Seed Dispersal of the Cocoplum (Chrysobalanus Icaco) by Gopher Tortoises (Gopherus

Home , Aldabrachelys, Chrysobalanus icaco, Gopher tortoise

Seed Dispersal of the Cocoplum (Chrysobalanus icaco) by Gopher Tortoises (Gopherus

polyphemus) in Southeastern Florida

Carolyn J. Hanish

A Thesis Submitted to the Faculty of

The Charles E. Schmidt College of Science

In Partial Fulfillment of the Requirements for the Degree of

Master of Science

Florida Atlantic University

Boca Raton, FL

May 2018

ii Seed Dispersal of the Cocoplum ( Chrysobalanus icaco) by Gopher Tortoises ( Gopherus

polyphemus) in Southeastern Florida

Carolyn J. Hanish

This thesis was prepared under the direction of the candidate's thesis advisor, Dr. Jon A. Moore, Department of Environmental Science and Harriet L. Wilkes Honors College, and has been approved by the members of her supervisory committee. It was submitted to the faculty of the Charles E. Schmidt College of Science and was accepted in partial fulfillment of the requirements for the degree of Master of Science.

Jon A. Moo , li. . Thesis Advisor

Brian~~~- Benscoter, Ph.D.

Dale Gawlik, Ph.D. Chair, Department of Environmental Sciences

Aeaifii,Ph.D.~t-- Dlt:e2. ~ofScience ();;zU 9. df)/9 Diane E. Alperi~,~' Date Interim Dean, Graduate College

111 Acknowledgements

Funding for this project was provided by the Harriet L. Wilkes Honors College. I am very grateful to Corey D. Anderson of Valdosta State University for collaborating with us on the Geospatial Components in this project. His help with the spatial analysis was crucial to our project impact. I am also thankful for the constructive ideas and field help I received from Amanda Hipps, Richard Jones, and Lauren Fremont. I am especially grateful for the help Sebastian Velez committed to this project, with a great sight for samples and reliable help with the germination trials. I’m thankful to my family for supporting my endeavors and my husband Kyle Hanish for his help as both a supporter and contributor to this project. Lastly I am grateful to Jon Moore for the support, technical assistance, laboratory space, and his guidance throughout this project.

iv Abstract

Author: Carolyn J. Hanish

Title: Seed Dispersal of the Cocoplum (Chrysobalanus icaco) by Gopher Tortoises (Gopherus polyphemus) in Southeastern Florida

Institution: Florida Atlantic University

Thesis Advisor: Dr. Jon A. Moore

Degree: Master of Science

Year: 2018

Gopher tortoises (Gopherus polyphemus) are keystone species mainly due to their burrow construction. Gopher tortoises can also impact the plants around them, but it is rarely quantifiable due to constraints in dispersal studies including time period and seasonality of fruit. The objective of this study was to measure the effect gut-passage has on a native Florida stone-pitted shrub, the cocoplum bush (Chrysobalanus icaco), as well as to attempt to model the relationship between the gopher tortoise and the plant using our unique field site. This study shows that gut-passage has a significant effect on the germination rate of the cocoplum, allowing it to germinate faster than control groups.

This study also found that a model involving covariates relating to tortoise movement as a predictor for cocoplum intensity was favored over a homogeneous null model. We believe the pattern of plants is nonrandom and relates to the gopher tortoise’s seed dispersal.

v Dedication

This manuscript is dedicated to my loving family, for supporting me in my endeavors to chase my dreams. I would like to dedicate this work especially to my late grandmother, Jeanne M. Smith, who was a brave and compassionate scientist throughout her life, never hesitating to challenge her obstacles. Lastly, to Gertrude, who never backed down from a fight, and was always ready to defend her home.

Seed Dispersal of the Cocoplum (Chrysobalanus icaco) by Gopher Tortoises (Gopherus

polyphemus) in Southeastern Florida

List of Tables ...... ix

List of Figures ...... x

Chapter One: Introduction ...... 1

Chapter Two: Materials and Methods ...... 7

Seed Collection ...... 7

Germination Experiment ...... 8

Seed Tray Configuration ...... 8

Protocol ...... 9

Analysis ...... 10

Geospatial Component ...... 12

Spatial data collection ...... 12

Protocol ...... 13

Point-pattern analysis ...... 15

Chapter Three: Results ...... 17

Germination Experiment ...... 17

Germinability Testing ...... 17

One-way Analysis of Variance on Ranks ...... 17

Survival Analysis ...... 18

Point Pattern analyses ...... 19

vii Chapter Four: Discussion ...... 21

Germination Experiment ...... 22

Geospatial Component ...... 25

Appendix ...... 31

References ...... 46

viii List of Tables

Table 1. Analysis of deviance table for nested inhomogeneous Poisson point

process models...... 38

Table 2. Germinability results across treatments with the event of successful

germination measured against the “right-censored” unknowns...... 39

Table 3. Chi-square results amongst final Germinability counts in all treatments...... 40

Table 4. Independent-samples Kruskal-Wallis test summary of median

germination times amongst treatments...... 40

Table 5. Pairwise Comparison of Treatment Using Dunn’s Test and

Bonferroni Correction...... 41

Table 6. Means and medians for survival time with Kaplan-Meier estimator...... 42

Table 7. Kaplan-Meier analysis of treatment groups using Log Rank and

Breslow Tests...... 43

Table 8. Pairwise comparisons across treatments groups with Breslow post-hoc...... 44

Table 9. Regression table for the inhomogeneous Poisson point process model

that was best supported by the data...... 45

Table 10. Day germinations in each treatment reached 50% of total germinations...... 45

ix List of Figures

Figure 1. Graphical results of the Kruskal-Wallis analysis relating to treatment...... 31

Figure 2. Abacoa Greenway site showing surveyed cocoplum bush locations,

tortoise burrows, and tortoise trails (High Traffic Trails)...... 32

Figure 3. Abacoa Greenway Site Covariates...... 33

Figure 4. Pairwise comparison of treatment in Kruskal-Wallis test...... 34

Figure 5. The Survival Distributions of the groups by treatment ...... 35

Figure 6. (A) Smoothed Pearson residual field for the inhomogeneous Poisson

process model that was best supported by the data. (B) Areas within the

study area were the absolute value of the smoothed Pearson residual

exceeded 2 standard deviations (= TRUE) are shown in orange...... 36

Figure 7. Partial residual plot for (A) the x-coordinate and (B) distance from

gopher tortoise trails, in meter (Trail_dist) indicating that the systematic

relationship between cocoplum intensity and these two predictors appears

to be log-cubic...... 37

x Chapter One: Introduction

Seed dispersal ensures genetic information will continue into the next generation of plants by allowing an adult plant to end its reproductive cycle with the establishment of offspring (Wang and Smith 2002). The dispersal of seeds can be a passive process relying on abiotic factors such as wind for light-weighted seeds or dispersal by water in floods for heavy seeds (Herrera 2002). Dispersal can also use biotic factors through the plant itself or through animals as vectors for physical travel. When seed dispersal is implemented through animals, seeds can be attached either externally (epizoochory) or seeds can be ingested and later excreted (endozoochory) (Janzen 1984, Herrera 2002).

The advantages of seed dispersal stem from the Janzen-Connell Hypothesis which proposes that traveling away from the seed shadow of a parent plant allows seeds to avoid the high density-dependent mortality rates which are often due to seed predators, pathogens, or seed competition under the parent plant (Janzen 1970; Connell 1971). The farther a seed can travel away from the parent, the higher the probability that the seed will mature, making dispersal of seeds a factor in the maintenance of biodiversity, colonization of habitat, and species conservation (Janzen 1970; Wang and Smith 2002).

All vertebrate taxa include members that have been identified as plant-dispersers, though a majority of well-known plant-dispersers are bird and mammal frugivores (fruit eaters) (Herrera 2002). The high diversity of seed adaptations seen in animal dispersal suggests that frugivory and the impacts of frugivory on seed dispersal have evolved independently over many vertebrate ancestries (Herrera 2002). The characteristics of

1 ingestion can have varying effects on seeds depending on the animal frugivore, with some animals causing damage to the seed or embryo during mastication, gut passage, or gut retention time and resulting in a failure for dispersal (Rick and Bowman 1961,

Herrera 2002). Dispersal through endozoochory is also related to plant characteristics; traits such as diaspore size, thickness or permeability of the seed coat, and seed dormancy all contribute to the success or failure of gut-passage (Milotić and Hoffmann 2016). The relationship between the seed and the plant-dispersers through endozoochory is generally a mutualistic one, where the animal receives a nutritional reward when ingesting the fruit, and the seeds benefit by traveling away from their parent’s shadow, potentially allowing them to root in new areas with available resources and less competition from other fallen seeds (Herrera 2002).

Tortoises and lizards are rarely the prominent herbivore in ecosystems, but have been well documented as major herbivores and frugivores in tropical, insular, or arid systems like those found on the Galápagos Islands (Rick and Bowman 1961), South

American dry Woodlands (Varela and Bucher 2002) or in the xeric scrub forests of

Florida (Diemer 1986; Herrera 2002; de Miranda 2017). Seed dispersal by reptiles

(saurochory) has also been documented as far back as 31 million years ago with fossil remains of Stylemes shells showing evidence of seeds inside the tortoise’s body, found in

South Dakota’s Oligocene formations (Marron and Moore 2013). The relationship between herbivorous reptiles and plants has likely been co-evolving through time, as early reptiles are purported to have impacted the morphology of modern fruit (Moll and

Jansen 1995). Seed passage through tortoises has been found to enhance germination of certain species of seed already, as popularly known in the Galápagos tomato

2 (Lycopersicon esculuntum var minor) after passage in the Galápagos tortoise

(Chelonoidis porteri), and also found with Ebenaceae Ebony trees (Diospyros egrettarum) passing through the Aldabra giant tortoise (Aldabrachelys gigantea) (Rick and Bowman 1961, Griffiths et al. 2011). Seed dispersal through tortoises may also provide distinct advantages over other animals based on their feeding characteristics.

Tortoises lack teeth and are less likely to damage the seed through mastication, they have a propensity for swallowing food whole, and are typically fully herbivorous animals

(Rick and Bowman 1961, Birkhead et al. 2005). As hind gut fermenters, tortoises have a relatively long gut retention time and gopher tortoises (Gopherus polyphemus) specifically have an average rate of 13 days for complete passage, which makes a large dispersal distance more likely when consumed by a tortoise than by frugivores with faster passage (Bjorndal 1987, Birkhead et al. 2005). Tortoises may also be imperative to large- seeded species dispersal, as they are less likely to be dispersed by other frugivores, more likely to be depredated by insects while fallen, and tortoises as reptilian frugivores target fruits that are colorful, fragrant, low-hanging, or fallen (Janzen 1971; Howe and

Smallwood 1982; Birkhead et al. 2005).

The gopher tortoise (Gopherus polyphemus) is a North American tortoise found in the southeastern United States which is linked closely with scrub forests, flatwoods, and xeric upland ridges (Ennen 2012). While the gopher tortoise is considered a keystone species primarily due to the benefits their large fossorial burrows provide to the flora and fauna around them, their grazing behavior may also merit their classification as keystone species or ecosystem engineers (Franz 1986; Jackson and Milstrey 1989; Birkhead et al.

2005). The gopher tortoises turn soil at their burrow entrances, returning leached

3 nutrients to the surface and providing a disturbed surface for plant colonization (Kaczor and Hartnett 1990). They are also known to pass seeds whole without visible damage, after they swallow the fruits whole, potentially allowing for endozoochory dispersal into areas the plants can’t reach alone (Birkhead et al. 2005).

The plant of interest in this study is the cocoplum bush (Chrysobalanus icaco) which is a native shrub in South Florida, though its range extends farther south into the

Caribbean, Central and South America, and tropical western Africa (Brown and

Cooprider 2011). The cocoplum has a history of human use in traditional medicine for parts of its range, but within South Florida it is a common food source for wildlife and used commercially as an ornamental shrub that withstands salt spray, flooding, and aggressive hedging (Francis 2004; Castilho and Kaplan 2011; Brown and Cooprider

2011). The fruit of the cocoplum is colorful and ranges from deep purple to white depending on variety. The fruit is considered a drupe as it has a soft slightly sweet flesh surrounding a large stone diaspore (Brown and Cooprider 2011). The cocoplum germinates mostly with the diaspore, which resembles a teardrop shaped brown stone with 5-6 ridges on the seed endocarp, and has a white seed protected inside (Brown and

Cooprider 2011). In ideal conditions, the cocoplum seed can germinate as soon as 34 days after sowing (Francis 2004). The cocoplum fruits ripen and fall to the ground, where they are known to disperse by water after heavy rains, and while many animals consume parts of the fruit, the size and weight of the fruit make it unlikely to be dispersed by birds or other small frugivores with gape limitations (Francis 2004; Brown and Cooprider

2011; Gawlik et al. 2012). These characteristics make them ideal candidates for dispersal by gopher tortoises.

4 The study site for this project was located in the Abacoa Greenway, a residential development in Jupiter, Florida (26.90°N, 80.11°W), which acts as a reserve intended to preserve gopher tortoise habitat (Wetterer and Moore 2005). The entire site encompasses

33.24 acres (0.135km2) and is surrounded by a perimeter chain-link fence 1.924km

(1.195miles) in length (GoogleMaps). The central habitat is a wooded range that is elevated and considered upland habitat approximately 23 acres in size (9.27 ha). A mowed pathway blanketed by bahiagrass (Paspalum notatum) and pusley (Richardia spp.) encircles the central area, and two straight-line paths crossing the northern and eastern sections are also occasionally maintained and cover an old (prior to 1996) cleared cattle fence line and a buried pipeline (circa 1999) (Wetterer and Moore 2005). The habitat of the range most closely resembles a xeric or scrubby flatwood habitat with sparse canopy consisting of mature slash pines (Pinus elliotii), some scrubby oaks

(Quercus spp.), and a thick understory of saw palmetto (Serenoa repens) (USFWS 1999).

While the site has occasional introductions of waif tortoises and escapes of resident tortoises, the fence acts as a barrier to travel by the tortoises and the population has been monitored since 2001 (J.A. Moore, personal communication).

The gopher tortoise burrows are primarily located along the upland wooded range but are occasionally found in the catchment basin that borders the southern and western portions of the range. The basin is lower in elevation and acts as a water retention site for the nearby housing development, though it rarely holds standing water outside of flooding events. Because of the thick understory of saw palmetto in the upland range, tortoises are primarily using the mowed pathways blanketed by bahiagrass and pusley as

5 basking and foraging areas, as well as using the narrow tortoise pathways throughout the interior vegetation that have been maintained by high tortoise activity over the years.

Uniquely, the cocoplum bushes on site have a known introduction location and date, as they were planted for aesthetic purposes to block the view of a cement drainage culvert. They were introduced in early spring of 2004 as four sub adult plants (approx.

1m in height) to the northeast corner of the site, three of the plants were of the red-tip variety (C. icaco var. icaco) which produces dark purple fruit, and one plant of the green- tip variety (C. icaco var. pellocarpus) which produces white fruits. In the 14 years since introduction to the site, the cocoplum has spread throughout the approximately 23 acres along the upland habitat, with many of the seedlings and new adults found in the aprons of active gopher tortoise burrows.

The objective of this study was to test the potential for seed dispersal of the cocoplum through endozoochory using the gopher tortoise as a vector. By examining whether cocoplum seeds could survive gut passage in the gopher tortoise, and by analyzing the spatial dispersal by gopher tortoises using the locations of the cocoplum and their distances to known gopher tortoise pathways and burrows, this study aimed to investigate the relationship between the two organisms. These outcomes could indicate whether a relationship between the tortoise and cocoplum is nonrandom and indicative of the pattern of seed dispersal by the gopher tortoise.

6 Chapter Two: Materials and Methods

Seed Collection

Primary sample collection was conducted in the upland 9.27 ha (26.90°N,

80.11°W) greenway segment within the “Abacoa” housing development in Jupiter,

Florida (Moore et al. 2009). All gut-passed seeds came from this field site, as well as most of the ripe fruit collected for the control groups, with some dried fruits having been collected from the Harbor Branch Oceanographic Institute’s campus of Florida Atlantic

University.

Fecal sampling occurred at the study site on a weekly basis from 3 August 2016 until 1 November 2016. Fecal samples were collected opportunistically from throughout the site, with preference for fresh samples that had a mucus sheen. Samples were still collected as long as they remained inside their packaged shape and retained some moisture, if the scat had lost shape integrity or was missing a part of the sample it was disregarded. When any fecal matter was found, field dissection was performed in order to determine whether cocoplum seeds had been ingested. In the field, fecal samples were dissected on portable trays in order to preserve as much of the original sample as possible while still allowing immediate investigation in lieu of transportation to the lab. If samples had positive results and contained a cocoplum seed, the fecal mass was saved along with the seeds found in the excrement. Each sample was stored in an individual paper bag to reduce the amount of trapped moisture that impedes the drying process and to prevent

7 undue fungal growth. Samples were labeled with distance to closest known burrow or given GPS coordinates.

Fecal samples were allowed to dry offsite and indoors. After the samples dried, each individual sample was organized into seed and scat separately along with date collected. No seeds were washed or introduced to water before the germination trials began. The seeds were stored in dry conditions until germination trials began, when they were sorted to their individual seedling trays.

Additional collection of ripe fruit occurred during the fruit bearing season. The fruits were fully ripe when they were prepared for the germination trials where they would act as the two controls to the gut-passed seeds. The first control group was defleshed or depulped using hemostat forceps, and the second control group was allowed to dry as a whole fruit surrounding the seed. In total for the C. icaco experiment, 108 Gut

Passed seeds, 104 Depulped seeds, and 106 Whole Fruits were prepared and organized into individual and labeled seed cells.

Germination Experiment

Seed Tray Configuration

Individual seedling starter cells measuring 1.5 inches square and 2.25 in deep were used to separate each seed during the germination trial. They were arranged in a

6×12 grid within five germination trays, with the fifth tray holding 36 individual cells, for a total of 324 seedling cells. The seeds were numbered individually based on date of collection and trial group, with each trial taking approximately one hundred numbers and moving consecutively from Gut Passed, to Depulped, and ending with Whole Fruits.

After the 318 seeds were assigned cells, the residual six cells remained empty and were

8 treated as a blind control for germination observations. Each seedling starter cell was assigned a random number between 1 and 324 using a Fisher-Yates shuffle algorithm executed through a computer program. This ensured that each tray of seeds had a random pattern and that no seedling neighbors were intentionally grouped by treatment. As the seed trials were implemented indoors in a laboratory setting, seed trays were positioned on a seedling heat mat with a thermostat controller and timer in order to provide a gradient between day 90°F (32°C) and night 70°F (21°C) temperatures for 12 hour day and night cycles throughout the experiment.

Protocol

On 27 January, 2017, all C. icaco seeds were planted in their respective seedling cells using 8-10 grams (9±1 g) of Pro-Mix HP Mycorrhizae soil, which was chosen for its high porosity (HP) for soil drainage and in order to standardized soil components across the experiment. Each seed was buried just below the surface of the soil, within 1 cm to reproduce natural conditions of sowing after fecal deposit or gravity dispersal. Each seedling cell received water until soil saturation occurred without standing water at the surface. The plants were monitored frequently in order to implement a watering regimen that allowed the cells to completely dry, and each tray was rotated every three days on the shelving unit and amongst themselves in order to control for microclimate bias (based on

Griffiths et al. 2011).

Observations were made daily after the first germination occurred on 14 March,

2017. Germination in this study was considered successful if any portion of the plant was observable from the surface, most often the hypocotyl or radicle of the germinating seed.

If no portion of the plant penetrated the seed coat at the termination of the experiment,

9 the germination was considered a failure and “right censored” in the data (McNair et al.

2012).

Analysis

After the initial delay in germination, the results of individual seeds were recorded on time intervals measured during 24-hour periods, and once germination occurred the seed was removed from the pool and data was recorded with reference to its unique identification and experimental treatment. Germination time (Days to

Germination) began once water was introduced into the seed cells on January 27th, 2017.

Germinability (percentage germination) was measured as the cumulative percentage of seeds that had germinated in each treatment at a given day in the trial, as well as by the end of the experiment. Mean time to germination (mDays) for each treatment was calculated based on Ranal and Garcia De Santana (2006):

∑ 푑 ×푛 mDays = 푖 푖 푁

For this equation, ni is the number of seeds having germinated in the time period i, di is the number of days in the experiment within the time period i, and N is the total number of seeds germinated in the treatment. The data collected in the germination trials was interval data as the exact timing of germination was unknown but assumed to occur between observations approximately 24 hours apart. Germinations not occurring by the end of the experiment were considered “right censored” as they had the potential to germinate outside the bounds of the experiment at unknown times.

Germinability was tested against a null hypothesis stating that no differences in

Germinability would exist between treatment groups. A chi-square test of homogeneity was conducted between the treatment types and their Germinability (percentage

10 germination) during the trial. Statistical analyses for germination results were conducted with SPSS software version 24 (IBM Corp 2016).

A nonparametric Kruskal-Wallis H test (α = 0.05) of median rank values was used to determine if differences in Time to Germination existed between Treatment groups in the experiment: the “Depulped (DP)” (n = 104), “Gut Passed (GP)” (n = 108), and the “Whole Fruit (WF)” (n = 106) (Kruskal and Wallis 1952). Distributions of germination dates were found to be similar across treatment groups based on visual examination of a boxplot (Fig. 1). A Dunn’s post hoc test with a Bonferroni correction (α

= 0.0167) was conducted as a pairwise test of significance amongst the treatments.

Additionally, time-to-event (survival) analysis was performed as a secondary method of analysis in order to compare the data to more contemporary germination research, as this statistical analysis manages germination data more accurately by being able to cope with delayed germination starting times and “right censoring” germinations not occurring within the bounds of the experiment more effectively (Altman 1999;

Hosmer et al. 2008; Onofri et al. 2010; McNair et al. 2012). This method equates survival functions with germination probability, where in a typical survival analyses, a death would be concurrent with a germination event.

Germination probability in the time-to-event analysis was estimated non- parametrically with the Kaplan-Meier survival estimator (Kaplan and Meier 1958):

푗 푑푗 S ( t ) = ∏푖=1 ( 1 − ) 푛푗

For this equation, the germination probability (survival) S (t), is estimated at any time t, and during the time interval j, dj represents the number of seeds having germinated in the given time interval, nj represents the number of seeds that are “at risk” to germinate in the

11 same time interval, which is the number of non-germinated samples remaining during each interval i which is measured in periods of one day each (Laerd Statistics 2015).

Two post hoc tests were used on the survival analysis data; a Log-Rank test (α =

0.05) was run initially to determine if differences in the survival curves existed for the three treatment types. Secondarily, a pairwise comparison of a Breslow (Generalized

Wilcoxon) test was run in order to determine if differences amongst treatment groups were significant, using survival distributions and an emphasis on earlier event

(germination) occurrences, as it relates to an important timeline for plant development

(McNair et al. 2012). A Bonferroni correction was again made to the alpha level to adjust for multiple comparisons (α = 0.0167). All statistical analyses referring to the survival analysis were conducted using SPSS version 24 (IBM Corp 2016).

Geospatial Component

Spatial data collection

Geospatial coordinates and relevant feature attributes were collected between 5

December 2016 and 7 July 2017 on a Trimble® Juno® 3 with a ProXH™ Global

Navigation Satellite System (GNSS) receiver. The GNSS receiver was set to collect 10 fixes per coordinate location, but varied based on daily conditions. The Trimble® hardware used Terrasync™ software for efficient field data measurements and collection as well as post-processing (i.e., differential correction using nearby base stations; CORS,

West Palm PBCH). Trimble GPS Pathfinder Office® was used to create data dictionaries and to export the spatial data into ESRI shapefile format. ArcGIS® version 10.1 was used to edit shapefiles and to create maps.

12 Protocol

The study site within the Abacoa Greenway in Jupiter, Florida, has a fenced perimeter that limits tortoise movement. Because there is an elevation change between the upland wooded range and the basin designed for water retention, the study area boundary was demarcated to exclude lower elevations habitats to the south and west and to include only the upland range (Fig. 2). Excluding lower elevation habitats precludes plant dispersal by gravity and flooding, which is the typical way the cocoplum bush disperses (Francis 2004; Brown and Cooprider 2011). The basin rarely holds water, even during the rainy season, but it is possible that past flood events have allowed for movement not attributable to tortoise activity. The study area boundary was mapped by travelling the perimeter with the GNSS receiver.

Gopher tortoise burrow (n = 132) and cocoplum bush (n = 99) coordinates were collected during the fruiting season to classify adult plants by their reproductive status.

Plants were also classified by their variety type (red-tip vs white-tip). Fruiting adults included any plant that showed signs of recent fruit development, or were fruiting

(flowering) during the survey. Non-fruiting adult plants bore no fruit or flowers but measured above 5 ft. (~1.5 m) in height; the approximate height of the smallest fruiting plants encountered. Sub-adult plants were measured at heights less than 5 ft. (~1.5 m) but above seedling height (2.5 ft. or ~ 0.80 m).

Tortoise burrows were targeted for coordinate collection based on archival maps of the site made during previous field observations (Moore, unpublished work). New burrows encountered during additional site surveys were assigned numerical identifiers and classified by tortoise activity level (following Cox et al. 1987 and Smith et al. 2005).

13 If a tortoise was in the burrow at the time of encounter, the burrow was considered active and classified as “Tortoise In Burrow” (TIB). Burrows that had evidence of very recent occupation (cleared entrances, tortoise tracks, tortoise activity in apron’s vicinity) were classified as “Active”. “Inactive” burrows were potentially occupied, as they were structurally sound, but showed no signs of recent activity and often had debris piling in the burrow entrance that indicated disuse. Burrows were considered to be “Abandoned” if they were obstructed by a permanent physical structure (new tree growth) or no longer had a usable entrance (previous collapse). Abandoned burrows could not be occupied by tortoises but had a history of use in the archival data.

The GNSS receiver was also used to map the boundaries of saw palmetto thickets

(n = 10). Saw palmetto thickets were mapped as polygons by tracing their perimeter as closely as possible. The saw palmetto thickets may act as ‘exclusion zones’ for both tortoise movement and cocoplum seed germination, as the recumbent trunks often made walls of vegetation where previously open areas once stood. The saw palmetto thickets were spared during the last intensive reduction mowing operation in 2006 as they had encircled known burrows, most of which were inactive at time of the present survey. To survey inside the thickets, machetes were used to cut entrances and paths.

Gopher tortoise movement trails (~ 20) were walked with the GNSS receiver and mapped as polyline files. As the site became overgrown in the decade since the reduction mowing was performed, tortoises have maintained pathways used to travel from areas of grazing to their burrow sites, as well as pathways to connect to high traffic areas and highly frequented female burrows. Those trails are identifiable in many ways: by archival data showing pathways created by motocross bikes which can still be seen in satellite

14 images, by identifying passages made through thick understories and maintained by constant animal traffic, and by personal observation of tortoises traversing pathways while roaming for grazing or mating. The gopher tortoise trails were often connected to the two paths maintained by county mowers as well as to the running paths that encircle the study boundary and provide for basking areas adjacent to the wooded understory.

Most pathways are easily traversed during surveying with the GNSS receiver, but a machete was occasionally required to clear the upper portions of the trail that restricted human movement but did not impede the tortoise trail below.

Point-pattern analysis

All spatial analyses were conducted in R version 3.4.2 (R Core Team 2017). The packages maptools and rgdal were used to convert ESRI shapefiles into spatial vector objects. The spatstat package (Baddeley et al. 2015) was used to process covariate data and fit point-process models, with cocoplum intensity as the response variable.

Continuous predictors included burrow intensity [estimated by nonparametric kernel smoothing of locations, with the smoothing bandwidth chosen via cross-validation

(Diggle 1985)], distance to gopher tortoise movement paths (in meters), and Cartesian coordinates (x and y, based on truncated UTM coordinates) (Fig. 3). A logical covariate – a binary mask representing areas inside or outside the saw palmetto patches – was also used in the model.

A likelihood-ratio test was used to evaluate the null hypothesis of a homogeneous

Poisson process against the alternative of an inhomogeneous Poisson process with intensity that is a log-linear function of a covariate. If the null model could be rejected, an additional covariate was added to the model and tested against the alternative “nested”

15 model (Table 1). Because plants often germinate in clusters, results for homogeneous and inhomogeneous Poisson process models were compared to results for homogeneous and inhomogeneous Thomas and Matern cluster models (Baddeley et al. 2015).

For the inhomogeneous Poisson model that was best supported by the data, the fit of the model to the data across the study area was performed by examining the spatial distribution of the smoothed Pearson residuals. To determine areas where the model fit was suboptimal, we calculated the standard deviation of the smoothed Pearson residuals and used the rule-of-thumb that a residual is large if its absolute value exceeds 2 standard deviations (Baddeley et al. 2015). To examine potential nonlinearity of predictors with respect to cocoplum intensity that may explain poor model fit in some areas, partial residuals plots were examined.

16 Chapter Three: Results

Germination Experiment

Germinability Testing

Germinability (percentage germination) of the three seed treatment groups was compared to a null hypothesis: there is no detectible difference in Germinability (percent successful germination) between treatment groups. Germinability did not differ significantly (p = 0.162) between groups, with 68 (63%) Gut Passed, 73 (70.2%)

Depulped, and 61 (57.5%) Whole Fruit seeds successfully germinating (Table 2).

Supported by Table 3, we fail to reject this hypothesis (χ² (2) = 3.645, p = 0.162).

One-way Analysis of Variance on Ranks

Median germination times differed significantly between groups (χ² (2) = 18.887, p = 0.00008), shown with the Kruskal-Wallis H test in Table 4. This supports rejecting the null hypothesis of an equal distribution of median day to germination across all treatments. Post-hoc pairwise comparisons were performed using Dunn’s test with a

Bonferroni correction for multiple comparisons and adjusted p-values (α = 0.016) (Dunn

1964). This analysis supported the Kruskal-Wallis test and showed that significant differences were found between the Gut Passed (GP, Mdn = 74) and Depulped (DP, Mdn

= 122) treatments (p = 0.0016) and Gut Passed and Whole Fruit (WF, Mdn = 146.5) (p =

0.0002) treatments, but no significant differences were found between the Depulped and

Whole Fruit treatments (p = 1.00) (Table 5). Figure 4 shows the pairwise comparison of

17 treatments graphically using the mean rank values, with orange lines pairing significant comparisons.

Survival Analysis

Using the Kaplan-Meier estimator, seeds within the Gut Passed treatment had a median time to germination of 73 days (95% CI, 61.4 to 84.7 days), which was faster than the groups not experiencing gut passage, with the treatments of Depulped and Whole

Fruit resulting in median times of 122 days (95% CI, 109.5 to 134.5 days) and 145 days

(95% CI, 119.8 to 170.2 days) respectively (Table 6). Similar numbers of seeds were

“right censored” across all treatments, as they failed to germinate within the time bounds of the study: Depulped n = 31 (29.8%), Gut Passed n = 40 (37.0%), and Whole Fruit n =

45 (42.5%). Censored seeds across all trials was n = 116 (36.5%).

The survival distributions (Fig. 5) for the three treatments were found to have statistically significant differences (Log Rank (Mantel-Cox) α = 0.05; χ² (2) = 6.794, p =

0.033). Additionally a Breslow (Generalized Wilcoxon) test was run on the survival curve data, as the Breslow test places greater weight on events occurring earlier in the study, which correlates to faster germinations (χ² (2) = 21.709, p < 0.005) (Table 7).

Statistically significant differences were found in the pairwise post hoc Breslow

(Generalized Wilcoxon) analysis of the survival distributions when comparing the Gut-

Passed treatment to both the Depulped (χ² (1) = 13.757, p < 0.0005) and Whole Fruit (χ²

(1) = 14.836, p < 0.005) treatments, but no significant differences were detected between the Depulped and Whole Fruit treatments (χ² (1) = 0.535, p = 0.464) (Table 8).

18 Geospatial Component

Point Pattern analyses

There was strong evidence against the null model of a homogeneous Poisson process in favor of an inhomogeneous Poisson process with intensity that is a log-linear function of any of the examined covariates except the kernel-smoothed intensity of gopher tortoise burrows and the truncated y-coordinate (Table 1). The model containing covariates representing the x-coordinate (“x”), distance to high traffic gopher tortoise movement paths (“Trail_dist”), and whether a cocoplum bush was located inside or outside of saw palmetto patches (“thick_mask”) was favored over nested sub-models.

Regression coefficients indicated a statistically significant increase in intensity of cocoplum from west to east (β = 0.86, SE = 0.25), a decrease in cocoplum intensity with increasing distance from gopher tortoise movement paths (β = −13.83, SE = 4.87), and lower cocoplum intensity inside (as compared to outside) saw palmetto patches (β =

−9.8, SE = 0.38) (Table 9). Homogeneous cluster models (Thomas, Matern) suggested that, if clusters exist, the average number of points per cluster is relatively low (~1.4); inhomogeneous cluster models yielded the same inferences as Poisson models with regard to covariates.

For the inhomogeneous Poisson model that was best supported by the data, a plot of the smoothed Pearson residual field revealed several areas within the study area where the model substantially underestimated or overestimated the intensity of the cocoplum

(Fig. 6). Partial residual analysis (Fig. 7) indicated that while cocoplum intensity increased from east to west within the study area, the systematic relationship was approximately log-cubic, suggesting undulations in intensity. Partial residual analysis

19 also supported a systematic decrease in cocoplum intensity with increasing distance from gopher tortoise movement trails up to about 50 to 60 m; beyond that distance the relationship undulates and the overall systematic relationship appears to be best explained by a log-cubic function.

20 Chapter Four: Discussion

Typical seed dispersal studies are unable to connect the germination results to a larger spatial scale because of the short periods of study possible when investigating seasonal fruits and their dispersers (Wang and Smith 2002). This study had a unique opportunity based on the history of the field site: because of the pattern of introduction of this plant and the subsequent lack of forest management in the following decade (J.A.

Moore, personal observation), a spatial pattern of dispersal was discreetly sown. This study quantified the dispersal effect gopher tortoises, Gopherus polyphemus, had on the cocoplum bush, Chrysobalanus icaco. While the gopher tortoise is well-known for its keystone impact through the construction of burrows, it is not often recognized for its role in the composition of the forest surrounding it. The cocoplum bush, which is less likely to be dispersed by common frugivores because of the size of its seed and fruit are able to successfully use the tortoises for endozoochory, with a benefit of distance dispersal and of significantly faster germination rates. This study was able to show that the spatial association between the tortoises and cocoplum bushes was quantifiable and that a model containing the x-coordinate (westward expansion), distance to high traffic tortoise trails, and whether the cocoplum was located inside or outside of the saw palmetto thickets was favored over nested sub-models.

21 Germination Experiment

This study found that gut-passage through the gopher tortoise significantly affected germination times, and that seeds that had passed through a tortoise prior to planting were able to germinate faster than control groups. In this experiment, this study determined that the impact of gut-passage on the seed led to an accelerated germination time. Although the Gut Passed group was able to germinate faster, the seeds in all three treatment groups had similar Germinability results, and no significant differences were detected. As a result, germination is quantifiably affected by gut-passage in terms of time-to-germination rather than Germinability.

Accelerated germination times are impactful for plant species as they give seedlings an opportunity to grow and gain a foothold in less dense clusters of cohorts

(Ross and Harper 1972). When plants emerge earlier, they have been shown to have competitive advantages in both excluding cohorts and by being able to grow larger in size

(Black and Wilkinson 1963). Previous literature with experimental plots of clover

(Trifolium subterraneum) found that a delay of even 5 days coincided with a 50% reduction in final yield, and a delay of 8 or 9 days resulting in a reduction of 75% compared to its competitors (Black and Wilkinson 1963). It is arguably more important for a seedling to germinate before its cohort, as timing will affect plant fitness, survival, and competitive interactions, rather than for the seeds to have a greater Germinability potential (Orrock and Christopher 2010). Our Gut Passed seeds had 50% of their germinations (compared to final amount) occur at day 61, which was 34 days before the

50% point of the Whole Fruit treatment and 47 days before the 50% point of the

22 Depulped treatment (Table 10), but data on the resulting fitness from head-starting in this species was not investigated.

The collection of the seeds occurred over a period of several months, and with the germination results we are able to show that the time in dormancy did not affect the date of germination. Collection date was negligible as long as water was not introduced

(imbibition) (Bewley 1997). Notable examples from the dataset that support seed dormancy include: Ci006 and Ci017; collected 63 days apart and germinated simultaneously on day 53, and Ci105 and Ci098; collected 82 days apart and both germinated on day 68. Imbibition began the germination cycle, an important aspect of the germination trial, and justified our not washing the fecal samples (Bewley 1997).

The scarification of the seed coat is purportedly an aid to germination, as the tissues surrounding the embryo must be eroded in order for the radicle to protrude and signal germination success (Bewley 1997, McNair et al. 2012). During the preparation of the Depulped treatment, flesh was stripped from the diaspore using hemostat forceps rather than a metal screen in an attempt to control and reduce the opportunity for unintentional scarification of the seed coat. Regardless of intention, it is still possible that scarification occurred and could contribute to the timing of the Depulped treatment’s germination dates. The Depulped control is used to show the best opportunity for the seed to germinate in laboratory settings, as it would not have to undergo the initial breakdown of the flesh that would prevent imbibition of the embryo, as modeled after Griffiths et al.’s (2011) similar study examining Aldabra tortoises and endangered Ebony trees. The

Whole Fruit control is used in a similar way to show how typical seeds would fare without the aid of dispersers, having dropped to the ground without any predators to

23 remove the flesh (Griffiths et al. 2011). With these justifications in mind, the results support the control preparation, as the fastest germinating control was the Depulped treatment. It also supports the idea that simply stripping the flesh off, either manually or in digestion, did not account for the total benefits experienced by the Gut Passed seeds

(Table 6).

While this study found significant evidence supporting the hypothesis that gut- passage would affect the germination of these seeds, it did not investigate which aspects of gut-passage are significant. Further work to find a connection to the treatment of gut- passage is needed, investigating the types of chemical digestion experienced in tortoises

(hind-gut fermenters), as well as passage time, water retention, and body temperatures as a few examples (Milotić and Hoffmann, 2016). Additionally, to support the line of thought connecting these plants to reptilian dispersers, more investigation into the physical differences these taxonomic groups have on plants through gut-passage is needed, including the obvious outlier of body temperature regulation differences between endothermic and exothermic organisms.

The Germinability of the seeds in this study was calculated during the pre- determined time period, ending after 165 days of observation. The seeds were watched casually for some time after the end of the trial, but only a small number of them germinated outside of those bounds. We did not investigate the viability of seeds not germinating within the bounds of the study, and conservatively considered them to be

“right-censored” in the dataset rather than inviable. We observed several seeds in the Gut

Passed trial that had been cracked open or partially damaged by passage, so some reduction in Germinability is evident, but considering that the rates among the groups

24 were not significantly different it appears that tortoise gut-passage does not have a significant negative impact on this species of seed (Table 2 and Table 3). Future projects involving this relationship would benefit from an investigation into seed viability among treatments, in order to investigate the effects on the embryo’s viability.

Geospatial Component

With strong evidence against the null model of a homogeneous Poisson process, we favor a model that uses intensity measured as a log-linear function of all covariates except the kernel-smoothed intensity of the burrow locations and the truncated y- coordinate (North & South distribution) (Table 1). These results indicate that the relationship seen at the study site is not likely a random pattern, and instead the model containing covariates such as x-coordinate (“x”), distance to high-traffic tortoise trails

(“Trail_dist”), and whether the plant location was inside or outside of the saw palmetto thickets (“thick_mask”) was favored. Additionally, a significant increase in intensity of cocoplum was measured from west to east (as it approaches origination point), and a significant decrease in cocoplum intensity as distance from tortoise trails increased, which relate to the tortoise movements (Table 9).

The unique study site allowed for these sort of conclusions to be drawn relating the tortoises to the plant. Typically, the cocoplum could be dispersed by mammals in the area, such as foxes, coyotes, and raccoons, but this site has not yet seen these seeds in mammal feces. Additionally, with the urban nature of the site, the populations of foxes and coyotes is very low with only transient individuals or small groups moving through the area (J.A. Moore, personal observation). The raccoons would be capable dispersers if not for a large hit to their population due to a breakout of rabies in 2009. During the

25 sampling of tortoise feces we rarely came across raccoon droppings, and those found did not contain cocoplum seeds even in peak season. Additional study into possible dispersal methods outside of the tortoises is needed, with the introduction of trail cams to the site suggested as a simple investigation method. More information on animal usage of the tortoise trails would also be useful, as often times the trails wind through the densest portions of the range and would likely be useful passages into and out of the core of the site for all animals.

The gopher tortoise burrow locations are used to relate to the tortoise movements, but should not be thought of as static representations of the tortoise. The gopher tortoises do have high site fidelity to their burrows, but the strength of the fidelity is sexually dimorphic, with females tending to stay in fewer burrows for many years with smaller home ranges, and males using more burrows in multiple locations relating to female courting opportunities (Diemer 1992; Karlin 2002; Eubanks et al. 2003). Although tortoises also graze in close proximity to their burrows (Alexy et al. 2003), males will graze while roving their ranges and some of our specific burrow sites have become too overgrown for grazing, prompting the use of tortoise trails to reach prime grazing areas.

With this in mind, the gopher tortoise burrow locations more closely resemble a “base of operations” for the tortoise activity, from which they leave to graze and socialize.

Nonetheless, we feel strongly that these markers are appropriate indicators of tortoise activity, though not static points.

The saw palmetto thickets on site were introduced later in sampling, as it became apparent that they restricted the movement of both the tortoises and the cocoplum bush.

These areas are incredibly dense due to both the clonal nature of saw palmetto

26 reproduction (Van Deelen 1991), and due to a management decision sparing clusters of saw palmetto surrounding several burrows in 2006, contributing to the unusually dense clumps seen now (J.A. Moore, personal observation). The results showed that the cocoplum had a lower intensity reading inside these thickets rather than outside of them, supporting the thought that their denseness would disallow for pioneering seedlings to gain footholds (Table 9). However, the results of the inhomogeneous Poisson model also indicated several areas where the model was substantially underestimating or overestimating the intensity of the cocoplum (Fig. 6). These anomalous areas are likely due to unmeasured covariates at play, as in portions of the Northeastern range where the model is skewed by a large gallberry patch (Ilex glabra) that has become overgrown, potentially shading out any competitors. There are also particularly dense colonies of tortoise burrows occupied by adult females in the southeastern region of the range, possibly leading to an overestimation of cocoplum intensity due to high tortoise activity.

Similarly, the southwestern deviations are likely due to a cluster of large adult female burrows that brings high tortoise traffic into the area. Future work on this system would benefit from more sampling of vegetation related covariates as well as a better understanding of the sexually dimorphic activity of the tortoises.

As mentioned earlier, the tortoise trail system is likely used by other animals at the site, but it is our opinion that the high traffic use by the tortoises has maintained these pathways in order to connect them to grazing and mating opportunities. Some of the trails began in human use, as seen by the aptly named “Motox” trails in the interior, having originated as motocross trails used by locals before the housing communities developed, but which went inactive in 1996 when fencing started going up for the greenway. Those

27 trails were maintained by the traffic of the tortoises and can still be identified using archival satellite imagery. These trails do not likely represent the sole activity of tortoises on the site, but we would argue that they are crucial to the daily activity of the tortoises based on personal observations of their use. Future endeavors to map the usage of the site would benefit from telemetry work, and if funding allowed for satellite tracking we believe many of these questions could be addressed.

The dispersal of the cocoplum bush by the gopher tortoise supports an idea that reptiles would still be able to affect the evolution of the vegetation around them (Moll and Jansen 1995). Undoubtedly, reptiles had an impact on the development of fruits as the primary taxonomic group for millennia, but their role as major herbivores in tropical systems remains especially crucial in isolated and insular systems like the Mascarene

Islands (Hansen et al. 2008; Griffiths et al. 2011) or the Galápagos Islands (Herrera 2002;

Rick and Bowman 1961; Moll and Jansen 1995; Marron and Moore 2013). The cocoplum seed is large enough to limit its dispersal options by gape limitations, but is also large enough to attract the attention of grazing tortoises (Janzen 1971; Howe and Smallwood

1982; Gawlik et al. 2002). Based on collection data, it is likely some tortoises are consuming the fruit as a dietary choice rather than ingesting it as bycatch, as our outlier scat sample had 19 seeds and the average positive scat sample had 3.1 seeds found within it. It is possible that the tortoises have a mutual relationship around ingestion of the fruit, though the parameters of that relationship have not been explored.

There is a great variety of other studies involving gut-passage of seeds through tortoises, including some famous examples of giants like the Galápagos tortoise (Testudo elephantopus porteri) (Rick and Bowman 1961) and the more recent revival of a tortoise-

28 plant relationship seen on Mauritius island (Mascarene islands) with the Aldabra tortoise

(Hansen et al. 2008; Griffiths et al. 2011). We placed an importance on the standardization of our germination experiment, in hopes that it would be easily reproducible and in order to control for as many variables as possible. While we had control over some aspects of the project, our unique study site has been affected recently by management. Several weeks after the final data collection survey, we returned to the site to find one of our largest cocoplum adults had been cut down entirely, as it was located near the popular running path, and other adults had been sheared back, reducing their fruit bearing potential. The managers have few options for intervening due to the close proximity to residential properties, but it is necessary to address the overgrown understory which may well include the shade out caused by the large number of cocoplum plants. While burrows remain used even after major shade out by the understory, eventually browse material may be affected and would limit the carrying capacity of this site.

It appears that some change is coming, as in July of 2017 after the site survey was completed the first recorded loss of an entire adult cocoplum, as well as severe pruning of other adults, was found and as a result the unique pattern we measured has been altered.

As recently as December of 2017, a fire likely set off unintentionally burned a portion of the interior, which will help address the dense saw palmetto thicket in that area but which also changes the pattern recorded during our survey. The saw palmettos do adjust well after burns and will likely be back in full force shortly. The potential for follow up on this site is still possible, as it has been designated a wildlife preservation area as well as a drainage site and will not likely be removed from the neighborhood, but the dynamics of

29 the interior will change as management addresses the issues on site and as natural occurrences change the landscape.

Future work relating to the dispersal of the cocoplum would benefit with an investigation into site specific dispersers possibly by using a trail cam system. This sort of work could also elucidate on the ecological importance and usage of the trail system measured in this study. More information relating to the anomalous patches in the model could also inform on the potential for dispersal hotspots and inactive zones. We suggest that tortoises are able to influence the habitat around them in more ways than just physically with their burrow use. More investigation into a tortoise’s impact on the plants around them would help relate the possibility for co-evolution between the plants and fruits using tortoises as a dispersal agent. For now, we are confident that their impact can be quantified, leading to a better understanding for their role in the ecosystem, and the potential for better management of policies relating to both gopher tortoises and forest management.

30 Appendix

Figure 1. Graphical results of the Kruskal-Wallis analysis relating to treatment. The boxplots show that distributions amongst the groups are similar, though standard deviations vary. Germination time is in number of days.

Figure 2. Abacoa Greenway site showing surveyed cocoplum bush locations, tortoise burrows, and tortoise trails (High Traffic Trails). The Abacoa site was surveyed between December 5th 2016 and July 7th 2017 in order to collect data attributing the cocoplum bushes, with a red oval in the north eastern corner of the plot indicating the original four plants introduced in 2004 as seedlings. The yellow squares represent the gopher tortoise burrow locations, each of which has information relating to its use patterns during the survey. Each cocoplum plant surveyed appears in green, and the high traffic tortoise pathways are outlined in white.

Figure 3. Abacoa Greenway Site Covariates. Location of cocoplum (black and green asterisks) within the 9.27 Ha (~23 acre) study area (in Jupiter, Florida) relative to ( A ) saw palmetto thickets (“thick_mask”)(dotted polygons), ( B ) the kernel smoothed intensity of gopher tortoise burrows (points/km), and ( C ) the distance (km) to high traffic gopher tortoise movement paths (“Trail_dist”).

33 Figure 4. Pairwise comparison of treatment in Kruskal-Wallis test. Points with orange lines connecting them show a significant relationships was found. The numbers below each node’s treatment name represent the mean rank value of the treatment. Median values were used throughout most of the analysis but we feel it is appropriate to show the mean values as well.

34 Figure 5. The Survival Distributions of the groups by treatment. The curve of each treatment represents the cumulative germination rates: as the y-axis approaches zero germinations approach 100%. Each group has relatively similar final Germinability but the curve of the Gut-Passed treatment (green) has a steeper curve initially. Survival time is in days.

B A

Figure 6. (A) Smoothed Pearson residual field for the inhomogeneous Poisson process model that was best supported by the data. (B) Areas within the study area were the absolute value of the smoothed Pearson residual exceeded 2 standard deviations (= TRUE) are shown in orange.

A B

Figure 7. Partial residual plot for (A) the x-coordinate and (B) distance from gopher tortoise trails, in meter (Trail_dist) indicating that the systematic relationship between cocoplum intensity and these two predictors appears to be log-cubic.

Table 1. Analysis of deviance table for nested inhomogeneous Poisson point process models. For each model the response variable is cocoplum intensity and the null model is an intercept only model ( ~ 1) representing a homogeneous Poisson point process. Additional predictors included: x: the easting based on a truncated UTM coordinate; Trail_dist: distance (in meters) from gopher tortoise movement trails; thick_mask: a binary mask representing whether points lie inside or outside saw palmetto thickets. Nested models were compared with a likelihood-ratio test and the significance level of the chi-square test statistic is denoted by the number of asterisks (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). The model containing all covariates is favored over the nested sub- models.

Number of Degrees of

Model parameters freedom Deviance P-value

~ 1 (null model) 1

~ x 2 1 18.7 ***

~ x + Trail_dist 3 1 14.5 ***

~ x + Trail_dist + 8.6 ** 4 1 thick_mask

Table 2. Germinability results across treatments with the event of successful germination measured against the “right-censored” unknowns. The table is used to show that Germinability amongst the treatments resulted in similar percentages of censored seeds, as well as the total “n” planted for each treatment.

Event status * Type of treatment Crosstabulation Type of treatment Depulped Gut Passed Whole Fruit Total Event status Censored Count 31 40 45 116 % within Type of treatment 29.8% 37.0% 42.5% 36.5% Germination Count 73 68 61 202 % within Type of treatment 70.2% 63.0% 57.5% 63.5% Total Count 104 108 106 318

39 % within Type of treatment 100.0% 100.0% 100.0% 100.0

Table 3. Chi-square results amongst final Germinability counts in all treatments. As seen in the subtext, the Chi-square assumptions were met for this data set without transformation. The results of the test show that the treatments are not significantly different from each other in the percentage of germinations occurring during the study.

Chi-Square Tests Asymptotic Significance Value df (2-sided) Pearson Chi- 3.645a 2 .162 Square Likelihood Ratio 3.673 2 .159 N of Valid Cases 318 a. 0 cells (0.0%) have expected count less than 5. The minimum expected count is 37.94.

Table 4. Independent-samples Kruskal-Wallis test summary of median germination times amongst treatments. The three treatments were tested for differences using the ranked Kruskal-Wallis test, and those median values showed significant differences were found in the treatments.

Independent-Samples Kruskal-Wallis Test Summary Total N 318 Test Statistic 18.887a Degree Of Freedom 2 Asymptotic Sig.(2-sided test) .00008 a. The test statistic is adjusted for ties.

Table 5. Pairwise Comparison of Treatment Using Dunn’s Test and Bonferroni Correction. The significance found in the first Kruskal-Wallis test was explored using pairwise comparisons and Dunn’s test. The α-value was adjusted using a Bonferroni test because of the multiple comparisons. The results are significant between the experimental group (Gut Passed) and the two control groups (Depulped & Whole Fruit) but not between the two control groups.

Pairwise Comparisons of Type of treatment

Std. Test Sample 1-Sample 2 Test Statistic Std. Error Statistic Sig. Adj. Sig.a Gut Passed-Depulped 42.629 12.321 3.460 .0005 .0016 Gut Passed-Whole Fruit -48.967 12.261 -3.994 .0001 .0002 Depulped-Whole Fruit -6.337 12.377 -.512 .6086 1.000 Each row tests the null hypothesis that the Sample 1 and Sample 2 distributions are the same. Asymptotic significances (2-sided tests) are displayed. The significance level is .05. a. Significance values have been adjusted by the Bonferroni correction for multiple tests.

Table 6. Means and medians for survival time with Kaplan-Meier estimator. The survival analysis estimator gave results on the median rank values that were similar to the Kruskal-Wallis test but still had discrepancies as the highest values were censored rather than simply out-ranked as in the Kruskal-Wallis test. The Estimate value below is the median value of total germination.

Means and Medians for Survival Time Meana Median 95% Confidence Interval 95% Confidence Interval Upper Lower Type of treatment Estimate Std. Error Lower Bound Bound Estimate Std. Error Bound Upper Bound

Depulped 124.779 3.385 118.144 131.413 122.000 6.373 109.510 134.490 Gut Passed 103.537 4.815 94.099 112.975 73.000 5.938 61.361 84.639 Whole Fruit 127.528 3.969 119.749 135.307 145.000 12.870 119.776 170.224 Overall 118.481 2.453 113.674 123.288 122.000 7.604 107.097 136.903

a. Estimation is limited to the largest survival time if it is censored.

Table 7. Kaplan-Meier analysis of treatment groups using Log Rank and Breslow Tests. The analysis of each group done below with two tests. The Log Rank is one of the more common tests, but the Breslow puts more emphasis on events happening early in the experiment, and as we are specifically looking for faster germination rates amongst groups we choose that test as well, though both results are significant.

Overall Comparisons Chi-Square df Sig. Log Rank (Mantel- 6.794 2 .033 Cox) Breslow (Generalized 21.709 2 .000019 Wilcoxon) Test of equality of survival distributions for the different levels of Type of treatment.

Table 8. Pairwise comparisons across treatments groups with Breslow post-hoc. This pairwise comparison was corrected with a Bonferroni correction as well so the new α-value would be 0.016. This test puts greater emphasis on the events (germinations) occurring earlier in the experiment, and it finds that the experimental Gut Passed group is significantly different from the two control groups, Depulped and Whole Fruit, though the controls are not significantly different from each other.

Pairwise Comparisons Depulped Gut Passed Whole Fruit Type of treatment Chi-Square Sig. Chi-Square Sig. Chi-Square Sig. Breslow (Generalized Depulped 13.757 .000208 .535 .464 Wilcoxon) Gut Passed 13.757 .000208 14.836 .000117 Whole Fruit .535 .464 14.836 .000117

Table 9. Regression table for the inhomogeneous Poisson point process model that was best supported by the data. The response variable was cocoplum intensity and predictors included the x-coordinate (the easting based on a truncated UTM coordinate), Trail_dist (distance in meters from gopher tortoise movement trails), and Thick_mask (a binary mask representing whether points lie inside or outside saw palmetto thickets). For each predictor, the regression coefficient for the log-linear model (β), the standard error for the estimated regression coefficient (SE), and the statistical significance of Z-statistic (β/SE) based on a Wald test are presented (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).

훽 SE Z-test

Intercept only -803.26 238.98 *** x-coordinate 0.86 0.25 ***

Trail_dist -13.83 4.87 **

Thick_maskTRUE -0.98 0.38 **

Table 10. Day germinations in each treatment reached 50% of total germinations. This table is used to show that the experimental group (Gut Passed) reached 50% total germination faster in time than the other group, as fast as 47 days before the Depulped group and 34 days before the Whole Fruit group.

Days at 50% of Difference in Total Germ. Days to GP Gut Passed 61 Depulped 108 47 Whole Fruit 95 34

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