ORIGIN and BIOGEOGRAPHY of the CUBAN TREEFROG Osteopilus Septentrionalis in the BAHAMAS

ORIGIN AND BIOGEOGRAPHY OF THE CUBAN TREEFROG Osteopilus septentrionalis IN THE BAHAMAS

JOSHUA ALAN RINGER

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

2019

To my beautiful parents, you have been nothing but supportive my entire life. Thank you for always letting me be me. Most importantly, thank you to my sister Virginia. You truly are my inspiration. Love you, always and forever

ACKNOWLEDGMENTS

I would like to thank my advisors Dr. Steve A. Johnson and Dr. David W.

Steadman, as well as Dr. David C. Blackburn for all the support throughout my graduate career. I appreciate all the valuable suggestions and recommendations which helped tremendously in shaping this thesis. I could not thank Dr. Steadman enough for the constant motivation, and truly inspiring me to become a better scientist and researcher.

Learning should be an adventure, and Dr. Steadman has always reminded me of that. I am also very grateful to Dr. David Reed, Dr. Verity Mathis, Dr. Aida Miro, Lauren

Rowan, Aditi Jayarajan and the rest of the Reed Lab, for all the support and assistance in and out of the field. I’d like to thank Dr. Angelo Soto-Centeno for his immense help and patience with the morphometric chapter. I thank the Blackburn Lab, especially

Danielle Hayes for always helping me with molecular techniques and analysis.

Last but certainly not least, I thank my family and friends. Without the constant motivation, love and encouragement, none of this would have been possible.

TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 6

LIST OF FIGURES ...... 7

ABSTRACT ...... 9

CHAPTER

1 THE BAHAMIAN ARCHIPELAGO AND LIFE HISTORY OF Osteopilus septentrionalis ...... 11

Background ...... 11 Species of Interest ...... 13 Objectives...... 15

2 USING POPULATION GENETICS TO EVALUATE THE NATIVE STATUS OF THE CUBAN TREEFROG IN THE BAHAMAS ...... 16

Introduction ...... 16 Methods ...... 17 Collection of Specimens and Tissue Extraction ...... 17 Mitochondrial DNA Extraction and PCR ...... 18 Results ...... 19 Phylogenetic Analysis ...... 19 Population Genetic Analysis ...... 19 Haplotype Network Reconstruction and Analysis ...... 20 Discussion ...... 21

3 ANCIENT AND MODERN MORPHOMETRICS OF CUBAN TREEFROGS IN THE BAHAMAS AND FLORIDA ...... 36

Introduction ...... 36 Methods ...... 37 Results ...... 38 Discussion ...... 40

4 DISCUSSION AND FUTURE DIRECTIONS ...... 50

LIST OF REFERENCES ...... 53

BIOGRAPHICAL SKETCH ...... 58

LIST OF TABLES

Table page

2-1 Locality of modern specimens of Osteopilus septentrionalis collected for this study...... 24

2-2 Matrix of pairwise FST values comparing genetic differences between populations...... 26

2-3 Average number of pairwise differences between population (above diagonal) and corrected average pairwise difference (below diagonal) ...... 27

2-4 Molecular Diversity Indexes...... 27

2-5 Mean number of pairwise differences and nucleotide diversity at the intra- population level for each locality...... 27

2-6 Distribution and number of individuals of the 21 haplotypes in seven populations of O. septentrionalis...... 28

LIST OF FIGURES

Figure page

2-1 Map showing the separation between Bahamian banks, ...... 29

2-2 Map of Abaco (LBB) showing the caves where anuran fossils were found...... 30

2-3 Map of Andros (GBB) indicating where Osteopilus septentrionalis specimens were collected...... 31

2-4 Map of Eleuthera (GBB) indicating where Osteopilus septentrionalis specimens were collected...... 32

2-5 Map of Eleuthera (GBB) indicating where Osteopilus septentrionalis specimens were collected...... 33

2-6 Haplotype tree inferred from all Osteopilus septentrionalis samples (n=146) in IQ-Tree 1.6.12; Osteopilus vastus (Genbank: AY843713.1), Osteopilus dominicensis (Genbank: AY843711.1), Osteopilus crucialis (Genbank: AY8437.10.1) were used as an outgroups. Numbers in parenthesis are the number of individuals from a given population that have that haplotype. Values above and to the left of the nodes represent maximum likelihood bootstrap scores (from IQ-Tree) using 1000 iterations...... 34

2-7 Haplotype network of O. septentrionalis 16s sequences from Florida, Cuba, and the Bahamas. Each circle represents a haplotype; size of the circle is proportional to number of individuals (larger size, more individuals). Mutations are indicated by hatch marks, and black dots represent inferred intermediate haplotypes...... 35

3-1 Eleutherodactylus fossil ilium from the Great Bahama Bank, Cuba and Florida...... 42

3-2 Intraspecific variation between fossil ilium from the Bahamas (left) and modern Osteopilus (right)...... 43

3-3 Selected ilial characters measured in this study ...... 44

3-4 Selected humeral characters measured in this study ...... 45

3-5 PCA of modern Osteopilus with complimentary ilium and humerus; all 6 measurements are included reflecting PC1 and PC2 representing the highest proportion of variance...... 46

3-6 Box plot of PCA plotting male vs female modern Osteopilus septentrionalis. Females on the left indicate a higher variation, where males appear to be much more restricted in size. Individual samples are indicated by black dots. .... 47

3-7 PCA of both modern and fossil ilia of Osteopilus from the Great Bahama Bank. PC1 and PC2 representing the highest proportion of variance...... 48

3-8 LDA histogram of modern and fossil GBB ilia showing the distribution of each measurement per locality...... 49

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

ORIGIN AND BIOGEOGRAPHY OF THE CUBAN TREEFROG Osteopilus septentrionalis IN THE BAHAMAS

Joshua Alan Ringer

December 2019

Chair: Steve A. Johnson Cochair: David W. Steadman Major: Wildlife Ecology and Conservation

Islands are significant hotspots of biodiversity and endemism. Around 980 km in length, the Bahamian Archipelago in the West Indies comprises ~700 islands and more than 2,000 cays. Lying on four large and 13 smaller shallow-water banks, the Bahamian islands were much more exposed during the late Pleistocene and Early Holocene

(Steadman et al., 2017). When sea level was 40 m lower (10-12 ka), the Little Bahama

Bank (LBB) and Great Bahama Bank (GBB) (Figure 2-1) formed two large islands and were separated by less than 34 km of ocean (now 90 km), and the Great Bahama Bank was only 20 km from Cuba (now 180 km) (Steadman and Franklin 2015). The Cuban treefrog (Osteopilus septentrionalis) is a purported native of the Bahamas, having colonized the GBB from Cuba during the low sea levels of the Last Glacial Maximum

(LGM) (Steadman and Franklin 2017) with recent fossil evidence to support this claim.

However, fossil excavations and radiocarbon dating on the Little Bahama Bank suggest that O. septentrionalis is a more recent colonizer, likely human-facilitated (Steadman and Franklin 2017). This species is highly prolific and has shown an incredible persistence in both urban and natural settings. The aim of my study is to use genetic

material and the fossil record to determine if and where O. septentrionalis is indigenous to the Bahamas. I used tissue samples from the LBB, GBB, Florida, and Cuba to complete a population genetic analysis of O. septentrionalis in this region. Additionally, I evaluated whether morphological changes have occurred in O. septentrionalis in the

Bahamas and Florida with morphometric analyses of modern and fossil specimens. My study advances our understanding of the historical biogeographic pathway of O. septentrionalis in the West Indies, and, investigates human-mediated dispersal and its implications on introgression, species diversity, and the radiation of persistent nonindigenous species.

CHAPTER 1 THE BAHAMIAN ARCHIPELAGO AND LIFE HISTORY OF Osteopilus septentrionalis

Background

The West Indian islands are a hotspot of biodiversity with high levels of species richness and endemism (Borroto-Páez et al., 2015). The West Indies has a complex geological history that has offered opportunities for both dispersal and vicariance events

(Hedges 2001). Located in the Atlantic Ocean, the West Indies comprise three major archipelagoes, the Greater Antilles, Lesser Antilles, and the Bahamian Archipelago. The

Bahamas are the northernmost islands in the West Indies and exist today as a series of

23 major islands and many smaller ones lying on shallow carbonate banks separated by deep water (Steadman and Franklin 2014; Figure 1 herein). This archipelago totals nearly 700 islands and stretches 980 km from northwest to southeast (Steadman and

Franklin 2014). These low islands are surrounded by shallow oceans with surfaces features dominated by limestone karst features (eroded surfaces, hills, sinkholes, cliffs, and caves).

During the Last Glacial Maximum (LGM; ~25 to 18 ka), sea levels were as low as

~120 m below present conditions, creating shorter dispersal distances between islands.

Even during the low LGM sea levels, the tectonically stable Bahamian islands never were connected to a continental landmass or to another island group (Oswald and

Steadman 2018). Until ~10-12 ka, when sea level was 40 m below present, the

Bahamian banks formed the “superislands” of the Little Bahama Bank (LBB) and Great

Bahama Bank (GBB), separated by ~34 km of ocean (Steadman and Franklin 2014).

The GBB was only 20 km from Cuba at the time, and had a land area of ~103,670 km2, exceeding that of Hispaniola and presumably facilitating the northwest dispersal of

plants and animals from Cuba (Steadman and Franklin 2014). Subsequent sea level rise resulted in fragmentation of the islands until the mid-Holocene, leading to their current configuration (Steadman et al., 2017). At present, the GBB includes the largest island Andros, as well as Bimini, Cat Island, Eleuthera, the Exumas, Long Island, the

Ragged Islands and New Providence.

The native herpetofauna of the Bahamas is believed to comprise 46 species, including 3 frogs, Osteopilus septentrionalis, Eleutherodactylus planirostris, and

Eleutherdactylus rogersi (Knapp et al., 2011). In addition to these, there are 22 introduced species of amphibans and reptiles (5 are frogs), among which ~16 are established and breeding (Knapp et al., 2011). For island groups such as the Bahamas, which never have been connected to any other landmass, the only possible mechanism of dispersal for non-volant species is over water (Hedges 2001). Frogs in general are known to be poor oceanic dispersers, although O. septentrionalis does have dispersal- facilitating attributes, including water conserving behaviors and a tolerance for saline conditions (Heinicke et al., 2011; Brown and Walls, 2013). While human-facilitated dispersal is currently the most common for modes of introductions and establishment, ancient frogs that successfully colonized the Caribbean islands may have hitched rides on floating mats of vegetation known as flotsam. Long-distance dispersal by flotsam has been documented, leading to the colonization and establishment of species throughout the Caribbean Islands (Cox et al., 2016).

Understanding dispersal characteristics of island species not only assists in gaining knowledge on historical colonizations and expansions, but also can aid in the control and management of “unwanted species.” Human travel rates continue to

increase, having implications on dispersing species. In the past, methods of reaching islands have been described hypothetically. Four prosed methods, from Tarbuck et al.

(1999), are: natural land-bridges, jump-dispersal, separation by plate tectonic processes, and rafting. Unlike large mammals or volant species, amphibians face physiological challenges when dispersing. Amphibians have highly permeable skin and while most amphibians are intolerant of high levels of salinity, some have the ability to tolerate saline conditions to some extent. Rafting on debris or vegetation is likely how certain frogs were and still are able to disperse across expanses of ocean. Knowing the mechanisms species use to move around when faced with vicariance or other selective pressures can influence decisions on managing invasive species, or species in need of protection.

Species of Interest

The Cuban treefrog (Osteopilus septentrionalis) is believed to be native to Cuba, the Cayman Islands, and the Bahamas (Meshaka et al., 2004). Accidentally introduced to Florida as a stowaway in cargo, these prolific breeders have spread to other places in the tropics (Johnson 2007). Osteopilus septentrionalis now has a broad distribution including Florida, Puerto Rico, US Virgin Islands, and the Lesser Antilles (Johnson

2007; Vargas-Salinas 2006; Perry & Platenberg 2007; Lindsay & Cooper 2008; Somma

& Graham 2015).

Osteopilus septentrionalis is the largest treefrog in North America and one of the largest anurans in the Caribbean with a snout-vent length (SVL) ranging 25.4-100 mm

(Johnson 2007). Sexual size dimorphism is pronounced in O. septentrionalis, with the

SVL of mature males being much smaller than mature females (Vargas-Salinas 2006).

Osteopilus septentrionalis has a wide range of color variations and mottles, and

individuals typically have rough or warty-textured skin. Fecundity is high in females; one with an SVL of up to 100 mm can produce around 16,000 eggs (Meshaka, 2001).

Osteopilus septentrionalis typically uses small pools of fresh water for oviposition but can also deposit eggs in swimming pools and other areas of stagnant water (Johnson

2007). In addition to high fecundity, O. septentrionalis exhibits explosive breeding behaviors that help to offset predation; the mating probabilities of large and small males seem to be similar increasing chances of reproductive success (Vargas-Salinas 2006).

A generalist feeder, O. septentrionalis consumes a wide variety of invertebrate and small vertebrate prey including other anurans. Cannibalism is common, but appears to be more facultative, associated with low alternative food availability or crowded conditions (Babbitt and Meshaka, 2000). While the value of eating conspecifics appears to depend on availability of alternative food sources, cannibalism has the potential for enhancing growth and survival of species with complex lifecycles in ephemeral habitats

(Babbitt and Meshaka, 2000). Naturally found in wet, humid environments, O. septentrionalis can easily adapt to its surroundings, occurring in areas of human development as well as natural environments. In urban and suburban settings Cuban treefrogs are most commonly found on and around homes and buildings, and in gardens and landscape plants (Johnson, 2007). In their natural environment, O. septentrionalis occurs in swamps, marshes, mangroves, and other flooded areas with some access to fresh water (Vargas-Salinas 2006). High fecundity, short larval period, generalist diet, and broad habitat and dietary niches account for the high invasiveness of O. septentrionalis (Meshaka 2001; Vargas-Salinas 2006).

Objectives

During the past century, Osteopilus septentrionalis has earned a reputation for being a successful human-assisted colonizer in Florida as well as parts of the

Caribbean. The species is considered to be native to the Bahamas, although there has been little support for this claim. Until recently, the lack of fossil evidence has made it difficult to assess the colonization history of O. septentrionalis in the Bahamas. Fossils of O. septentrionalis collected by David Steadman (Florida Museum of Natural History,

Gainesville, FL. and colleagues on Abaco (LBB) (Figure 2-2) have yielded no radiocarbon dates older than ~700 bp, which is several centuries after the arrival of the

Lucayan people, thus suggesting human-mediated introduction. On the other hand, abundant Cuban treefrog fossils were collected from older (pre-cultural) contexts on

Long Island (GBB) in December 2017, thereby providing evidence of an older colonization event on the GBB that could not have been aided by humans. These fossils are late Pleistocene in age (>10,000 bp) and therefore substantially pre-date the arrival of the Lucayans.

The aim of my study is to use the fossil record, morphological data, and genetic data to determine if O. septentrionalis is indigenous to the Bahamas. My specific objectives are to: 1) use modern tissue samples from O. septentrionalis to compare mtDNA sequence data between specimens from the Little Bahama Bank (LBB), Great

Bahama Bank (GBB), Cuba and Florida to examine modern population genetics; and 2) use modern and fossil osteological specimens to evaluate possible morphologic changes over time in the Bahamas and Florida.

CHAPTER 2 USING POPULATION GENETICS TO EVALUATE THE NATIVE STATUS OF THE CUBAN TREEFROG IN THE BAHAMAS

Introduction

An introduced species in Florida, Osteopilus septentrionalis is a purported native to Cuba, the Cayman Islands, and the Bahamas (Meshaka et al., 2004). Reports of O. septentrionalis both from the Florida Keys date to the early 1900’s, and mainland

Florida by the mid-1900s, (Barbour 1931; Heinicke et al., 2011). Edificarian species such as Cuban treefrogs benefit from the growth of human populations and therefore have expanded their range in modern times. While phylogenetic analyses of O. septentrionalis trace their origin to at least two Cuban sources (Heinicke et al., 2011), analyses of Bahamian population genetics has yet to be explored. Due to population bottlenecks and founder effects during introduction, within-population genetic variation in invasive species is believed to be greatly reduced when compared to their native founding population. This overall reduction in genetic diversity may hinder establishment, reduce population size, and ultimately lead to extinction. However, if individuals from multiple native-range sources interact, admixture can increase within- population genetic variation despite a reduction in genetic variation experienced by each founding propagule (Kolbe et al., 2007). This compliments the Bridgehead effect phenomena, which could be occurring with more recent introduced populations

(Lombaert et al., 2010). Successful invasive populations could serve as the source of additional invasive colonizations in other areas.

In this chapter, I evaluated population genetics of O. septentrionalis throughout the Bahamas, Florida, and Cuba. Molecular data from O. septentrionalis were analyzed with the intent of comparing modern genetics of specimens from the Little Bahama Bank

(LBB), Great Bahama Bank (GBB), Cuba and Florida. I hypothesized that O. septentrionalis was able to colonize the GBB from Cuba during the LGM, leading to a prehistoric native population of Cuban treefrogs on Great Bahama Bank and less genetic diversity between populations. On the other hand, I hypothesize that the populations in other parts of the Bahamas (Little Bahama Bank) and Florida represent more recent, human-mediated invasions. These predictions should result in specimens from Cuba and the Great Bahama Bank forming separate clades, representing indigenous populations and those in the LBB and Florida representing recent, human- mediated colonizations.

Methods

Collection of Specimens and Tissue Extraction

I obtained tissue samples from specimens of O. septentrionalis captured by hand on the Bahamian islands of Andros (GBB), Eleuthera (GBB), Long Island (GBB), Abaco

(LBB), and mainland Florida (Figures 2-3, 2-4, 2-5; Table 2-1). Frogs from Gainesville,

Florida were also captured using PVC pipe refugia. Using ground-placed PVC pipes is an easy, inexpensive, and effective method to capture treefrogs (Zacharow et al., 2003).

Multiple species were identified in the PVC with Hyla cinerea and H. squirella outnumbering Osteopilus. Frogs were shaken from the PVC into plastic bags for proper identification. A large wooden dowel was used to “plunge” frogs from pipes when they were unable to be shaken out. Native species were placed back into their associated pipes. Captured Cuban treefrogs were euthanized using MS-222 following guidelines approved by the UF animal committee (IACUC Protocol #201709995). Tissue samples

(liver and skeletal muscle) were collected from euthanized frogs and stored in RNAlater storage buffer (Qiagen, USA). Individuals were given an ID, weighed, sexed, and SVL

measurements were taken. Initially frogs were externally inspected for dark/rough nuptial pads or visible eggs. Individuals were also internally sexed based on gonad location and descriptions following Duellman and Trueb (1994). Skeletal specimens were prepared at the Special Projects Lab at The University Florida, and deposited in the collection of the Florida Museum of Natural History. Tissues from Cuba were obtained on loan from the Smithsonian (NMNH).

Mitochondrial DNA Extraction and PCR

Genomic DNA was extracted from each sample using QIAGEN DNeasy Blood and

Tissue Kit following the manufacturer’s standard protocol for DNA isolation using liver tissue. Nucleotide sequences of the mitochondrial gene 16s r RNA, were obtained from

146 specimens of O. septentrionalis. I used PCR to amplify a 572 base-pair segment of the mitochondrial 16s rRNA gene for all samples. Primers 16sa-l 5’-

CGCCTGTTTATCAAAAACAT-3’ and 16sb-h 5’-CCGGTCTGAACTCAGATCACGT-3’ were used in each PCR reaction. The 16s rRNA gene was amplified for all samples, using 35 cycles and the oligonucleotide primers 16sa-l and 16sb-h (Palumbi et al., 1991). PCR amplification methods of the 16s rRNA are described in the steps listed below. An initial denaturing step at 95°C (10 min) was followed by 35 cycles of denaturing at 96°C (3 s), annealing at 54°C and extending at 68°C (15 s). A final extension at 72°C (10 s) completed the cycle.

I visually inspected all mtDNA sequences for errors, then trimmed, and aligned using Geneious R9. I used IQ-TREE multicore version 1.6.12 to find the best-fit nucleotide substitution model (TIM2e+G4) and to infer a phylogenetic tree using a maximum likelihood (ML) approach with 1,000 ML bootstrap replicates used to assess nodal support. I used Arlequin 3.5.2.2 to evaluate summary statistics including number

of polymorphic sites and nucleotide diversity. In order to determine the level of population structure within and among defined groups, an analysis of molecular variance (AMOVA) was implemented on mtDNA. Population groups were defined as

Cuba, LBB, GBB, and Florida. I used PopART 1.7 to create a TCS haplotype network based on statistical parsimony. Because estimating genealogical relationships at the population level presents many challenges, it is unwise to interpret the haplotype network as a reconstruction of evolutionary history (Clement et al., 2000).

Results

Phylogenetic Analysis

Examination of Osteopilus septentrionalis mtDNA revealed a total of 21 unique haplotypes. Levels of nucleotide diversity and mean number of pairwise differences at the intra-population level are shown in Table 2-5. I used IQ-TREE multicore version

1.6.12 to infer a haplotype tree under the best-fit model using a maximum likelihood approach with 1,000 ML boostrap iterations to assess nodal support (Figure 2-6).

Osteopilus crucialis was included as the outgroup in analysis. Within the O. septentrionalis clade, there is phylogenetic structuring between Great Bahama Bank populations with the exception of two North Florida individuals. A stronger phylogenetic relationship is seen between purported ancestral populations in Cuba and younger populations in Florida and the LBB. With the caveat of only having four Cuban samples, my tree assumes that this is a true representation of Cuba’s population. However, I cannot dismiss the possibility of reintroductions to Cuba from non-native populations.

Population Genetic Analysis

I used Arlequin 3.5.2.2 to analyze population diversity based on proportion of differentiation. Population pairwise FST values were overall high between populations

(Table 2-2). Mean pairwise differences were highest in Andros (6.3% +/- 3.1%), and

Florida populations (5.1% +/- 2.5%). A matrix of significant FST P values showed high differentiation between populations except for Eleuthera vs. Long Island and Eleuthera vs. Abaco. The highest differentiation was seen between Cuba and islands of the GBB

(Andros; FST = 0.83, Eleuthera; FST = 0.86, Long Island; FST = 0.87). An AMOVA test was run using 1,000 permutations to assess percentage of variation among groups and among populations within groups. As noted earlier, populations were defined as Cuba,

Bahamas, and Florida. Among-group percentage of variation was significant (44.39%) as well as among-populations within groups (6.67%). Total locus expected heterozygosity indicated a mean of 0.24 and a standard deviation of 0.21. I ran an additional AMOVA comparing sample populations in the Bahamas (Abaco, Andros,

Eleuthera, Long Island) (n=43) to Cuban populations (n=4). Among-group percentage of variation values also were high but not statistically significant (58.5%, P = 0.2+-0.01).

Percentage of variation among populations within groups, however, was significant

(10.2%, P = 0.00098). Population average pairwise differences were surprisingly highest between Andros and Cuba at 2% (Table 2-3). Garg and Biju (2016) considered minimum 3% divergence value as reliable for delineating candidate species, which implies genetic divergence between these populations is rather significant. Populations were close to 2% between Eleuthera and Cuba, Long Island and Cuba, and Florida and

Andros. Molecular diversity indexes including number of transitions, transversions, polymorphic sites and averages are shown in Table 2-4.

Haplotype Network Reconstruction and Analysis

The total dataset consisted of 147 individual sequences of 572 bp from the 16s rRNA gene. I used PopART 1.7 to create a haplotype network of unique sequences

(Figure 2-7). Examination of mtDNA revealed 21 unique haplotypes for O. septentrionalis (Table 2-6). All four Cuban samples displayed unique haplotypes, however they were more nested with individuals from Florida and the Little Bahama

Bank. Andros(GBB) was unique as well, not sharing haplotypes with any other population. Unique haplotypes also were seen in Abaco and Florida samples. The

Cuban samples, surprisingly clustered more closely with individuals from not only

Florida, but Abaco (LBB) as well. Furthermore, every sample from the GBB (Andros,

Eleuthera, Long) clustered together, with the addition of some from Abaco.

Discussion

The goal of this chapter was to evaluate the population genetics of Osteopilus septentrionalis throughout the Bahamas, Florida, and Cuba using the16s rRNA gene. I predicted higher structure between Cuba and the GBB, and higher structure between

Florida and the LBB according to not only fossil evidence, but also the physical and biogeographical history of the Bahamas. The structure of my haplotype network however, appears to contradict my hypothesis pertaining to possible relatedness between defined populations. Certain caveats need to be considered when interpreting haplotype networks. The TCS method uses a specific algorithm and calculates the number of mutational steps by which pairwise haplotypes differ, computing the probability of parsimony for pairwise differences until the probability exceeds 0.95

(Clement et al., 2000). Unique haplotypes from Cuba and Andros indicate the possibility of several ancestral populations. However, although four unique haplotypes only seen in specimens from Florida make it difficult to state the source for these individuals. Again, since these networks do not infer ancestral haplotypes, it is difficult to interpret with confidence the mechanisms driving these relationships. While the amount of variability

is surprising, introgression seems to be apparent. Gene flow and repeated backcrossing between populations seems to be occurring.

Population pairwise FST values were highest among Cuban samples, and all GBB samples (Andros (0.83239), Eleuthera (0.86344), and Long Island (0.86575). Again, this pattern contradicts predicted relationships among populations. What this seems to indicate is lack of much contemporary gene flow between Cuba and islands of the GBB.

Subsequent sea level rise has drastically increased distances between Cuba and the

Bahamas making it more difficult for the species to disperse between the two islands across open sea. Also, as indicated by Barbour (1931), the probability of Osteopilus introduction from Cuba was vastly greater when cargo and passenger ferries traveled between Florida and Havana daily. Although ancestral populations should exhibit higher levels of genetic diversity, invasive populations can actually become more genetically variable enhancing their invasiveness. Also, introduced populations that are widespread and have the ability to transport themselves easily through human facilitation can become more genetically diverse with continuous introductions of individuals from different areas (Kolbe et al., 2004). This could be what is driving the pattern seen in the haplotype network. Four unique haplotypes were detected only in Florida, which presents the possibility of multiple introductions and admixture within the peninsula.

While Osteopilus septentrionalis in Florida is said to have descended from at least two Cuban sources (Heinicke et al., 2011), we cannot discount the possibility that some Florida populations also originated in the Bahamas. Sequences from the GBB are clustered with GBB, LBB, and Florida individuals even though all individuals from

Andros possess unique haplotypes. With Pleistocene fossils from the GBB indicative of

more ancestral populations, results support the notion that O. septentrionalis is native to the Great Bahama Bank. While fossils from the LBB do not present evidence of an older colonization, current genetics support introgression concurrent with gene flow between populations. While this is not surprising, the amount of phylogenetic structure is unexpected. Although population-level processes such as founder effects and population bottlenecks are thought to decrease overall genetic diversity, admixture from multiple native-range sources could in turn increase overall genetic diversity and potentially phenotypic diversity if selective pressures are high enough (Kolbe et al.,

2007).

Table 2-1. Locality of modern specimens of Osteopilus septentrionalis collected for this study. ID Field Number Locality JR 11 0163 Gainesville, Florida JR 12 0164 Gainesville, Florida JR 13 0165 Gainesville, Florida JR 14 0166 Gainesville, Florida JR 15 0167 Gainesville, Florida JR 16 0168 Gainesville, Florida JR 17 0169 Gainesville, Florida JR 18 0170 Gainesville, Florida JR 19 0171 Gainesville, Florida JR 20 0172 Gainesville, Florida JR 21 0173 Gainesville, Florida JR 22 0174 Gainesville, Florida JR 23 0175 Gainesville, Florida JR 24 0176 Gainesville, Florida JR25 0380 Central Eleuthera, Bahamas JR26 0381 Central Eleuthera, Bahamas JR27 0382 Central Eleuthera, Bahamas JR28 0383 Central Eleuthera, Bahamas JR29 0384 Central Eleuthera, Bahamas JR30 0385 Central Eleuthera, Bahamas JR31 0386 Central Eleuthera, Bahamas JR32 0387 Central Eleuthera, Bahamas JR33 0388 Central Eleuthera, Bahamas JR34 0389 Central Eleuthera, Bahamas JR35 0390 Long Island, Bahamas JR36 0391 Long Island, Bahamas JR37 0392 Long Island, Bahamas JR38 0393 Long Island, Bahamas JR39 0394 Long Island, Bahamas JR40 0395 Long Island, Bahamas JR41 0396 Long Island, Bahamas JR42 0397 Long Island, Bahamas JR43 0398 Long Island, Bahamas JR44 0399 Long Island, Bahamas JR45 0400 Gainesville, Florida JR46 0401 Gainesville, Florida JR47 0402 Gainesville, Florida JR48 UF 184686 Highlands Hammock State Park, Sebring, Florida JR49 UF 184685 Highlands Hammock State Park, Sebring, Florida JR50 UF 184789 224 SW 40 Street, Gainesville Florida JR51 UF 184790 225 SW 40 Street, Gainesville Florida JR 52 0403 Gainesville, Florida JR 53 0404 Gainesville, Florida JR 54 UF-H-184802 Gainesville, Florida JR 55 0405 Gainesville, Florida JR 56 0406 Gainesville, Florida JR 57 0407 Gainesville, Florida JR 58 0408 Gainesville, Florida JR 59 0409 Gainesville, Florida JR 60 0410 Gainesville, Florida JR 61 0411 Gainesville, Florida JR 62 0412 Gainesville, Florida JR 63 0413 Gainesville, Florida JR 64 0414 Gainesville, Florida JR 65 0415 Gainesville, Florida JR 66 0416 Gainesville, Florida JR 67 0417 Gainesville, Florida JR 68 0418 Gainesville, Florida

Table 2-1. Continued ID Field Number Locality JR 69 0419 Gainesville, Florida JR 70 0420 Gainesville, Florida JR 71 0421 Gainesville, Florida JR 72 0422 Gainesville, Florida JR 73 0423 Gainesville, Florida JR 74 0424 Gainesville, Florida JR 75 0425 Gainesville, Florida JR 76 0426 Gainesville, Florida JR 77 0427 Gainesville, Florida JR 78 0428 Gainesville, Florida JR 79 0429 Gainesville, Florida JR 80 0430 Gainesville, Florida JR 81 0871 Gainesville, Florida JR 82 0872 Gainesville, Florida JR 83 0873 Gainesville, Florida JR 84 0874 Gainesville, Florida JR 85 0875 Gainesville, Florida JR 86 0876 Gainesville, Florida JR 87 0877 Gainesville, Florida JR 88 0878 Gainesville, Florida JR 89 0879 Gainesville, Florida JR 90 0880 Gainesville, Florida JR 91 0881 Gainesville, Florida JR 92 0882 Gainesville, Florida JR 93 0883 Gainesville, Florida JR 94 0884 Gainesville, Florida JR 95 0885 Gainesville, Florida JR 96 0886 Gainesville, Florida JR 97 UF 185151 Gainesville, Florida JR 98/DWS-AB-1 UF 185141 Abaco, Bahamas JR 99/DWS-AB-3 UF 185142 Abaco, Bahamas JR 100/DWS-AB-4 UF 185143 Abaco, Bahamas JR 101/DWS-AB-5 UF 185144 Abaco, Bahamas JR 102/DWS-AB-6 UF 185145 Abaco, Bahamas JR 103/DWS-AB-7 UF 185146 Abaco, Bahamas JR 104/DWS-AB-8 UF 185147 Abaco, Bahamas JR 105/DWS-AB-9 UF 185148 Abaco, Bahamas JR 106/DWS-AB-10 UF 185149 Abaco, Bahamas JR 107 0887 Gainesville, Florida JR 108 0888 Gainesville, Florida JR 109 0889 Gainesville, Florida JR 110 0890 Gainesville, Florida JR 111 0891 Gainesville, Florida JR 112 0892 Gainesville, Florida JR 113 0893 Gainesville, Florida JR 114 0894 Gainesville, Florida JR 115 0895 Gainesville, Florida JR 116 0896 Gainesville, Florida JR 117 0897 Gainesville, Florida JR 118 0898 Gainesville, Florida JR 119 0899 Gainesville, Florida JR 120 0900 Gainesville, Florida JR 121 01137 Gainesville, Florida JR 122 01138 Gainesville, Florida JR 123 01139 Gainesville, Florida JR 124 01140 Gainesville, Florida JR 125 01141 Gainesville, Florida JR 126 01142 Gainesville, Florida JR 127 01143 Gainesville, Florida JR 128 01127 NW 118th Dr, Gainesville, FL 32606

Table 2-1. Continued ID Field Number Locality JR 129 01125 NW 118th Dr, Gainesville, FL 32607 JR 130 01128 NW 118th Dr, Gainesville, FL 32608 JR 131 01129 NW 118th Dr, Gainesville, FL 32609 JR 132 01136 NW 118th Dr, Gainesville, FL 32610 JR 133 01124 NW 118th Dr, Gainesville, FL 32611 JR 134 01120 NW 118th Dr, Gainesville, FL 32612 JR 135 01126 NW 118th Dr, Gainesville, FL 32613 JR 136 01135 NW 118th Dr, Gainesville, FL 32614 JR 137 01130 NW 118th Dr, Gainesville, FL 32615 JR 138 01122 2801 NW 23rd Blvd, Gainesville, FL 32605 JR 139 01123 2802 NW 23rd Blvd, Gainesville, FL 32605 JR 140 01133 2803 NW 23rd Blvd, Gainesville, FL 32605 JR 141 01121 2804 NW 23rd Blvd, Gainesville, FL 32605 JR 142 01194 Palm beach, Florida JR 143 01195 Palm beach, Florida JR 144 01196 Palm beach, Florida JR 145 01197 Palm beach, Florida JR 146 01198 Palm beach, Florida JR 147 01199 Palm beach, Florida JR 148 01200 Palm beach, Florida JR 149 01201 Palm beach, Florida JR 150 01202 Palm beach, Florida JR 151 01203 Miami-Dade, Florida JR 152 01204 Miami-Dade, Florida JR 153 01205 428 NW 96th way Gainesville Florida, 32607 JR 154 USNM 497935 Matanzas cuba JR 156 USNM 317830 Guantánamo JR 157 USNM 317831 Guantánamo JR 158 USNM 317832 Guantánamo JR 161 UF 168852 Abaco Islands, Great Abaco Island, Marsh Harbour, Regattas of Abaco JR 162 UF 168853 Abaco Islands, Great Abaco Island, Marsh Harbour, Regattas of Abaco JR 163 UF168854 Abaco Islands, Great Abaco Island, Marsh Harbour, Regattas of Abaco JR 164 UF 168864 Abaco Islands, Great Abaco Island, Gilpin Point JR 165 UF 168874 Abaco Islands, Great Abaco Island, Marsh Harbour, Regattas of Abaco JR 166 UF 168875 Abaco Islands, Great Abaco Island, Marsh Harbour, Regattas of Abaco JR 167 UF 168876 Abaco Islands, Great Abaco Island, Marsh Harbour, Regattas of Abaco

Table 2-2. Matrix of pairwise FST values comparing genetic differences between populations. Cuba Abaco Andros Eleuthera Long Island Florida Cuba - Abaco 0.34126 - Andros 0.83239 0.27073 - Eleuthera 0.86344 0.19959 0.27160 - Long Island 0.86575 0.22244 0.26117 0.15067 - Florida 0.31779 0.27948 0.59033 0.55596 0.57730 -

Table 2-3. Average number of pairwise differences between population (above diagonal) and corrected average pairwise difference (below diagonal) Cuba Abaco Andros Eleuthera Long Island Florida Cuba - 0.0146 0.0205 0.0181 0.0194 0.0135 Abaco 0.0058 - 0.0101 0.0084 0.0090 0.0138 Andros 0.0167 0.0031 - 0.0032 0.0031 0.0182 Eleuthera 0.0149 0.0020 0.0009 - 0.0019 0.0159 Long Island 0.0163 0.0028 0.0009 0.0003 - 0.0172 Florida 0.0046 0.0035 0.0123 0.0117 0.0120 -

Table 2-4. Molecular Diversity Indexes. Statistics Cuba Abaco Andros Eleuthera Long Island Florida No. of transitions 5 13 3 3 2 17 No. of transversions 0 2 2 0 0 3 No. of substitutions 5 15 5 3 2 20

Table 2-5. Mean number of pairwise differences and nucleotide diversity at the intra- population level for each locality. Location Mean # of pairwise differences Nucleotide diversity Cuba 2.666667 +/- 1.778499 0.004662 +/- 0.003713 Abaco 6.258333 +/- 3.135825 0.010941 +/- 0.006141 Andros 1.688889 +/- 1.077124 0.002953 +/- 0.002130 Eleuthera 0.933333 +/- 0.698088 0.006132 +/- 0.001380 Long Island 0.857143 +/- 0.681989 0.001499 +/- 0.001366 Florida 5.052979 +/- 2.474190 0.008834 +/- 0.004791

Table 2-6. Distribution and number of individuals of the 21 haplotypes in seven populations of O. septentrionalis. Population Haplotype Cuba Abaco Andros Eleuthera Long Island Florida A - - - - 3 B - 4 - - - - C - - - - - 7 D - - - - - 1 E - - - - - 1 F 1 - - - - - G 1 - - - - - H 1 - - - - - I - 1 - - - 29 J 1 - - - - - K - - - - - 1 L - 2 - - - 54 M - 1 - 4 - - N - - - 1 3 2 O - - - 1 1 - P - - 1 - - - Q - - 4 - - - R - - 1 - - - S - - 1 - - - T - - 3 - - - U - 8 - 4 3 1

FigureFig. 1 .2 M-1a.p Mapshow ishowingng the sep thearat iseparationons between between Bahamian Bahamian banks, Cub abanks, and Florida.

Sawmill sink Ralph’s cave

Fig. 3. Map of Abaco (LBB) showing the caves where anuran fossils Figurewer 2e -f2o.u nMapd. of Abaco (LBB) showing the caves where anuran fossils were found.

Nathan’s Lodge

Fig. 4. Map of Andros (GBB) indicating where Osteopilus Figure 2-3. Map of Andros (GBB) indicating where Osteopilus septentrionalis septenspecimenstrionalis s pwereecim collectedens wer.e collected.

The Sunset Cove

Pascal’s Oceanfront Leisure Lee Restaurant house

Fig. 5. Map of Eleuthera (GBB) indicating where Osteopilus Figurese p2-t4.en tMaprion aofli sEleuthera specime n(GBB)s wer eindicating collecte dw. here Osteopilus septentrionalis specimens were collected.

Palmirage

FigureFig .2 6-5.. M Mapap o fof L oEleutherang Island (GBB)(GBB) iindicatingndicating wwherehere OsteopilusOsteopilus septentrionalis septenspecimenstrionalis sp wereecim ecollectedns were .c ollected.

Figure 2-6. Haplotype tree inferred from all Osteopilus septentrionalis samples (n=146) in IQ-Tree 1.6.12; Osteopilus vastus (GenBank: AY843713.1), Osteopilus dominicensis (GenBank: AY843711.1), Osteopilus crucialis (GenBank: AY843710.1) were used as outgroups. Numbers in parenthesis are the number of individuals from a given population that have that haplotype. Values above and to the left of the nodes represent maximum likelihood bootstrap scores (from IQ-Tree) using 1000 iterations.

Figure 2-7. Haplotype network of O. septentrionalis 16s sequences from Florida, Cuba, and the Bahamas. Each circle represents a haplotype; size of the circle is proportional to number of individuals (larger size, more individuals). Mutations are indicated by hatch marks, and black dots represent inferred intermediate haplotypes.

CHAPTER 3 ANCIENT AND MODERN MORPHOMETRICS OF CUBAN TREEFROGS IN THE BAHAMAS AND FLORIDA

Introduction

Skull and pelvic bones are typically used when identifying species of frogs, with the ilium often being the most common diagnostic element (Georgiev 2008). A combination of osteological characters from both the ilium and humerus are useful in genus-level identifications. Despite clear sexual dimorphism in modern specimens of

Osteopilus septentrionalis, it is difficult and often impossible to assign a sex to fragmentary fossils. Unless there are clear structural differences in osteology, whether individual elements came from males or females presents a challenge. However, based on statistical analysis we can see whether the size distribution of these frogs was different in the past. I examined osteological data to determine if morphometric changes occurred between ancient and modern specimens of Cuban Treefrogs within the

Bahamas (changes over time in the same locality) or if changes occurred within modern frogs between the Bahamas and Florida (changes occurring in modern times after new invasions). Exploring morphology can be useful for understanding evolutionary processes, such as how new functions can arise as the result of changes in size or habitat (Koehl, 1996). Based on measurements of the fossil ilia and humeri, one can infer whether there is a difference between mainland and island populations. Another purported native frog, Eleutherodactylus planirostris, is a small to medium-sized neotropical frog said to be native to Cuba and the Bahamas as well. Fossil evidence has not been available to validate this claim until now. Eleutherodactylus fossil ilia were also found in Long Island in the same context as the Osteopilus fossils (Figure 3-1).

Stochastic processes have the ability to facilitate changes in morphology. Although

male and female Osteopilus display sexual-size dimorphism, It is clear based on visual inspection, that fossil ilia and humeri exhibit a large amount of variance compared to extant Osteopilus. I believe this will also be reflected in statistical analysis.

Methods

Sampling and analysis: Most specimens captured in the Bahamas and Florida for genetic analyses were skeletonized for the purpose of morphometric analysis. Methods of collection, euthanasia (following guidelines approved by the UF animal ethics committee (IACUC Protocol #201709995), and storage are described in Chapter 2. I measured 133 modern frog specimens with corresponding ilia and humeri along with

122 ilia and 304 humeri from the fossil collections from Long Island, Andros, Great

Exuma and Abaco. In order to ensure that all fossil samples were Osteopilus, comparisons of ilia and humeri were made using Rana grylio and Eleutherodactylus planirostris, which are known in the Bahamas. I used the following distinctive features in my morphometric analysis of Osteopilus skeletal elements. ilium (nomenclature from

Turazzini et al. 2016, Bever 2005): 1, height of pars ascendens; 2, height of acetabular fossa; and 3, greatest height of ilial shaft (Figure 3-3). For humeri (nomenclature from

Delfino 2017): 1, greatest height of humeral head; 2, widest point of distal end; 3, width of humeral shaft (Figure 3-4). I measured elements with an ocular micrometer and took measurements twice in order to minimize error. I performed principal component analysis (PCA) in order to evaluate the variation within the dataset. Next, I performed a linear discriminant analysis (LDA) not only to validate the results of my PCA, but also to reduce dimensionality while preserving as much of the class discrimination information as possible. Linear discriminant analysis finds linear combinations of parameters in our dataset that give the best possible separation between different groups.

Results

Morphometric analysis: My multivariate statistical analysis included modern specimens from Florida, and the Bahamas (Abaco, Andros, Eleuthera, Long Island), and fossil elements from Abaco, Andros, Exuma, and Long Island. I made multiple comparisons were made using a principal component analysis (PCA) and linear discriminant analysis (LDA) based on the above measurements.

The first comparison incuded modern specimens from the LBB, GBB, and

Florida (Figure 3-5). This comparison addressed the following question: Did Cuban treefrogs respond differently to an island human-mediated colonization versus a mainland, human-mediated colonization? Snout-vent length and sex of all individuals was measured for this data set. Including SVL into the analysis, the first principal component (PC1) explained 94% of the total variance. Removing SVL as a contributing variable changed the output, although it did not change the results. LDA analysis, which creates a histogram of the distribution of each measurement category per locality, shows how much these distributions overlap. Linear discriminant analysis confirms the results from my PCA supporting the idea of introgression and connectivity.

Morphologically, these populations cannot be teased apart. A box plot (Figure 3-6) further compliments the trend of extreme sexual dimorphism within Osteopilus septentrionalis. Although female sample size was double that of males, females seem to be driving most of the spread, whereas males are more constrained in size. An independent 2-group t-test run on male versus female data further supported this dimorphism, with males significantly smaller than females, as expected (P > 3.59e-07,

M mean 48.3, n = 41, F mean 59.2, n =79).

Within-bank comparisons to evaluate both fossil and modern elements address the following questions: Have frogs changed in size and shape from the Pleistocene to the present in the GBB? Have frogs changed in size and shape from the foundation populations in the late Holocene to present in the LBB? Although analysis of fossil and modern elements from the LBB yielded insignificant results within both PCA and LDA, differences were mainly attributed to the acetabular fossa and shaft (87.3% of total variation in PCA). However, analysis of fossil and modern ilia from GBB showed slight separation between fossil ilia from Long Island, and modern specimens from the island of Andros (Figure 3-7). Principal component analysis revealed that PC1 (89.4% of total variation) describes differences mainly attributed to the acetabular fossa and shaft.

Proportion of trace in LDA is the percentage of separation achieved by each discriminant function. First linear discriminant (LD1) explained 98.8% of the variance in the dataset. A histogram of my results indicates a similar trend with fossil ilia from Long

Island (and the Exumas) being larger than any of the modern samples (Figure 3-8). This suggests that individuals from Andros may have arrived from a subset of a possibly different population.

Between-bank comparisons looked at morphometric data between the LBB and

GBB, addressing the following question: Were the founding populations in the LBB more similar to the Pleistocene GBB frogs? Results of PCA and LDA were insignificant, indicating overlap between individuals from the LBB and GBB. Although populations appear to be inseparable morphologically, I also take into consideration relative available sample size between banks. Both ilial and humeral comparisons compliment

the theory that morphological changes within a species not only take a long time, but also may involve processes such as bottlenecks and founder events.

Discussion

Analyses of the potential morphometric variation in Osteopilus septetrionalis populations did not concur with original predictions. Smaller sample sizes in modern

Bahamian specimens potentially were not true representatives of the total variation in O. septentrionalis. Fossil elements were clearly larger than modern samples included in this study. Mean SVL in Florida ranged from 29 to 93 mm, while pooled data from the modern Bahamas samples ranged from 34-84 mm. Principal component analysis on size-adjusted data indicated differences were equally variable among linear measurements. Results still showed extensive overlap between samples with rotation values being very similar; features seemed to be equally contributing to the little variation seen.

Extreme sexual size dimorphism is clear in this species and is typical for many other anuran species (Monnet and Cherry 2002). Although male and female O. septentrionalis do not appear to be structurally different (in terms of ilia and humeri only) other than in SVL, the Long Island fossils include individuals that clearly differ in other features that were not included in the analysis (Figure 2-14). Since analyses such as

PCA require all features to be present in individuals, only elements that contained all three measurements were included, thus excluding a large subset of the fossil data.

Data imput on missing SVL and gender data were not possible simply because we did not have a large enough sample size from our modern specimen dataset.

Exploratory analyses such as these make it possible to infer relationships, and analyses such as PCA and LDA can tell us only part of the story. Further analysis is

needed to accurately explain intraspecific differences seen in O. septentrionalis. Three- dimensional geometric morphometric analysis of fossil elements could potentially display greater discrimination among samples. This species is the only member of

Osteopilus reported in the Bahamas and it is difficult to truly describe species from fragmentary fossils unless clear diagnostic differences are seen. Although I was unable to distinguish clear separations between populations, it is evident that the intraspecific variation needs to be further examined (Figure 3-2). Again, there appears to be some phylogenetic structure within O. septentrionalis (see Chapter 2) that could potentially help explain patterns seen. And while morphometric and molecular trends don’t necessarily mirror one another, deeper investigation into Bahamian anuran fossils may help better understand the history of O. septentrionalis in the Bahamas.

While there is a significant fossil record to examine from the Bahamas, comparable samples from Cuba are lacking. I cannot dismiss the fact that Osteopilus fossils from the Bahamas could include species not yet described. Without the addition of Cuban fossils, it is even more difficult to predict the origin of O. septentrionalis. Only the ilium and humerus were included in my morphometric comparisons, but additional associated anuran fossils are available to compliment data for further analysis. It is rare to have such a robust data set for fossil elements like Long Island, and even more uncommon to have fossil elements from multiple sites to include. Fossils discovered by

David Steadman present a unique opportunity to gain a deeper understanding into a species that has become a very successful invader because of its high plasticity and resilience.

10 mm

Figure 3-1. Eleutherodactylus fossil ilium from the Great Bahama Bank, Cuba and Florida.

Figure 3-2. Intraspecific variation between fossil ilium from the Bahamas (left) and modern Osteopilus (right).

Figure 3-3. Selected ilial characters measured in this study. A) Height of pars ascendens. B) Height of acetabular fossa. C) Widest width of shaft.

Figure 3-4. Selected humeral characters measured in this study. A) Height of radial condyle. B) Distal width. C) Width of shaft.

Figure 3-5. PCA of modern Osteopilus with complimentary ilium and humerus; all 6 measurements are included reflecting PC1 and PC2 representing the highest proportion of variance.

Figure 3-6. Box plot of PCA plotting male vs female modern Osteopilus septentrionalis. Females on the left indicate a higher variation, where males appear to be much more restricted in size. Individual samples are indicated by black dots.

Figure 3-7. PCA of both modern and fossil ilia of Osteopilus from the Great Bahama Bank. PC1 and PC2 representing the highest proportion of variance.

. 0

−6 −4 −2 0 2

group Andros_GBB_Mdrn

. 0

−6 −4 −2 0 2

group Eleuthera_GBB_Mdrn

. 0

−6 −4 −2 0 2

group Exuma_GBB_Fos

. 0

−6 −4 −2 0 2

group Long_GBB_Fos

. 0

−6 −4 −2 0 2 group Long_GBB_Mdrn

Figure 3-8. LDA histogram of modern and fossil GBB ilia showing the distribution of each measurement per locality.

CHAPTER 4 DISCUSSION AND FUTURE DIRECTIONS

Molecular analysis allowed me to infer biogeographical patterns associated with gene flow between populations using mtDNA from Osteopilus septentrionalis.

Presented with the unique opportunity to compliment these data by including a robust data set from fossil elements to compare to modern osteological specimens, I was able to explore potential changes in morphology through time. As mentioned in Heinicke et al. (2011), fossil or historical evidence of O. septentrionalis have always been lacking until Bahamian fossils were discovered recently. By incorporating various molecular and morphometric techniques, I was not only able to compare results, but look at possible relationships with the intent of inferring the most parsimonious story.

For molecular analysis interpretation, it is difficult to explain with confidence the differences in variation reflected by my haplotype network and the ML tree. My objective was to look at population structure within Osteopilus given the very limited number of samples available to use as reference sequence material. Given the challenges faced with permitting and logistics outside of Florida, samples sizes were as evenly distributed as possible, with the understanding that Florida samples are logistically much more feasible to collect. Locations were as dispersed as possible to capture as much variability as possible. Interestingly, my haplotype network revealed unanticipatged relationships. During the LGM, when distances between Cuba and the Bahamas were only ~20 km, it seems likely that frogs were able to disperse on their own to the GBB.

This supports the hypotheis that frogs from the GBB would be more clustered with individuals of Cuba. With the caveat that only four Cuban samples were included, the haplotype network may be missing the true variation within Cuban Osteopilus. Each

sample from Cuba, however, was independently unique among haplotypes, and was more structurally clustered with those from Florida and Abaco (LBB) than with the GBB.

Developing information from a rich fossil site on Long Island allowed me to form hypotheses pertaining to ancient and modern specimens. In exploratory analysis such as these, one can only come up with the best inference of observed patterns. Many factors can contribute to trends that are seen or potentially missed. If we had complete fossils to compare with, results could’ve indicated differences that were not picked up in my analysis. Only three characters per element were chosen due to this limitation. The taxonomic scale by default showed high overlap in analysis because they appear to be too closely related. Again, although changes in morphology can occur in rapidly in shorter periods of time (Stuart et al., 0214) significant processes and pressures are needed for changes in morphology to occur. I also hypothesize ancestral populations derived from Cuba, although I did not have the opportunity to study Cuban fossils of

Osteopilus. Incorporating molecular and morphological samples from the Cayman

Islands in future analyses would also be informative to help help further resolve relationships of Osteopilus in its assumed native range.

Osteopilus septentrionalis is an exceptional species of amphibian that with human assistance, evolved to be an excellent colonizer of islands, and continents.

Fossil elements (ilium, humerus) on average were from larger individuals than in modern specimens. Even though PCA and LDA did not present results that truly differentiate populations, substantial variation in size is evident among elements. Three- dimensional morphometric analyses and additional statistical analysis looking at variation among variables should be pursued in further analyses of Great Bahama Bank

and Little Bahama Bank fossils. Understanding these relationships are essential when trying to make sense of evolutionary patterns.

Osteopilus septenrionalis is a resilient species that has been able to expand its distribution northward toward temperatures much cooler than in its native West Indian range. While morphometric analysis was limited to ilia and humeri, thousands of additional fragmentary and complete fossil elements are waiting to be included in the story of Osteopilus in the Bahamas.

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BIOGRAPHICAL SKETCH

Joshua Alan Ringer received his BS in 2016 from the School of Forest

Resources and Conservation from the University of Florida. He received his Master of

Science in Wildlife Ecology and Conservation from the University of Florida in 2019.

Joshua has been working with the Florida Museum of Natural History and FWRI (Florida

Wildlife Research Institute; FWC) while completing his undergraduate and graduate career. While pursuing his master’s degree in wildlife ecology and conservation, Josh was advised by Dr. Steve A. Johnson, and co-advised by Dr. David W. Steadman. His research focuses on Cuban Treefrog population genetics, and morphology in the

Bahamas, Cuba, and Florida.