Citation
Assessing Critical Thermal Minima to Determine the Thermal Limits of the Invasive Cuban Treefrog (Osteopilus Septentrionalis)

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Title:
Assessing Critical Thermal Minima to Determine the Thermal Limits of the Invasive Cuban Treefrog (Osteopilus Septentrionalis)
Creator:
Simpson, Suzanne E
Place of Publication:
[Gainesville, Fla.]
Florida
Publisher:
University of Florida
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Language:
english
Physical Description:
1 online resource (64 p.)

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Interdisciplinary Ecology
Committee Chair:
Johnson, Steven Albert
Committee Members:
Cameron Devitt, Susan Elizabeth
Walls, Susan Claire
Graduation Date:
5/4/2013

Subjects

Subjects / Keywords:
Acclimatization ( jstor )
Amphibians ( jstor )
Climate change ( jstor )
Climate models ( jstor )
Cold tolerance ( jstor )
Ecology ( jstor )
Frogs ( jstor )
Invasive species ( jstor )
Species ( jstor )
Toads ( jstor )
Interdisciplinary Ecology -- Dissertations, Academic -- UF
change -- climate -- ctmin -- cuban -- ecology -- florida -- invasive -- thermal -- treefrog
City of Gainesville ( local )
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Interdisciplinary Ecology thesis, M.S.

Notes

Abstract:
Invasive species have detrimentally impacted ecosystems worldwide. Amphibian invaders have affected ecological, environmental, and human health by establishing non-native populations in naïve ecosystems. Understanding the physiological barriers to species expansion is essential to predicting the ultimate range distribution of invasive species. The Cuban treefrog (Osteopilus septentrionalis) is an invasive amphibian in Florida with established populations throughout the Peninsula. Their size and predatory nature coupled with high fecundity gives them a competitive advantage over some native treefrogs. In order to inform management decisions to alleviate the impacts of CTFs, more accurate predictions of their potential range expansion are needed. Temperature niche, driven by the thermal tolerance of a species, is an important factor that affects an animal’s ability to extend its current distribution. Understanding the thermal limits of CTFs will aid in forecasting their range limit when the effects of climate change are considered. I used critical thermal minimum (CTMin) tests with and without an acclimation period to determine the temperature at which CTFs lost vital locomotor abilities, thus rendering them vulnerable to predators and environmental stress. Frogs from North Florida had consistently lower CTMins than frogs from Central and South Florida in both the presence and absence of acclimation, indicating an ability to adapt to the cooler temperatures in North Florida and continue their expansion northward and westward. The knowledge gained by testing the CTMin of CTFs will help predict the extent of their impacts on U.S. ecosystems. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (M.S.)--University of Florida, 2013.
Local:
Adviser: Johnson, Steven Albert.
Statement of Responsibility:
by Suzanne E Simpson.

Record Information

Source Institution:
UFRGP
Rights Management:
Copyright Simpson, Suzanne E. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
890156570 ( OCLC )
Classification:
LD1780 2013 ( lcc )

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1 ASSESSING CRITICAL THERMAL MINIMA TO DETERMINE THE THERMAL LIMITS OF THE INVASIVE CUBAN TREEFROG ( OSTEOPILUS SEPTENTRIONALIS ) By SUZANNE ELYSE SIMPSON 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 2013

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2 2013 Suzanne Elyse Simpson

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3 To Maddie: I h

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4 ACKNOWLEDGMENTS First, I want to thank my committee c hair, Dr. Steve Johnson, for his guidance and enthusiasm with my project. This opportunity would not have been possible without his faith in my research. Thanks to Dr s Susan Cameron Devitt and Susan Walls for being valuable committee member s and to Dr. W alls for granting me access to the USGS research facility. My committee members have each played an essential role in my research, and I could not have completed this without them. I am grateful to Dr. Stephen Humphrey for providing me funding through a gr aduate assistantship. Many thanks are also due to Mary Brown and William J. Barichivich of USGS Southeast Ecological Science Center for allowing me to crowd their research areas with my many frogs and for assisting with frog husbandry They were incredibly generous and helpful with their time and ideas. Thanks also to Shane Ruessler and William Hyde for their tutorials on the Fitotron thermal chambers and allowing me to access them whenever I needed I am indebt ed to the USGS SESC for providing me with top notch facilities for my research. Thanks to the Florida Park Service for providing my collection permit #04021210. I am very grateful to those who allowed me to install PVC pipes around their property for Cuban t reefrog collection. Thank you to Liz Golden of Bill Baggs Cape Florida State Park and Rick Magee of English Creek Nature Preserve for their field a ssistance and enthusiasm help in the field and the lab. Thanks to Pete Scalco of Fort Clinch State Park, Meghan Knappe of Pumpkin Hill State Park, Dustin Smith of Zoo Miami, Dr. Jerry Johnston of Santa Fe College and Dr. Ken Langeland for allowing me to install PVC pipes and

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5 collect samples on their land. Last but not least, a big thank you to Emily Leary for lending me her statistical knowledge and navigating me through my first analysis.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 10 ABSTRACT ................................ ................................ ................................ ................... 11 CHAPTER 1 BACKGROUND ON SPECIES INVASIONS, CLIMATE CHAN GE, AND THE CUBAN TREEFROG ................................ ................................ .............................. 13 General Species Invasions and Impacts ................................ ................................ 13 Amphibian Invasions ................................ ................................ ............................... 14 Temperature as a Limitation on Amphibian Distribution ................................ .......... 15 ................................ .............. 16 Testing Thermal Limits of Ectotherms ................................ ................................ ..... 17 Cuban Treefrog Life History ................................ ................................ .................... 17 2 ASSESSING THERMAL LIMITS TO DETERMINE THE RANGE EXP ANSION POTENTIAL OF AN INVASIVE VERTEBRATE ................................ ...................... 19 Introduction ................................ ................................ ................................ ............. 19 The Cuban Treefrog: An Invasive Amphibian ................................ ................... 20 Evolution and Adaptation of Invasive Species ................................ .................. 20 Acclimation Presence as an Experimental Treatment ................................ ...... 21 Thermal Isoclines of Florida ................................ ................................ ............. 22 Objective, Hypotheses, and Predictions ................................ ........................... 23 Objective ................................ ................................ ................................ .... 23 Hypotheses ................................ ................................ ................................ 23 Predictions ................................ ................................ ................................ 23 Methods ................................ ................................ ................................ .................. 23 Specimen Collection and Husbandry ................................ ............................... 23 Sampling Design ................................ ................................ .............................. 24 Testing Critical Thermal Minimum ................................ ................................ .... 25 Critical Thermal Minimum Without Acclimation ................................ ................ 26 Critical Thermal Mimimum With Acclimation ................................ .................... 27 Data Ana lysis ................................ ................................ ................................ ... 27 Correlation between snout vent length, mass, and critical thermal minimum ................................ ................................ ................................ 27 Split plot analysis of variance ................................ ................................ ..... 28

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7 Accounting for weather differences ................................ ............................ 28 Pairwise comparisons of critical thermal minimum values ......................... 29 Results ................................ ................................ ................................ .................... 29 CTMin Trends ................................ ................................ ................................ ... 29 Weather Differences Between Rounds ................................ ............................ 30 Discussion ................................ ................................ ................................ .............. 30 Significance of Acclimation and Location ................................ ......................... 31 Significance of Round ................................ ................................ ...................... 32 Biological Significance of CTMin ................................ ................................ ...... 33 Implications For Cuban Treefrog Invasive Potential ................................ ......... 34 3 FUT URE DIRECTIONS ................................ ................................ .......................... 50 APPENDIX A RAW DATA ................................ ................................ ................................ ............. 53 LIST OF REFERENCES ................................ ................................ ............................... 58 BIOG RAPHICAL SKETCH ................................ ................................ ............................ 64

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8 LIST OF TABLES Table page 2 1 Sampling distribution for each treatment by location ................................ .......... 46 2 2 Correlation analysis for CTMin, mass, and SVL ................................ ................. 46 2 3 CTMin mean, median, and standard deviation values ................................ ........ 46 2 4 Average CTMin for non acclimated frogs by Round ................................ ........... 47 2 5 Average CTMin for acclimated frogs by Round ................................ .................. 47 2 6 ANOVA valu es for split plot analysis ................................ ................................ .. 47 2 7 Means and medians by FAWN Station and Round ................................ ............ 48 2 8 Temperature comparisons between rounds ................................ ....................... 48 2 9 Pairwise comparisons using t tests between locations and treatments. ............. 49 2 10 Pairwise comparisons using t tests between location s and treatments within Round 2. ................................ ................................ ................................ ............. 49

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9 LIST OF FIGURES Figure page 2 1 Confirmed CTF populations and thermal isoclines. ................................ ............ 36 2 2 Map of my field sites and nearby cities ................................ ............................... 37 2 3 Conducting the research experiment. ................................ ................................ 38 2 4 Plot of CTMin values vs. BCI. ................................ ................................ ............. 39 2 5 Boxplot of CTMin means by location ................................ ................................ .. 40 2 6 CTMin means by location and treatment ................................ ............................ 41 2 7 Graph of the studentized residuals vs. predicted values of CTMin data ............. 42 2 8 Graphic representation of statistical split plot d esign ................................ .......... 43 2 9 Boxplots of FAWN temperature data ................................ ................................ .. 44 2 10 Potential CTF dispersal under two climate change scenarios. ........................... 45

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10 LIST OF ABBREVIATION S ANOVA Analysis of variance CTF Cuban treefrog ( Osteopilus septentrionalis) CTM IN Critical thermal minimum FAWN Florida Automated Weather Network FLMNH Florida Museum of Natural History GNV Gainesville, Flo rida IUCN International Union for Conservation of Nature LDI Least difference of interest MIA Miami, Florida NATL Natural Area Teaching Laboratory NOAA NCDC Climate Data Center SERCC Southeast Reg ional Climate Center SVL Snout vent length TPA Tampa, Florida USGS United States Geological Survey YOY Young of year

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11 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirem ents for the Degree of Master of Science ASSESSING CRITICAL T HERMAL MINIMA TO DET ERMINE THE THERMAL L IMITS OF THE INVASIVE CUBA N TREEFROG ( OSTEOPILUS SEPTENTRI ONALIS ) By Suzanne Elyse Simpson May 2013 Chair: Steve A. Johnson Major: Interdisciplinary Ec ology Invasive species have detrimentally impacted ecosystems worldwide. Amphibian invaders have affected ecological environmental, and human health by establishing populations in non native ecosystems. Understanding the physiological barriers to specie s expansion is essential to predicting the ultimate range distribution of invasive species. The Cuban treefrog ( Osteopilus septentrionalis ) is an invasive amphibian in Florida with established populations through out the P eninsula. Their size and predatory nature coupled with high fecundity elicits a competitive advantage over some native treefrogs In order to inform management decisions that alleviate the impacts of CTFs, more accurate predictions of their potential range expansion are needed. Temperature niche, driven by the thermal tolerance of a species, is an important factor that affects an CTFs will aid in forecasting their range limit when the effects of climate change are considered. I used critical thermal minimum (CTMin) tests with and without an acclimation period to determine the temperature at which CTFs lost vital locomotor abilities thus rende ring them vulnerable to predators and environmental stress. Cub an

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12 treefrogs withstood much cooler temperatures than previously reported, and f rogs from North Florida had consistently lower CTMins than frogs from Central and South Florida in both the presence and a bsence of acclimation. These results possibly indicate an ability of CTFs to adjust to the cooler temperatures in North Florida and continue their e xpansion northward and westward

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13 CHAPTER 1 BACKGROUND ON SPECIES INVASIONS, CLIMATE CHANGE, AND THE CUBAN TREEFROG General Species Invasions and Impacts Species i nvasions are a worldwide problem with broad economic and ecological impacts (Kalin Arroyo et al. 2000) Species introduced to foreign ecosystems by human activity are considered invasive if they are able to establish a population that causes ecological or environmental harm or harm to human health (Clinton 1999) Not all non native or exotic species are considered invasive; many do not become established or remain localized with lit tle ecological impact (Colautti & MacIsaac 2004) Invasive species are diverse and include plant, animal, fungus, and virus taxa (Lowe et al. 2000) Predicting the likelihood of a species becoming invasive remains a stumbling block for ecologists (Kolar & Lodge 2001) but fecundity, niche specialization, and generation time are all thought to factor into invasive potential (Sakai et al. 2010) Invasive species have had considerable ecological, economic, and human health c onsequences They can catalyze an increase in biomass, alter growth and survivorship of native species, incur significant economic costs, and disrupt human quality of life (Sakai et al. 2010; Ehrenfeld 2010) For example, plant invasions in Florida have regimes (Gordon 1998; Platt & Gottschalk 2001) while black spiny tailed iguanas ( Cte nosaura similis ) have become a nuisance to South Florida homeowners by preying on native plants and damaging landscape vegetation (Krysko et al. 2003) The invasive mosquito Aedes aegypti served as a disease vector responsible for the introduction of yellow and dengue fever in the Americas and Asia (Juliano & Lounibos 2005) and feral pigs ( Sus scrofa ) have damaged native landscapes worldwide, posing a serious

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14 management challenge to landowners and policy makers (Lowe et al. 2000; Campbell & Long 2009) The estimated annual cost incurred by invasive species is nearly $120 billion in the United States alone (Pimentel et al. 2005) Several species of amphibians are known to be invasive in their introduced ranges Some of those were intentionally introduced, like the cane toad ( Rhinella marina [Bufo marinus] ), whereas others, like the African clawed frog ( Xenopus laevis ), were released through the pet trade or human mediated transport (Lobos & Jaksic 2005; Phillips et al. 2007) Controlling and understanding species invasions is a complex but necessary endeavor to preserve the integrity of ecosystems and protect economic and human interests. A mphibian Invasions Invasive amphibians have had significant detrimental impacts worldwide The cane toad ( R. marina ), originally introduced to control insects in sugar cane fields, has become so damaging to the ecosystems and native fauna of Australia that the International Union for Conservation of Nature ( IUCN ) has listed it as one of the 10 0 worst alien species in the world (Lowe et al. 2000) Cane toads have led to morpholog ical changes in native fauna (Phillips 2004; Shine 2011) and continue to persistently establish populations with increasing severity (Phillips et al. 2006) The North American bullfrog ( Lithobates catesbaianus ) is also listed as one of the 100 worst invaders by IUCN and has invaded regions ranging from South America to China (Laufer et al. 2008; Li et al. 2011) Likewise, the coqui frog ( Eleutherodactylus coqui ), nati ve to Puerto Rico, has affected Hawaiian floriculture and tourism (Beard & Pitt 2005) and are also known to alter nutrient cycles (Sin et al. 2007) Coqui frogs emit a disruptive call at night that lowers property values in invaded are as (Kaiser & Burnett

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15 2006) The African clawed frog ( X. laevis ), introduced via exotic pet trade, has spread at an alarmin g rate through regions of Chile, raising concerns about their ability to outcompete native fauna (Lobos & Jaksic 2005) In order to inform decisions about mitigating the impacts of invasive amphibian species, limits to the range expansion of ectotherms must be explored. Ectotherms depend on their environment for their heat source so their thermal physiology should be understood to provide insight of the effect of temperature on their distribution. Temperature as a Limitation on Amphibian Distribution B iotic and abiotic factors both play a role in determining the range limits of species (Mott 2010) Geographic barriers, such as mou ntain ranges and rivers, can preclude a population from establishing beyond a certain border whereas soil quality and rainfall can restrict suitable breeding sites (Wyman 1988; Walther et al. 2002) Habitat fragmentation and climate gaps, or areas of suitable climate interrupted by regions of unsuitable climate present additional barriers for dispers al Competition, predation, and other species interactions can limit invasions and also work synergistically with abiotic factors (Stanton Geddes et al. 2012) Because ectotherms rely on ambient sources for heat rather than internal physiology, thermal gradients a re among the most important abiotic influences in their distribution (Bomford et al. 2008; van Wilgen et al. 2009) Amphibians cannot thrive in environments that exceed their temperature tolerance wit hout access to microclimates that alleviate the effects of excessive heat or cold. However, global climate change is expected to alter thermal isoclines and could affect the distribution limits of ectotherms (Davis 2001)

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16 Climate change has been implicated in contributing to increased extinction rates (Davis 2001; Thomas et al. 2004) as well as catalyzing the range expansion of invasive species (Carroll & Taylor 2003; Walther et al. 2009; Wilso n 2010) The u ltimate ramifications of climate change are still debated among conservation biologists as improved climate m o deling techniques are explored (Davis 2001; Leuzinger et al. 2011) H owever, even the most conservative estimates predict that clim ate change will potentially increase the extinction rate of species by 18% (Thomas et al. 2004) Ecologists remain gravely concerned about the ability of organisms to adapt at a rate consistent with historical temp erature swings (Davis 2001; Walther et al. 2002; Thomas et al. 2004; Sinervo et al. 2010) Anthropogenic climate modification has already affected the ranges of native species and could increase the effects of invasive popula tions on indigenous fauna (Carroll & Taylor 2003; Thomas et al. 2004; Walther et al. 2009) For example, climate change enabled the mountain pine beetl e ( Dendroctonus ponderosae ) to significantly expand its breeding range from 1970 2000 (Carroll & Taylor 2003) D. ponderosae populations are at epidemic levels in British Colum bia and will have more opportunity to expand with an increase in amiable climate. Invasive species that originate in tropical regions gain a particular advantage from the ramifications of climate warming. As the average temperatures increase, tropical ect otherms may expand their range northward and establish populations in previously inhospitable environments (Tewksbury et al. 2008) This expansive potential lends urgency to the task of understanding the thermal lim its of invasive reptiles and amphibians.

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17 Testing Thermal Limits of Ectotherms T o make predictions about the possible range expansion of a population, its thermal limits must be e xplored. Critical thermal minimum (CTMin) is a widely used method for determin ing physiological temperature tolerance in ectotherms (Layne & Romano 1985; John Alder et al. 1988; Doughty 1994; Wilson 2010) Critical thermal minim um is defined as the temperature at which over when placed on its back locomotor abilities become significantly impaired and prevent it from escaping predators, lethal environmental conditions, or performing basic motor tasks, such as perching (Layne & Romano 1985; Doughty 1994) Several biological or geographical characteristics can affect thermal limits. Previous studies suggest that body size and latitudinal gradient d (John Alder et al. 1988; Doughty 1994; Huang et al. 2006; 2007; McGarrity & Johnson 2008; Wilson 2010; Johnson & McGarrity 2011 unpublished data ) While habitat fragmentation and climate gaps can also dictate a species distribution, many invasive amphibians, including the Cuban treefrog ( Osteopilus septentrionalis ), spread by jump dispersal as stowaways on vehicles and cargo shipments thus dimin ishing the impact of other range restrictors (Johnson 2007; Johnson & McGarrity 2011 unpublished data ) Cuban Treefrog Life Histor y The Cuban treefrog ( O septentrionalis ; CTF) is an invasive amphibian that has detrimentally impacted its non native habitat. Cuban treefrogs are native to Cuba, the Cayman islands, and the Bahamas, but have established their range throughout much of Flo rida, exclu ding only the w estern Panhan dle, via jump dispersal (Fig. 1; Meshaka 2001; Johnson 2007) They are the largest treefrog in Flor ida with fe males known to

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18 exceed 6 inches. C uban treefrog s can be distinguished from native frogs by their larger earance, and rough skin (Fig. 2; Meshaka 2001) Their diet includes an array of insects, smaller anurans (including other CTFs), lizards, and small snakes (Glorioso et al. 2012) Cuban t reefrogs have an irritating skin secretion that can adversely affect mucosal membranes and exacerbate asthma or allergies (Johnson 2007) S nake s ar e their primary predator but p redator pressure has never limited their range expansion in Florida (Meshaka 2001) Cuban treefrogs impact native anuran populations through predation and competition (Smith 2005) Thei r large size and vagility enable them to prey upon native species including the green treefrog ( Hyla cinerea ) and squirrel treefrog ( Hyla squirella ) (Waddle et al. 2010) O. septentrionalis tadpoles also reduce growth rates and delay metamorphosis in green treefrog and southern toad ( Bufo terrestris ) larvae (Smith 2005) They are highly fecund; large females may lay more than 15,000 eggs in one breeding season (Meshaka 2001) Although the ultimate CTF range limit is unknown, preliminary research indicates future expansion may exte nd northward to the Florida Panhandle and westward along the Gulf Coast (Rdder & Weinsheimer 2009) However, predicted climatic variation makes it difficult to understand exactly where they will disperse and what climates will be suitable to establis h populations My study focuses on temperature tolerance, one of the key limits to range expansion, and the future directions that must be considered to gain a firm grasp on the limits of the CTF invasion.

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19 CHAPTER 2 ASSESSING THERMAL LIMITS TO DETERMINE TH E RANGE EXPANSION POTENTIAL OF AN INVASIVE VERTEBRATE Introduction limits and potentially informing management decisions. These data are especially useful for understanding t he temperature tolerances of invasive species. The Cuban treefrog ( Osteopilus septentrionalis ) is an invasive amphibian in Florida with established populations through out the Peninsula. Their size and predatory nature coupled with high fecundity gives them a competitive advantage over some native treefrogs and can reduce native populations. In order to inform management decisions to alleviate the impacts of CTFs, more accurate predictions of their potential range expansion are needed Temperature niche dri ven by the thermal tolerance of a species, is one Understanding the thermal limits of CTFs will aid in predicting their range limit when the effects of climate change are considered. I used critical thermal minimum (CTMin) to determine the temperature at which CTFs lose vital locomotor abilities, thus rende ring them vulnerable to predators and environmental stress. Frogs from North Florida had consistently lower CTMins tha n frogs from Central and South Florida in both the presence and absence of acclimation, indicating an ability to adjust to the cooler temperatures in North Florida and continue their expansion northward and westward. The knowledge gained by testing the CTM in of CTFs will help predict the extent of their impacts on U.S. ecosystems.

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20 The Cuban Treefrog : An Invasive Amphibian Cuban treefrogs are native to Cuba, the Bahamas, and the Cayman Islands, but have established invasive populations throughout much of Fl orida (Johnson 2007) Cuban treefrogs were likely introduced as stowaways in Caribbean shipping crates The first published record of CTF presence in Florida was in 1931 in the F lorida Keys (Barbour 1931). By the mid 1970s, CTFs had established populations in most of southern Florida and continued their expansion northward into Jacksonville by the early 2010s. Frogs have been observed traveling on vehicles, which has likely aided in their expansion northward (Johnson, pers. comm.). This invasive amphibian is highly predacious and extremely fecund, enabling it to negatively impact native anuran populations (Rice et al. 2011) Its ability to adjust to new climates might suggest an evolutionary adaptation to its new environment, or the presence of phenotypic plasticity, which differentially expresses a genotype in response to varying environmental conditions. Evolution and Adaptation of Invasive Species There are numerous examples of invasive species successfully ev olving and adapting to their new environments. A broad ecological niche and an ability to adjust to variation are two recurring characteristics of many effective invaders (Crowl et al. 2008) The cane toad invasion in Australia has been studied ex tensively and provides an excellent example of invasive adaptation. The rate of cane toad dispersal has increased since the early 2000s (Phillips et al. 2007; Urban & Phillips 2008) and has been facilitated by the evolution of lo nger legs in newer populations (Phillips et al. 2006) Ca ne toads have also induced evolutionary adaptations in native species. Populations of r ed bellied black snakes ( Pseudechis porphyriacus ) inhabiting areas affected by the

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21 cane toad invasion have displayed a higher toxin tolerance and decreased preference fo r cane toads as prey (Phillips & Shine 2006) This behavior likely has a genetic component since it could not be learned in nave snakes. Cuban treefrog populations could conceivably undergo physiological and genetic ada ptations in their introduced range in Florida similar to cane toads in Australia. As CTF dispersal progresses northward and westward, frogs are experiencing cooler temperatures than those in their native range. In order for expansion to continue, CTFs will have to quickly adapt to this new environment. Adaptations in introduced herpetofauna have been observed in as little as 10 generations (Losos et al. 1997) s o the CTF population in Gainesville, FL that has successfully bred since 2002 (Krysko et al. 2005) could exhibit physiological adjustments to cooler temperatures However, thermal adaptations could be counteracted by gene flow from CTFs inhabiting the southern parts of Florida. Since CTFs are primarily distributed through jump dispersal, frogs from South Florida could frequently migrate to northern populations. If robustness to cold is heritable, then mating between these geographically isolated po pulations of treefrogs could dilute the efficacy of a genetic adaptation. Additionally, CTFs may alleviate the effects cold weather by seek ing refuge in the warmer microclimates of their habitat. I tested for a physiological tolerance to cold that may exis t in CTF populations. To account for the different weather patterns throughout CTF range, I used 3 different field sites that each represented a different type of thermal isocline Acclimation Presence as an Experimental Treatment The ability of animals t o withstand extreme temperatures i s affected by their previous experience with warm or cold weather (Brattstrom & Lawrence 1962; Wilson & Franklin 1999; Wilson 2010) Brief or long term exposures to abnormally low or high

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22 temperatures can induce a compensatory reaction in an organism that affects locomotor ski lls, metabolism, and minimum or maximum temperature thresholds. Acclimation ability can vary along longitudinal gradients, like the northern versus southern range of a species, as well as altitudinal gradients, such as mountain versus lowland populations (Brattstrom 1968; Navas 1997; Huang et al. 2006) I used acclimation as a treatment to determine if previous exposure to cold induce d a physiological response in CTFs. If an acclimation period removes latitudinal differences between CTMins, then a CT response to cold is more likely a result of phenotypic plasticity, rather than adaptation. If differences between locations still exist in the presence of acclimation, then there is evidence for an evolutionary adaptation to cold temperatures in CTFs. Thermal Isoclines of Florida The state of Florida is home to great bio logical and environmental diversity (Whitney et al. 2004) The warm sub tropical climate of South Florida is contrasted with the cooler, slightly drier climate of North Florida and the Panhandle ( South east Regional Climate Center 2012 ). climate can be categorized by the mean number of days between freeze event s (Fig. 2 1). Freezes are rare in southern Florida and increasingly more common in North Florida and the Panhandle The distribution of established C TF populations in Fig. 2 1 coincides with warmer isoclines where freezes are less frequent Frogs have continued to progress northward and westward but have not yet established steady populations where freezes are most common in Florida. This coul d be due to a factor of time, or because CTFs cannot withstand yearly freezes A greater understanding of CTF thermal tolerance will elucidate whether North Florida temperatures are a constraint on their population expansion.

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23 Objective, Hypotheses an d Predictions Objective The objective of my research was to d etermine if CTFs could withstand cooler temperatures than previously reported, and to detect if from North Florida have a significantly lowe r CTM in than those from Central and South Florida in th e presence and absence of an acclimation period. Hypotheses Cuban treefrogs can tolerate cooler temperatures than previously assumed. Cuban treefrogs in North and Central Florida can tolerate colder temperatures in their new environments A previous exposu re to cold will enable a frog to withstand cooler temperatures. Predictions The overall CTMin of Cuban treefrogs will be lower than described in prior studies. Cuban treefrogs in North Florida will have a lower average CTM in than CTFs in Central and South Florida in the presence and absence of an acclimation period Cuban treefrogs in Central Florida will have a lower average CTMin than CTFs in South Florida in the presence and absence of an acclimation period. Frogs in the acclimation treatment group will have a lower average CTMin than those not acclimated. Methods Specimen Collection and Husbandry I chose field sites near the cities of Gainesville, Tampa, and Miami to represent each thermal isocline where CTFs are established (Fig. 2 2) All of these citi es are surrounded by I 75, a major highway in Florida, so they are more vulnerable to CTF invasions via jump d ispersal. Gainesville represented the n orthernmost breeding population of CTFs and Miam i the southernmost. Tampa served as a central location.

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24 Col lection from these sites provided me with CTMin data from key locations of the CTF invasion and afforded significant insight to the progression of cold tolerance in this species. I collected frogs by hand from PVC pipe refugia (Boughton et al. 2000) I cap tured CTFs on three different occasions, referred to as Rounds. Gainesville specimens were taken from pipes at ponds around Santa Fe College and the Natural Area Teaching Laboratory (NATL) on the University of Florida campus. Tampa frogs were collected at English Creek, located about six kilometers southeast of Plant City and Miami frogs were taken from pipes placed in Bill Baggs State Park on Key Biscayne. Collections occurred on 12 September and 2 and 27 October 2012. The majority of captures occurred in Rounds 1 and 2 Round 1 had 65 specimens, Round 2 had 90, and Round 3 had 25. After capture, I transferred the frogs to a plastic zipped bag and transported them in a cooler to the USGS Southeast Ecological Science Center in Gainesville, where I measured their SVL. Frogs were kept in dividually in 32 oz. mason jar s covered with mesh netting at ambient room temp erature with a photoperiod of 12 hours of light and 12 hours of dark. They were given a moist paper towel and fed small to medium sized crickets twic e a week. Towels were watered and changed as needed. Sampling Design I tested 180 frogs, approximately 60 from each location and 90 for each treatment (acclimated vs. not acclimated) Acclimation took place over a period of 48 72 hours at 13C. This sampl ing design allowed 30 replicates of each treatment by location (Gainesville, Tampa, Miami) and exceed ed the minimum sample size recommended by an a prior i power analysis (Table 2 1). Frogs from each region were randomly assigned to testing group CTMin with out acclimation or CTMin with acclimation I used stratified

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25 random sampling to ensure that each testing group contained an equal representation of specimens from each collection site (Gainesville, Tampa, and Miami) Within each treatment frogs were also randomly assigned to a n environmental chamber (A or B) and a level in the testing compartment (1, 2, or 3; Fig. 2 3 ). All randomizations were done using Microsoft Excel or www.random.org (RANDOM.ORG 1998 2012) The t wo environmental chambers were the same make and model, but they were included in the analysis to account for any variations in cooling rate between them. Testing Critical Thermal Minimum I used a common garden experiment to test critical thermal minima in nave frogs in the presence or absence of an acclimation period. T esting occurred at the U nited S tates G eological S urvey Southeast Ecological Science Center (USGS SESC) about a week after capture. T o lend greater assurance that frogs were nave (no prev ious exposure to cold temperatures ) specimens greater than 50 mm SVL were rarely used in the experiment Since a dult CTFs can measure in excess of 100 mm, frogs less than 50 mm SVL are likely young of year ( YOY ; Johnson 2007) Frogs less than 25mm SVL were eliminated from testing to prevent potential fatalities in very young juveniles as a result of overexposure to cold. T esting occurred in a Weiss Gallenkamp Fitotron Plant Growt h Chamber SGC 120 and followed the methods established in Wilson ( 2010 ) and John Alder et al. ( 1988 ) but the environmental chamber was used in place of an ice bath. The chamber had a digital thermometer that indicated the set temperature and the actual te mperature inside the chamber. The actua l temperature, labeled as CTMdis was recorded for the critical thermal minima (see Appendix A for raw data). Food was withheld from individuals for 72 hours prior to trials to remove effects of feeding (W ilson 2010) Frogs were weighed in a zipped bag with a Pesola balance to

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26 the nearest 0.25 g immediately before testing. All specimens were kept at room temperature (23 25 C) when not undergoing acclimation or experimental tests. Room temperature was equiv alent to the average springtime temperatures (April May) in Miami, Tampa, and Gainesville ( FAWN; http://fawn.ifas.ufl.edu/data/reports/) Frogs were teste d in a plastic testin g compartment that held 3 frogs and placed on a moist paper towel as seen in Figu re 2 3c after being placed on its backside in extreme temperatures. To test righting behavior, specimens were briefly removed from the environmental chamber and fl ipped on their back in the testing compartment They were stroked twice on their ventral side with a small brush to stimulate movement. The CTMin was defined as the temperature at which a frog could not right itself after thirty seconds, indicating a loss of viable locomotor abilities (Brattstrom 1968; Layne & Romano 1985) Individuals were only tested once and monitored for 24 hours after trials to check for survival. They were then euthanized by applying a small amount of tricaine mesylate (MS 222) solution to their vent ral side and placed in a freezer. Critical Thermal Minimum Without Acclimation Frogs in the testing compartment were transferred to the environmental chamber, where they were exposed to a temperature of 13C for 10 minutes to gain familiarity with their su rroundings. This temperature represents a common winter temperature at my sample sites ( FAWN; http://fawn.ifas.ufl.edu/data/reports/ ). The temperature was then lowered slowly at approximately 0.17C per minute. P revious studies verify that an nternal temperature changes will mirror those of the ambient temperature

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27 when the rate of change is less than 1C per minute (Layne & Claussen 1987; John Alder et al. 1988) Righting behavior was tested in increments of 2C beginning when the first signs of thermal distress appeare d, indicated by slower movements and decreased ability to right themselves This usually began around 7 9C. The testing increments decreased to every 1C when t hermal distress became profound Frogs were ret urned to their mason jars in the secondary conta inment area post test. Critical Thermal Mimimum With Acclimation Frogs were placed in an environmental chamber held at 13C for 48 72 hours since previous studies have shown the effects of acclimat ion do not change after one day ( Bra ttstrom & Lawrence 1962; Wilson 2010) Acclimation has significantly affected CTM in in CTFs at both 15C and 20C, so an acclimation temperature of 13C ensured that the subjects could elicit a response to the acclimation period (Wilson 2010) The frogs were then tested using the previously described methods, and they were returned to their mason jars in the secondary containment area post test Data Analysis The minimum temperature threshold of the thermal chambers used in this experim ent was 3C. In less than 4% of trials (7 instances), the frog had not yet reached its CTMin when the temperature was set at 3C. These values were recorded as 4C for use in the analysis with the understanding that the CTMin averages may be conservativ e given that some thermal minimums had not yet been reached. Correlation b etween snout vent l ength m ass, and critical thermal m inimum To ensure that any relationships between CTMin and location were not due to size bias, a body condition index (BCI) was c reated using methods established by van Berkum, et. al (1989) and empl oyed in Howard and Young (1998). BCI, defined as

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28 mass 1/3 /SVL, was plotted against CTMin values to determine if there was a significant relationship between the two variables (Fig. 2 4 ; J MP 8.0.2, 2009 ) A correlation analysis for BCI and CTMin was also run in SAS software (SAS 9.3 1989 2007) The results in Figure 2 4 and Table 2 2 indicate that BCI was not significantly cor related to CTMin. This was an expected result because only a li mited size range of frogs was used in the tests. Therefore, body size was not used as a covariate in subsequent analyses. Split plot analysis of variance Boxplots of CTMin means were made using JMP software ( 8.0.2, 2009; Fig. 2 5 and 2 6 ; Tables 2 3, 2 4, and 2 5 ). Before analyzing the data, normality was confirmed using a residual vs. predicted values plot. Fig. 2 7 illustrates that the residuals are randomly distributed around 0, verifying normality of the data. Critical thermal minima data were analyzed using a split plot design with the Chamber (A or B) as a block and the time of testing, or Round (1, 2, or 3), as th e whole plot (Fig. 2 8 ). The two treatments, acclimated vs. not acclimated, and the three locations (GNV, TPA, and MIA) were combined to fo rm six treatment levels comprising a composite treatment variable, l abeled TRT The creation of a composite treatment variable enabled pairwise comparisons between treatments and locations and facilitated the data analysis. An analysis of variance (ANOVA) test was run with SAS software (SAS 9.3, 2012). The blocking variable, Chamber, was treated as a fixed effect and had no significant effect on the CTMin of frogs, so it was not included in pairwise comparisons. Accounting for weather d ifferences To inves tigate the significance of the Round variable, I examined daily minimum temperatures from weather stations near my field sites for 30 days prior to each

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29 specimen collection. Data were collected from the Florida Automated Weather Network (FAWN). The Alachua station was near Gainesville (23 km) the Dover station near Tampa (40 km) and the Homestead station near Miami (55 km). I calculated the means and medians for each Round and conducted pairwise comparisons using t tests between Rounds within each station using a Bonferroni adjustment Pairwise c omparisons of critical thermal minimum v alues Though TRT was not significant overall, I conducted pairwise comparisons for key TRT and Round variables to uncover any significant differences between treatments by lo cation. P values were adjusted with a SAS simulated adjustment for multiple comparisons to avoid rejecting a correct null hypothesis (SAS 9.3, 2012). The SA S simulated adjustment was used to prevent over adjusting p value s, as might occur with the Scheffe and Bonferroni adjustment since only some comparisons are considered, rather than all (Edwards & Berry 1987) Since Round exhibited high significance, I also made comp arisons within Round 2, eliminating any variance that would be a result of comparing between Rounds. Round 2 was chosen because it had the highest overall sample size (n = 90) of any Round. Results CTMin Trends Frogs from Gainesville had the lowest overall CTMin mean/median ( 1.01C / 1.40C ) indicating the highest tolerance for cold, followed by Tampa ( 0.86C / 1.35C ) and Miami ( 0.14 C / 0.4C ; Table 2 3; Figure 2 5 ) Gainesville had the lowest mean and median non ac climated CTMin values ( 0.87C/ 1.25C; Table 2 4) whereas Tampa had the lowest mean and median ac climated CTMin ( 1.18 C/ 1.80C; Table 2 5; Figure 2 6 )

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30 Table 2 6 displays the ANOVA table of the split plot design. Round was significant at = 0.05, but TRT (treatment | loca tion) was not TRT*Round*Chamber was also statistically significant but was not a n interesting interaction for this experiment. Round was not expected to be an important factor, but its significance indicates that the time of testing (September, October, or November) was a key variable in predicting a minimums 30 days before each specimen collection were explored. Comparisons between acclimate d and non acclimated treatments within and between locations were not statistically significant (Table 2 9 ). While the comparisons in Round 2 between GNV/acc vs. MIA/acc and GNV/non vs. MIA/non initially have significant p values (p = 0.0507 and p = 0.1075 ), neither was significant after the SAS simulated adjustment (p = 0.2290 and p = 0.4241). Weather Differences Between Rounds Significant differences were found in Alachua between Rounds 2 & 3 and Rounds 1 & 3, as well as in Dover between Rounds 2 & 3 and Rounds 1 & 3 (Tables 2 7 and 2 8) There were no differences between any Rounds in Homestead nor were differences found comparing Rounds 1 & 2 in Alachua and Dover. Even in the significant comparisons, the difference in minimum temperature means was never more than 7 C. Discussion T hermal minima were considerably lower from those of Gainesville frogs acclimated at 15C had an average CTMin of 7.8 0.8C, and the Tampa frogs averaged a CTMin of 7.7 0.6C. My acclimation temperatu re of 13C was slightly lower, but even non acclimated frogs in my trials had a decrease in CTMin

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31 from those reported just a few years ago. Wilson ( 2010 ) that an ice bath and not a thermal chamber was used for testing, and my sample size was much larger The results of this study indicate that CTFs have a lower thermal tolerance than was previously reported. Even though treatment /location did not have a statistically significant effect on CTMin, some consistent patterns were ev ident Frogs in the acclimation treatment elicited a lower CTMin than non acclimated specimens, with the exception of Tampa in Round 3. Though the differences in CTMin between treatments were not statistically significant, the results are consistent with s tudies that show that previous exposure to (Brattstrom & Lawrence 1962; Brattstrom 1968; Feder 1985; Layne & Claussen 1987; Wilson & Franklin 1999; Wilson 2010) Anoth er trend in the data was that t he overall CTMin means and medians decreased as northward latitude increased, which is consistent with my predictions This indicates that frogs in northern areas of Florida are becoming more adept at resisting cooler tempera tures than their southern counterparts. However, even frogs from Miami populations that have never experienced a freeze were able to consistently withstand freezing temperatures in CTMin tests. These results suggest that CTFs are much more resistant to col d than previously thought. This increased resistance has substantial ramificiations for the management of CTF invasion. Significance of Acclimation and Location The lack of significance for the TRT variable can be attributed to a number of factors. The d ata had high variance and several outliers that had to be included in order to yield p value estimates A fair portion of these outliers were from Gainesville in Round 3, suggesting a high amount of variability in cold tolerance from that location.

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32 This ma y be because Gainesville is the most recently populated area out of any of my sample sites and is less likely to have a population with stable and consistent traits. Some of the frogs I tested could have recently dispersed to Gainesville, whereas others ma y have been the offspring of frogs that had inhabited the area for several generations. The Alachua FAWN weather station also experienced the highest degree of variation before Round 3 frogs were collected which could have affected their expression of col d tolerance (Fig. 2 9 ). Additionally, adaptations to temperature in CTFs may include a degree of plasticity that allows the trait to be profoundly expressed in one animal and less expressed in another. However, despite these variations, Gainesville still h ad the lowest CTMin mean and median, indicating the possibility of an evolutionary adaptation. Comparisons within the Round with the largest sample size, Round 2, revealed a stronger tendency for Gainesville frogs to have a more substantial tolerance for c old than frogs from other locations, particularly after acclimation. This may be the population has onl y reproduced for about 10 generations (Krysko et al. 2005 ) Repeating this experiment in about 5 10 more years may yield more stark contrasts between locations. L atitude does not appear to have a biologically significant effect on the tolerance of CTFs to cold w eather. However, this may be a premature conclusi on based on the consistent ability of Gainesville frogs to tolerate cold better than Tampa or Miami frogs in Rounds 1 and 2 where sample sizes were greatest (n = 155) Significance of Round Round proved to be the most significant predictor of CTMin in m y experiment, which was an unexpected result. This reveals that the time of year Cuban treefrogs are

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33 tested can significantly alter the values of their temperature tolerance The pairwise comparisons between the FAWN data revealed sigificant differences in Gainesville and Tampa temperatures during Rounds 2 & 3 and 1 & 3. However, the difference in mean minimum temperature between Rounds 2 & 3 in Gainesville was only 4.75C and 4.16C in Tampa The difference in mean between Rounds 1 & 3 was less than 7C in both stations. These small temperature decreases might indicate that CTFs are very sensitive to changes in their ambient environment and compensate by making swift adjustments to counteract ecological variations ; however, the acclimation and control treat ments in the experiment were not significantly different, so this explanation is doubtful It is more likely that physiological adaptations to cold temperatures become more apparent in CTFs as winter approaches. This pattern is similar to hibernation or br umation behaviors observed in animals such as bears and reptiles. Biological Significance of CTMin Another factor of the CTMin test that must be explored is its biological significance. While there was a consistent yet non significant, geographic trend i n the data, it is unclear whether this trend will actually have ecological impacts. The difference in CTMin means between Gainesville and Miami is less than 1C. Although the CTMin values are a bit conservative because some frogs never reached their therma l minimum, there were frogs from each location that displayed this extreme resistance to cold temperatures, so one location is not underestimated more than another. However, behavioral adaptations to cold must also be considered, and this experiment does n ot account for them. It could be that, along with a slightly more robust physiological tolerance to cooler temperatures CTFs in North Florida are also able to circumvent the effects of winter by taking advantage of warmer microclimates in their environmen t.

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34 Populations in disturbed areas hide around homes and buildings and seek artificial heat sources. test its absolute thermal limits until it is unable to retain vital locom otor skills. Even though the temperature was lowered very gradually during the experiment (<1C/min) it is unlikely that a CTF would actually experience a temperature drop from 13C to below freezing in a matter of hours. The extra time allotted to reach a freezing temperature could allow frogs to gradually acclimate to the colder environment and enable them to withstand winter temperatures for a longer period of time than in a CTMin test. Conversely, the weather could elicit freezing temperatures for days though this is uncommon in Florida. Under these extreme circumstances, CTFs may not be able to survive, and populations could experience die offs (Johnson and McGarrity 2011, unpublished data ). However, even after successive cold winters in Gainesville, CTFs rebounded and continue to thrive in the area (Johnson, pers. comm.). Implications For Cuban Treefrog Invasive Potential The presence of an adaptation to cold weather will have serious impacts on CTF invasive potential. Rodder and Weinsheimer ( 2009 ) pr edicted that CTFs may spread throughout the Gulf Coast by 2020 given climate change scenarios, but the dispersal of this species could be quicker because their temperature tolerance will not limit t hem as significantly (Figure 2 10 ). Cuban treefrog populat ions need to be monitored and eliminated if possible, and ecosystems vulnerable to invasion should be protected from the threat of this species. Once Cuban treefrogs have established an invasive population, they are difficult to eradicate, so mitigation ma y be the best solution to curbing CTF invasions. Managers should be vigilant about the presence of CTFs on

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35 their land, and ornamental plants need to be inspected for the presence of frogs before they are shipped to limit jump dispersal. The invasiveness of CTFs could be affected by gene flow between frogs from North and South Florida. Gainesville is home to the University of Florida and Santa Fe College, both of which enroll students from South Florida. Gainesville is also near I 75, a major highway that ru ns from Miami to the Florida Georgia border. The large amount of commuting traffic that enters Gainesville will bring more frogs from southern Florida that could breed with existing CTF populations and dilute their genetic adaptation to cold temperatures. Repeating my experiment in several years will reveal if this constant gene flow between geographic areas will significantly limit the ability of CTFs to continue expanding northward. While my study did not unveil any statistically significant comparisons i n cold tolerance among the range of invasive Cuban treefrog populations in Florida, it did reveal that CTFs are able to withstand much cooler conditions than previously thought. It also uncovered some important patterns in the temperature thresholds of thi s species. CTMin values were lower in frogs captured from northern field sites than they were in frogs further south. These trends indicate the further CTF expansion northward is likely and may occur more quickly than predicted.

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36 Figure 2 1. Confirmed C TF populations and thermal isoclines. Dark gray shading represents current breeding limits of CTF range ; light gray shading indicates isolated reports. Thermal isoclines show annual mean freeze free period. Range map based on McGarrity & Johnson 2009 and m ore recent records from the Florida Museum of Natural History; climate data from NOAA NCDC 2005. Map credit: Monica McGarrity, 2011.

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37 Figure 2 2. Map of my field sites and nearby cities

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38 A B C D Figu re 2 3. Conducting the research experiment A) Suzanne Simpson c hecks PVC pipes for CTFs B) a Fitotron chamber used for CTMin testing C ) a CTF flipped on its dorsal side in the testing compartment during a CTMin test and D) the secondary containment facility at USGS SESC conta ining the research specimens. All photos courtesy of Steve Johnson, University of Florida.

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39 Figure 2 4. Plot of CTMin values vs. BCI The red horizontal line is the trendline of the data. R 2 = 0.0 00621 indicates that no significant correlation betwee n the variables exists.

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40 Figure 2 5 Boxplot of CTMin means by location The gray horizontal line indicates the overall mean The top line of each box is Q3 and the bottom line is Q1. The horizontal line in the middle of each box represents the sample me dian. Error bars are determined by the upper and lower data point values, excluding outliers.

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41 Figure 2 6 CTMin means by location and treatment Boxplots are defined by the same parameters outlined in Fig. 2 4.

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42 Figure 2 7 Graph of the studentized re siduals vs. predicted values of CTMin data

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43 Figure 2 8 Graphic representation of statistical split plot design. This is not a representation of the spatial arrangement of the experiment, but rather how it was analyzed.

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44 Figure 2 9 Box plots of FAWN temperature data Boxplots are defined by the same parameters outlined in Fig. 2 4.

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45 Figure 2 10 Potential CTF dispersal under two climate change scenarios. Credit: Rodder and Weinsheimer 2009.

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46 Table 2 1. Sampling distribution for each treatmen t by location Table 2 2. Correlation analysis for CTMin, m ass, and SVL CTMin (C) BCI CTMin (C) Pearson correlat ion c oefficient 1.000 0.025 p value at 0.740 BCI Pearson correlation coefficient 0.025 1.000 p value at 0.740 Table 2 3. CTMin mean, median, and standard deviation values Location/Treatment Mean (C) Median (C) St d. Dev. Miami 0.1 4 0.40 2.0 9 Not acclimated 0.0 1 0.10 2.0 6 Acclimated 0.29 0.50 2.15 Tampa 0.8 6 1.35 1.93 Not acclimated 0.5 4 0.40 1.82 Acclimated 1.18 1.80 2.0 1 Gainesville 1.0 1 1.40 2.17 Not acclimated 0.87 1.25 2.0 1 Acclimated 1.1 4 1.50 2.34 Location Number per t reatment Total Acclimated Not acclimated Gainesville 31 30 6 1 Tampa 31 31 62 Miami 27 30 57 Total 89 91 180

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47 Table 2 4. Average CTMin for non a cclimated frogs by Round Locatio n Round Avg CTMin (C) Miami 1 0.63 Miami 2 0.35 Miami 3 0.35 Tampa 1 0.53 Tampa 2 0.98 Tampa 3 1.56 Gainesville 1 0.27 Gainesville 2 1.47 Gainesville 3 0.25 Table 2 5. Average CTMin for acclimate d frogs by Round Location Round Avg CTMin (C) Miami 1 0.02 Miami 2 0.43 Miami 3 0.85 Tampa 1 0.78 Tampa 2 1.41 Tampa 3 1.38 Gainesville 1 0.25 Gainesville 2 1.81 Gainesville 3 1.06 Table 2 6. ANOVA values for split plot analys is Source Num DF Den DF F Value Pr>F Chamber 1 144 0.17 0.6795 Round 2 144 4.54 0.0123 Chamber Round 2 144 0.62 0.5414 TRT 5 144 1.04 0.3963 TRT*Round 10 144 0.47 0.9071 TRT*Chamber 5 144 1.74 0.1286 TRT*Chamber*Round 10 144 2.30 0.0153 S tatis tically significant at = 0.05

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48 Table 2 7. Means and medians by FAWN Station and Round FAWN Station Round Median (F) Mean (F) Homestead 1 73.19 73.24 Homestead 2 72.43 72.39 Homestead 3 71.67 71.32 Dover 1 72.21 72.19 Dover 2 71.19 70.88 Dover 3 68.9 0 66.72 Alachua 1 70.84 70.39 Alachua 2 68.88 68.27 Alachua 3 63.61 63.52 Table 2 8. Temperature comparisons between rounds FAWN Station Round Min Temp Avg (F) FAWN Station Round Min Temp Avg (F) Pr > |t| Adjusted p value Homestead 1 73.24 Homestead 2 70.88 0.3101 0.9183 Homestead 2 70.88 Homestead 3 71.32 0.2009 0.7898 Homestead 1 73.24 Homestead 3 71.32 0.0217 0.1599 Dover 1 72.19 Dover 2 70.88 0.1180 0.5930 Dover 2 70.88 Dover 3 66.72 <0.0001 <0.0001 Dover 1 72.19 Dover 3 6 6.72 <0.0001 <0.0001 Alachua 1 70.39 Alachua 2 68.27 0.0111 0.0888 Alachua 2 68.27 Alachua 3 63.52 <0.0001 <0.0001 Alachua 1 70.39 Alachua 3 63.52 <0.0001 <0.0001 *Statistically significant at = 0.05.

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49 Table 2 9. Pairwise comparisons using t te sts between locations and treatments TRT 1 and TRT 2 indicate the two variables being compared. TRT 1 TRT 2 Estimate SE DF t Value Pr > |t| Adjusted p value Acclimated Non Acclimated 0.4241 0.3579 1.18 0.2380 0.8035 GNV/acc GNV/non 0.2078 0.5877 144 0.35 0.7241 0.9998 GNV/acc MIA/acc 0.4371 0.6799 144 0.64 0.5213 0.9851 GNV/acc TPA/acc 0.3717 0.5717 144 0.65 0.5166 0.9839 GNV/non MIA/non 0.6968 0.6033 144 1.16 0.2500 0.8195 GNV/non TPA/non 0.0126 0.5877 144 0.02 0.9829 1.0000 MIA/acc MIA/non 0.4724 0.6934 144 0.68 0.4968 0.9796 MIA/acc TPA/acc 0.8088 0.6799 144 1.19 0.2362 0.8006 MIA/non TPA/non 0.6890 0.5877 144 1.17 0.2430 0.8106 TPA/acc TPA/non 0.5922 0.5717 144 1.04 0.3020 0.8811 Table 2 10. Pairwise comparisons using t tests between loc ations and treatments within Round 2 TRT 1 and TRT 2 indicate the two variables being compared. TRT 1 TRT 2 Estimate SE DF t Value Pr > |t| Adjusted p value GNV/acc MIA/acc 1.4438 0.7326 144 1.97 0.0507 0.2290 GNV/acc TPA/acc 0.4232 0.7326 144 0.58 0.5644 0 .9702 GNV/non MIA/non 1.1866 0.7326 144 1.62 0.1075 0.4241 GNV/non TPA/non 0.5562 0.7326 144 0.76 0.4489 0.9231 TPA/acc MIA/acc 1.0205 0.7326 144 1.39 0.1658 0.5714 TPA/non MIA/non 1.0205 0.7326 144 0.86 0.3910 0.8830

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50 CHAPTER 3 FUTURE DIRECTIONS Th e results of my study illuminate several areas of research that should be undertaken to increase scientific knowledge about the invasiveness of the Cuban treefrog. Repeating my experiment in 5 10 years could provide valuable insight to the progression of t hermal adaptations in CTF. Ideally, the target sample size would be collected in one trip to the field sites rather than three separate occasions, as was done in this study. This would incr ease the power of CTMin results; h owever, collecting and maintainin g at least 180 CTFs will require a large facility, lots of resources, and assistance with testing and husbandry. Alternatively, time of testing could be explored further to investigate the apparent plasticity of temperature tolerance. Critical thermal mini mum could be tested at different times of year, perhaps during summer, fall, winter, and spring, and the results compared to determine if thermal resistance expresses itself most profoundly in winter months. If differential expression of cold tolerance is consistently reported, exploring the genetic component that leads to these physiological diffe rences will be useful Future studies comparing CTMin may also benefit from sampling a larger geographic range of CTF populations. Collecting frogs from their nat ive habitat in Cuba will expand our knowledge of the extent of temperature evolution and adaptation in this species. Since frogs from South Florida are able to consistently withstand freezing temperatures though they have likely never experienced these in nature, it would be interesting to explore whether native Cuban treefrogs also have a tolerance for extreme cold. Also, a s CTFs continue to disperse northward and westward and establish breeding populations, sites like Jacksonville or Tallahassee could bec ome focal areas to

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51 research the evolution of temperature tolerance. More generations are required before Jacksonville data become useful, and CTFs are just beginning to establish permanent residence in the Panhandle area (see Fig. 2 1). Repeating this stud y in 5 10 years may also reveal more significant patterns in physiological adaptation between populations. After more generations reproduce CTFs occupying the northern area of Florida could exhibit a more profound evolutionary response to their new enviro nment, furthering the invasive impacts of this species. Conversely, gene flow from South Florida frogs transported on cars or ornamental plants could effectively counteract any evolutionary adaptations in northern populations. Testing CTMins of Gainesville frogs inhabiting areas near and far from campuses may reveal a difference attributed to gene flow effects. Revisiting this CTMin test in the future could provide useful information regarding the gradual progression of temperature tolerance in CTFs. An alt ernative method to monitoring temperature tolerance would involve mimicking the natural environment of captured CTFs in the lab oratory and observe how they respond to temperature swings that often occur in Florida winters. The FAWN data for each location a nd month would have to be closely examined to recreate the weather measure their ability to withstand consistent cooler temperatures for a long amount of time. This exp eriment would require long term access to thermal chambers and resources like food and lab space. the Cuban treefrog. Temperature patterns indicate that CTFs may be evolvin g to the cooler weather they experience in North Florida. Cuban treefrog management and

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52 eradication must be a priority for landowners to preserve the ecological integrity of

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53 APPEN DIX RAW DATA R oun d Loc ation ID Cha mber Tr e atmen t CTMset CTMdis 1 Miami M01 B non 3 3 1 Miami M02 A non 2 2.1 1 Miami M03 B non 0 0.4 1 Miami M04 B non 1 0.2 1 Miami M05 A non 1 1 1 Miami M06 B non 2 1.6 1 Miami M07 B acc 1 0.4 1 Miami M09 A acc 1 0.5 1 Miami M10 A non 7 6.3 1 Miami M11 A acc 1 1.1 1 Miami M12 A acc 2 2 1 Miami M13 A acc 2 2.3 1 Miami M14 B acc 1 0.7 1 Miami M15 B acc 0 0.5 1 Miami M16 B acc 3 2.7 1 Miami M17 B non 1 0.7 1 Miami M18 A non 1 0.6 1 Miami M19 B acc 1 0.4 1 Miami M20 B acc 2 1.4 1 Miami M 21 A non 1 1 1 Miami M22 A non 1 1 1 Tampa T01 A non 1 0.6 1 Tampa T02 B acc 1 1.3 1 Tampa T03 B non 1 0.2 1 Tampa T04 A non 0 0.1 1 Tampa T05 A non 2 1.7 1 Tampa T06 A acc 2 2.1 1 Tampa T07 B non 2 2 1 Tampa T08 A non 4 3.7 1 Tampa T0 9 B non 2 1.7 1 Tampa T10 B acc 2 2.1 1 Tampa T11 B acc 3 2.7 1 Tampa T12 A acc 3 2.7 1 Tampa T13 A acc 1 1.1 1 Tampa T14 B acc 3 2.4 1 Tampa T15 B acc 3 3.3 1 Tampa T16 A acc 3 3.2 1 Tampa T17 B acc 1 1.4 1 Tampa T19 A non 0 0.2

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54 1 Tam pa T20 A non 1 0.2 1 Tampa T25 B non 0 0.4 1 Tampa T26 B non 1 0.4 1 Tampa T27 A acc 2 1.9 1 Gainesville G01 A acc 1 1.1 1 Gainesville G02 A non 0 0.2 1 Gainesville G03 A non 2 2.1 1 Gainesville G04 B non 3 3.1 1 Gainesville G05 B non 1 1.4 1 Gainesville G06 B non 1 1.2 1 Gainesville G07 A acc 0 0.1 1 Gainesville G08 A non 1 0.6 1 Gainesville G09 A acc 3 2.7 1 Gainesville G10 A non 7 6.3 1 Gainesville G11 A acc 2 2.1 1 Gainesville G12 B acc 1 0.4 1 Gainesville G13 A non 0 0 .1 1 Gainesville G14 A non 1 1 1 Gainesville G15 B acc 2 2.3 1 Gainesville G16 B acc 1 1.1 1 Gainesville G17 A acc 1 1.2 1 Gainesville G18 B acc 3 3.1 1 Gainesville G19 B acc 3 2.7 1 Gainesville G20 B non 1 0.5 1 Gainesville G29 B non 1 1.3 1 Gainesville G30 B acc 3 2.4 2 Miami M02 B acc 1 0.5 2 Miami M03 A non 2 1 2 Miami M05 A acc 0 0.6 2 Miami M06 A non 2 2.2 2 Miami M07 A acc 1 1.6 2 Miami M08 B non 1 0.7 2 Miami M09 A acc 5 4.2 2 Miami M10 A acc 0 0.5 2 Miami M12 B no n 1 0.3 2 Miami M13 A non 3 3 2 Miami M14 B acc 5 4.3 2 Miami M15 A non 2 1.4 2 Miami M16 B acc 2 2.3 2 Miami M17 A non 0 0.5 2 Miami M18 A acc 1 0.3 2 Miami M19 A non 0 0.6 2 Miami M20 B non 5 4.4

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55 2 Miami M22 A acc 0 0.5 2 Miami M24 A non 4 4 2 Miami M25 B non 1 0.2 2 Miami M26 B non 2 2.3 2 Miami M27 B acc 3 3.3 2 Miami M28 B non 1 0.3 2 Miami M29 B acc 1 1.5 2 Miami M30 B non 0 0.4 2 Miami M31 B acc 3 3 2 Miami M32 B acc 3 3.5 2 Miami M33 A non 0 0.6 2 Miami M34 B acc 0 0.7 2 Miami M35 A acc 1 0.6 2 Tampa T02 A non 2 2.2 2 Tampa T04 A non 4 4 2 Tampa T05 A acc 1 1.5 2 Tampa T07 A non 1 0.3 2 Tampa T08 A acc 0 0.6 2 Tampa T09 B acc 2 2.5 2 Tampa T10 B acc 3 2.2 2 Tampa T11 A acc 3 3 2 Tampa T12 A acc 2 2.4 2 Tampa T14 B acc 2 2.2 2 Tampa T15 A non 2 2.3 2 Tampa T17 A non 3 3 2 Tampa T18 A acc 3 2.4 2 Tampa T19 B non 0 0.4 2 Tampa T20 B non 1 1.5 2 Tampa T21 B non 1 1.5 2 Tampa T22 B non 1 0.2 2 Tampa T23 B non 5 4.4 2 Tam pa T24 A non 1 0.3 2 Tampa T25 B acc 3 3.1 2 Tampa T26 A non 1 1.1 2 Tampa T27 B acc 4 4 2 Tampa T28 B acc 1 0.5 2 Tampa T29 B non 0 0.5 2 Tampa T31 A acc 1 0.9 2 Tampa T32 B non 2 2.3 2 Tampa T33 A non 1 1.1 2 Tampa T35 A acc 0 0.5 2 Tampa T36 B acc 2 2.3 2 Tampa T37 B acc 1 1.4

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56 2 Gainesville G01 B acc 1 0.5 2 Gainesville G02 B non 3 3.2 2 Gainesville G03 B non 1 0.3 2 Gainesville G04 B acc 2 2.2 2 Gainesville G05 A acc 3 3.2 2 Gainesville G06 B acc 1 1.4 2 Gainesv ille G07 A acc 1 1.5 2 Gainesville G08 A non 2 2 2 Gainesville G09 A non 3 3 2 Gainesville G10 B acc 3 3.5 2 Gainesville G11 B non 1 0.7 2 Gainesville G12 B non 2 2.3 2 Gainesville G13 A acc 0 0.5 2 Gainesville G14 B non 3 3 2 Gainesvi lle G15 A acc 0 1 2 Gainesville G16 B non 2 2.3 2 Gainesville G17 B acc 2 2.4 2 Gainesville G18 B non 1 0.2 2 Gainesville G19 A acc 1 1.5 2 Gainesville G20 B acc 1 0.2 2 Gainesville G21 A non 2 2.2 2 Gainesville G22 A non 2 1 2 Gainesville G23 A acc 2 2.1 2 Gainesville G24 A non 0 0.6 2 Gainesville G25 A non 2 2.2 2 Gainesville G26 A non 3 3.1 2 Gainesville G27 A acc 3 3.2 2 Gainesville G28 B non 0 0.4 2 Gainesville G29 B acc 4 4 2 Gainesville G31 B acc 1 1.4 3 Miami M 04 B non 0 0.6 3 Miami M05 B non 1 0.6 3 Miami M06 A acc 3 2.3 3 Miami M08 A non 1 0.3 3 Miami M09 A non 1 1.7 3 Miami M10 B acc 4 4 3 Tampa T01 B acc 0 0.7 3 Tampa T02 A non 1 0.3 3 Tampa T03 B non 2 2.1 3 Tampa T04 B non 1 1.6 3 Tampa T05 B acc 2 2.7 3 Tampa T06 A non 1 1.7 3 Tampa T07 A non 2 2.7

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57 3 Tampa T08 A acc 1 1.8 3 Tampa T09 A acc 4 4 3 Tampa T10 B acc 3 2.3 3 Gainesville G01 B acc 2 2.7 3 Gainesville G03 A acc 7 6.9 3 Gainesville G04 B non 0 0.6 3 Gainesv ille G05 B non 3 3 3 Gainesville G06 A non 1 1.7 3 Gainesville G07 B acc 1 1.9 3 Gainesville G08 B acc 3 3.6 3 Gainesville G09 A acc 4 4 3 Gainesville G10 A non 1 1.7 Note: CTMset indicated the temperature at which the freezer was set and C TMdis r ecorded the actual temperature inside the chamber.

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58 LIST OF REFERENCES Barbour, T. 1931. Another introduced frog in North America. Copeia 1931 : 140. Beard, K. H., and W. C. Pitt. 2005. Potential cons equences of the coqui frog invasion in Hawaii. Diversity and Distributions 11 :427 433. Bomford, M., F. Kraus, S. C. Barry, and E. Lawrence. 2008. Predicting establishment success for alien reptiles and amphibians: a role for climate matching. Biological In vasions 11 :713 724. Boughton, R.G., J. Staiger, and R. Franz. 2000. Use of PVC pipe refugia as a sampling technique for hylid treefrogs. The American Naturalist 144 : 168 177. Brattstrom, B. H. 1968. Thermal acclimation in anuran amphibians as a function of lati tude and altitude. Comparative Biochemistry and P hysiology 24 :93 111. Brattstrom, B. H., and P. Lawrence. 1962. The rate of thermal acclimation in an uran amphibians. Physiological Z oology 35 :148 156. Campbell, T. A., and D. B. Long. 2009. Feral swine damage and damage management in forested ecosystems. Forest Ecology and Management 257 :2319 2326. Carroll, A., S. Taylor, J. Regniere, and L. Safranyik. 2003. Effect of climate change on range expansion by the mountain pine beetle in British Columbia. The Bark Beetles, Fuels, and Fire Bibliography. Available from http://digitalcommons.usu.edu/barkbeetles/195 (accessed November 6, 2011) Clinton, W. J. 1999. Executive order 13112: Invasive species. Weekly Compilation of Presidential Documents 355 :185 189. Su perintendent of Documents. species. Diversity and Distributions 10 :135 141. Crowl, T. A., T. O. Crist, R. R. Parmenter, G. Belovsky, and A. E. Lugo. 2008. The spread of i nvasive species and infectious disease as drivers of ecosystem change. Frontiers in Ecology and the Environment 6 :238 246. Davis, M. B. 2001. Range Shifts and Adaptive Responses to Quaternary Climate Change. Science 292 :673 679. Doughty, P. 1994. Critical Thermal Minima of Garter Snakes ( Thamnophis ) Depend on Species and Body Size. Copeia 1994 :537 540. Edwards, D., and J. J. Berry. 1987. The efficiency of simulation based multiple comparisons. Biometrics 43 :913 928.

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59 Ehrenfeld, J. G. 2010. Ecosystem Conseque nces of Biological Invasions. Annual Review of Ecology, Evolution, and Systematics 41 :59 80. Feder, M. E. 1985. Acclimation to Constant and Variable Temperatures in Plethodontid Salamanders. II. Time Course of Acclimation to Cool and Warm Temperatures. Her petologica 41 :241 245. Glorioso, B. M., J. H. Waddle, M. E. Crockett, K. G. Rice, and H. F. Percival. 2012. Diet of the invasive Cuban Treefrog ( Osteopilus septentrionalis ) in pine rockland and mangrove habitats in South Florida. Caribbean Journal of Scie nce 46 :346 355. Gordon, D. R. 1998. Effects of invasive, non indigenous plant species on ecosystem processes: lessons from Florida. Ecological Applications 8 :975 989. Howard, R.D. and J. R. Young. 1998. Individual variation in male vocal traits and female mating preferences in Bufo americanus Animal Behaviour 55 : 1165 1179. Huang, S. M., S. P. Huang, Y. H. Chen, and M. C. Tu. 2007. Thermal tolerance and Altitudinal distribution of three Trimeresurus snakes (Viperidae : Crotalinae) in Taiwan. Zoological Stu dies 46 :592 599. Huang, S. P., Y. Hsu, and M. C. Tu. 2006. Thermal tolerance and altitudinal distribution of two Sphenomorphus lizards in Taiwan. Journal of Thermal Biology 31 :378 385. John Alder, H. B., P. J. Morin, and S. Lawler. 1988. Thermal physiology phenology, and distribut ion of tree frogs. The Ame rican Naturalist 132 :506 520. Johnson, S. 2007. The Cuban Treefrog ( Osteopilus septentrionalis ) in Florida. Publication #WEC218. Gainesville: University of Florida Institute of Food and Agricultural Scien ces. Available from http://edis.ifas.ufl.edu/uw259 (accessed October 13, 2011). Johnson, S. A., and M. E. McGarrity. 2011 Cold induced mortality of invasive Cuban treefrogs ( Osteopilus septentri onalis ) in Central Florida. Unpublished data. JMP, Version 8. SAS Institute Inc.,Cary, NC, 1989 2007. Juliano, S. A., and L. P. Lounibos. 2005. Ecology of invasive mosquitoes: effects on resident species and on human health. Ecology L etters 8 :558 574. Kais er, B. A., and K. Burnett. 2006. Economic impacts of E. coqui frogs in Hawaii. Interdisciplinary Environmental Review 8 :1 11. Kalin Arroyo, M. T., C. Marticorena, O. Matthei, and L. Cavieres. 2000. Plant Invasions in Chile: Present Patterns and Future Pred ictions. in H. A. Mooney and R. J. Hobbs, editors. Invasive Species in a Changing World. Island Press.

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60 Kolar, C. S., and D. M. Lodge. 2001. Progress in invasion biology: predicting invaders. Trends in Ecology & Evolution 16 :199 204. Krysko, K. L., K. M. En ge, and J. H. Townsend. 2005. New county records of amphibians and reptiles from Florida. Herpetological Review 1 :85 87. Krysko, K. L., F. W. King, K. M. Enge, and A. T. Reppas. 2003. Distribution of the introduced black spiny tailed iguana ( Ctenosaura sim ilis ) on the southwestern coast of Florida. Florida Scientist 66 :141 146. Laufer, G., A. Canavero, D. Nez, and R. Maneyro. 2008. Bullfrog ( Lithobates catesbeianus ) invasion in Uruguay. Biological Invasions 10 :1183 1189. Layne, J. R., and D. L. Claussen. 1987. Time courses of thermal acclimation for critical thermal minima in the salamanders Desmognathus quadramaculatus, Desmognathus monticola, Desmognathus ochrophaeus and Plethodon jordani Comparative Biochemistry and Physiology Part A: Physiology 87 :89 5 898. Layne, J. R., Jr, and M. A. Romano. 1985. Critical Thermal Minima of Hyla chrysoscelis H. cinerea, H. gratiosa and Natural Hybrids ( H. cinerea H. gratiosa ). Herpetologica 41 :216 221 Leuizinger, S., Y. Luo, C. Beier, W. Dieleman, S. Vicca, and C. Korner. 2011. Do global change experiments overestimate impacts on terrestrial ecosystems? Trends in Ecology and Evolution 26 :236 241. Li, Y., Z. Ke, Y. Wang, and T. M. Blackburn. 2011. Frog community responses to recent American bullfrog invasions. Curren t Zoology 57 :83 92. Lobos, G., and F. M. Jaksic. 2005. The ongoing invasion of African clawed frogs ( Xenopus laevis ) in Chile: causes of concern. Biodiversity and Conservation 14 :429 439. Losos, J. B., K. I. Warheit, and T. W. Schoener. 1997. Adaptive diff erentiation following experimental island colonization in Anolis lizards. Nature 387 :70 73. Lowe, S., M. Browne, S. Boudjelas, and M. De Poorter. 2000. 100 of the World's Worst Invasive Alien Species: A Selection From the Global Invasive Species Database. Pages 1 12. Invasive Species Specialist Group. Available from http://www.issg.org/booklet.pdf (accessed November 16, 2011) McGarrity, M. E., and S. A. Johnson. 2008. Geographic trend in sexual size dimorphism and body size of Osteopilus septentrionalis (C uban treefrog): implications for invasion of the southeastern United States. Biological Invasions 11 :1411 1420. Meshaka, W. 2001. The Cuban treefrog in Florida: life history of a successful colonizing species. University Press of Florida, Gainsville, FL.

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61 M ott, C. L. 2010. Environmental Constraints to the Geographic Expansion of Plant and Animal Species. Nature Education Knowledge 3 :72. Navas, C. A. 1997. Thermal extremes at high elevations in the Andes: physiological ecology of frogs. Journal of Thermal Bio logy 22 :467 477. Phillips, B. L. 2004. Adapting to an invasive species: Toxic cane toads induce morphological change in Australian snakes. Proceedings of the National Academy of Sciences 101 :17150 17155. Phillips, B. L., G. P. Brown, M. Greenlees, J. K. We bb, and R. Shine. 2007. Rapid expansion of the cane toad ( Bufo marinus ) invasion front in tropical Australia. Austral Ecology 32 :169 176. Phillips, B. L., G. P. Brown, J. K. Webb, and R. Shine. 2006. Invasion and the evolution of speed in toads. Nature 439 :803. Phillips, B. L., and R. Shine. 2006. An invasive species induces rapid adaptive change in a native predator: cane toads and black snakes in Australia. Proceedings of the Royal Society B: Biological Sciences 273 :1545 1550. Pimentel, D., R. Zuniga, and D. Morrison. 2005. Update on the environmental and economic costs associated with alien invasive species in the United States. Ecological Economics 52 :273 288 Platt, W. J., and R. M. Gottschalk. 2001. Effects of exotic grasses on potential fine fuel loa ds in the groundcover of south Florida slash pine savannas. International Journal of Wildland Fire 10 :155 159. Rice, K. G., J. H. Waddle, M. W. Miller, M. E. Crockett, F. J. Mazzotti, and H. F. Percival. 2011. Recovery of Native Treefrogs After Removal of Nonindigenous Cuban Treefrogs, Osteopilus septentrionalis Herpetologica 67 :105 117. Rdder, D., and F. Weinsheimer. 2009. Will future anthropogenic climate change increase the potential distribution of the alien invasive Cuban treefrog (Anura: Hylidae)? Journal of Natural History 43 :1207 1217. Sakai, A. K., F. W. Allendorf, J. S. Holt, D. M. Lodge, J. Molofsky, K. A. With, S. Baughman, R. J. Cabin, J. E. Cohen, and N. C. Ellstrand. 2010. The Population Biology of Invasive Species. Annual Review of Ecology and Systematics 32 :305 332. SAS, Version 9.3. SAS Institute Inc., Cary, NC, 1989 2007. Shine, R. 2011. Invasive species as drivers of evolutionary change: cane toads in tropical Australia. Evolutionary Applications 5 :107 116.

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62 Sin, H., K. H. Beard, W. C. P itt. 2007. An invasive frog, Eleutherodactylus coqui increases new leaf production and leaf litter decomposition rates through nutrient cycling in Hawaii. Biological Invasions 10 :335 345. Sinervo, B. et al. 2010. Erosion of Lizard Diversity by Climate Cha nge and Altered Thermal Niches. Science 328 :894 899. Smith, K. G. 2005. Effects of nonindigenous tadpoles on native tadpoles in Florida: evidence of competition. Biological Conservation 123 :433 441 Southeast Regional Climate Center (SERCC). 2012. Florida Climate Normals. SERCC, Chapel Hill, NC. Stanton Geddes, J., P. Tiffin, and R. G. Shaw. 2012. Role of climate and competitors in limiting fitness across range edges of an annual plant. Ecology 93 :1604 1613. Tewksbury, J. J., R. B. Huey, and C. A. Deutsch. 2008. Ecology. Putting the heat on tropical animals. Science 320 :1296 1297. Thomas, C. D., A. Cameron, R. E. Green, M. Bakkenes, L. J. Beaumont, Y. C. Collingham, B. F. N. Erasmus, M. F. De Siqueira, A. Grainger, and L. Hannah. 2004. Extinction risk from c limate change. Nature 427 :145 148. Urban, M., and B. Phillips. 2008. A toad more traveled: the heterogeneous invasion dynamics of cane toads in Australia. The American Naturalist 171 :E134 E148. van Berkum, F.H., R. B. Huey, J. S. Tsuji, and T. Garland. 19 89. Repeatability of individual differences in locomotor performance and body size during early ontogeny of the lizard Sceloperus occidentalis (Baird and Girard). Functional Ecology 3 : 97 105. van Wilgen, N. J., N. Roura Pascual, and D. M. Richardson. 2009 A quantitative climate match score for risk assessment screening of reptile and amphibia n introductions. Environmental M anagement 44 :590 607. Waddle, J. H., R. M. Dorazio, S. C. Walls, K. G. Rice, J. Beauchamp, M. J. Schuman, and F. J. Mazzotti. 2010. A new parameterization for estimating co occurrence of interacting species. Ecological Applications 20 :1467 1475. Walther, G., E. Post, P. Convey, and A. Menzel. 2002. Ecological responses to recent climate change. Nature 416 :389 395. Walther, G. R., A. Roqu Bacher, Z. Botta Dukt, and H. Bugmann. 2009. Alien species in a warmer world: risks and opportunities. Trends in Ecology & Evolution 24 :686 693. Whitney, E. N., D. B. Means, and A. Rudloe. 2004. Priceless Florida. Pineapple Press Inc., Sarasota, FL.

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63 Wilson, L. 2010, May 3. Diet, Critical Thermal Minimum, and Occurrence of Batrachochytrium Dendrobatidis in Cuban Treefrogs ( Osteopilus Septentrionalis ). Valdosta State University. Wilson, R. S., and C. E. Franklin. 1999. Thermal acclimation of locomotor performance in tadpoles of the frog Limnodynastes peronii Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology 169 : 445 451. Wyman, R. L. 1988. Soil acidity and moisture and the distribution of amphibians in five forests of southcentral New York. Copeia 1988 :394 399.

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64 BIOGRAPHICAL SKETCH Suzanne was born in Arlington Heights, Illinois, but grew up in Kingwood, Texa s. She received a B.S. in biology from Texas A&M University and then contacted Dr. Steve Johnson at the University of F lorida about pursuing a Master of Science in Interdisciplinary Ecology under his advisement. Suzanne looks forward to starting her profes sional career in wildlife biology after graduating in May 2013.