement followed similar methods put forth by Folk (1974).

Each sample was weighed, dried for 16 h at 105° C, and re-weighed. The dried sample was placed on a sieve stack (mesh sizes 5, 10, 35, 60, 120, and 230uM) with running water to allow the single grains to fall through. After running through the sieve, the grains were dried again for a minimum of 12 h. The dry weight of single grains that fell through was recorded for

each sieve size. This procedure was continued until the entire sample ran through the sieve stack and could not fall through anymore. Percentage of the grain size distribution in both of the seagrass habitats, Sawyer Key and Channel Key, was calculated with Eq. 2-7:

Corresponding Mesh Size Sample Weight ( ) * 100 (2-7) Total Sample Weight

Subsequently, a weighted average was calculated for each sample using Eq. 2-8:

Mesh Weight ( )+…+k Mesh Size (mm) (2-8) Total Sample Weight (g)

Where K is the respective number of mesh sizes in sample.

Results

Objective 1: Ossicle and Reproductive Comparison between Hard-bottom and Seagrass Morphologies of Holothuria floridana

Ossicles extracted from both small and large H. floridana were of similar size and shape

(Figure A-5). H. floridana has a rosette that is found only within this species, and it was found in all 12 specimens extracted. Dissection of the small and large H. floridana showed that both appeared reproductively mature (Figure A-1). Engstrom (1980) provide minimum size (cm) and weight (g) at sexual maturation, and all H. floridana processed were found to be above each threshold.

Objective 2: Relative Predation on H. floridana

H. floridana suffered higher mortality within the lower Keys than the middle Keys, high mortality within hard-bottom compared to seagrass, and higher mortality among the smaller size animals (Figure 2-2). Log linear analysis revealed no significant interaction with a all variables paired (χ2=2.5784; df=1; p-value=0.10835), but significant interactions between predation events against location, habitat and size, individually (χ2=19.102; df=4; p-value=0.0007).

Objective 3: Effect of H. floridana Foraging on Sediment Characteristics

Total organic content at Sawyer Key decreased with both addition and exclusion of H. floridana, whereas, TOC at Channel Key decreases over time with addition of H. floridana and increases slightly with exclusion of H. floridana (Table 2-1, Figure 2-3, 2-4, 2-5). Change in average chlorophyll a (ug/mL), was plotted against site and sampling time, similar to TOC, where chlorophyll a increased in addition and exclusion of H. floridana at Channel Key, but an opposite change in chlorophyll a occurred at Sawyer Key (Figure 2-6). Chlorophyll a decreased at Sawyer Key (Figure 2-7), with both addition and exclusion of H. floridana, whereas chlorophyll a at Channel Key increased (Figure 2-8). Chlorophyll a increased only slightly over sampling times with addition of H. floridana at Channel Key. Unfortunately, during the weekly check at Sawyer Key, H. floridana had to be removed from control enclosures multiple times. It is not clear how they gained access but this potentially biased chlorophyll a measurements within the control enclosures.

Grain size distribution was significantly different between sites (F=10.792; df=1.000; p- value=0.002), however not significantly different between treatments or time at Sawyer Key

(Figure 2-9) and Channel Key (Figure 2-10), which is why it was entered as a covariate in the analysis. Grain size distribution had minimal change over time at Sawyer Key (Figure 2-11) and

Channel Key (Figure 2-12). However, the gravel portion decreased and fine sand increased over time at Channel Key.

Table 2-1. Results of a repeated measures MANCOVA analysis summarize the effect of H. floridana on the chlorophyll a and total organic content within the sediment. Grain size distribution was used as a covariate. Significant values (α < 0.05) are in bold. Type III Sum Source Measure of Squares df Mean Square F p-value Time CHLA Sphericity Assumed 3.559E-5 1.000 3.559E-5 0.190 0.666 Greenhouse-Geisser 3.559E-5 1.000 3.559E-5 0.190 0.666 Huynh-Feldt 3.559E-5 1.000 3.559E-5 0.190 0.666 Lower-bound 3.559E-5 1.000 3.559E-5 0.190 0.666 TOC Sphericity Assumed 23.049 1.000 23.049 0.207 0.652 Greenhouse-Geisser 23.049 1.000 23.049 0.207 0.652 Huynh-Feldt 23.049 1.000 23.049 0.207 0.652 Lower-bound 23.049 1.000 23.049 0.207 0.652 Time * Size CHLA Sphericity Assumed 0.000 1.000 0.000 0.797 0.378 Greenhouse-Geisser 0.000 1.000 0.000 0.797 0.378 Huynh-Feldt 0.000 1.000 0.000 0.797 0.378 Lower-bound 0.000 1.000 0.000 0.797 0.378 TOC Sphericity Assumed 10.019 1.000 10.019 0.090 0.766 Greenhouse-Geisser 10.019 1.000 10.019 0.090 0.766 Huynh-Feldt 10.019 1.000 10.019 0.090 0.766 Lower-bound 10.019 1.000 10.019 0.090 0.766

Table 2-1. Continued. Type III Sum Source Measure of Squares Df Mean Square F p-value

Time * TRT CHLA Sphericity Assumed 0.001 1.000 0.001 7.233 0.011 Greenhouse-Geisser 0.001 1.000 0.001 7.233 0.011 Huynh-Feldt 0.001 1.000 0.001 7.233 0.011 Lower-bound 0.001 1.000 0.001 7.233 0.011 TOC Sphericity Assumed 7.507 1.000 7.507 0.067 0.797 Greenhouse-Geisser 7.507 1.000 7.507 0.067 0.797 Huynh-Feldt 7.507 1.000 7.507 0.067 0.797 Lower-bound 7.507 1.000 7.507 0.067 0.797 Time * Site CHLA Sphericity Assumed 0.001 1.000 0.001 4.841 0.035 Greenhouse-Geisser 0.001 1.000 0.001 4.841 0.035 Huynh-Feldt 0.001 1.000 0.001 4.841 0.035 Lower-bound 0.001 1.000 0.001 4.841 0.035 TOC Sphericity Assumed 3.788 1.000 3.788 0.034 0.855 Greenhouse-Geisser 3.788 1.000 3.788 0.034 0.855 Huynh-Feldt 3.788 1.000 3.788 0.034 0.855 Lower-bound 3.788 1.000 3.788 0.034 0.855 Time * TRT * Site CHLA Sphericity Assumed 0.000 1.000 0.000 1.600 0.215 Greenhouse-Geisser 0.000 1.000 0.000 1.600 0.215 Huynh-Feldt 0.000 1.000 0.000 1.600 0.215 Lower-bound 0.000 1.000 0.000 1.600 0.215 TOC Sphericity Assumed 21.030 1.000 21.030 0.189 0.667 Greenhouse-Geisser 21.030 1.000 21.030 0.189 0.667 Huynh-Feldt 21.030 1.000 21.030 0.189 0.667 Lower-bound 21.030 1.000 21.030 0.189 0.667

Figure 2-1. Hard-bottom and seagrass sites where holothurians were tethered in A) the middle Keys and B) the lower Keys.

Figure 2-2. The percent mortality of larger and smaller H. floridana was captured within and between seagrass and hard-bottom habitats, in the lower and middle Keys.

Figure 2-3. The mean total organic content (%) at Sawyer Key and Channel Key, before and after the 1-month trial. Treatment = addition of H. floridana; Control = exclusion of H. floridana. Error bars represent ± 1 SE.

Addition/Exclusion of H. floridana

Figure 2-4. The mean total organic content (%) sampled at Sawyer Key, before and after the 1- month trial. Treatment = addition of H. floridana; Control = exclusion of H. floridana. Error bars represent ± 1 SE.

Addition/Exclusion of H. floridana

Figure 2-5. The mean total organic content (%) sampled at Channel Key, before and after the 1- month trial. Treatment = addition of H. floridana; Control = exclusion of H. floridana. Error bars represent ± 1 SE.

Figure 2-6. The mean change in chlorophyll a (ug/mL) at Sawyer Key and Channel Key, before and after the 1-month trial. Error bars represent ± 1 SE.

Addition/Exclusion of H. floridana

Figure 2-7. The mean chlorophyll a (ug/mL) at Sawyer Key, before and after the 1-month trial. Treatment = addition of H. floridana; Control = exclusion of H. floridana. Error bars represent ± 1 SE.

Addition/Exclusion of H. floridana

Figure 2-8. The mean chlorophyll a (ug/mL) at Channel Key, before and after the 1-month trial. Treatment = addition of H. floridana; Control = exclusion of H. floridana. Error bars represent ± 1 SE.

Figure 2-9. A) Initial and B) final grain sizes of the sediment at Sawyer Key. Error bars represent ± 1 SE.

Figure 2-10. A) Initial and B) final grain sizes of the sediment at Channel Key. Error bars represent ± 1 SE.

Figure 2-11. Percent change in each grain size category at Sawyer Key over 1-month trial.

Figure 2-12. Percent change in each grain size category at Channel Key over 1-month trial.

Discussion

Holothuria floridana was found to play an important role in the food web, where predation occurred more frequently in the middle Keys, in hard-bottom habitats, and on the small morphology. Alternatively, H. floridana reduced chlorophyll a in the sediment, but surprisingly had little to no effect on the total organic content.

Relative Predation on H. floridana

Predator assemblages could differ considerably between seagrass and hard-bottom habitats due to different predatory guilds within each. This may have caused the difference in relative predation observed within these habitats. Considering they appear to be the same species the difference in predation may also explain the difference in morphology between small and large H. floridana in the middle and lower Keys. Greater predation in the middle Keys could be due a greater number of predators in this area, which includes the loggerhead sea turtle, Caretta caretta, and the green sea turtle, Chelonia mydas. The middle Keys are adjacent to the

Everglades National Park where sea turtles frequent when nesting (Davis and Whiting 1977;

Hart and Fujisaki 2010) and where a loggerhead sea turtle was found to have a gut full of small

H. floridana following necropsy (Brian Stacy pers. comm., Figure A-4). This could drive the distributional patterns observed in H. floridana where they are cryptic in hard-bottom habitats and, displaying a camouflage color adaptation, hiding diurnally. A similar color adaptation has been documented as an adaptive response to predator avoidance in Australostichopus mollis

(Slater et al. 2010). Large H. floridana may not experience many predators or may have also adapted an unpalatable body wall through production of secondary metabolites, documented in

Holothuria forskali, which secreted more saponins within the body wall and Cuvierian tubules when introduced to predatory fish (Van Dyck et al. 2011).

In preliminary laboratory trials I found that stone crabs fed on small H. floridana, but the stone crab was starved, which may have induced the behavior. Induced feeding was also documented in Francour (1997), where crustaceans began feeding on holothurians through starvation. The stone crab feeding trials documented them feeding on 100% of the smaller H. floridana (n=5) and never the larger (n=5). Alternatively, the defensive mechanisms have developed so well in holothurians that predation on adults is infrequent, whereas predation on early life stages can attribute to the scarcity of the smaller (Francour 1997). The larger size of H. floridana may also be attributed to a greater nutrient intake. This trend was found at high nutrient sites where large A. mollis frequently occur and when small A. mollis were transplanted to these areas their specific growth rates increased (Slater and Jeffs 2010). Alternatively, gamete synthesis of small Cucumaria frondosa found that shorter feeding bursts over a longer period of time used more energetic reserves, resulting in their respective size (So et al. 2010).

Effect of H. floridana Foraging on Sediment Characteristics

The seagrass habitats in which large H. floridana reside in the Florida Keys have experienced decades of decline (Hall et al. 1999; Zieman et al. 1999; Borum et al. 2005).

Foraging by holothurians, such as H. scabra, has been documented to reduce microalgal biomass and the total organic content in the sediment, and, when excluded, seagrass growth declined

(Wolkenhauer et al. 2010). This similar trend is seen in H. floridana where chlorophyll a was reduced at Sawyer Key and markedly so at Channel Key. It is important to note that H. floridana burrowed under the cages and into the control enclosures on numerous occasions at Sawyer Key.

This may explain the lack of increase in chlorophyll a in the treatment enclosures at Sawyer Key.

This reduction in benthic microalgae could enhance seagrass health within these areas by higher oxygenation of the sediment and an increase in sunlight penetration. H. floridana may also be

recycling nitrogen and phosphorous through their defecation which has been found to increase productivity in seagrasses (Wolkenhauer et al. 2010).

Anecdotal observations have long suggested that H. floridana is a detritovore but results of this study suggest they are at least partially herbivorous. Their foraging decreased chlorophyll a but had little effect on total organic content. Experiments involving C. frondosa documented an increase in weight and body size when feeding on algal supplements, which supports selective feeding on benthic microalgae in that species as well (So et al. 2010) However, H. floridana may be omnivorous as opposed to only feeding on microalgae, as found for Stichopus chloronotus and Holothuria atra, both of which are important components in a benthic recycling system

(Massin 1982; Uthick 2001b). Alternatively, it may be that they are detritivorous in their more cryptic, small morphology but this requires future studies on their foraging behavior.

Trophic Importance

This study has shown that H. floridana is important both as a prey item for higher trophic levels, but also as a primary consumer, grazing microalgae. Alternatively, these interactions help in understanding species interactions, which gives a baseline of knowledge to sustainable management of the holothurian fishery in Florida. Overexploitation of holothurian populations can prove to be detrimental to the environment, where a shift in seagrass productivity and a change in nutrient cycling through the food web may occur (Uthicke et al. 2004, Purcell et al.

2014). A decrease in holothurian populations could cause an increase in benthic microalgae, which could create a hypoxic/anoxic sediment boundary layer, where greater anoxic sediment has detrimental impacts on seagrass growth and increased seagrass mortality. The more anoxic the sediment is, the higher the growth inhibition potential and the higher the seagrass mortality

(Terrados et al. 1999). Sulphide invasions have also played a detrimental role in seagrass health, where Thalassia testudinum in the Florida Keys has experienced die off through hypoxic/anoxic

sediment (Borum et al. 2005). Alternatively, with H. floridana having importance as a prey item, overfishing could be detrimental to the energy moving through the trophic food web or animals that specialize on them may be disproportionally affected, such as C. caretta.

CHAPTER 3 ENVIRONMENTAL CORRELATES WITH THE DISTRIBUTION AND ABUNDANCE OF HOLOTHURIA FLORIDANA

Distribution of Holothuria floridana

Environmental variables can determine the distribution and abundance of plants and animals (Jones et al. 2006). Although, it is important to understand the spatial and temporal patterns of these factors, understanding their local patterns can provide a baseline of knowledge.

Localized estuarine studies about macrofaunal distributional patterns have found that faunal biomass, faunal density, species richness and productivity are affected by changing environmental variables such as nutrient loadings, variable anoxic sediment, freshwater flushing and salinity, variable seagrass biomass, and sediment particle size (Howard et al 1989; Diaz and

Rosenberg 1995; Heck et al. 1995; Heip et al. 1995; Ardisson and Bourget 1997; Mannino and

Montagna 1997; Edgar and Barrett 2002). Vegetative cover, in particular, can provide important ecological information on the relationship between flora and associated fauna where coastal heterogeneity and topographical differences in coastal ecosystems can influence diversity and distribution of organisms (Wikum and Shanholtzer 1978; Blanchard and Bourget 1999).

Recently, sediment characteristics have been found to be important in driving holothurian distribution (Sloan and von Bodungen 1980; Slater and Jeffs 2010).

H. floridana is dispersed throughout the middle and lower Florida Keys, in both seagrass and hard-bottom habitats. Small H. floridana have a mottled coloration and show cryptic behavior by hiding diurnally under structure in hard-bottom habitats, whereas large H. floridana lie exposed in seagrass beds diurnally and are found in high abundances, however do not show different color morphologies or cryptic behavioral traits. The cryptic hiding behavior of the small morph has also been seen in other species of holothurians where the environmental characteristics may be driving the spatial distributional patterns between the small and large

holothurians (Purcell 2004; Yamana et al. 2006; Slater and Jeffs 2010). Environmental characteristics such as total organic content, chlorophyll a, grain size distribution, canopy height, sediment depth, and vegetative cover may be important distributional factors influencing holothurian distributional patterns.

Holothuria atra, a holothurian found in high abundance in the Indo-Pacific, was found distributed throughout seagrass beds where the sediments were characterized by 2-3.5% organic content with 15-25% gravel and coarse sand (Dissanayake and Stefansson 2012). H. atra was also found at higher abundances within a protected site where organic content, organic matter, and nitrogen concentrations were all higher (Asha et al. 2015). However, Holothuria edulis were found in rocky habitats where the sediment characteristics were similar to the seagrass habitats

H. atra preferred, but organic content was not important (Uthicke 1999; Dissanayake and

Stefansson 2012). The deposit-feeding Australasian sea cucumber Australostichopus mollis, is mainly found in habitats where the sediments are characterized by lower total organic and nitrogen content, high chlorophyll a content, and coarser grain size distributions (Slater and Jeffs

2010).

The physical complexity of a habitat can also be important in understanding species distributional patterns. A more shallow sediment depth can lead to a higher anoxic/hypoxic layer, ultimately causing problems for seagrass growth (Terrados et al. 1999), where the height of the canopy or the structure and richness of the seagrass may dampen the wave energy and slow the tidal flow of water (Gambi et al. 1990). These factors, including predation from higher trophic species, potentially affected amphipod and decapod abundance and distribution, which were found to be significantly different when leaf height and density were manipulated in seagrass beds of Thalassia testudinum (Leber 1985; Bell and Westoby 1986). The Braun-Blanquet

method has not been used to determine the distribution or abundance of holothurians however is an important addition, as it will help determine the seagrass species richness and diversity and the abundance of macroalgae (Braun-Blanquet 1932). Large and small H. floridana differ in their spatial distribution and apparent habitat preference, so gathering important environmental variables will help us to better understand their population dynamics.

Methods

Survey sites were chosen near mangrove islands, in middle of islands and randomly

(Figure 3-1). A total of 34 sites were used; 17 in the lower Keys and 17 in the middle Keys.

Variables recorded included: biogeographic location (middle Keys vs. lower Keys), total organic content (TOC), chlorophyll a (CHLA), grain size distribution (SIZE), canopy height (CH), sediment depth (SD), vegetative cover (total seagrass cover (TS), total calcareous algae cover

(TC), total macroalgae cover (TM), Thalassia testudinum cover (TT), Syringodium filiforme cover (SF), and Halodule wrightii cover (HW)) and abundance of H. floridana at each site.

A 50 m transect was laid out at each site. I initially swam up and down the transect line, with a 1.0 m PVC pipe, and counted number of H. floridana. The transect line was then followed with a 1.0 m2 quadrat and every 5 m vegetative cover was measured using the Braun-Blanquet method (Braun-Blanquet 1932). The sediment penetration depth (m) and height of the canopy

(m) were measured within each quadrat with a meter stick, and the holothurians inside each quadrat were counted.

The Braun-Blanquet method was used to measure percent seagrass and macroalgae cover.

Thepercentage of each vegetation type was measured in each quadrat, where: 0.1 = < 5% solitary; 0.5 = 5% sparse; 1 = < 5% numerous; 2 = 5 - 25%; 3 = 25 - 50%; 4 = 50 - 75%; 5 =

>75%. This technique was originally designed to measure terrestrial cover for land development studies, however it has been applied to vegetative cover in marine studies (Braun-Blanquet 1932;

Wikum and Shanholtzer 1978). Finally, I swam back down the transect line and took three, 5.08 cm diameter sediment cores at 15 m, 30 m and 45 m. The cores were taken to 2.5 cm depth, since

H. floridana feed at the surface of the sediment with their peltate tentacles. The cores were placed in ziplock bags and iced for later analysis following the same sediment processing methodology described in Chapter 2.

Analysis

To determine if any correlations existed between the abundance of holothurians and the environmental variables a stepwise multiple linear regression was used. The assumptions of independence of observations, linear relationships, homoscedasticity, an absence of multicollinearity in the data, no significant outliers, and a normal distribution met with the raw data. The number of H. floridana was the dependent variable, whereas, location (L), canopy height (CH), sediment depth (SD), total seagrass (TS), Halodule wrightii (HW), Thalassia testudinum (TT), Syringodium filiforme (SF), total macroalgae (TM), total calcareous algae (TC), chlorophyll a (CHLA), total organic content (TOC), and grain size distribution (Size) were the independent variables.

Results

The variables in the full model explained 47.5% of the variance in the abundance of H. floridana (F =1.674; df = 20; p-value = 0.008; Table 3-1). In the stepwise regression, the independent variables that were removed included CH, TC, Size, TS, SF, HW, and TOC, in respective order. In SPSS, significance of variables are reported, where I removed each variable that was least significant and ran the respective variables through multiple stepwise regression models. The final model included L, SD, TT, TM, S, and CHLA, which explained 57.9% of the variance in the abundance of H. floridana, which was run through a MANOVA with location as a fixed factor (F = 1.71; df = 27; p-value < 0.001).

Each site sampled was plotted against the total number of H. floridana along the transect

(Figure 3-2) and then sites were pooled and location (lower Keys, middle Keys) was plotted against the mean number of H. floridana counted along each transect (Figure 3-3). The lower

Keys had a greater abundance of H. floridana than the middle Keys (Figure 3-3) and sediment depth (m) was deeper in areas with abundant H. floridana (Figure 3-4). Mean chlorophyll a

(ug/mL) was higher in the middle Keys where fewer H. floridana were found (Figure 3-5). T. testudinum was more evenly distributed among the sites in the lower Keys where more H. floridana are found (Figure 3-6), but the total macroalgae was similar in distribution between locations (Figure 3-7). Total organic content was slightly higher in the lower Keys (p-value =

0.338; Figure 3-8) and grain size was slightly coarser in the middle Keys (p-value = 0.764;

Figure 3-9), but neither differed significantly between regions.

Table 3-1. Results of the full multiple linear regression model of environmental variables that correlate with H. floridana distribution. Contribution strength of each variable is denoted in standardized coefficients beta column, which determines if the respective variable is removed or not through a stepwise fashion. Unstandardized Standardized Coefficients Coefficients Correlations Model B Std. Error Beta t p-value Zero-order 1 (Constant) 11.545 16.097 0.717 0.482 Location -20.415 7.624 -0.534 -2.678 0.014 -0.457 CH -2.554 51.654 -0.017 -0.049 0.961 0.160 SD 86.449 23.557 0.788 3.670 0.002 0.678 TS -1.863 4.776 -0.124 -0.390 0.701 0.325 HW 5.129 5.121 0.193 1.002 0.329 -0.150 TT 4.181 3.104 0.298 1.347 0.193 0.396 SF 2.062 3.599 0.174 0.573 0.573 0.144 TM -6.574 4.977 -0.316 -1.321 0.201 -0.261 TC -0.949 5.304 -0.036 -0.179 0.860 -0.150 CHLA 324.349 183.432 0.361 1.768 0.092 -0.367 TOC -0.236 0.241 -0.180 -0.981 0.338 -0.039 Size 3.671 12.058 0.053 0.304 0.764 -0.357

Figure 3-1. Survey sites in the A) middle and B) lower Florida Keys. Yellow pins mark sites and the numbers in parentheses reference the number of H. floridana counted along the 50 m x 1m belt transect.

Figure 3-2. The total number of H. floridana counted at each site within the lower and middle Florida Keys.

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H. floridana H. Mean Number of Number Mean

Figure 3-3. The mean number of H. floridana counted along 50m x 1 m belt transect at sites in the lower and middle Keys. Error bars represent ± 1 SE of the mean.

Figure 3-4. The mean sediment depth (m) at lower and middle Keys survey sites. Error bars represent ± 1 SE of the mean.

Figure 3-5. The mean chlorophyll a (ug/mL) in lower and middle Keys survey sites. Error bars represent ± 1 SE of the mean.

Figure 3-6. The percent cover of Thalassia testudinum in the A) lower Keys and the B) middle Keys using Braun-Blanquet methodology. The x-axis represents the percentage in categorical number variables.

Figure 3-7. The percent cover of total macroalgae in the A) lower Keys and the B) middle Keys using Braun-Blanquet methodology. The x-axis represents the percentage in categorical number variables.

Figure 3-8. The mean total organic content (%) at lower and middle Keys survey sites. Error bars represent ± 1 SE of the mean.

Figure 3-9. The weighted mean of grain size distribution (mm) at the lower and middle Keys survey sites. Error bars represent ± 1 SE of the mean.

Discussion

The abundance of H. floridana was much higher in the lower than the middle Keys. H. floridana abundance was correlated with deeper sediment depth, lower average chlorophyll a, even cover of T. testudinum, and even cover of total macroalgae. While other species of holothurians have been documented to prefer habitats rich in total organic content and coarser sand (Uthicke 1999; Dissanayake and Stefansson 2012; Asha et al. 2015), they were not important for H. floridana in this study.

Holothurians throughout the world have been documented to diurnally burrow in the sediment as they forage, which may increase the depth of the sediment and aerate it (Mercier et al. 1999; Purcell et al. 2016). Oxygenation of the sediment ultimately increases growth of seagrass blades (Terrados et al. 1999). As these factors are shown to correlate in distribution of

H. floridana, it may also be that they are affecting these characteristics of the environment through their movement. Alternatively, Chapter 2 showed that H. floridana likely feed on the overlain benthic microalgae, which if left to overgrow, can cause a photosynthetic block, inhibiting seagrass growth. In contrast to other species of holothurians, H. floridana are found in areas of lower average chlorophyll a, which may be occurring through their feeding on the benthic microalgae.

Total macroalgae, differing from benthic microalgae, does play a role in distribution of

H. floridana, where preference is towards a more even cover. Macroalgal cover is important, as it has been found to increase productivity within a system (Bruno et al. 2005). Asha et al. (2015) document that holothurian abundances in seagrass beds and macroalgal habitats can be attributed to the nutrients and richness of the detritus, which relates to findings and observations made by

Moriarty (1982) and Massin and Doumen (1986) and more recently, H. floridana. However this

detritus may be accumulated more often with H. floridana herbivory, if they are in fact only feeding on the microalgal content of the sediment.

It has been documented that coarse sediment rich in total organic content are two preferential qualities for holothurian distribution, however these characteristics were not correlated with H. floridana abundance. This is supported by the prior work that showed the selective feeding by H. sanctori on sediment rich in organic matter. Consumption of organic matter was ultimately found to increase, as more organic content was available within the sediment (Navarro et al. 2013). Sediment grain size is an important factor to consider in determining the habitat preference of holothurians, because of the close association between organic content and grain size (Asha et al. 2015).

There also seems to be an association with habitat choice near mangrove islands. This may be because of the influx of terrestrial nutrients fueling microalgae growth. Furthermore, many mangrove islands serve as rookeries, which may provide an additional input of nutrients from guano, which may fuel microalgae growth. Underlying the mangrove islands where H. floridana were found in high abundances are seagrass beds, which ultimately reduce wave exposure and tidal water flow (Gambi et al. 1990). This reduction in water energy may allow the nutrients from the guano to settle into the benthos, where H. floridana forages, ultimately increasing the nutrients within these habitats. Higher nutrient input from guano can attribute to the higher abundances of holothurians as multiple species have been found to increase productivity of primary producers through nutrient release in their feces (Purcell et al. 2016).

Furthermore, as benthic microalgae increases from nutrient input and is a main food source of other holothurians, potentially including H. floridana, these seagrass beds near rookeries may be a potential hotspot for H. floridana populations.

One site that was considerably different in environmental variables and had higher occurrence of H. floridana than any other site in the middle Keys was at the southern end of

Bamboo Key. When compared to the other middle Keys sites, Bamboo Key had high abundances of H. floridana, deeper sediment depth, no macroalgae cover, 25-50% average seagrass cover, low total organic content, coarse grain size, and lower mean chlorophyll a content. These characteristics were consistent with those observed in the lower Keys.

Overall, important factors in considering habitat preference of H. floridana include sediment depth, chlorophyll a content, abundance of T. testudinum and total macroalgae. The deeper sediment depth may be attributed to burrowing cycles of H. floridana, whereas the lower average chlorophyll a may be attributed to their feeding. Alternatively, the even cover of T. testudinum and macroalgae may provide a structural complexity necessary for H. floridana to survive, or can help with nutrient stock in the sediment, through tidal water flow reduction and settlement of nutrients from guano.

CHAPTER 4 CONCLUSIONS

Holothurians play essential ecological roles through bioturbation of sediments (Uthicke

1999), regeneration of nutrients (Uthicke 2001), enhanced production of seagrass systems

(Michio et al. 2003; Wolkenhauer et al. 2010), dissolution and recycling of CaCO3 (Sneider et al.

2011), as food for higher trophic level species (Larson et al. 2013), and by providing a symbiotic shelter for the juvenile lithodid crab, Neolithodes diomedeae (Barry et al. 2016). Purcell et al.

2016 strengthened these studies in a review of the ecological roles of commercially exploited species. Subsequently, holothurians are distributed often relative to these factors and understanding how and what distributes them is imperative to manage and conserve their populations, where overexploitation has detrimental impacts on their respective ecosystems.

Within the Florida Keys, Holothuria floridana resides in both seagrass and hard-bottom habitats, however a distinct morphological difference in size and color exists between H. floridana found in each habitat. The larger are uniformly dark in color and reside in seagrass beds, whereas the smaller reside in hard-bottom habitats and are mottled in coloration with cryptic diurnal behavior. This difference in habitat preference has been documented in other species of holothurians (Dissanayake and Stefansson 2012), and is documented here for H. floridana. Multiple factors could explain this distinct distribution including predator avoidance

(Slater et al. 2010; So et al. 2010) or the influence of environmental characteristics associated with each habitat. The difference in habitat preference between H. atra and H. edulis occurs through a link between their foraging and shelter (Dissanayake and Stefansson 2012). This similar link is found in H. floridana, where they significantly reduce the benthic microalgae through foraging in seagrass habitats and show cryptic behavior in hard-bottom habitats.

Overexploitation of holothurians has taken place in many Caribbean and Indo-Pacific species and fishing intensity has increased on holothurians in Florida. Therefore, understanding their trophic importance, their affect on the sediment characteristics, and how these two topics correspond with the environmental variables that correlate with their abundance are important in regulations for sustainable management and informed decision making. H. floridana has been documented as a prey item for higher trophic predators, however further studies to investigate what predators are necessary. H. floridana also potentially controls the microalgal growth in seagrass meadows. Furthermore, potential food sources for higher trophic predators, such as sea turtles, and seagrass health, which has already been declining in the Florida Bay, will be negatively impacted with overharvesting.

Overall, these studies aimed at understanding the ecological importance of the Florida sea cucumber, Holothuria floridana, while bridging the gap of knowledge on what distributes them throughout the Florida Keys. As H. floridana is susceptible to predation and affects the sediment characteristics in which it forages, it may be a critical organism and play a significant role in its respective ecosystem. The variables that distribute them also play a significant role in their habitat preference. Furthermore, these studies can be applied in other holothurian species as it may strengthen regulations that can promote sustainability in the Florida holothurian fishery.

APPENDIX SUPPLEMENTARY MATERIALS

Figure A-1. A portion of the body wall from H. floridana was obtained for extraction of ossicles. Photo courtesy of author. Gainesville, Florida. Fisheries and Aquatic Sciences.

Figure A-2. A dissection of H. floridana showing reproductively mature gonads. The dorsal body wall was cut, posterior to anterior, to keep mesentery in tact. Photo courtesy of author. Gainesville, Florida. Fisheries and Aquatic Sciences.

Figure A-3. Enclosure used for foraging study. Foam rubber was used to prevent holothurians from escaping or entering, while allowing light penetration. Photo courtesy of author. Long Key, Florida. Keys Marine Lab.

Figure A-4. Gut of a necropsied Loggerhead sea turtle Caretta caretta, which appears to contain many H. floridana. Photo courtesy of Brian Stacy (NOAA).

Figure A-5. Light microscopy images of the ossicles extracted from the body wall of H. floridana. A) Table extracted from smaller H. floridana from the middle Keys. B) Rosettes extracted from smaller H. floridana from the middle Keys. These were identical to those extracted from large H. floridana from the lower keys. Photo courtesy of author. Gainesville, Florida. Fisheries and Aquatic Sciences.

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BIOGRAPHICAL SKETCH

Nathan grew up in Fairfax, Virginia, where his interest in marine life began with frequent trips to Virginia Beach and his attendance at Old Dominion University (ODU). He began his undergraduate career at ODU in 2009 and became incredibly intrigued by marine sciences.

During his first year at ODU, he became the vice president of the recently organized, Marine

Biology Student Association (MBSA). This was his first introduction with Dr. Butler, and an immediate relationship was formed. Nathan obtained an internship as an undergraduate at ODU in 2011. This introduction ultimately led to his hire as a research technician following graduation from ODU in 2013. While working with Dr. Behringer, Nathan developed a particular interest in the holothurians inhabiting the Florida Keys. He noted their distinct distributions in abundance and the few ecological studies about them. This ultimately led to a focus for his thesis in 2014.