Distribution and Environmental Correlates Between Amphibians and the Fungal Pathogen, Batrachochytrium Dendrobatidis

Distribution and environmental correlates between amphibians and the fungal pathogen, Batrachochytrium dendrobatidis

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By Chelsea Anne Korfel Graduate Program in Evolution, Ecology and Organismal Biology

The Ohio State University 2012

Dissertation Committee: Thomas Hetherington, Advisor Stanley Gehrt, Thomas Mitchell, David Stetson

Abstract

Amphibian populations worldwide are vulnerable to a variety of threats, and one serious cause of population declines and extinctions is the pathogenic fungus Batrachochytrium dendrobatidis (Bd). Amphibian species of tropical, montane regions have suffered the greatest impacts of Bd- related declines, extirpations, and extinctions. Bd is also present in temperate regions, but its effects on temperate amphibian species appear to be less severe, although they remain poorly understood. Bd impacts populations, species, and individuals differentially and environmental factors affect host-pathogen relationships. Temperature, specifically, is a major factor impacting the growth and spread of Bd and the ability of amphibians to resist disease. Temperature varies along seasonal, altitudinal, and landscape gradients. Interactions of hosts and the pathogen in tropical and temperate regions along these environmental gradients are explored here.

Chapter One: I compiled a thorough review of previous research on the influence of temperature on the interaction between amphibian hosts and the fungal pathogen Bd. The review includes both laboratory and field studies, which explore the effects of microhabitat and regional temperature and the effects of global climate change on amphibian populations. Despite thorough laboratory studies, complete understanding regarding the influence of temperature on the interaction between amphibian hosts and Bd in the natural environment is necessary for understanding dynamics of disease and designing appropriate conservation strategies.

Chapter Two: I conducted a survey of Bd prevalence and infection load in amphibian communities in the temperate landscape of central Ohio and engaged other amphibian biologists to create a statewide survey. Bd is widespread throughout Ohio, infecting the ii skin of 19.5% of all amphibians sampled in this study. Prevalence was highest among bullfrogs (59.2%, Lithobates catesbeianus) and the highest infection intensity, 1,946,586 ZE, was in a cricket frog (Acris crepitans). Infection intensities among green frogs (L. clamitans) were relatively high (19,328 ZE) and infection prevalence was moderate (27.3%). No obvious symptoms of infection were observed, even for amphibians with high intensity fungal loads. This survey established that Bd is well-established in a variety of habitats in Ohio and could impact amphibian populations.

Chapter Three: I studied Bd prevalence and fungal load intensity with respect to both season and habitat in two common amphibian hosts (green frogs, Lithobates clamitans, and American bullfrogs, L. catesbeianus) in central Ohio, USA. Bd prevalence, the percent of infected animals sampled, was highest among samples collected in spring (89%) and from forested streams (54%). Both were associated with cooler temperatures. Bd infection intensities, the severity of infection per individual, were highest among samples collected in summer (8,240 ± 31,852 ZE) and from emergent streams (72,645 ± 18,092 ZE). Both summer and emergent streams were associated with warm temperatures. These relationships may result from tradeoffs in the effect of temperature on parameters of Bd life history and amphibian behaviors. For example, amphibians congregate in breeding pools in the spring, which could facilitate the spread of disease. However, in summer amphibians in open canopy habitat can bask at temperatures above the lethal temperature for Bd and minimize infections. Despite these factors, amphibians with poor immune function are likely to develop severe infections.

Chapter Four: I studied Bd prevalence in marsupial frog (Gastrotheca pseustes) tadpoles in mid to high altitude pools (2500-4200 masl) in Ecuador’s Cajas National Park and the effects of Bd on infected tadpoles. While other field studies have examined the high- temperature climatic threshold for Bd associated with low altitudes, this work investigates a lower critical temperature for Bd prevalence at mid to high elevations. At the upper end of its altitudinal distribution, G. pseustes e eriences tem eratures ithin

iii the same ran e as the la orator -determined tem eratures - 0 C) at hich d ro th and development slows. I found widespread but low prevalence of Bd throughout the altitudinal range of our study, and no difference in prevalence between high and mid altitude pools along this gradient. Body condition of infected tadpoles was poor compared to disease-free conspecifics, and there was no difference in body condition of infected tadpoles at lower versus higher altitude sites. Despite experiencing temperatures cooler than the ideal range predicted for Bd, the fungus impacts G. pseustes tadpoles at both mid and high altitudes equally. Bd prevalence at mid to high altitudes was comparatively lower than Bd prevalence identified in other studies conducted at low elevations, which may suggest that Bd is of less concern for amphibians with mid to high altitudinal distributions.

Chapter Five: Atelopus exiguus is one of the few persisting species of harlequin frogs (genus Atelopus), a genus that has suffered severe declines and extirpations from epizootic Bd in Latin America. I hypothesize that a series of hot and dry years in the early 990’s in s ner ith the arrival of d to the area in the late 980’s ma e res onsi le for the initial population decline. However, the population remains small and all sampled individuals in the current population were not Bd positive despite the detection of Bd in nearby populations of Gastrotheca pseustes. I suggest that this population has adapted to persist with endemic Bd and provides hope for the persistence of global amphibian species.

Research in this dissertation provides significant insight into the relationship between amphibians and the fungal pathogen Bd in a variety of environments, and contributes recommendations to inform future efforts in global amphibian conservation.

Dedication

For the frogs, all the people who care about them, and especially the people who taught me to care about them (especially you, Grampa).

Frogs do for the night what birds do for the day... they give it a voice. And that voice is a varied and stirring thing that ought to be better known. Archie Carr

Acknowledgments

The accumulation of data, variety of perspectives, thoughtful insights, and writing process demonstrated here could not have been completed without the help of many amazing people in my life: some who knew me before I began, others who entered my life because of this work, and others who I’ve met alon the a . Your time, energy, and expertise are appreciated. You have been blessings in my life!

M famil has een a constant source of su ort as I’ve follo ed m heart and ursued a somewhat unpredictable route towards a yet unknown career. Mom and Dad, thanks for your encouragement, your patience with my free-spirited ways, your hugs, and for seeing me throu h nearl 25 ears of schoolin . Grann ird, ou’ll never kno ho much our middle-of-the-day phone calls have been my sanity throughout all of this, I love you! Ashle and Lindse , ou’re stuck ith me as a sister, ut I’m so lad ou’re m est friends! Rick, Diane, Tiffan , Parker, Grandmother and Grandadd , I’m so lad ou’re a part of who I call family. Thanks for sharing Brad with me. He’s an amazing field assistant, I’m an unconventional ife, and I appreciate your patience and understanding.

With much gratitude, I acknowledge the expertise of my PhD adviser, my committee, and my undergraduate adviser. Tom Hetherington, thank you for letting me pursue my passion, for giving me space to work independently but always being there to guide me, for many hours of planning, thinking, field work, “hmmm….s,” discussin , and editin . My committee members, Dave Stetson, Tom Mitchell, and Stan Gehrt challenged me to

vi think critically about my work. I am indebted to them for their varied perspectives and contri utions. Kell , ou’ve su orted me from the da e first met hen I stum led into the UD biology office, you have guided and encouraged me through graduate school, and I hope you continue to do so as I pursue my (or maybe your!) career.

For the many friendships and sources of spiritual support that have brought joy to my life. The Branches and the Marianist family, my UD girlfriends, my herp friends, SBS Little Rock, Glene Mynhardt, Dan Fink, Charlie Pizanis, Carrie Petruso, Elizabeth Goussetis, Lauren Kelley. You are the people who have prayed for me and reminded me that there is a reason I’m on this ath and the lon and meanderin journey is all worthwhile.

My dream was to work in the tropics, but the work came with many unforeseen challenges that could only be faced with the guidance and support of friends I met along the way. Thank you to Carlos Martinez, David Salazar, Santiago Ron, Luis Coloma, and Elicio Tapia for starting me off and contributing to my research along the way. Thank you to Ernesto Arbelaez and Amanda Vega for being my ground team, for helping me open a bank account, buy a car, slide down a mountain in the car, learn to speak and understand Cuencano, and showing me the beautiful country of Ecuador in spur-of-the- moment adventures, all while literally running a zoo. I appreciate your dedication to conservation and wish you the best with Bioparque Amaru. Thank you to Jose Caceres for being both a friend and my research coordinator for Cajas National Park. Thank you to Giovanni Onore, entomologist, Marianist, and chef extraordinaire!

Thank you to my husband. Brad, you have loved me, kept me on my path, and pushed me to be a better scientist. I am grateful to you for sharing in this crazy adventure with me, for entertaining my wild ideas, and for grounding me when I needed it. For your patience in carefully planning our careers and our lives together, for appreciating my ambitions and making sacrifices so that I could pursue them. Thanks for being a happy camper and for both asking and affirming this most important question, “Would ou o ith me?”

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Vita

Januar 984…………………….…………… orn, Stron sville, Ohio June 2002 ...... Westerville North High School, Ohio 2006 ...... B.S. Environmental Biology and Geology, University of Dayton, Ohio 2007 ...... M.S. Natural Resources, The Ohio State University 2011-2012 ...... Graduate Teaching Associate, Departments of Evolution, Ecology and Organismal Biology and Center for Life Science Education. Graduate Research Fellow, National Science Foundation Graduate Research Fellowship Program. Presidential Fellow, Department of Evolution, Ecology and Organismal Biology, The Ohio State University

Publications

Korfel, CA, TE Hetherington, JJ Mack, and WJ Mitsch. 2010. Hydrology, physiochemistry, and amphibians in natural and created vernal pool wetlands. Restoration Ecology. 18(6): 843- 854.

Fields of Study Major Field: Evolution, Ecology and Organismal Biology

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Table of Contents

A stract……………………………………………………………………………………ii Dedication……………………………………………………………………….….……..v Ackno led ements………………………………………………………………....…….vi Vita…………………………………………………………….…………………….….viii List of Ta les…………………………………………………………………...... …….xv List of Fi ures……………………………………………………………………..…..xviii

Chapter 1: Review of the effects of temperature on Batrachochytrium dendrobatidis (Bd) and amphibian biology and the role of temperature-mediated effects on host and pathogen …………………….……….………………………………………….…………...………1 Introduction…..……………………………………………………………...…….1 d descri tion………..…………………………………………………………....2 Potential ori in of d, its lo al s read, strains…………………………………..2 General im acts…………………………………………………..……………….4 Effects of temperature on d iolo ……………………………..………………4 Effects of tem erature on am hi ian susce ti ilit to d………..………...……..6 Ex-situ studies of amphibian – Bd (host – pathogen) interactions in the framework of tem erature………………………………………………..…..………………..8 In-situ studies of amphibian Bd (host – pathogen) interactions in the framework of tem erature………………………………………………………..…...………….9

Influence of lo al scale tem erature atterns on ch trid e idemics………..…..12 Conclusion……………………………………………………………………….15 Tables and fi ures……………………………………………….....……………

Chapter 2: Review of Batrachochytrium dendrobatidis (Bd) in Ohio, with references to ne data………………………………………………………………………...………..21 Introduction………………………………………………………………………21 Materials and methods………………………………………………………...…24 Sample collection…………………………………………………….…..24 Fro collection and s a sam lin …………………………………...... 24 PCR anal sis and zoos ore equivalents ZE)……………………..……..24 Results……………………………………………………………………………25 Distri ution and overall revalence of d……………………………….25 Ta onomic distri ution of d…………………………………………....26 Am hi ian infection intensities……………………………………….…26 Ha itat distri ution of d………………………………………………..26 Seasonal atterns of d distri ution………………………………….….27 Geo ra hic distri ution of d……………………………………….…..27 Discussion………………………………………………………………………..27 Tables and fi ures…………………………………..……………...……………30

Chapter 3: Seasonal and microhabitat correlates of the distribution of the amphibian fungal pathogen Batrachochytrium dendrobatidis (Bd) in a temperate landsca e……....38 Introduction………………………………………………………………..……..38 Materials and methods…………………………………………………………...43 Sam lin sites and schedule……………………………………………..43 Frog collection and swab sampling………….………………………..….44 PCR analysis and zoospore equivalents ZE)………………………..…..45 Tests of the relia ilit of ZE measurements……………………………..45

Tem erature data………………………………………………………...46 Statistical anal sis………………………………………………………..46 Results……………………………………………….…………………………...47 S ecies differences………………………….………………………..…..47 Results from dead and / or s m tomatic fro s…………………..………47 Ha itat differences……………………………………………………….48 Seasonal differences………………………………………………….…..48 Data from reca tured fro s……………………………………………....49 Life sta e differences……………………….…………………………....49 Tem erature……………………………………………………………...49 Discussion………………………………………………………………………..50 Prevalence and intensities of d infections in sam led fro s………..…..50 Habitat related patterns of Bd infection rates and intensities……………51 Seasonal atterns on d infection rates and intensities……………...…..52 Life histor related atterns in d infection rates and intensities………..52 Seasonal and ha itat associated tem erature variation…………………..54 Impacts of temperature on host – Bd interactions…………………….....54 Conservation implications……………………………………………………….56 Tables and fi ures………………………………………………...……………..59

Chapter 4: Effects of altitude on Batrachochytrium dendrobatidis (Bd) prevalence in the hi h Andes of Ecuador………………………………………………………………...…66 Introduction………………………………………………………………….…...66 Materials and methods………………………………………………………...…69 Sam lin schedule…………………………………………………...…..69 Tem erature data………………………………………………………... Data anal sis……………………………………………………………..71 Results……………………………………………………………………………72 Altitude and d revalence…………………………………………...….72

Season and d revalence……………………………………………….72 Tem erature rofiles……………………………………………………..72 Tem erature and d revalence………………………………………… 3 Tadpole lifestage and body condition and d revalence……………….73 Discussion and conservation implications………………………………………. 3 Altitude atterns……………………………………………………….....73 Season…………………………………………………………………....74 Tem erature…………………………...... …..75 Tad ole ro th and develo ment………………………………………..75 d as an enzootic atho en in the Ecuadorean Andes………………...... 76 Conclusion…………………………………………………………………….…77 Tables and fi ures………………………………………………...……………..79

Chapter 5: Conservation status of the Cajas green harlequin frog, Atelopus exiguus.…..85 Introduction………………………………………………………………………85 Materials and methods……………………………………………………..…….90 S ecies descri tion……………………………………………………….91 Site descri tion………………………………………………………...... 91 Land use histor ………………………………………………………….92 Tem erature collection…………………………………………………..92 d s a sam les…………………………………………………………93 Results……………………………………………………………………………94 Descri tion of s ecies……………………………………………..……..94 Natural histor and ha itat characteristics………………………….……95 Chan es in the o ulation………………………………………………..96 Threats to the o ulation………………………………………………...97 Discussion………………………………………………………………………..97 Conclusion……………………………………………………………………... 00 Tables and fi ures………………………………………………...……………101

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Conclusion: Insights to environmental factors influencing impacts of Bd on global amphibian po ulations…………………...……………………………………………. 06 Overvie ………………………………………………………………………..106 Rationale………………………………….…………………………………….107

Bd dynamics in a temperate environment and implications for amphibian conservation…………………………………………………………………….107 Bd dynamics in a high altitude tropical environment and implications for amphibian conservation………………………………………………………...108 A s nthesis of stud findin s………………….…………………..…………… 0 Tables and fi ures………………………………………………...….…………112 References……………………………………………………………………………… 4

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List of Tables

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Table 1.1. Results of laboratory studies of the interaction between temperature and Bd infections in various amphibian species……………………………………………...….18

Ta le 2. . Distri ution of d in Ohio’s adult and juvenile amphibians, grouped by family. Includes breeding habitat association, total sample size, Bd prevalence rate (% positive), mean infection intensity (ZE), and sample size of infected amphibians.……..31

Ta le 2.2. Distri ution of d in Ohio’s larval am hi ians, rou ed famil . Includes breeding habitat association, total sample size, Bd prevalence rate (% positive), mean infection intensity (ZE), and sample size of infected am hi ians……………………..34

Table 2.3. Distribution of Bd among aquatic amphibian habitats in Ohio……………....35

Table 2.4. Seasonal (spring, summer, autumn) distribution of Bd among Ohio amphibians……………………………………………………………………………….35

Table 2.5. Geographic variation between populations of the same species for Ambystoma maculatum, Anaxyrus americanus, and Lithobates sylvaticus…………………………...36

Table 3.1. Infection intensities (ZE) for left-side/right-side swab replicates for ten individual green frogs…………………………………………………………………....60

Table 3.2. Infection prevalence and intensity (ZE) for symptomatic and dead frogs……60

Table 3.3. Seasonal trends in infection prevalence and intensity (ZE) of recaptured green frogs………………………………………………………………………………...……60

Table 3.4. Aquatic and terrestrial temperatures including mean, maximum, minimum and standard deviation by season for forested stream and emergent wetland habitats………61

Table 4.1. Percentage of Gastrotheca pseustes tadpoles infected with Bd in the localities sampled in this study………….…………………………………………………………80

Table 4.2. Aquatic and terrestrial temperatures from select tadpole collection pools ranging from 2500-4200 masl……………………………………………………….…..82

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List of Figures

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Figure 1.1. Batrachochytrium dendrobatidis growth, development, and infection of amphibian skin. A) Life cycle of Bd including substrate dependent and independent stages. Diagram Rosenblum et al. 2010. B) Development of a zoosporangium in amphibian tissue and the production of zoospores, zoospore release. Available: http://www.esf.edu/efb/brunner/research.html. C) In order: zoospores, zoosporangium with thread-like rhizoids, zoosporangium after zoospores are discharged, newly released zoospores, Bd infection on Leopard frog (Lithobates pipiens) skin. Available: http://www.broadinstitute.org/annotation/genome/batrachochytrium_dendrobatidis/Multi Home.html D) Cross section of zoosporangium in amphibian skin. Berger et al. 1998. E) Mild, medium, and heavy Bd infection loads on amphibian skin. Available: http://www.umaine.edu/chytrids/Index-Batrachochytrium.html...... 19

Figure 1.2. Diagram of effects of temperature on Bd and the interaction of Bd with amphibians. References: Forrest and Schlaepfer 2011, Piotrowski et al. 2004, Woodhams et al. 2003 Bradley 2002, Longcore et al. 1999, Kriger and Hero 2007, Hossack et al. 2010, Johnson et al. 2011, Seimon et al. 2006, Pullen et al. 2010, Savage et al. 2011, Manzano 2010…………………………………………………………………………....20

Figure 2.1. Bd prevalence (% positive) from amphibians collected from regions in Ohio and the distribution of species collected in each region. Central Ohio samples were largely dominated by Lithobates clamitans and L. catesbeianus, species known to carry high Bd prevalence among populations………………………………………………….3

Figure 3.1. A) Log transformed Bd infection intensities (in zoospore equivalents, ZE) in green frogs (Lithobates clamitans) by habitat. Emergent pool (n=4), emergent stream (n=11), forested stream (n=130). B) Bd prevalence in green frogs by habitat. Emergent pool (n=48), emergent stream (n=30), forested stream (n=254)………………………..62

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Figure 3.2. A) Seasonal pattern of Bd infection intensities (in zoospore equivalents, ZE) in green frogs (Lithobates clamitans) and mean water temperature in forested streams. Spring (n=32), summer (n=39), autumn (n=59). B) Seasonal pattern of Bd prevalence in green frogs and water temperature variation (standard deviation of hourly temperature measurements for each 24-hour day) in forested streams. Spring (n=36), summer (n=101), autumn (n=106)……………………………………………………………….63

Figure 3.3. Seasonal aquatic temperatures and temperature variation collected from representative forested streams and emergent pools…………………………………....64

Figure 3.4. Aquatic temperatures (means and ranges) of representative forested stream and emergent wetland sites in relation to optimal and critical temperature range for Bd (Piotrowski et al.2004, Voyles et al 2011, Woodhams et al.2003, 2008)……………....65

Figure 4.1. Correlates of Bd prevalence in G. pseustes tadpoles with A) altitude, B) time of year, and C) water temperature C ). ………………………………………………..80

Figure 4.2. Temperature profiles of select sites in Cajas National Park. A) Aquatic pool water temperatures along an altitudinal gradient (2500, 3000, 4000, 4200 masl). Laboratory findings suggest that Bd growth ecomes im aired elo a out 0 C line (shaded area). B) Seasonal stream water temperature and average rainfall (Instituto Nacional de Meterologia y Hidrologia, Ecuador (INAMHI)) at 3000 masl. C) Seasonal profile of stream water temperatures from March –May 2011 at select pools from 2500, 3000, 4000, and 4200 masl………………………………………………………………83

Figure 4.3. Bd prevalence and Gastrotheca pseustes tadpole growth and development. A) Graph of Bd prevalence plotted against Gosner (1960) developmental stages (B) Bd prevalence in three categories of tadpole body condition ((weight/length)*100): 0-1.99

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(n=256), 2.00-3.99 (n=143), and 4.00-5.99 (n=26) and (C) Body condition of Bd positive tadpoles by altitude……………………………………………………………….……..84

Figure 5.1. A. Ventral view of Atelopus exiguus. B. Ventral view of gravid female A. exiguus. C. Adult male A. exiguus exhibiting normal posture. D. Ventral view of juvenile A. exiguus with black patch at base of hind limbs. E. Dorsal view of A. exiguus (normal green). F. Dorsal view of A. exiguus (dark mottled)………………………………...…. 03

Figure 5.2. A. Atelopus exiguus with white feet, B. A.exiguus with black feet………...104

Figure 5.3. Characteristic habitat where Atelopus exiguus are found. A. River B. Siksalis plant……………………………………………………………………………………..104

Figure 5.4. Daily average temperatures of Atelopus exiguus refuge site and riverine aquatic habitat…………………………………………………………………………..105

Figure 6.1. Summary of temperature effects on Bd and amphibian–Bd infections based on laboratory and field based studies. Contributions from the research described in this dissertation are in red…………………………………………………………………..113

Chapter 1: Review of the effects of temperature on Batrachochytrium dendrobatidis (Bd) and amphibian biology and the role of temperature-mediated effects on host and pathogen

INTRODUCTION Batrachochytrium dendrobatidis (Bd) is a fungal amphibian pathogen responsible for the disease chytridiomycosis that has contributed to global amphibian declines. Many aspects of the biology of Bd are heavily influenced by temperature. Bd is a keratinophilic fungus that has two life stages, a motile zoospore and a sessile sporangium which implants in amphibian skin. To infect an amphibian, Bd must overcome defenses of the amphibian skin mucus (antimicrobial peptides, symbiotic bacteria) and lymphocytes in the tissues (Rollins-Smith et al. 2011). The Bd sporangia form inside epidermal cells and can parasitize cells up to a few layers deep. The development of the sporangium coincides with amphibian cell maturation so that the cell moves outward and keratinization occurs when zoospores are ready to be released (Berger et al. 2005). Symptoms of chytridiomycosis include skin sloughing, lethargy, and poor righting reflex (Pessier 1999). Susceptibility to chytridiomycosis varies greatly among different amphibian species. Some species are asymptomatic even when heavily infected while others die within two days of infection. An infection threshold of 10,000 zoospore equivalents typically causes death in the mountain yellow legged frog (L. muscosa) (Vredenburg et al. 2010) and crawfish frogs (L. areolatus) (Kinney et al. 2011). Here, I review laboratory and field studies in the literature to assess scientific knowledge on the interaction between temperature, Bd pathogenicity, amphibian susceptibility to infection, and the influence of global scale temperature patterns on chytrid epidemics.

For the sake of this dissertation, the term “infection” ill e used to describe amphibians that are colonized d. In most fields, the term “infestation” ould e more appropriate. However, infection is commonly used among amphibian biologists who study Bd. Severity of an “infection” refers to the quantified number of zoospores detected by PCR analysis.

BD DESCRIPTION Batrachochytrium dendrobatidis (Fungi: Chytridiomycota: Chytridiomycetes: Rhizophydiales, incertae cedis: Batrachochytrium dendrobatidis), commonly called the amphibian chytrid or Bd, lives in keratinized cells in the superficial layers (stratum corneum and stratum granulosum) of the amphibian epidermis (Figure 1.1). Chytrids are unique among fungi because they have a flagellated zoospore and Bd is unique among the chytrids because it parasitizes a vertebrate. Bd is diploid and reproduces asexually. It has two lifestages (Figure 1.1), a sessile zoosporangium and an aquatic, motile zoospore. The zoosporangium is spherical to subspherical in shape, ranges from 10-40µm in diameter, and has thread-like rhizoids. It produces a single discharge tube that protrudes out of the skin and is used for dispersing zoospores (Figure 1.1). The zoospores are 0.7 to 6µm in diameter, elongate to ovid in shape, and have a singular posterior flagellum for mobility in water (Longcore et al. 1999). Zoospores have one to two days to find a host or they will die (Piotrowski et al. 2001, Woohhams et al. 2008).

POTENTIAL ORIGIN OF BD, ITS GLOBAL SPREAD, STRAINS Batrachochytrium dendrobatidis is nearly global in distribution, occurring on every continent amphibians inhabit (Fisher et al. 2009), and often in remote, relatively pristine areas. It is likely that Bd emerged as a novel pathogen and was introduced to different geographic locations. Its emergence is hypothesized to have begun in Africa and spread 2

by the African clawed frog (Xenopus laevis). Three points support its emergence from Africa. First, the earliest record of occurrence is from Africa in 1938. Second, the African clawed frog (X. laevis) shows no symptoms of disease when inoculated with the pathogen. Finally, international trade of X. laevis for scientific and medical research could explain global movement and multiple introductions of the pathogen (Weldon et al. 2004). Bd was introduced on separate occasions and spread quickly through amphibian populations in Latin America (25-282 km/yr), the Sierra Nevada Mountains, USA (700 m/yr), and Australia (17 km/yr), devastating upland amphibian populations in a short period of time (Lips et al. 2008, Vredenburg et al. 2010, Laurance et al. 1996).

Additional support for the novel pathogen hypothesis comes from genetic work. James et al. (2009) sampled 59 strains of Bd collected from 5 continents and 31 host species and found a low rate of DNA polymorphism, but a high diversity of the diploid genotype. The genetic diversity of a Bd isolate collected from a bullfrog (Lithobates catesbeianus) was greater than the global mean. These findings support the hypothesis that the epidemic is resulting from a novel pathogen undergoing recent and rapid range expansion. Other studies suggest that strains may differ in virulence. Gahl et al. (2012) found that green frogs (L. clamitans) exposed to a local strain from northeast North America (JEL404) did not die, but when exposed to the strain responsible for amphibian dieoffs in Panama (JEL423), 60% of green frogs died after 15 days of exposure. Retallick and Miera (2007) exposed chorus frogs (Pseudacris triseriata) to two isolates of Bd collected from dead frogs in proximally located lakes, and found that a larger percent of frogs survived when exposed to one isolate compared to the other. Similarly, Berger et al. (2005) exposed green tree frogs (Litoria caerulea) to three isolates of Bd and found that time to death varied with isolate.

GENERAL IMPACTS Batrachochytrium dendrobatidis was first recognized as a cause of amphibian declines in 1997 (Pessier et al. 1999). Chytridiomycosis, the amphibian disease associated with Bd, has been implicated in global amphibian declines (Kiesecker 2011, Blaustein and Johnson 2010). Upon introduction, the novel pathogen has caused severe declines and in some cases localized extirpations or extinctions (Morgan et al. 2007, James et al. 2009). Tropical montane habitats are most suitable for Bd survival and persistence, and, because these areas also are iodiversit “hots ots” for am hi ians, the disease has had the most devastating impacts in these areas (Wake et al. 2008). Bd prevalence within a population can vary from 0-100% infected (Rosenblum et al. 2010), different infection intensities in different species range from asymptomatic to death within a few weeks (Kilpatrick 2009), and individuals within a species may vary significantly in susceptibility (Rosenblum et al. 2010). Chytridiomycosis impacts populations that are already impacted by other threats such as increased UV radiation, disease, and climate change (Wake et al. 2008). Some populations have recovered and persist with endemic infections (Blaustein et al. 2010, Kriger and Hero 2006, Retallick et al. 2004, Lampo et al. 2012). There is some evidence that selection pressure alters an amphibians population by favoring individuals with stronger immune defenses against Bd which may include antimicrobial skin peptides, mucosal antibodies, complement factors, and the involvement of cytokine receptors (Richmond et al. 2009). Symptoms of chytridiomycosis include lethargy, frequent skin sloughing, and an inhibited righting reflex (Pessier 2002), with Bd eventually killing animals by heart failure (Voyles et al. 2007, 2010, 2011).

EFFECTS OF TEMPERATURE ON Bd BIOLOGY Batrachochytrium dendrobatidis tolerates a wide temperature range (Woodhams et al. 2008). Temperatures between 4 -28 °C are suitable for Bd growth (Piotrowski et al. 2004, 4

Woodhams et al. 2003). Growth is slow below 10°C, above 25°C, and ceases at 28°C (Figure 1.2) (Piotrowski et al. 2004, Bradley 2002). Optimal growth occurs between 17- 25°C (Piotrowski et al. 2004), with fastest development of implanted zoospores occurring at 23°C (Figure 1.2) (Longcore et al. 1999, Bradley 2002). Bd can survive on hosts inhabiting pools that approach freezing temperatures (Seimon et al. 2006). At cool temperatures (10°C), Bd produces more zoospores per sporangium and zoospores live longer (Woodhams et al. 2008). For example, at 23°C zoospores are motile for 24 hrs and can move approximately two centimeters (Piotrowski et al. 2004), but at cooler temperatures (4-14°C) zoospores are motile for 48 hours and likely move farther (Woodhams et al. 2008). Although Bd growth is slowed at cool temperatures, it can persist in the environment. As temperatures increase, the rate of Bd mortality increases. Plated Bd replicates kept at 30°C for 8 days resulted in mortality of 50% of the cultures (Piotrowski et al. 2004). As temperature increases, lifespan decreases. 100% mortality occurred after 96 hours at 32°C, after 4 hours at 37°C, after 5 minutes at 60°C, and after one minute at 100°C (Johnson and Speare 2003). However, temperatures at the higher end of the spectrum are not relevant in natural systems. At 23°C there is a higher rate of zoospore encystment and a shorter time from encystment to sporulation (Woodhams et al. 2008). Whereas at 10°C zoospore mortality is decreased and the number of viable spores produced per sporangium is higher (Woodhams et al. 2008). The combination of these factors results in greater Bd population growth at 23°C than 10°C (Woodhams et al. 2008). Overall, these laboratory studies indicate that Bd can maintain a relatively high growth rate over the range of temperatures most commonly experienced by amphibians (Woodhams et al. 2008).

EFFECTS OF TEMPERATURE ON AMPHIBIAN SUSCEPTABILITY TO BD Many aspects of amphibian biology are affected by temperature, including physiological and immunological responses to pathogens such as Bd. Amphibians can thermoregulate by seeking shelter or basking in the sun. Ouelett et al. (2005) found lower Bd prevalence among amphibians in North America collected between July and September and suggested that this may be evidence of increased amphibian immune system function during warmer months. Warm temperatures increase the rate of metabolism and the rate of epithelial turnover (Berger et al. 2005). Berger et al. (2005) suggested that a high rate of epithelial turnover could assist amphibians in clearing Bd infections because Bd would not have time to complete its lifecycle before the skin layers are shed. At cooler temperatures, the amphibian immune system is less competent and Bd growth may be favored (Forrest and Schlaepfer 2011). For example, African clawed frogs (Xenopus laevis) displayed increased establishment of Helminth parasites at cooler temperatures (Jackson and Tinsley 2002) and red spotted newts (Notopthalmus viridescens) decreased leukocyte production and lysozyme activity at cool temperatures (Raffel et al. 2006). Among ranids, wood frogs (Lithobates sylvaticus) stopped synthesis of antimicrobial peptides at cooler temperatures (<7°C) (Matutte et al. 2000). Leopard frogs (Lithobates pipiens) could not create lymphocytes at cold temperatures (5°C) experienced during hibernation and this decreased their ability to recognize and respond to an antigen (Maniero and Carey 1997). When leopard frogs were returned to normal temperature (22°C), their immune competence was quickly restored (Maniero and Carey 1997, Cooper et al. 1992). Amphibian immune systems undergo involution during hibernation and cold temperatures, and this may allow rates of Bd infection in populations to increase (Rollins-Smith et al. 2011).

Amphibian species occurring in different environments and at different altitudes display adaptations to different temperature regimes. For example, studies of tropical species found that low elevation species were impaired and unable to move at 5°C whereas high elevation species were able to achieve 80% of peak performance at the same temperature (Navas 1996). Snyder and Weathers (1975) found that as species distributions of amphibians below 1000 masl shift away from the equator, they can tolerate a broader range of temperature variation. Hutchinson et al (1976) found that the Lake Titicaca frog (Telmatobius culeus) which inhabits cold lakes (10°C) at high elevations, has a particularly small erythrocyte volume and high erythrocyte count, elevated oxygen capacity, hemoglobin, and hematocrit compared to most amphibians, and among the lowest metabolic rate reported for amphibians. Immune function may display similar temperature adaptations such that the immunological capabilities of species living in cool environments may remain competent despite low temperatures.

In addition to the impacts of mean temperature, the degree of temperature fluctuation may also impact amphibian biology. Woodhams et al. (2003) suggest that an 8°C temperature fluctuation within a 24 hour period may favor amphibians because amphibians placed in enclosures with fluctuating temperatures survived longer than amphibians held at constant temperature. Raffel et al. (2012) disagreed and found that immune response was compromised in red spotted newts (Notopthalmus viridescens) experiencing widely varying temperatures in their natural habitat. Finally, frogs are able to control their temperature behaviorally by basking or seeking refuge. This thermoregulation may favor amphibians in some cases and Bd in others. Amphibians are unlikely to expose themselves to extreme temperatures that could cause dessication or freezing. Navas (1996) noted that amphibian species occurring at high altitudes bask or seek refuge under large rocks. Amphibians behaviorally maintaining their body 7

temperature within an ideal range may assist Bd growth by providing an environment matching the optimal temperature range for the fungus (Lauch et al. 2008). However, amphibians may also increase their body temperature above the optimal (and even lethal) temperatures for Bd by basking. In Panamanian golden frogs (Atelopus zeteki). Bd infections were less common on frogs with warmer body temperatures (Richards- Zawacki 2009).

EX-SITU STUDIES OF AMPHIBIAN-BD (HOST-PATHOGEN) INTERACTIONS IN THE FRAMEWORK OF TEMPERATURE Several laboratory studies have increased our knowledge of how temperature affects the host pathogen dynamic between amphibians and Bd. In addition, models developed from these studies have made predictions regarding this relationship for application to in-situ conservation. Several studies have analyzed survivability of infected amphibians at different temperatures. For example, Bd exhibited increased pathogenicity to the great barred frog (Mixophytes fasciolatus) at 17°C compared to 27°C (Table 1.1)(Berger et al. 2004), and Panamanian golden frogs (Atelopus zeteki) exposed to Bd survived longer at 23°C than at 17°C (Table 1.1)(Bustamante et al. 2010). Several lab experiments have analyzed how increased temperatures might assist amphibians in clearing infections (Table 1.1). In midwife tadpoles (Alytes obstetricans), most individuals lost infections when left in water warmer than 26°C for 5 days but not in cooler water (21.4°C) (Geiger et al. 2011), and orange-eyed tree frogs (Litoria chloris) housed at the relatively high temperature of 37°C for 16 hrs were able to clear Bd infections (Woodhams et al. 2003). Another experiment compared the effects of constant versus variable temperatures on the ability of L. chloris to survive Bd infections (Woodhams et al. 2003). Frogs at a constant 20°C were short-lived compared to those experiencing daily temperatures fluctuating between 13.5-23.5°C. Inducing a temperature spike also affects the interaction between 8

amphibians and Bd. An imposed temperature spike to mimic basking reduced Bd growth which authors hypothesized to be the result of either lower Bd survival at warmer temperatures or lower production of zoospores per sporangium (Daskin et al. 2011). Woodhams et al. (2008) found both lower Bd survival and fewer zoospores produced per sporangium at warmer temperatures but also established that Bd reproduction is disrupted by temperatures which exceed the critical threshold (30°C). Similar studies described above, combined with field studies of Bd prevalence in natural populations, have led to the creation of models to predict Bd distribution and survivability of infected amphibians. Woodhams et al. (2008) predicted how temperatures will impact survival of infected amphibians suggesting that infected amphibians will live longer at 10°C than 23°C because at warmer temperature they will reach their species-specific infection threshold sooner. However, thresholds vary among species, populations, and individuals (Rosenblum et al. 2010), so predictions from one study may not be generalizable.

IN-SITU STUDIES OF AMPHIBIAN-BD INTERACTIONS IN THE FRAMEWORK OF TEMPERATURE Studies of several factors correlated with temperature, including altitude, season, and habitat type, have focused on field studies of the interaction between amphibians and Bd. Low Bd prevalence has been found among amphibians inhabiting very cool (near 0°C) or very warm (30°C) waters. Kriger and Hero (2007) found a significant negative relationship between water temperature and the number of zoospores in the water. Among samples collected from lowland leopard frogs (Lithobates yavapaensis) in Arizona, Forrest and Schlaepfer (2011) compared Bd prevalence in pools fed by hot springs to regular pools and found very low Bd prevalence in geothermally influenced pools. Bd prevalence in water below 15°C was 75-100% whereas prevalence in water above 30°C was less than 10%. Alternately, Hossack et al. (2010) identified low 9

prevalence (3%) from a variety of species sampled in streams with snow-dominated hydrology collected in the Appalachian, Rocky, Cascade, and west coastal mountain ranges with temperatures less than 10°C for most of the year. Findings from the above studies suggest that Bd prevalence is highest in water above 10°C but below 30°C.

Studies of the influence of altitude on Bd prevalence include samples collected from sea level up to 2600 masl and generally agree that Bd prevalence is highest at mid-range sites but occurs at lower altitudes and up to the highest altitudes amphibian hosts inhabit. The rancho grande harlequin frog (Atelopus cruciger) in Venezuela was historically distributed from sea level to 2400 masl; today only remnant populations exist below 320 masl where Bd persists at low infection rates (10%) within the population (Lampo et al. 2012). This indicates that the amphibian species distribution is now restricted to a zone outside the optimal temperatures for Bd. In Panama, a study between 80-760 masl found higher elevation frogs were nine times as likely to be infected as amphibians at the lower elevation sites (Brem et al. 2008). In Costa Rica, samples collected from 30 different amphibian species over the altitudinal range 100-2600 masl identified Bd prevalence as 17.6% between 100-399 masl, 14.3% between 1000-1299 masl, and 14.3% between 1900-2200 masl (Puschendorf et al. 2006). In contrast, a study in Australia found no evidence of an altitudinal effect of Bd among amphibians collected from 90-885 masl (overall frequency 35% Bd positive) (Kriger and Hero 2008). In Colorado, USA northern leopard frogs (Lithobates pipiens) were studied over their historical distribution and none were found above 2200 masl which may be the result of Bd-related declines at these higher altitudes. At lower elevations, Bd infections were not observed in northern leopard frogs (Johnson et al. 2011). Bd can persist among high altitude amphibians at sites where they endure both extreme daily temperature variation (30°C), and tolerate freezing temperatures (-13.5 °C). Seimon et al. (2006) identified Bd among samples collected 10

from Pleurodema marmorata, Bufo spinulosus, and Telmatobius marmoratus in Peru at 5400 masl.

Seasonally, Bd is most common during the cooler seasons of spring, fall, and in winter in places with moderate climates. Bd prevalence generally declines during the warmest part of the year. A study of museum specimens collected in North America between 1960- 1990 identified a seasonal pattern where Bd was more common in amphibians collected during the spring (April, May, and June) and autumn (October) (Ouelett et al. 2005). Several studies of more recently collected samples support a similar seasonal pattern. In Australia, Kriger and Hero (2007) noted that disease prevalence varied significantly between seasons such that it was inversely related to the previous 30-day mean air temperature. Kinney et al. (2011) found that Bd prevalence was much lower in adult Lithobates areolatus coming to breeding pools (27% positive) than in adults exiting pools after breeding activity (46% positive). This may be an artifact of both increased contact during mating activities and warming from below to within the ideal temperature range for Bd. In Virginia, USA Bd prevalence was associated with season with the highest prevalence (45%) occurring in late May (average temperature 18-19°C) while prevalence declined substantially (to 2%) during the warmer months of late summer (Pullen et al. 2010). Gaertner et al. (2009) found that individual cricket frogs (Acris crepitans blanchardi) that tested positive in the cooler spring tested negative for Bd during summer months. In Lithobates yavapaensis in Arizona, Bd prevalence during summer months was low compared to mild winter months where Bd prevalence was high resulting in the majority of Bd-associated mortalities (Savage et al. 2011). Higher temperatures may enable individual amphibians to clear infections by behavioral warming in these environments. In contrast, in a much cooler climate, Bd prevalence was highest (85% positive) during the warmest part of the year (17°C) among marsupial frogs (Gastrotheca 11

riobambae) in Quito, Ecuador (Manzano 2010). In general, Bd prevalence is positively correlated with seasonal temperatures that correspond to the optimal range for Bd despite temperatures varying with season, altitude and latitude.

Habitat associations of Bd prevalence have found higher infection rates in cooler, more moderate temperatures. In a study that compared Bd prevalence in ponds with different periods of inundation, higher rates of Bd were detected in Lithobates areolatus from samples collected in deeper, cooler, more permanent pools than shallow, warmer, semipermanent pools (Kinney et al. 2011). Raffel et al. (2011) noted a general pattern of higher Bd prevalence among newts (Notopthalmus viridescens) in cooler, shaded terrestrial sites.

In summary, several studies of the influence of temperature, and the influence of factors correlated with temperature (e.g., altitude, season, and habitat) on Bd dynamics have found significant temperature impacts on amphibian susceptibility to Bd. However, other factors such as timing of amphibian breeding, precipitation, and cloud cover, and other environmental factors may facilitate the spread of Bd.

INFLUENCE OF GLOBAL SCALE TEMPERATURE PATTERNS ON CHYTRID EPIDEMICS Global scale climate change has led to disease outbreaks in a growing number of species worldwide. For example, warm years in the Caribbean have triggered outbreaks of the bacterial Caribbean yellow band disease which resulted in widespread coral bleaching (Weil and Croquer 2009). In addition, abalones (Haliotis) on the west coast of California, USA show increased transmission of the rickettsial agent causing withering syndrome due to warm water (Vilchis et al. 2005). Extended breeding seasons have 12

increased the time of parasite exposure as in the case of Ophryocystis elektroschirrha, the protozoan parasite on monarch butterflies (Harvell et al. 2002). Parasite range expansion has increased in some species, making host species that historically exceeded the parasite range more susceptible. This appears to be the case for the mountain pine beetle (Dendroctonus ponderosae) parasitism on lodgepole pines in Colorado USA (Carroll et al. 2003) and the protozoan Perkinsus marinus parasitism on oysters along the Atlantic coast, USA (Harvell et al. 1999). Finally, populations of lions stressed by canine distemper disease and exposed to unusually high levels of tick-infested prey in warm years led to increased mortality rates (Munson et al. 2008).

Climate change is impacting amphibian species in a variety of ways. For example, degraded body condition of common toads (Bufo bufo) in England are associated with rising temperatures (Reading 2007). Also, climate change has triggered earlier breeding in the natterjack toad (B. calamita) at the northern range of its distribution in England at a rate of 9-10 days per 1°C increase in maximum temperature (Beebee 1995). Amphibians (Lithobates sylvaticus, L. catesbeianus, L. clamitans, and Pseudacris crucifer) in New York, USA are calling 10-13 days earlier than they were a century ago (Gibbs et al. 2001). Increased mortality has been reported among amphibians due to increased UV-B penetration in aquatic habitats caused by decreased cloud cover which is associated with climate change (Blaustein et al. 1995).

Two hypotheses regarding climate and chytridiomycosis have been proposed to explain widespread amphibian declines. The climate-linked epidemic hypothesis suggests that climate is promoting disease and thus decreasing biodiversity. Large scale warming has been proposed to be a key factor in the disappearance of amphibians (Pounds et al. 2006). The chytrid-thermal optimum hypothesis suggests that temperatures at highland localities 13

are shifting toward the growth optimum for Bd such that increased vapor creates increased cloud cover which makes nights warmer and days cooler resulting in reduced temperature variation (Pounds et al. 2006). In Spain, rising temperature is linked to the occurrence of chytrid-related disease in central Spain (Bosch et al. 2007). A warmer overwintering regime increased the probability of infection of common toads (Bufo bufo), supporting the concept that the dynamics of infection are altered by changing environmental temperature profiles (Garner et al. 2011).

Climate change is increasing stress experienced by amphibians in several ways, and chytridiomycosis may be exacerbating this stress. Pounds et al. (2006) and Blaustein and Dobson (2006) noted that episodic loss of frogs occurred in years that were unusually warm across the tropics. Later, Pounds and Coloma (2008) added that regional scale temperatures have tended to peak the year before the last observation of declining populations was made in the tropics. Pounds et al. (1999) suggested that climate change has impacted the interaction between amphibians and Bd, and that the stress created by climate change favors the pathogenic fungus and has led to amphibian population declines.

Declines observed in amphibian populations are non-random regarding species ecological preferences and geographic distributions (Stuart et al. 2004). Amphibian species occurring in tropical montane regions have been most impacted, and tropical montane regions are also highly influenced by climate change. In Ecuador, temperatures in the Andes have increased 2°C or four times the global average (Ron et al. 2003). Many tropical montane amphibian species have small population distributions and are much more vulnerable to extinction. Wide-ranging temperate amphibians may suffer localized extirpations, but because of their distribution they are less vulnerable to extinction. 14

Puschendorf et al. 20 ) said “Species that are tolerant of broad environmental gradients may be less vulnerable to epizootic outbreaks of disease.” Widel distri uted species will persist in refuges beyond the thermal optimum for Bd while species with small, isolated populations occurring within the thermal optimum for Bd will suffer declines and perhaps extinctions.

CONCLUSION In natural populations, high rates of Bd transmission among amphibians at cooler temperatures are the result of the interplay of various aspects of the biology of Bd and amphibians. In temperate environments, amphibians congregate at breeding pools in the spring. Cold-induced suppression of the immune system combined with increased skin- to-skin contact during breeding activities work together to increase prevalence among conspecifics. As the weather warms, amphibian immune systems become more competent, and most amphibians are likely to clear infections. However, in moderate temperature conditions Bd produces more zoospores per sporangium and zoospores live longer and move longer distances increasing the probability of infection (Woodhams et al. 2008). For the individual amphibians with poor immune systems, Bd is able to grow exponentially and infection intensities increase. This combination of factors enables Bd to colonize weakened amphibian hosts and dramatically increase Bd intensity. Bd temperature sensitivity, amphibian behavior, and amphibian immune system competence may be key factors in explaining and predicting outbreaks of chytridiomycosis.

In the following chapters, I present the findings of several studies which contribute to our understanding of how temperature affects the interaction between amphibians and Bd. I assess the dynamics of Bd infections in both temperate and tropical environments. Individually, the studies address specific questions regarding the distribution and 15

interaction between amphibians and Bd. Together, the studies broaden our understanding of Bd dynamics and provide the groundwork for future work in amphibian conservation.

Tables and Figures

Table 1.1. Results of laboratory studies of the interaction between temperature and Bd infections in various amphibian species. Species Temperature (°C) Time of Exposure % cleared N Source infection Mixophytes fasciolatus 27 98 days 50 8 Berger et al. 2004 Litoria chloris 37 16 hrs 100 10 Woodhams et al. 2003 Lithobates catesbeianus 30 10 days 100 12 Chatfield et al. 2011 Acris crepitans 30 10 days 94 16 Chatfield et al. 2011 Pseudacris triseriata 32 5 days 100 6 Retallick et al. 2007 Alytes obstetricans 26 5 days 88 8 Geiger et al. 2011 Plates 30 8 days 50 ? Piotrowski et al. 2004 Mixophytes fasciolatus 17 25-29 days 0 8 Berger et al. 2004 Mixophytes fasciolatus 23 29-76 days 0 8 Berger et al. 2004

Figure 1.1. Batrachochytrium dendrobatidis growth, development, and infection of amphibian skin. A) Life cycle of Bd including substrate dependent and independent stages. Diagram Rosenblum et al. 2010. B) Development of a zoosporangium in amphibian tissue and the production of zoospores, zoospore release. Available: http://www.esf.edu/efb/brunner/research.html. C)In order: zoospores, zoosporangium with thread-like rhizoids, zoosporangium after zoospores are discharged, newly released zoospores, Bd infection on Leopard frog (Lithobates pipiens) skin. Available: http://www.broadinstitute.org/annotation/genome/batrachochytrium_dendrobatidis/Multi Home.html D) Cross section of zoosporangium in amphibian skin. Berger et al. 1998. E) Mild, medium, and heavy Bd infection loads on amphibian skin. Available: http://www.umaine.edu/chytrids/Index-Batrachochytrium.html.

Chapter 2: Review of Batrachochytrium dendrobatidis (Bd) in Ohio, with references to new data

INTRODUCTION An estimated 32% (1856) of amphibian species are threatened with extinction and 43% (2469 species) have populations that are declining (Kiesecker 2011). In the Midwest region of the USA, reports have documented the decline or local extirpation of species including Lithobates sylvaticus in the southern part of their range (Thurow 1994), and L. pipiens (Hine et al. 1981) and Acris crepitans in the northern part of their range (Lehtinen and Skinner 2006). Some populations of these species have disappeared from remote and protected areas (Lehtinen and Skinner 2008). Declines and loss of amphibian populations from pristine and protected areas worldwide have occurred frequently in recent decades (Collins and Storfer 2003) and in many cases the cause of these losses is unknown.

One suspected factor involved in such declines and extirpations is the amphibian pathogenic fungus Batrachochytrium dendrobatidis (Bd). It is nearly global in distribution (Bd spatial epidemiology). Bd differentially impacts amphibian host species (Gahl et al. 2012, Rosenblum et al. 2010) and varies in virulence according to fungal strain and local environmental characteristics (Kilpatrick et al. 2009). Bd prevalence within a population can vary from 0-100% infected (Rosenblum et al. 2010), different species vary greatly in their response to a given infection intensity level (from asymptomatic to lethal (Kilpatrick 2009)), and individuals within a species may vary significantly in susceptibility (Rosenblum et al. 2010). Bd has an aquatic zoospore stage 21

that allows it to spread in moist or aquatic environments, and spread is facilitated by direct contact between amphibians during mating, territorial encounters, or from parent to offspring in species that provide parental care (Belden et al. 2007). Symptoms of chytridiomycosis include lethargy, frequent skin sloughing, and an inhibited righting reflex (Pessier 2002), and Bd eventually kills animals by heart failure (Voyles et al. 2007, 2010, 2011).

Environmental characteristics may affect the dynamics of infection (Blaustein et al. 2010). Known factors include ambient temperature (Berger et al. 2004, Bosch et al. 2007, Conradie et al. 2011, Daskin et al. 2011, Forrest et al. 2011, Gaertner et al. 2009, Gieger et al. 2011, Kilpatrick et al. 2009, Lowe et al. 2009, Morgan et al. 2007, Piotrowski et al. 2004, Puschendorf et al. 2009, Richards-Zawacki et al. 2009, Rollins-Smith et al. 2011, Rohr et al. 2008, Woodhams et al. 2003), substrate moisture (Raffel et al. 2011) and rainfall (Daszak et al. 2005, Kriger and Hero 2007,Puschendorf et al. 2005). In addition to environmental conditions, characteristics of the amphibian immune system and behavior can affect severity of Bd infections (Rollins-Smith et al. 2011). Amphibian immune systems can be depressed by environmental factors such as UV-B radiation and chemical pollutants (Rollins-Smith et al. 2011), by cold temperatures experienced during hibernation (Rollins-Smith et al. 2011), and during the changes that take place as tadpoles metamorphose (Rollins-Smith et al. 2011). Savage et al. (2011) found that many hibernating L. yavapaeinsis died from chytridiomycosis in overwintering retreats. Amphibians in temperate zones worldwide have not been impacted as severely by Bd as their counterparts in tropical montane habitats, and this may be related to climatic differences.

Bd has been identified in amphibian populations throughout most of the continental USA (Brodman and Briggler 2008, Campbell et al. 2008, Chatfield et al. 2009, Chinnadurai et al. 2000, Gaertner et al. 2009, Hossack et al. 2010, Kinney et al. 2011, Krynak et al. 2012, Lauch et al. 2008, Longcore et al. 2007, Lowe 2009, Ouelett et al. 2005, Pullen et

al. 2010, Raffel 2011, Rothermel et al. 2008, Steiner and Lehtinen 2008, Todd-Thompson et al. 2009, Venesky and Brem 2008, Zelmer et al. 2008, Zippel and Tabaka 2008, Hayes et al. 2009, Harner et al. 2011). The impacts of Bd are as varied as the species and landscapes of the United States. In the Sierra Nevada mountain range of California, populations of mountain yellow legged frogs (Lithobates muscosa) inhabiting alpine pools have successively declined as Bd spread from one pool to the next (Vredenburg et al. 2010). In the pacific northwest, Bd was identified in a population of Oregon spotted frogs (Rana pretiosa) in Washington. In the Southwest, it has been documented among Bufo boreas in the Rocky Mountains of Colorado from 1030-3550 masl (Muths et al. 2008). In lowland leopard frogs, L. yavapaiensis, high Bd prevalence (75-100%) was detected in cooler pools while much lower prevalence (less than 10%) was detected in thermal pools at temperatures above 30°C (Forrest et al. 2011). In the Midwest, higher Bd prevalence was identified among newt populations in cooler, more shaded areas (Raffel 2011). In Virginia, an overall Bd prevalence of 11.9% infected was identified. A study of cricket frogs Acris crepitans identified the presence of Bd at every site where 10 or more samples were collected, but infection prevalence was low and the frogs seemed healthy (Steiner and Lehtinen, 2008). In the Northeastern United States, Bd is widespread but no Bd-related declines have been documented (Longcore et al. 2007). In the Southeast, the result of seven years sampling to collect 1200 samples from 30 sites identified widespread, subclinical Bd infections among amphibians during breeding season (Rothermel et al. 2008). In the Smoky Mountains, chytridiomycosis was determined to be the cause of death for an L. palustris specimen (Todd-Thompson et al. 2009).

There is evidence that d arrived in North America in the 960’s Ouelett et al. 2005). The purpose of this paper is to document the taxonomic and geographic distribution of Bd in Ohio, a temperate region of North America with significant seasonal changes in temperature. It is unclear how broadly distributed the fungus was initially in Ohio, whether or not there were significant epizootic impacts on any species, and whether or

not Bd now is best considered enzootic in Ohio. Our aims of this study were to determine the geographic and taxonomic extent of Bd in Ohio and the level of infection intensities for each species. Here we show that it is widespread in Ohio.

MATERIALS AND METHODS Sample collection We collected 1497 samples from 25 species in 8 families (see Table 2.1) collected from seven different aquatic habitats (forested vernal pool, emergent seasonal wetland, marsh, riverine wetland, permanent pond, river, ephemeral stream) and terrestrial sites. Our samples were distributed throughout the season of amphibian activity (approximately March through November) and included adults and juveniles (n=1259), and larvae (n=256). Sample collection occurred throughout Ohio, but most samples were taken in the central and eastern parts of the state. Data reported in this paper include data published in Lehtinen and Steiner (2008), and Krynak et al. (2012). Pooled data from a number of sampling efforts provide the best opportunity to analyze the statewide distri ution of d amon Ohio’s am hi ians.

Frog collection and swab sampling Frogs were captured by hand or dip net and precautions were taken to minimize sample contamination. Adult and juvenile frogs were swabbed following the techniques of Brem et al. (2007) that concentrate on the feet, hind limbs, and drink patch. Tadpole mouthparts were swabbed (Stockwell et al. 2010) by 10 strokes over the beak and salamander larvae were swabbed on their feet and belly. Swabs were air dried, labeled, and stored in the freezer until they were processed by PCR analysis.

PCR analysis and zoospore equivalents (ZE) Some samples were analyzed by quantitative Taqman real-time PCR following a modified protocol of Boyle et al. (2004) while other samples were analyzed by conventional PCR (Annis et al. 2004). Quantitative TaqMan PCR measures the

amplified products directly during the exponential phase of amplification cycle using fluorescent hybridization probes and in addition to providing quantified data, it is more sensitive than conventional PCR (Hyatt et al 2007). TaqMan PCR used the primers ITS1- 3 Ch tr 5’-CCTTGATATAATACAGTGTGCCATATGTC-3’) and 5.8S-Ch tr 5’- AGCCAAGAGATCCGTTGTCAAA-3’) o le et al. 2004). The am lification conditions were carried out on 25 µl containing 12.5 µl 2X TaqMan Master Mix, PCR primers at concentrations of 900 nM, MGB probe at 250 nM and 5 µL of DNA (diluted 10-1 in water) and run in triplicate (Boyle et al. 2004). Default ABI Prism 7700 amplification conditions (2 minutes at 50°C, 10 minutes at 95°C) followed by 15 seconds at 95°C and 1 minute at 60°C for 50 cycles were used (Boyle et al. 2004). PCR analysis was run in triplicate and zoospore equivalents (ZE) were calculated by averaging the three samples and multiplying by a factor of 100 to account for dilution. Conventional PCR, which measures end-point products of PCR and can detect at a minimum of 10 zoospores or 10 of DNA, used the rimers d a 5’-CAGTGTGCCATATGTCACG- 3’) and d2a 5”-CATGGTTCATATCGTCCAG-3’), each 300 . Am lification reactions were carried out in 25 µl which included 1µl of each primer, 0.9 mM MgCl2, 1 X Taq buffer, and 0.2 nM of each dNTP and 0.8 units of Platinum, and Taq polymerase. The amplification conditions incudes an initial denaturation at 93°C for 10 minutes followed by 30 cycles of 45 seconds at 93°C, 45 seconds at 60°C, 1 minute at 72°C and a final extension of 10 minutes at 72°C. 10 µl of the reaction were then run on 1.2% agarose to visualize the bands (Annis et al. 2004).

RESULTS Distribution and overall prevalence of Bd Bd was widely distributed throughout the state (Figure 2.1). We found an overall Bd prevalence of 19.5% positive among 1259 adult and juvenile samples from 23 species (Table 1) and a prevalence of 3.5% positive among 256 larval samples of 6 species of frogs and salamanders (Table 2.2).

Taxonomic distribution of Bd We detected Bd among nearly all anuran and caudate families sampled in this study: Bufonidae, Hylidae, Ranidae, Ambystomatidae, Plethodontidae, Proteidae and Salamandridae. None of the hellbender salamanders (Cryptobranchus alleganiensis, family Cryptobranchidae) tested positive for Bd. Prevalence was highest among bullfrogs (Lithobates catesbeianus) 59.2% , Jefferson’s salamanders Ambysotma jeffersonianum) 35.5% , red spotted newts (Notophthalmus viridescens) 32.0% (Table 2.1). Among sampled larvae, only bullfrogs (L. catesbeianus) and green frogs (L. clamitans) tested positive for Bd. Samples collected from ambystomatid larvae (A. maculatum (n=13), A. texanum (n=6) and A. tigrinum (n=1) were all negative.

Amphibian infection intensities Mean infection intensities were highest among Acris crepitans (1,722,094 ZE), Lithobates clamitans (19,328 ZE) and Notopthalmus viridescens (19,045 ZE) (Table 2.1). Although the second highest intensity was detected among L. clamitans samples, most animals tested had low level infections. 73% of green frogs had infection intensities less than 100 ZE and 85% (n=151) had infection intensities less than 1000 ZE. Infection intensity patterns by family were strongly influenced by individual species as mentioned above (Table 2.1). Infection intensities were higher among anurans than caudates.

Habitat distribution of Bd Bd was detected in all wetland habitats tested, including: permanent and ephemeral, forested and open-canopy, and streams and ponds. Bd prevalence was highest among amphibians at emergent pools (40.2%) and lowest among forested pools (12.9%) (Table 2.3). Among salamanders, Bd prevalence was slightly higher among pond species (Ambystomatids 16.2%, Salamandrids 32%) than stream species (Cryptobranchids 0%, Plethodontids 8.1%, and Proteids 25%) (Table 2.1).

Seasonal patterns in Bd distribution Bd prevalence was highest among autumn samples (33.7%) followed by spring (26.3%) and summer (15.6%) (Table 2.4). We removed bullfrogs and green frogs because we suspected large sample sizes and high prevalence were driving these results. We found Bd prevalence was highest among spring samples (17.9%), followed by summer (11.0%) and autumn (0.0%).

Geographic distribution of Bd No pattern emerged in Bd prevalence distributed across the state when all samples were examined. Regions ranged from 9.9-26.4% Bd positive, and the mean infection prevalence for the state was 19.5% Bd positive (Figure 2.1). To remove the bias of bull frogs (Lithobates catesbeianus) and green frogs (L. clamitans), we examined three species, Ambystoma maculatum, Anaxyrus americanus, and L. sylvaticus, for possible patterns of infection across the state and found higher Bd prevalence among amphibian populations in the northern counties than in the central part of the state (Table 2.5).

DISCUSSION Bd is widely distributed throughout the state of Ohio. Prevalence was highest in central Ohio, but the greatest number of bullfrogs (Lithobates catesbeianus) and green frogs (L. clamitans), species with the highest prevalence of Bd, were collected from this region Fi ure 2. ). d has een in North America since the 960’s Ouelett et al. 2005). Perha s the ersistence of Ohio’s am hi ians des ite the resence of d is an e am le for other parts of the world where amphibians have recently declined and are currently declinin due to d e izootics. Althou h e don’t kno ith certaint that Ohio’s amphibians did or did not decline upon arrival of Bd, we do know that they persist together in the environment today. There are examples of rebounding populations where declines recently occurred in Australia, South and Central America (Retallick et al. 2004, Lampo et al. 2012, Briggs et al. 2010, Richards-Zawacki et al. 2009). The status of Ohio’s am hi ians and evidence of more recent o ulation declines and recoveries

demonstrates the resiliency of amphibian populations in recovering from epizootic Bd outbreaks and may provide an example for what we should expect to happen with other amphibian species following the arrival of Bd. We found no evidence of symptomatic chytridiomycosis in animals tested in our study. We would have expected high intensity infections to bring forth symptoms in individuals, however, none were identified. Without recapture data on amphibians with high intensity infections, it is impossible to say if these animals survived.

Particular species of interest in Ohio include the hellbender (Cryptobranchus alleganiensis), cricket frogs (Acris crepitans), and bullfrogs (Lithobates catesbeianus). These species represent three different scenarios: a declining species with no evidence of Bd infections (hellbenders), high intensity infections but no symptoms (cricket frogs), and high prevalence but relatively low intensity infections (bullfrogs). Hellbenders live in large, cold water streams with summertime temperatures which are ideal for Bd growth and reproduction. Their populations have declined as much as 80% in Ohio (Pfingsten 1989). Our surveys did not detect Bd in hellbenders. Regester et al. (2012) found Bd on hellbenders in four of eight streams in the Susquehanna and Allegheny-Ohio drainages in Pennsylvania, and Gonynor et al. 2011 detected Bd on hellbenders in Georgia. Neither study observed symptoms associated with chytridiomycosis. Bd does infect hellbenders and may be an additional stressor involved in declines. Cricket frogs are unique for the high intensity infections which we encountered while sampling. One and a half to two million ZE is several orders of magnitude higher than Vredenburg’s 0,000 ZE threshold for L. muscosa (Vredenburg et al. 2010), and specimens we collected were asymptomatic. Our results suggest that cricket frogs can persist with high intensity infections and may be vectors for Bd. Cricket frogs show neither effective immune capabilities to limit infections to low intensities or susceptibility to chytridiomycosis by demonstrating symptoms of disease. It may be interesting to study this host-pathogen relationship more closely. Without recapture information of high intensity infection frogs, we cannot be sure that cricket frogs, a short-lived species, survive their typical lifespan. While cricket

frogs had notable infection intensities, bullfrogs are noteworthy for the highest Bd prevalence, 59.2% positive. Other studies have found Bd prevalence among asymptomatic bullfrogs as high as 96% positive (Kiesecker 2011). Bd prevalence is much hi her for ullfro s 59.2%) than overall revalence amon Ohio’s am hi ians 9.5%), so bullfrogs are also likely vectors.

Environmental characteristics of Ohio paired with the broad distribution of most amphibian species in the state may help explain the persistence of amphibians with significant Bd infection rates. Ohio has significant seasonal variation in temperatures characterized by cold winters, extreme temperature variation in the spring, and warm summers. Cold temperatures slow or stop Bd reproduction (Woodhams et al. 2003), variable temperatures facilitate Bd reproduction, and warm temperatures (above 30C, Woodhams et al. 2003) are lethal to Bd. Amphibian immune systems are least competent at cold temperatures and during metamorphosis (Rollins-Smith et al. 2011) that usually occurs during the warmest parts of the year when high temperatures may tend to limit Bd. There may be a small window of time, such as spring which is suggested by our data when bias from bullfrogs (Lithobates catesbeianus) and green frogs (L. clamitans) is removed, when amphibians are vulnerable and likely to experience high intensity Bd infections. Ohio hosts 40 amphibian species of which we sampled 23 species. All of the species we sampled here are widely distributed throughout Ohio and occur across several states (National Amphibian Atlas). Puschendorf et al. (2011) suggested that amphibian species tolerant of a broad range of environmental conditions may be less vulnerable to epizootic outbreaks of chytridiomycosis. While amphibians in other parts of the world (and the United States) are disappearing quickly, amphibians in Ohio seem to be successfully co-existing with Bd. However, we remain cautious because Bd is present amon Ohio’s am hi ians and ith variable weather patterns and threats of climate change it remains a concern to the persistence of temperate populations and species.

Tables and Figures

Table 2.1. Distribution of Bd in Ohio’s adult and juvenile amphibians, grouped by family. Includes breeding habitat association, total sample size, Bd prevalence rate (% positive), mean infection intensity (ZE), and sample size associated for infection intensity. Family Species Habitat* n % Bd Mean Intensity n positive (ZE)

Ambystoma jeffersonianum F, E, W 31 35.5 3958 11

Ambystoma maculatum F, E, W 57 15.8 2303 10

Ambystoma opacum F, E, W 5 0

Ambystoma texanum F, E, W 30 0

Ambystomatidae 123 16.2 3213 21

Bufonidae Anaxyrus americanus O-F, E, W 115 11.3 12140 9

Cryptobranchidae Cryptobranchus alleganiensis O, P, S 17 0

Hylidae Acris crepitans O, P, W-S 86 18.6 1722094 2

Hyla chrysoscelis F, E, W 15 26.7 675 4

Hyla versicolor F, E, W 24 0

Pseudacris crucifer F, E, W 27 0

Pseudacris triseriata O, P, W 1 0

153 13.1 574481 6

Plethodontidae Desmognathus fuscus F, E, S 41 2.4 382 1

Desmognathus ochrophaeus F, E, S 22 13.6 140 3

Eurycea bislineata F, E, S 72 5.6 174 4

Eurycea longicauda F, E, S 8 0

Plethodon cinereus F, T 79 11.4 590 10

Plethodon glutinosus F, T 9 0

Pseudotriton ruber F, E, S 16 18.8 991 3

247 8.1 494 21

Proteidae Necturus maculosus O-F, P, W-S 8 25.0

Ranidae Lithobates catesbeianus O, P, W 49 59.2 10544 34

Lithobates clamitans O, P, W 480 27.3 19328 122

Lithobates palustris O-F, E, W-S 12 25 459 3

Lithobates pipiens O, E-P, W 14 7.1 33 1

Lithobates sylvaticus F, E, W 75 21.3 8435 16

630 28.6 16302 175

Salamandridae Notophthalmus viridescens O-F, P, W 25 32.0 19045 8

*Generalized habitats. We used specific habitat information for individual amphibians in analyses. Habitat association key: F=forested, O= open canopy emergent, E= ephemeral (seasonally inundated), P= permanently inundated, W= wetland (marshes and ponds), S= stream or T= terrestrial. Source: ohioamphibians.com.

Table 2.2. Distribution of Bd in Ohio’s larval amphibians, grouped by family. Includes breeding habitat association, total sample size, Bd prevalence rate (% positive), mean infection intensity (ZE), and sample size associated for infection intensity. Family Species Habitat* n % Bd Mean Intensity n positive (ZE)

Ambystomatidae Ambystoma maculatum Forested pool 14 0

Ambystoma tigrinum Forested pool 1 0

Ambystoma texanum Forested pool 6 0

21 0

Hylidae Hyla versicolor Emergent pool 2 0

Ranidae Lithobates catesbeianus Emergent pool 142 3.8 570 4

Lithobates clamitans Emergent pool 91 4.4 1 4

233 3.5 286 8

Table 2.3. Distribution of Bd among aquatic amphibian habitats in Ohio. Habitat n % Bd positive

Emergent pools 391 40.2

Forested pools 442 12.9

Streams 509 33.4

Table 2.4. Seasonal (spring, summer, autumn) distribution of Bd among Ohio amphibians. Season N % Bd Positive

Spring 490 26.3

Summer 544 15.6

Autumn 208 33.7

Table 2.5. Geographic variation between populations of the same species for Ambystoma maculatum, Anaxyrus americanus, and Lithobates sylvaticus. County Ambystoma maculatum Anaxyrus americanus Lithobates sylvaticus

+ - % + - % + - %

Northern 9 22 29 5 19 21 16 22 42

Cuyahoga 9 15 5 7 3 8

Geauga 0 5 0 7

Lake 0 2 0 3 12 3

Medina 0 7 1 4

Summit 0 2

Central 0 25 0 7 83 8 0 55 0

Delaware 1 1

Franklin 0 25 6 81 0 55

Hardin 0 1

Eastern 0 1 0

Stark 0 1

Chapter 3: Seasonal and microhabitat correlates of the distribution of the amphibian fungal pathogen Batrachochytrium dendrobatidis in a temperate landscape

INTRODUCTION Chytridiomycosis, a cutaneous disease of amphibians caused by the fungal pathogen Batracohchytrium dendrobatidis (Bd), is an important factor involved in the declines of amphibian populations worldwide (IUCN, Blaustein and Johnson 2010, Kiesecker 2011). Bd is a chytridiomycete fungus (Longcore 1999) that has two life stages - an aquatic, mobile zoospore stage and a sessile zoosporangium which implants in the skin of amphibians, grows, and releases more zoospores into the environment (Longcore 1999, Piotrowski et al. 2004). It is a keratinophilic species and amphibians are its only known host. The pathogen infects both aquatic larvae and metamorphic individuals. In anuran amphibians (frogs and toads), it infects the keratinized mouthparts of tadpoles, later spreading to toe-webbing as the limbs and feet develop (Marantelli 2004). Spread of zoospores occurs in moist or aquatic environments and by direct contact between individuals (during mating or territorial encounters, and from parent to offspring in the case of parental care). Tadpoles are a suspected reservoir because they are less susceptible to the disease and remain in aquatic environments for months to years at a time (Rachowicz and Vredenburg 2004). Without a host, the motile zoospores can move approximately two centimeters and survive about 24 hours before they die (Piotrowski et al. 2004).

Symptomatic chytridiomycosis is characterized by lethargy, frequent skin sloughing, and an inhibited righting reflex (Pessier 2002). Bd colonization of the epidermal skin layers leads to hyperkeratosis and inhibits electrolyte transport across the epidermis. If sodium and potassium concentrations in the blood are significantly reduced, chytridiomycosis results in heart failure (Voyles et al. 2007, 2010, 2011). A critical infection level has been determined for only one species, the mountain yellow-legged frog (Lithobates muscosa). In this species, most frogs succumb to chytridiomycosis when a threshold of 10,000 zoospore equivalents is reached (Vredenburg et al. 2010). Zoospore equivalents are the unit of Bd zoospores detected and quantified in qPCR analysis.

The impact of Bd on both species and on different individuals within a species ranges from highly lethal to asymptomatic (Kilpatrick 2009). The functional basis of these varying effects remains under study (Voyles et al. 2007, 2009, 2011, Carver 2010). Research suggests that symbiotic bacteria on amphibian skin (Woodhams et al. 2007, Harris et al. 2006, Brucker et al. 2008), antimicrobial peptides (Woodhams et al. 2006, 2007, Richmond et al. 2009, Lam et al. 2011, Ramsey et al. 2010, Rollins-Smith et al. 2005), and behaviorally induced fevers (Lowe 2009, Richards-Zawacki 2009) may play a part in reducing susceptibility while environmental stressors such as pollution, increased UV-B, climate change, and other pathogens may increase susceptibility (Kiesecker 2011).

Temperature has been demonstrated to be a major factor affecting the interaction of Bd with amphibian hosts (Berger et al. 2004, Bosch et al. 2007, Conradie et al. 2011, Daskin et al. 2011, Forrest et al. 2011, Gaertner et al. 2009, Gieger et al. 2011, Kilpatrick et al. 2009, Lowe et al. 2009, Morgan et al. 2007, Piotrowski et al. 2004, Puschendorf et al. 2009, Richards-Zawacki et al. 2009, Rollins-Smith et al. 2011, Rohr et al. 2008, Woodhams et al. 2003). d has an o timal tem erature ran e of -23 C Piotro ski et al. 2004, Vo les et al. 20 ) and an o erative tem erature ran e of 4-28 C Woodhams et al. 2003). d dies at tem eratures e ceedin 30 C Woodhams et al. 2003). On the

cooler end of the s ectrum, re roduction slo s at - 0 C Vo les et al. 20 ) and sto s around 4 C Woodhams et al. 2003) ut the fun us can over inter at tem eratures approaching freezing on frogs in temperate regions (Garner et al. 20 , Sava e et al. 20 ). La orator studies have demonstrated that d re roduces faster i.e., roduces more zoos ores er unit time), is colonial, and is more infective at 23 C com ared to 0 C, hereas it roduces more zoos ores er s oran ium and is more often monocentric at the cooler tem erature Woodhams et al. 2008). The atho eneticit of d increases as tem erature cools from 2 C to C er er et al. 2004). Ho ever, these la orator results need to be extended to the field to understand how the fungus interacts with its amphibian hosts in natural environments. Bd-related amphibian declines have occurred on the lar est scale in tro ical montane re ions that re resent “hot s ots” for am hi ian diversity (Lips et al. 2006, Pounds et al. 2006). The climate of these regions seems well- matched to the optimal temperature range of the pathogen (Ron 2005). Impacts of Bd appear less at lowland tropical areas where temperatures often may exceed the thermal tolerance of Bd (Blaustein and Dobson 2006, Brem et al. 2008). Amphibians in temperate zones worldwide have not been impacted as severely by Bd as their counterparts in tropical montane habitats, and this may be related to climatic differences.

Records of Bd in North America date back to 1960 (Oulett et al. 2005) and it is widespread on the continent (Campbell-Grant et al. 2008, Gaertner et al. 2009, Lauch et al. 2007, Pullen et al. 2010, Rothermel et al. 2008. Savage et al. 2011, Steiner and Lehtinen 2008, Todd-Thompson et al. 2009). Significant impacts of Bd have been documented on North American species such as the mountain yellow-legged frogs (Lithobates muscosa) in California and the boreal toad (Anaxyrus boreas) in Colorado (Wixson and Rogers 2009, Vredenburg et al. 2010). Several studies have identified Bd in the Midwestern region of the USA (Brodman and Briggler 2008, Chatfield et al. 2009, Zelmer et al. 208) and Ohio (Steiner and Lehtinen 2008, Zippel 2008), although none have documented significant levels of symptomatic chytridiomycosis or Bd-related population declines.

The overall aim of this study was to examine the dynamics of Bd in amphibian populations in a temperate environment and to gain insight into the potential impact of the fungus on the amphibian communities in such areas. Our study focused on analyzing the effect of season, habitat, and lifestage on Bd infections in two species of frogs, American bullfrogs (Lithobates catesbeianus) and green frogs (L. clamitans). Bd infection rates and intensities were studied in frogs during their entire annual period of activity in central Ohio (approximately March through November) and focused on three habitat types, open emergent wetlands, open streams (no tree canopy), and forested, ravine streams. Bullfrogs and green frogs were the target species of this study because preliminary sampling for Bd in amphibian communities in central Ohio found that these species had the highest infection rates (Chapter 2). These two species also are common, generalist species that tolerate a broad temperature gradient, inhabit a wide variety of aquatic environments, and spend considerable time both in water and on land (Martof et al. 1952, 1953A, 1953B, Schroeder 1976, Shepard 2002, Bohnsack 2002, Lamoreaux et al. 2002). Both species are fairly tolerant of Bd (Gahl et al. 2012, James et al. 2009, Kiesecker et al. 2011, Schlogel et al. 2009). Bullfrogs are a suspected vector for spreading the disease because they are farmed globally (Kiesecker 2011) and Forzan et al. (2010) reported that green frogs are a reliable test species for detecting Bd in the environment. These two species therefore provided an excellent opportunity to test hypotheses regarding the impact of habitat, season, and temperature on Bd prevalence in the study area.

The strong seasonal variation in the climate of many temperate environments may have significant effects on the population dynamics of Bd. As discussed above, laboratory studies have found that temperature affects many aspects of the biology of Bd. There is evidence that Bd infection intensity may increase in amphibians over-wintering in cool hibernacula (Garner et al. 2011, Savage et al. 2011, Maniero and Carey 1997, reviewed by Rollins Smith et al. 2011), and this may facilitate spread of the fungus during

subsequent spring mating behavior, increasing infection rates in the spring (Belden and Harris 2007). Kinney et al. (2011) found that 27% of gopher frogs (Lithobates areolatus) sampled were Bd positive when they entered breeding pools, whereas 46% were Bd positive when they left the pools. It is likely that summer temperatures in emergent sites in temperate climates occasionally exceed the lethal temperature for Bd. Cricket frogs (Acris crepitans) in Texas, USA which tested positive in May had cleared infections when they were tested a month later (Gaertner et al. 2009). Kriger and Hero (2007) found that Bd prevalence peaked in early spring, then dropped in summer and fall in the Stony Creek Frog (Litoria wilcoxii) in south-east Queensland, and Pullen et al. (2010) identified a similar trend in our target species (L. clamitans and L. catesbeianus) in Virginia, USA. Based on the general thermal tolerance of Bd, we hypothesized that infection prevalence and intensities in our central Ohio study sites will be higher at cooler times of year (spring and autumn) than during the warmer summer.

Different types of microhabitats utilized by amphibians may display different temperature profiles and different patterns of seasonal temperature change that may influence Bd population dynamics. In Australia, Kriger and Hero (2007) tested amphibians from different breeding guilds to determine differences in Bd prevalence by habitat and found low Bd prevalence in species breeding in ephemeral ponds, increased prevalence in permanent pond breeders, and the highest infection rates in permanent stream breeders. In Indiana, USA, Kinney et al. (2011) found higher rates of Bd in deeper, cooler, permanent pools than in shallow, warmer, semipermanent systems, and Conradie et al. (2011) found Bd in South Africa to be most prevalent in gently flowing streams than in non-moving bodies of water or fast-moving streams. Foothill yellow-legged frogs (L. boylii) in central California displayed higher Bd prevalence in pool habitats with cooler water temperatures (Lowe 2009). In a study looking at headwater streams in mountainous regions of the United States, Bd was detected in only 3% of 1322 individuals of 21 species, suggesting that Bd is not common in headwater streams (Hossack et al. 2010). Our study in central Ohio focused on three aquatic habitats,

emergent wetlands, open canopy streams, and forested streams, which differed in mean temperature, daily temperature variation and seasonal temperature variation. For example, emergent wetlands with standing water and limited canopy warm more dramatically during spring and summer than shaded stream sites with dense canopy and flowing water. Based on the thermal tolerance of Bd and the characteristics of our habitats, we hypothesized that infection prevalence and intensities would be higher in the habitat we expect to experience cooler, more constant temperatures (forested streams), than the habitat we expect to experience warmer, more variable temperatures (emergent wetlands).

Amphibian lifestages are differentially impacted by Bd. There is evidence that newly metamorphosed frogs are immunologically more vulnerable to Bd infections (reviewed by Rollins Smith et al. 2011). In a California study on L. boylii, Lowe (2009) found Bd in recently metamorphosed frogs but none in adult animals. Forzan (2010) found that Bd prevalence was higher among L. clamitans juveniles than adults. To more fully understand Bd dynamics at our field sites, Bd infection rates and infection intensities in different life stages of L. catesbeianus and L. clamitans (tadpoles, juveniles, and adult) were analyzed. We hypothesized that different life stages may preferentially use different microhabitats and display different patterns of Bd infection. If juveniles are indeed more sensitive to Bd than adults, we expected to find higher Bd prevalence and infection intensities in juvenile individuals.

MATERIALS AND METHODS Sampling sites and schedule Sampling was conducted in protected, natural areas in central Ohio (Batelle Darby Metropark in Madison county, Blacklick Woods Metropark in Franklin county, Glacier Ridge Metropark in Union county, Highbanks Metropark in Delaware county, and Sharon Woods Metropark in Franklin county). Sampling was performed spring through autumn in both 2010 (March 13 To November 11) and 2011 (April 14 To October 24)

during the period of surface activity of bullfrogs and green frogs. Three distinct habitats were sampled: emergent wetlands, emergent streams without tree canopy, and forested streams. Sites were visited approximately 4 times each month from April through October 2010 and 2011. Due to larger sample sizes, we relied on green frog samples for habitat and seasonal analyses. Tadpoles were collected from two emergent wetlands during the spring, summer, and fall of 2011.

Frog collection and swab sampling Frogs were captured by hand or dip net. Animals were caught individually in plastic bags that were first inverted to cover the hand, then turned right side out while the amphibian was grasped in the bag-covered hand. This procedure minimizes the chance of contamination of samples by preventing contact between researchers and amphibians, equipment and amphibians, or amphibians and amphibians. Each frog was placed in the bag temporarily until all sampling was completed in the general area to ensure that the same individual was not captured twice. The snout-vent length (SVL) of frogs was measured with calipers and sampling for Bd was performed on frogs while they remained confined in the plastic bags. The frog was held such that the hind limbs and lower ventral surface were exposed and a fine point cotton medical swab was used to collect the skin samples. Each frog was swabbed following the general techniques of Brem et al. (2007) and Briggs and Burgin (2003), modified such that he ventral drink patch was swabbed 10 times and the left thigh, left foot, right thigh, and right foot swabbed 5 times each (total of 25 strokes per individual). This standardized technique allows comparison of results between individuals. To test for variation in sampling results potentially produced by this swabbing method, samples taken from the left and right sides of ten individual green frogs were independently tested. Swabs were air dried, labeled, and stored in the freezer until they were shipped for PCR analysis. Frogs were also marked by toe clipping (Heyer et al. 1994) so that some Bd infections could be followed through time. A total of 315 green frogs and 33 bullfrogs were sampled during the 2010 and 2011 seasons.

Tadpoles sampled in this study were collected with a dip net, measured (snout-vent length and total length), evaluated for Gosner stage (1960), and swabbed. Tadpole mouthparts were swabbed (Stockwell et al. 2010) by 10 strokes over the beak. Swabs were allowed to air dry and were stored in individual, labeled vials in the freezer. A total of 91 green frog and 145 bullfrog tadpoles were sampled.

PCR analysis and zoospore equivalents (ZE) Taqman real-time PCR analyses of swab samples were conducted by the San Diego Zoo’s Am hi ian Disease La orator and follo ed a modified rotocol of o le et al. (2004). Quantitative TaqMan PCR measures the amplified products directly during the exponential phase of amplification cycle using fluorescent hybridization probes and in addition to providing quantified data, it is more sensitive than conventional PCR (Hyatt et al 2007). TaqMan PCR used the primers ITS1-3 Ch tr 5’- CCTTGATATAATACAGTGTGCCATATGTC-3’) and 5.8S-Ch tr 5’- AGCCAAGAGATCCGTTGTCAAA-3’) o le et al. 2004). The amplification conditions were carried out on 25 µl containing 12.5 µl 2X TaqMan Master Mix, PCR primers at concentrations of 900 nM, MGB probe at 250 nM and 5 µL of DNA (diluted 10-1 in water) and run in triplicate (Boyle et al. 2004). Default ABI Prism 7700 amplification conditions (2 minutes at 50°C, 10 minutes at 95°C) followed by 15 seconds at 95°C and 1 minute at 60°C for 50 cycles were used (Boyle et al. 2004). If two of three wells were positive then the sample was determined to be positive and indicative of a low level infection. Zoospore equivalents (ZE) were calculated by averaging the resultant number of genomic equivalents detected by quantitative PCR detected in the three wells and multiplying by 100 to account for the dilution of the sample.

Tests of the reliability of zoospore equivalents (ZE) measurements ZE values were found to vary dramatically between animals (see Results). To test for the reliability of these measurements, results of swabs taken from the left and right sides of 10 individual green frogs were compared. The presence or absence of Bd was consistent

for swab results from both sides of each of the frogs (8 of 10 tested positive). ZE counts for the left and right sides of the ten animals are shown below in Table 3.1. A paired t- test demonstrated that there was no significant difference between swabs collected from the left and right of individual animals (t-val= -1.04, p=0.327).

Temperature Data Temperature data loggers (Onset Computer Corporation) were deployed at several representative emergent wetland and forested stream sampling locations in 2011. Two loggers were placed at each site, one to record air (ground surface) temperature and the other water temperature. Loggers were set to collect hourly data. Loggers recording air temperature were positioned on the ground surface along the shore of the wetland and covered with a thin layer of leaf litter to shade it from direct sunlight. Aquatic data loggers were positioned on the bottom of the wetland, typically held in place by tying them to submerged roots or covering them with small rocks. Aquatic logger depth ranged from about 10-50 cm, depending on water level. Data loggers also recorded light intensity and this information was referenced to ensure that direct sunlight was not significantly impacting the recorded temperatures.

Statistical analysis Disease prevalence was calculated as the simple percentage of sampled frogs that tested positive for Bd. Infection intensity was determined to be the number of zoospore equivalents obtained from qPCR analysis. Statistical tests were run in MiniTab 10 using one-way ANOVA tests. ZE results were log transformed to normalize the data to meet the test assumptions for one-way ANOVA. Standard deviation was calculated for each 24-hour day to assess temperature variation.

RESULTS Species differences Rates of Bd infection were higher in bullfrogs (L. catesbeianus) (67%, n=33) than in green frogs (L. clamitans) (50%, n=315). Bd prevalence in green frogs was similar in both 2010 and 2011, 44% (n=70) and 50% (n=245) respectively. Yearly sample sizes for bullfrogs were much smaller, but Bd prevalence was 78% (n=23) in 2010 and 50% (n=10) in 2011. Combined infection prevalence for both species were very similar over the two years, 52% (n=86) in 2010 and 51% (n=261) in 2011.

Infection intensities were higher in bullfrogs (11494 ±1902 ZE, n=23) than in green frogs (860 ± 46509 ZE, n=146). Infection intensities in individual frogs ranged dramatically in both species, from less than 1 to 475,277 zoospore equivalents (ZE). Green frogs exhibited more dramatic variation in Bd infection intensities than bullfrogs. Combined infection intensities for both species were significantly higher in 2010 (3.51±1.13 ZE, n=45) than in 2011 (0.63±1.27 ZE, n=132) (log transformed, one-way ANOVA p<0.001). 2010 was slightly warmer than 2011 throughout the months of amphibian activity (Ohio State University Airport temperature records).

Results from dead and/or symptomatic frogs During the course of the study five green frogs were sampled that displayed symptoms associated with chytridiomycosis, as well as other diseases. Of the symptomatic frogs, only two of five were positive for Bd (Table 3.2). None of the other 173 frogs that tested positive for Bd during this study exhibited symptoms. Asymptomatic frogs ranged in infection intensity from less than one to 475,277 ZE, so asymptomatic frogs were sampled that carried much heavier infection loads than the symptomatic frogs represented in Table 3.2. Seven frogs that likely had been dead from one to several days also were sampled during the course of the study. One tested positive for Bd (Table 3.2). Samples collected from dead frogs rarely test positive for Bd because the fungus does not survive

long on dead tissue (Savage et al. 2011). A single frog with a limb deformity (extra hind limb) also tested positive for the fungus (Table 3.2).

Habitat differences We examined green frog samples to discern patterns among the habitat types. Bd prevalence was higher in forested streams (54% positive, n=244) than in emergent wetlands (38%, n=44) or emergent streams (37%, n=27) (Figure 3.1B). In contrast, infection intensities were higher in emergent streams (72645± 18092 ZE, n=11) and emergent wetlands (18164±36207 ZE, n=4) than forested stream habitats (2939±18092 ZE, n=130) (Figure 3.1A). Differences were significant between emergent and forested stream habitats (one-way ANOVA, log transformed, p<0.001).

The habitat correlations observed for green frogs may not apply to bullfrogs, although the smaller sample of bullfrogs limits comparisons. Most bullfrogs sampled were captured in emergent wetlands, and in these sites Bd prevalence was much higher among bullfrogs (64%, n=22) than green frogs (15%, n=26). However, there was no difference in infection intensities between bullfrogs (14904±21652, n=15) and green frogs (18164±36207, n=3) collected from emergent pools (log transformed one-way ANOVA p=0.155).

Seasonal differences Green frogs captured in forested streams provided sufficient sample sizes for a seasonal analysis of Bd prevalence and infection intensity in this habitat. Sampling data were grouped as follows: spring - March 13-May 19, summer - June 8-August 29, and autumn - September 12-November 11. Prevalence peaked in spring (89%, n=36), was lowest in the summer (39%, n=101), and increased again in the autumn (57%, n=106) (Figure 3.2). Infection intensities also varied with the season but showed a different pattern that was statistically significant (one-way ANOVA (log transformed) p<0.001). Zoospore equivalents were highest among summer samples (8240±31852, n=39), autumn (1023±6402, n=59) and spring (72±230, n=32) (Figure 3.2).

Data from recaptured frogs Seven green frogs were recaptured during the course of this study. All of the recaptured frogs carried infections in either the spring or autumn, but four of them were infection- free in the summer (Table 3.3). Three tested positive in spring and subsequently cleared infections by summer. Two positive spring animals remained positive in summer, one negative summer frog tested positive in autumn, and one positive summer capture remained positive in the autumn. Infection intensities were low (less than 115 ZE, mean 22.7 ZE) for all of the recaptured frogs.

Life stage differences Green frogs provided a sufficient sample size to compare infection rates and intensities of juveniles (SVL<50.0mm) and adult individuals (SVL>50.0 mm). Bd prevalence was higher among adults (62%, n=156) than juveniles (34%, n=113). No significant difference in infection intensity was identified between adult and juvenile green frogs (adults 4656±22084, n=97; juveniles 160±577, n=38) (One-way ANOVA, p=0.229). Infection rates and intensities were much lower in tadpoles than in metamorphosed individuals. Prevalence was 3% (n=93) in green frog tadpoles and 6% (n=145) in bullfrog tadpoles. Infection intensities were higher in bullfrog (327.4±853.5, n=7) than green frog tadpoles (0.7±0.9, n=3).

Temperature Temperatures recorded by data loggers at representative emergent wetland and forested stream sites from April through November 2011 (the general period of amphibian activity) were significantly different between the different seasons and different habitats (Table 3.4, Figure 3.3). Mean aquatic temperatures for the two wetland types were generally warmer than the paired mean terrestrial temperatures, and mean emergent aquatic temperatures were significantly higher than mean stream aquatic temperatures (one-way ANOVA p<0.001) (Figure 3.3). As expected, summer was the warmest period,

and autumn was the coolest (Table 3.4). Based on weather records for Columbus, Ohio (NOAA), both 2010 and 2011 were slightly warmer than usual during the months of amphibian activity (March-October), and 2011 received approximately 140% the usual precipitation (Table 3.5).

Water tem eratures varied the most in emer ent sites in the s rin avera e standard deviation of dail tem erature, 2.48 C) and the least in streams in the autumn avera e standard deviation of dail tem erature, 0.3 C) Fi ure 3.3). The ater tem erature ran e in emer ent etlands as a out 28 C over the entire monitorin eriod of am hi ian activit , hereas forested stream tem erature varied over a 22 C ran e for the same period. In both habitats, daily temperature variation decreased from spring to summer to autumn (Figure 3.3). Temperature shifts occurred more rapidly in the emergent wetlands compared to the stream sites, and these more sudden shifts likely were related to the greater degree of solar radiation in the emergent sites (Figure 3.3). The greatest aquatic daily temperature fluctuation occurred in an emergent pool on May 5, 2011 3.9 C, . -2 .6 C).

In forested streams, the aquatic temperatures remained in the critical range for Bd throughout the seasons, and the mean was in the optimal range for Bd in the summer (Figure 3.4). In emergent pools, the aquatic temperature exceeded 30 C lethal temperature for Bd) in both spring and summer. Summer mean aquatic temperature in emergent pools was above the optimal temperature range for Bd, whereas spring mean temperature was in the optimal range (Figure 3.4).

DISCUSSION Prevalence and intensities of Bd infections in sampled frogs This study documented significant Bd infection rates and intensities in frog populations in central Ohio, and demonstrated effects of habitat and season on these infection parameters. American bullfrogs (Lithobates catesbeianus) and green frogs (L.

clamitans) can maintain very high infection intensities (zoospore equivalent (ZE) counts greater than 4 x 106 ) without displaying any symptoms of chytridiomycosis. With respect to lethal ZE thresholds of Bd in frogs, some studies have supported the lethal ZE threshold of 104 observed in mountain yellow-legged frogs (L. muscosa) by Vredenburg et al. (2011) as generalizable to frogs (Kinney et al. 2011). The high infections intensities observed in asymptomatic frogs in our study does not support this generalization. Although a few sampled frogs were lethargic, a symptom that may indicate chytridiomycosis, there were many frogs with infection intensities greater than 104 that displayed no visible symptoms.

Although ZE counts in our study varied widely between animals, our analysis comparing swab results from the left and right sides of individual frogs demonstrated the validity of these measurements. The variation found in ZE counts between left/right samples was not any greater than the range between most PCR replicates for individual animals run in triplicate. The left/right comparisons also demonstrated consistency in detecting presence of the fungus even at very low infection intensities, so it is unlikely there was an appreciable number of false negatives in our study.

Habitat-related patterns of Bd infection rates and intensities Bd prevalence was highest among frogs collected from forested streams (Figure 3.1), suggesting that species living in habitats which experience typically cooler, more moderate temperatures may be at greater risk of infection in the central Ohio region. Other studies have found Bd to be more prevalent in cooler, more shaded habitat in the temperate USA (Raffel 2011) and gently flowing streams compared to non-flowing water in African grasslands (Conradie et al. 2011). It is likely that shaded habitats and flowing water maintain cooler temperatures more suitable for Bd survival (Figure 3.4) (Raffel 2011). In contrast to the high infection rates in forested streams, results here indicate that infection intensities were higher in warmer, emergent habitats (Figure 3.1). Our temperature recordings documented that the emergent sites experienced overall warmer

conditions with greater daily fluctuations in temperature (Figure 3.3). Warm and variable temperatures as experienced in emergent habitats may negatively affect Bd prevalence while facilitating infection intensity.

Seasonal patterns in Bd infection rates and intensities In the s rin , ater tem eratures in oth the forested stream mean 5. C) and emer ent ools mean .8 C) ere in the lo to o timal ran e for d ro th (Piotrowski et al. 2004, Voyles et al. 2011), whereas daily high water temperatures in the summer exceeded the o timal ran e in forested streams 2 .6 C) and e ceeded the critical ran e 33. C) in emer ent ools Fi ure 3.4). There as a shift from ides read, lo intensity infections in the spring to higher intensity infections in fewer individuals during the summer. As hypothesized, Bd prevalence was highest in the cooler spring and lowest in the warmer summer months (Figure 3.2). This seasonal pattern matches the habitat correlations described above of increased infection rates in the cooler stream habitats compared to warmer emergent sites. In contrast to infection rates, Bd infection intensities were highest in summer, corresponding to the habitat-associated finding described above (Figure 3.2) that infection intensities were greater in the warmer emergent wetland sites. All seven recaptured frogs in this study had low intensity infections at some point in the year, suggesting that many frogs were constantly gaining and losing low-intensity infections. Most of the recaptured frogs lost infections from spring into summer, matching the seasonal trend of overall infection rates and suggesting that warmer temperatures may help some frogs clear infections (Table 3.3).

Life history-related patterns in Bd infection rates and intensities Bd prevalence was higher in adults than juvenile green frogs, but there was no difference in infection intensities between the two life stages. This is contrary to the findings of Forzan et al. (2010) of higher Bd prevalence in juvenile compared to adult green frogs. Juveniles were expected to have greater Bd prevalence and intensities because their immune system function is compromised as they metamorphose (Rollins-Smith et al.

2011). In contrast, adult frogs have more capable immune function that allows them to gain but effectively clear infections, thus maintaining low infection prevalence and intensities (Rollins-Smith et al. 2011). Some of the juvenile green frogs sampled in this study were well past metamorphosis and probably better able to handle infections, and this would lower the overall intensity of infections in our juvenile sample. Also, juveniles with heavy infections may have died, further lowering the intensity level observed in our sample of surviving juveniles and also lowering the overall juvenile infection rate. Perhaps the adult green frogs sampled had higher infection rates because adults are more likely to gain infections through increased contact with conspecifics during breeding and territorial behavior. Besides not engaging in breeding activities, green frog juveniles also typically are ignored by territorial adults (Martof 1953). Therefore adults may be more prone to infection via skin to skin contact with other infected individuals.

The very low incidence of Bd infections in tadpoles of both bullfrogs and green frogs is likely an artifact of the different sampling techniques used for tadpoles compared to frogs. In a controlled experiment Manzano (2011) found that the mouthpart swabbing technique used here generated a high rate of false negatives in PCR analysis compared to a technique of excising mouthparts for use in the analysis. Despite low prevalence detected among tadpoles in our study sites, bullfrog and green frog tadpoles may serve as a reservoir for Bd at our field sites. Tadpoles of several species (Lithobates muscosa, Rachowicz and Vredenburg 2004; Litoria wilcoxii and Taudactylus eungellensis, Retallick et al. 2004) have been proposed as likely reservoirs for Bd. However, because the bullfrog and green frog tadpoles sampled exclusively inhabited emergent wetland sites, if tadpoles do serve as a reservoir at our localities adults and juveniles captured in emergent pools should have displayed higher Bd infection rates compared to forested streams that lacked tadpoles. This was not observed, although as discussed above, thermal properties of emergent wetlands may have allowed frogs to more readily clear infections.

Seasonal and habitat-associated temperature variation Based on weather records for Columbus, Ohio (within about 30 km of most of the field sites) temperatures during the months of amphibian activity in both 2010 and 2011 were slightly warmer than historic average temperatures, and 2011 received considerably more precipitation than usual (about 50 cm) (Table 3.5). Besides the obvious seasonal temperature trend expected for a temperate zone, there were distinct differences between our different habitats in mean temperature, maximum and minimum temperatures, and daily temperature fluctuation during the 2011 field season. Temperatures were consistently warmer in emergent pools than forested streams throughout the period of amphibian activity (March - October). Daily temperature fluctuations were greater in spring than in summer or autumn, and emergent wetlands displayed more variable temperatures than forested streams through most of the activity season (Figure 3.4).

Impact of temperature on host-Bd interactions Temperature is a major factor in the interaction between Bd, amphibian host susceptibility, and amphibian declines (Berger et al. 2004, Bosch et al. 2007, Conradie et al. 2011, Daskin et al. 2011, Forrest et al. 2011, Gaertner et al. 2009, Gieger et al. 2011, Kilpatrick et al. 2009, Lowe et al. 2009, Morgan et al. 2007, Piotrowski et al. 2004, Puschendorf et al. 2009, Richards-Zawacki et al. 2009, Rollins-Smith et al. 2011, Rohr et al. 2008, Woodhams et al. 2003). In this study, Bd infection rates were correlated both with cooler mean daily water temperatures and with greater daily water temperature variation. Springtime emer ent ool tem eratures fluctuated as much as 3 C on a dail basis (Figure 3.2); enough to cause a cold-shock and trigger zoospore release from sporangia (Woodhams 2008). The finding that infection rates were highest in the spring when daily temperature variation was greatest may concur, although on a much smaller scale, with the findings of Pounds and Coloma (2008) that large-scale temperature peaks often occurred the year before the extirpation of amphibian populations by Bd. Temperature likely impacts Bd dynamics and these dynamics may be different in years with different conditions. In cooler years, the dynamic between Bd and amphibians may 54

be altered such that a high Bd prevalence would be maintained throughout the seasons. Dynamics may also be affected by precipitation patterns. Long term studies of Bd dynamics, analyses of temperature effects on Bd infection intensity, documentation of seasonal trends of zoospore abundance in the water, and a laboratory study of the effects of temperature on the Ohio strain of Bd are needed for a more complete understanding of Bd dynamics in this type of temperate environment.

Both season and habitat impact temperature conditions in amphibian habitats and affect the interactions between amphibian hosts and the Bd atho en. Forrest et al. 20 ) found 5- 00% d revalence in am hi ians in ater at tem eratures elo 5 C as e erienced in s rin and autumn in our stud ) and much lo er revalence 0%) in ater at tem eratures a ove 30 C similar to maximum water temperatures of the emergent sites during the summer in our study). At cooler and more variable temperatures, amphibian immune function is reduced (Maniero and Carey 1997, Raffel et al. in press). Increased susceptibility to acquiring Bd infections increases the spread of disease within a population. During springtime, many amphibian species congregate at wetland breeding sites and this likely facilitates transmission both through exposure to water borne zoospores and contact between conspecifics. Kinney et al. (2011) found an increase in infection prevalence from 27% to 46% in Lithobates areolatus before and after visiting breeding pools. The greatest daily temperature variation in our study wetlands occurred during the spring (Table 3.4, Figure 3.2). Bd responds to cold shocks by temporarily increasing zoospore production and lives longer in cooler water, and these responses would increase the odds of infecting new hosts (Woodhams et al. 2008). As temperatures increase and become less variable, amphibian immune systems are more competent at clearing Bd infections and thermal conditions are less favorable for Bd zoospore survival (Woodhams et al. 2008). Woodhams et al. (2008) found that Bd reproduced faster at warmer temperatures and that zoospores were faster to enc st hich resulted in increased infectivit at 23 C com ared to 0 C. Ho ever, as ater tem eratures continue to rise, zoos ores are shorter lived and die a ove 30 C. Warm

temperatures which are characteristic of emergent pools and summer months may result in re-encystment (increased infection intensity) but reduce the chance of Bd finding a new host (decreased prevalence). Bd infected amphibians may thermoregulate by basking in the sun (Richards-Zawacki 2009) and this temperature spike may decrease zoospore production by directly increasing Bd mortality (Daskin et al. 2011). In warm conditions, the differences in both prevalence and infection intensity may be the result of differential susceptibility among individuals of the same species that are acclimated to a warmer environment. Whereas cooler conditions facilitate widespread Bd but do not favor high rates of encystment on individual amphibians.

CONSERVATION IMPLICATIONS The effects of Bd on amphibian populations are dependent on many environmental and species-specific factors. Here the relationship of seasonal, habitat, and thermal factors to Bd infection rates and intensities in two common species of amphibians in central Ohio are emphasized. All three factors combine to create microhabitat conditions, and together they likely have a major impact on Bd dynamics in the temperate environment of the region. Although the species sampled in this study can carry sub-lethal infections of Bd and effectively co-exist with the fungus, impacts to these species and other more sensitive species may have gone unobserved. Also, healthy frogs with low intensity infections may serve as vectors and reservoirs of the fungus and exacerbate impacts of Bd on other less resistant species of amphibians.

Many species in central Ohio, including wood frogs (Lithobates sylvaticus), gray tree frogs (Hyla versicolor), spring peepers (Pseudacris crucifer), chorus frogs (P. triseriata), cricket frogs (Acris crepitans), American toads (Anaxyrus americanus), green frogs (L. clamitans) and bullfrogs (L. catesbeianus) congregate in breeding pools in spring. High rates of Bd prevalence in bullfrogs and green frogs in the spring suggest that Bd is abundant in these populations and these other species are also likely to experience high rates of infection. There is evidence that infection prevalence increases significantly from

the time amphibians enter breeding pools until they leave (Kinney et al. 2011). Some of the Ohio species, such as spring peepers (Pseudacris crucifer) and chorus frogs (P. triseriata), typically breed earlier than bullfrogs and green frogs and regularly breed in small vernal pools that may be rarely used by breeding bullfrogs and green frogs. However, even in these species, tadpoles may be exposed to waterborne zoospores released by infected bullfrogs and green frogs. Although green frogs may avoid vernal pools for breeding, we often observed them in these wetlands in the general study area. Here, Bd was detected in over-wintered frogs, was widespread in spring, and continued to reproduce throughout the summer and autumn.

Infection intensity may involve a more complex synergy of factors including host immune system, timing of exposure, and fecundity of Bd in addition to temperature. Frogs with high intensity infections did not exhibit any symptoms of disease; however, none of these frogs were recaptured to determine if they survived. Frogs with low intensity infections were recaptured throughout the seasons, and provide evidence that these species can gain and clear low intensity infections. High intensity infections may be of greater concern than widespread prevalence because it is likely high intensity infections that result in mortality. Mortality associated with Bd may have gone unnoticed by sampling techniques used in this study. If any Bd-related mortality occurred among green frogs and bullfrogs, it is likely that it occurred in emergent pools during the summer months.

Based on our findings, we suggest that future monitoring in temperate areas such as Ohio for the presence of Bd should focus on springtime when temperatures are cool and fluctuating while monitoring of potential impacts of Bd on vulnerable species should focus on warm microhabitats during the warmest months of the year. Amphibians emerging from overwintering sites may have reduced immune function in cool and fluctuating temperatures, and skin-to-skin contact during breeding events, combined with optimal temperatures for Bd reproduction and decreased mortality, may exacerbate Bd

spread. In contrast, mortalities are most likely to occur when infection intensities are highest and here that was observed in emergent habitat in the summertime.

Tables and Figures

Table 3.1. Infection intensities (ZE) for left-side/right-side swab replicates for ten individual green frogs. Sample Left side, ZE Right side, ZE 1 0.107 19.201 2 0.218 1.657 3 40.706 331.827 4 0 0 5 0.117 0.688 6 9.025 6.339 7 0.165 0.171 8 19.453 11.363 9 0 0 10 1.740 1.554

Table 3.2. Infection status and intensity (ZE) for symptomatic and dead frogs. Status Species ZE Lethargic Lithobates catesbeianus 11.2 Lethargic Lithobates catesbeianus 3766.9 Lethargic Lithobates catesbeianus 0 Lethargic Lithobates clamitans 0 Lethargic Lithobates clamitans 0 Deformity* Lithobates clamitans 0.02 Dead Lithobates clamitans 0 Dead Lithobates clamitans 0 Dead Lithobates clamitans 0 Dead Lithobates clamitans 0 Dead Lithobates clamitans 0 Dead Lithobates clamitans 0 Dead Lithobates clamitans 58.0 *Extra hind limb

Table 3.3. Seasonal trends in infection status and intensity (ZE) of recaptured green frogs Sample April May June July Aug Sept 1 95.97 NEGATIVE 2 0.22 1.17 3 113.15 NEGATIVE 4 NEGATIVE 1.21 5 2.58 0.06 6 1.42 NEGATIVE 7 1.70 9.01

Table 3.4. Aquatic and terrestrial temperatures including mean, maximum, minimum and standard deviation by season for forested stream and emergent wetland habitats. uatic Terrestrial Season* N Mean Max Min StDev** Mean Max Min Forested Spring 1284 15.14 23.77 7.48 1.21 12.71 21.47 4.62 Stream Summer 2140 20.35 27.57 16.33 0.63 19.82 25.90 13.37 Autumn 2186 12.20 19.66 4.73 0.37 11.50 23.77 0.23 Winter 707 4.85 9.37 1.00 0.64 3.08 11.43 -2.15 Annual 6317 14.74 27.57 1.00 0.66 13.61 25.90 -2.15 Emergent Spring 1284 17.76 33.95 6.57 2.48 15.61 39.05 0.78 Wetland Summer 2208 24.72 33.12 18.33 0.95 22.79 37.94 11.72 Autumn 2185 13.83 28.85 5.96 0.56 12.03 26.68 -0.66 Winter 707 5.70 7.38 4.00 0.16 3.01 10.45 -2.73 Annual 6317 17.49 33.95 4.00 1.03 15.47 39.05 -2.73 *Spring (April 9-May 31), summer (June 1-August 31), autumn (September 1-November 30), winter (December1- December 30). **Average standard deviation of daily temperatures calculated to determine the variation among temperatures experienced over each 24 hour day.

Table 3.5. Average monthly temperatures (C) and precipitation (cm) for Columbus, Ohio, including normal readings (average for years 1996-2011) and records for 2010 and 2011 (www.erh.noaa.gov). Mean Temperature, Celsius Total precipitation (cm) 2010 2011 normal 2010 2011 normal January -3.33 -4.17 -2.06 5.59 3.73 6.43 February -2.94 0.33 0.00 6.15 10.80 5.59 March 6.78 5.06 5.56 6.99 11.63 7.34 April 14.50 12.78 11.11 6.38 18.14 8.26 May 18.67 17.89 17.00 9.88 14.99 9.86 June 23.50 22.50 21.78 13.67 7.70 10.36 July 25.00 26.78 23.94 15.27 14.40 11.73 August 24.61 23.61 23.28 5.66 6.30 8.43 September 20.22 18.67 19.33 4.22 16.64 7.21 October 13.44 12.56 12.78 3.91 9.35 6.63 November 6.50 9.28 6.89 11.02 12.12 8.13 December -3.56 3.83 0.83 3.20 13.82 7.54 Annual 11.95 12.43 11.70 91.92 139.60 97.51

Figure 3.1. A) Log transformed Bd infection intensities (in zoospore equivalents, ZE) in green frogs (Lithobates clamitans) by habitat. Emergent pool (n=4), emergent stream (n=11), forested stream (n=130). B) Bd prevalence in green frogs by habitat. Emergent pool (n=44), emergent stream (n=27), forested stream (n=244).

Figure 3.3. Seasonal aquatic temperatures and temperature variation collected from representative forested streams and emergent pools.

Chapter 4: Effects of altitude on Batrachochytrium dendrobatidis (Bd) prevalence in the high Andes of Ecuador

INTRODUCTION Chytridiomycosis is a threat to amphibians worldwide, and the fungal pathogen which causes the disease, Batrachochytrium dendrobatidis (Bd), was first recognized as a cause of amphibian declines in 1997 (Pessier et al. 1999). One third of all amphibian species are threatened with extinction, and chytridiomycosis is listed as a threat for many of those species (Mendelson et al. 2006). Tropical montane habitats are most suitable for Bd survival and ersistence, and, ecause these areas also are iodiversit “hots ots” for amphibians, the disease has had the most devastating impacts in these areas (Wake et al. 2008). Ecuador alone hosts 443 described amphibian species, many of which are endemic (AmphibiaWeb Ecuador) and also at risk of extinction due, in part, to the impact of Bd (IUCN).

Bd is a chytridiomycete fungus and the first of its kind known to be pathogenic to a vertebrate (Longcore et al. 1999). Its lifecycle begins as a zoospore which encysts on the amphibian skin, develops into a zoosporangium, and then releases more zoospores (Longcore et al. 1999). It is known to have an optimal temperature range of -23 C Vo les et al. 20 ), ro in culture from 4-28 C Woodhams et al. 2003), die at 30 C Woodhams et al. 2003) and dis la slo er enc stment, maturit , and ro a ule roduction from - 0 C Vo les et al. 20 ). These la orator -determined temperature ranges are guidelines for understanding the biology of the pathogen, but field studies are

necessary to determine the effects of temperature on the interaction between host and pathogen. Temperature may play a critical role in the difference between declining versus sustained amphibian populations.

Bd infects amphibians by implanting in keratinized skin (Longcore et al. 1999). As infection intensity increases, Bd also infects prekeratin layers of the skin (Reeder et al. 2011). In adults, the majority of the skin is keratinized; however, in tadpoles the keratin is limited to the mouthparts (Heatwold 1994). Tadpoles develop keratin first in their jaw epithelium at Gosner stage 24, and keratinization of the skin is delayed until stage 39 (Marantelli et al. 2004). At stage 42 the keratinized mouthparts are lost, and at stage 43 significant amounts of keratin have formed in the skin of the body, legs, and tail (Marantelli et al. 2004). There is a short period during metamorphosis when infection levels are greatly reduced due to this change in distribution of keratin (Marantelli et al. 2004). Whereas adults with severe infections eventually suffer death by electrolyte depletion which leads to asystolic heart failure (Carver et al. 2010, Voyles et al. 2009, 2007), tadpoles typically do not experience mortality but persist with low intensity infections. There is evidence that Bd may impact feeding kinematics (Venesky et al. 2009), slow rates of metamorphosis, and reduce body size at metamorphosis (Venesky 2010, Garner et al. 2009, Parris 2004a, 2004b). Due to long larval periods and moderated environmental temperatures in many aquatic habitats, tadpoles may be a reservoir for sustaining Bd in the environment even when amphibian densities are low.

Gastrotheca pseustes is IUCN listed as “endan ered.” Althou h man s m atric am hi ian s ecies are “criticall endan ered” and ver rare, o ulations of G. pseustes persist along most of the central spine of the Ecuadorian Andes at altitudes between 2200 – 4200 masl (Arbelaez and Toral 2008, Duellman 1987). These altitudes include Andean cloudforest and paramo (high grassland) ecosystems. The oldest known record of Bd in our study area, Cajas National Park, was from G. pseustes in 1989 (Toral, 2001). We chose G. pseustes as our study species because it is a fairly common species throughout

the park, spans a broad altitudinal range, utilizes both aquatic and terrestrial habitat, and is known to host Bd. Because tadpoles are abundant and more easily sampled than adults, the tadpoles provide an excellent opportunity to sample large numbers of animals over the entire altitudinal gradient. G. pseustes may serve as an effective amphibian model for the study of Bd dynamics in the high Andes.

Niche modeling for the neotropics based on temperature characteristics predicts that Bd would likely occur above 1000 masl with a median altitude of 1714 masl and maximum altitude of 4112 masl (Ron 2005). The chytrid thermal optimum hypothesis suggests that in the neotropics, areas below 1000 masl and above 3500 masl are outside the thermal optimum for Bd (Pounds et al. 2006). Bd-susceptible amphibian species distributed within and beyond the higher or lower end would most likely be eliminated from their historical distribution occurring within the thermal temperature range. One example of this is Atelopus cruciger which was historically distributed from 0-2400masl. Now all persisting populations occur below 320 masl (Lampo et al. 2012).

Previous studies of Bd in wild amphibian populations have explored the impacts of altitude on the relationship between ampibians and Bd from sea level to 2600 masl. In Panama, a study between 80-760 masl found higher elevation frogs were nine times as likely to be infected as amphibians at the lower elevation sites (Brem et al. 2008). In Costa Rica, samples collected from 30 different amphibian species over the altitudinal range 100-2600 masl identified the highest frequency of Bd between 100-399 masl and the second highest frequencies between 1000-1299 and 1900-2200 (Puschendorf et al. 2006). A study in Australia found no evidence of an altitudinal effect of Bd among amphibians collected from 90-885 masl (Kriger and Hero 2008). In the high Andes of Peru Bd has been found at 5400 masl on Pleurodema marmorata, Bufo spinulosus, and Telmatobius marmoratus inhabiting pools and streams fed by snow-melt (Seimon et al. 2006). Bd has been identified among amphibians from a broad altitudinal distribution, but prevalence among high altitude populations remains to be explored.

In this study we examined Bd infection rates from 2500-4200 masl in the high Andes of Ecuador to better understand the distribution of the fungus in a high montane environment. Because the growth and reproduction of Bd in the laboratory slows at tem eratures elo 4- 0 C Woodhams et al. 2003; Vo les et al. 20 ), e h othesized that the fungus will be either absent or less prevalent at higher, cooler altitudes along the gradient. We also examined the effects of Bd on tadpole developmental stage and tadpole body condition. We expected Bd to be more prevalent among advanced-stage tadpoles because their skin is more keratinized and hypothesized that Bd infections may decrease body condition by impacting tadpole foraging. We expected infected tadpoles to maintain better body condition at higher altitude at temperatures cooler than the thermal optimum for Bd.

MATERIALS AND METHODS Sampling schedule We collected Gastrotheca pseustes tadpoles from semipermanent pools in and around Cajas National Park, Ecuador. Permits were obtained from Empresa municipial de telecomunicaciones, agua potable y saneamiento de Cuenca (ETAPA) to sample and collect amphibians in Cajas National Park. 10-15 tadpoles were collected per pool, although fewer were collected at certain sites where tadpoles were scarce. Pools from which tadpoles were collected ranged in size from 0.06 to 400 m2, 5 to 100 cm deep, and were typically partially vegetated. Pools were located along an altitudinal gradient of 2500 – 4200 masl and samples were collected from October – November 2010 and February – May 2011. A set of pools with large populations of G. pseustes tadpoles were selected as regular collecting sites as close to the following altitudinal increments as possible: 2500, 3000, 3500, 4000, 4200 masl. One-time-only samples of G. pseustes tadpoles were also collected at additional pools which were distributed randomly throughout the entire altitudinal range. Dip nets used to capture tadpoles were sprayed with bleach and air dried before being used at another site.

Sampled tadpoles ranged from Gosner stage 27 to 45 (Gosner, 1960). Tadpoles were euthanized by submersion in 95% ethanol. Tadpoles subsequently were measured (total length), weighed, assigned a Gosner stage (1960), and their condition was visually assessed (tail damage, mouthpart discoloration, discernible parasites). The beak and mouthparts were then excised using forceps and scissors which were sterilized over a flame for 15 seconds between each specimen. Tadpole mouthparts were excised instead of swabbed because mouthpart swab samples can lead to a high rate of false negatives (Conradie et al. 2011, Manzano 2010). In advanced stage tadpoles, feet were also excised and this tissue was processed along with the mouthpart sample.

When encountered, adult frogs were also captured and swabbed to collect samples for Bd testing. Swabs were collected from the drink patch, thighs, and webbing between the toes (technique adapted from Hyatt et al. 2007). Adults were captured in individual bags to prevent contamination (Mendez 2008) and released immediately after skin swabs were taken.

Each tadpole tissue sample was placed in a small labeled vial of 95% ethanol stored in a freezer at 4 C. Skin swabs collected from adult frogs were air dried, placed in a small labeled vial, and also stored in a freezer. Samples were delivered to the amphibian laboratory at Pontifica Universidad Catolica del Ecuador in Quito, Ecuador for processing. All samples were processed using qualitative PCR to detect the presence of Bd (Hyatt et al. 2007, Annis et al. 2004). Conventional PCR, which measures end-point products of PCR and can detect at a minimum of 10 zoospores or 10 pg of DNA, used the rimers d a 5’-CAGTGTGCCATATGTCACG-3’) and d2a 5’- CATGGTTCATATCGTCCAG-3’), each 300 Annis et al. 2004). Am lification reactions were carried out in 25 µl which included 1µl of each primer, 0.9 mM MgCl2, 1 X Taq buffer, and 0.2 nM of each dNTP and 0.8 units of Platinum, and Taq polymerase. The amplification conditions incudes an initial denaturation at 93°C for 10 minutes

followed by 30 cycles of 45 seconds at 93°C, 45 seconds at 60°C, 1 minute at 72°C and a final extension of 10 minutes at 72°C. 10 µl of the reaction were then run on 1.2% agarose to visualize the bands (Annis et al. 2004).

Temperature data We deployed aquatic temperature data loggers (Onset Temperature) at four locations: 2500, 3000, 4000, and 4200 masl. A data logger also was deployed at 3500 masl but it was subsequently lost. Loggers were tied to a rock and placed underneath a small rock pile to ensure that they would remain submerged and at the bottom of the pool and to ensure that sunlight was not directly hitting them. Each logger was set to collect hourly temperature data. Additionally, data loggers were deployed at terrestrial locations beneath rocks and vegetation near the pools at 3000 and 4200 masl. These terrestrial locations approximated daytime refuge sites for adult Gastrotheca pseustes. Recorded temperatures were later analyzed to compare differences in mean temperature and daily temperature fluctuations at the different altitudes.

Data Analysis Any sample in which Bd was detected was determined to be positive. Bd prevalence reported below represents the percent of samples that tested positive for Bd from a given sample set. G-tests were applied to detect differences in prevalence between groups along the altitudinal gradient, seasonally, by tadpole lifestage and body condition. Tadpoles were divided into groups based on development of keratin in the Gosner (1960) stages as explained in Marantelli et al. (2004). These grouped stages were: stages 27-33 (keratinized jaw epithelium present), stages 34-39 (larger tadpoles but keratin is still restricted to the mouthparts), and stages 40-45 (loss of keratinized mouthparts and formation of keratin in the skin, body, legs, and tail). Body condition was calculated as: (weight (g)/total length (mm))*100. We also calculated linear regressions to analyze the relationship of Bd prevalence to altitude, time of the year, and aquatic temperature.

RESULTS Gastrotheca pseustes tadpoles infected with Bd were collected throughout the geographic extent of Cajas National Park and the surrounding area. The overall Bd infection rate was 7.85% of the total of 446 Gastrotheca pseustes tadpoles sampled. Bd was found at 16 of the 42 collection sites, and Bd infections were observed over the entire altitudinal gradient examined (2500-4200 masl). Infection rates at the 16 sites varied from 7-40% (Table 4.1). We also tested 7 adult G. pseustes collected throughout the park, none of which tested positive for Bd.

Altitude and Bd prevalence There was no significant relationship between Bd infection prevalence and altitude, although there was a weak trend of decreasing prevalence with increasing altitudes (Figure 4.1). More than half (5 of 9) of the localities below 3500 masl contained Bd- infected tadpoles while only on third (8 of 24) pools above 3500 masl contained infected tadpoles (Table 4.1).

Season and Bd prevalence We did not identify a seasonal trend in Bd prevalence. We compared Bd prevalence between seasons (October-November, February-March, April-May) and found no seasonal differences (G=3.509, df=2, p=0.173, n=446) (Figure 4.2B).

Temperature profiles Average aquatic temperatures decreased with increasing altitude from 2500 to 4200 masl (Table 4.2), and there were significant differences in aquatic temperatures between each of the altitudes where data were collected (2500 vs 3000 masl ANOVA p<0.001, 3000 vs 4000 masl p< 0.001, and 4000 vs 4200 masl p<0.001). Aquatic temperatures were significantly warmer than terrestrial temperatures (ANOVA: 2500 masl p<0.001; 3000 masl p<0.001; 4000 masl p<0.001; 4200 masl p<0.001). Aquatic temperatures from all altitudes showed little fluctuation daily or seasonally, with an exception at 4200 masl

where a shallow pool experienced high temperatures when warmed by exposure to sunlight (Table 4.2). At 3000 masl, the overall seasonal temperature range from November 2010 through May 2011 as onl 3.4 C (Table 4.2).

Temperature and Bd prevalence Rates of Bd prevalence increased slightly with increasing temperature at our study sites, but the relationship was not statistically significant (Figure 4.2C).

Tadpole lifestage and body condition and Bd prevalence There was no significant relationship between lifestage category (grouped Gosner stages) and Bd prevalence (G=1.781, df=2, p=4.692, n=446) (Fig.4). There was a negative relationship between body condition and Bd prevalence. Tadpoles with lower body conditions were more likely to have Bd infections (G=15.229, df=2, p<0.001, n=446) (Figure 4). There was no significant relationship between body condition and altitude for infected tadpoles (Figure 4).

DISCUSSION AND CONSERVATION IMPLICATIONS Altitudinal patterns As expected, aquatic pool temperatures decreased as altitude increased (Figure 4.3). Bd prevalence, however, remained low throughout our entire altitudinal gradient (7.9%). Bd continues to persist in Gastrotheca pseustes tadpoles collected from high altitude pools below 10 C. The interaction between temperature and Bd has been established by la orator studies such that our lo er zone 2500-3 00 masl, mean aquatic tem erature 4.5 C) should be most suitable for Bd growth and reproduction (Piotrowski et al. 2004) whereas at temperatures characterizing the high zone (3900-4200 masl, mean aquatic tem erature 9.5 C ) spores encyst, mature, and produce propagules more slowly (Voyles et al. 2011). Stable, warm temperatures facilitate better fungal growth compared to cooler temperatures (Woodhams et al. 2008). In accordance with laboratory studies which suggest Bd is poorly adapted to persist at cool temeratures such as those experienced at

high altitudes, we identified low prevalence in our study. We considered that Bd persistence outside its predicted thermal range may be due to weak amphibian immune function. Some species of amphibian exhibit reduced immune response at cooler temperatures (Maniero and Carey 1997), but Navas (1996) found that high altitude amphibians are physiologically adapted to a broad temperature range. Perhaps Bd in the Andes is better adapted to cooler temperatures than expected.

We identified Bd among amphibians inhabiting pools at 4196 masl, higher than was predicted by either Ron (2005) or Pounds et al. (2006). In Peru, Bd was detected among amphibians at 5400 masl inhabiting streams and pools fed by melting glaciers (Seimon et al. 2006), so it is not surprising that we identified Bd along our entire altitudinal range. Despite cool temperatures below the ideal range for Bd, the fungus persists in high altitude amphibian populations in Andean Ecuador at prevalences comparable to mid altitude levels. Cool temperatures therefore do not appear to create an environmental refuge from chytridiomycosis. We anticipated finding a trend of decreasing prevalence with increasing altitude. We found a higher proportion of pools containing Bd-infected tadpoles at lower altitudes than at higher altitudes, but there was no difference in prevalence between pools. We also expected to find that body condition of infected tadpoles would improve at higher altitudes, but no such pattern was observed. Although our study does not identify a relationship between altitude and Bd susceptibility, it remains possible that certain impacts of Bd on amphibians do vary with altitude. Future studies should attempt to measure infection intensity, amphibian immune competence, and variation in Bd virulence over an altitudinal range.

Season We found no seasonal variation in Bd prevalence throughout our six month study period (Figure 4.2). Our temperature data demonstrates that there was little change in aquatic temperatures between November 2010 and May 2011 (mean: 12.6 C, min: 10.9 C, ma 4.4 C at 3000 masl) (Table 4.2), so if temperature is an important factor impacting Bd

abundance, the lack of a seasonal pattern in Bd prevalence is not surprising. Other factors such as precipitation may influence Bd prevalence. Kriger and Hero (2007) found a significantly negative relationship between rainfall over the preceding 30 days and zoospore prevalence; however, their high rainfall coincided with warmer temperatures. Out study was conducted mainly during the rainy season so the impacts of precipitation were not investigated.

Temperature We found very little seasonal variation in temperature (Table 4.2). Daily temperature variation increased with altitude (Figure 4.3). Seimon et al. (2006) found that neither freezing nor high diurnal temperature variation excluded Bd from the environment. Our findings agree with Seimon et al. (2006) that Bd can persist at cool and variable temperatures.

Tadpole growth and development We did not observe a trend in Bd prevalence by Gosner tadpole stage (1960) (Figure 4), although our study included a broad range of developmental stages: 27-45. Other studies indicate that tadpoles of more advanced developmental stages were more likely to test positive for Bd (Smith et al. 2007), including a closely related species, G. riobambae (Manzano 2010). Our results do not corroborate a relationship between Bd prevalence and developmental stage. However, we did identify a relationship between Bd prevalence and body condition (Figure 4). We found higher prevalence of Bd in thinner animals (i.e., lower body condition). Some reports suggest that Bd infections in keratinized mouthparts may interfere with tadpole feeding (Venesky 2010, 2009) and that Bd- exposed tadpoles metamorphose at smaller sizes than their counterparts (Venesky et al. 2011, Garner et al. 2009, Smith et al. 2007). Our findings support reduced body condition in Bd-infected G. pseustes tadpoles, which suggests that either tadpoles with poor body condition are more susceptible to Bd infections or that Bd impacts tadpoles in a way which reduces body condition (such as interfering with feeding). We expected that size

difference between infected and uninfected tadpoles at lower elevations would be greater than the size difference between infected and uninfected tadpoles at higher altitude because Bd has greater infectivify at warmer temperatures. Uninfected tadpoles below 3500 masl were more robust in body condition than higher altitude tadpoles, and there was only a slight difference where low altitude infected tadpoles were of slightly greater body condition than at higher altitude sites. Bd is equally harmful to tadpoles along the entire altitudinal gradient of our study.

Bd as an enzootic pathogen in the Ecuadorean Andes It is likel that d is enzootic to our stud sites. It ro a l arrived in the late 980’s or earl 990’s Merino-Viteri 2001) and severely impacted populations of many species of amphibian. Various amphibian species can co-exist with enzootic Bd, maintaining mild infections or gaining and clearing infections (Carver et al. 2010, Blaustein et al. 2010, Kriger et al. 2006). In adult frogs, Bd presence can be distinguished from the disease state (chytridiomycosis) by visible symptoms such as lethargy, skin sloughing, and peculiar behaviors (Pessier 2002). Documented symptoms of disease in tadpoles include mouthpart degradation (Venesky et al. 2010, Marantelli et al. 2004, Rachowicz et al. 2004), poor foraging ability (Venesky et al. 2009, 2010), and reduced size at metamorphosis (Venesky et al. 2011, Smith et al. 2007, Parris et al. 2004a,b). In the past 20 years at our study area, amphibian populations (including Gastrotheca pseustes) have decreased, while other species have become locally extirpated (Arbelaez and Toral, 2008). In our study, Bd prevalence among G. pseustes tadpoles in 2010-2011 was low (7.9%) compared to prevalence rates found in samples collected from G. riobambae at a 2800 masl pool in Quito, Ecuador (34%) (Manzano 2010). Compared to Bd prevalence identified in previous studies (Manzano 2010), the prevalence we found above 3500 masl would suggest that Bd prevalence does indeed decrease with altitude. In contrast, Puschendorf et al. 2006) found an overall prevalence of only 6% from 30 species between 100-2600 masl in Costa Rica. Our results for Bd prevalence were much lower than we expected at the lower end of our gradient (2500-3000 masl), and much closer to

the findings of Puschendorf et al. (2006). Perhaps G. pseustes is either more tolerant of Bd than other species, tadpoles are less likely to be infected, or G. pseustes populations have adapted well to Bd, resulting in low prevalence in our study. Other authors have suggested that amphibian breeding pools are reservoirs as tadpoles that are able to host Bd remain in the pools for a long time and adults also visit the pool to deposit eggs (or tadpoles) (Rachowicz and Vredenburg 2004, Briggs et al. 2005). This facilitates the harboring and spread of disease. However, at the pools we sampled, we typically found low prevalence among pools that contained hundreds to thousands of tadpoles. This suggests that the transmission rate between tadpoles may be low. Two factors which may generate low infection rates in G. pseustes tadpoles are host-mediated resistance and environmentally mediated pathogen control (such as cool temperatures). These possible factors, in synergy with low amphibian population densities, and slowed maturation and reproduction of Bd, reduce the potential for the spread of Bd infections among G. pseustes and other amphibians which share the same habitat (Briggs 2010, Greer 2008).

CONCLUSION We found that Bd persists at low prevalence among Gastrotheca pseustes from 2500- 4200 masl and similarly affects body condition of infected tadpoles in a consistent manner along the same gradient. Bd continues to persist at high altitudes where it experiences cool temperatures below its optimal temperature range as indicated by laboratory studies. Throughout the world, studies have been done on Bd prevalence at lower altitudes (80-760 masl in Brem et al. 2008, 90-885 masl in Kriger and Hero 2008, and 100-2600 masl in Puschendorf et al. 2006). Most studies concluded that there is an upper temperature threshold (correlated with lower altitudes) at which Bd impact on amphibian populations is inconsequential. This study sought to determine a lo er tem erature threshold for d in the hi h Andes. La orator studies su est that d ma e oorl ada ted to tem eratures elo 0 C, and although we identified it among tadpoles in cooler pools with mean temperatures of 9.5 C, prevalence was low. In the example of Atelopus cruciger, Bd restricted the historical altitudinal range of the species

so that now it occurs only at temperatures warmer than the optimal range for Bd (from 0- 2200 masl historically to a current distribution of 0-320 masl, Lampo et al. 2012). We concur with Puschendorf et al. (2007) that researchers should search the extremes of a rare am hi ian s ecies’ altitudinal distri ution. Our results su est that the lo altitude extremes that may occur at temperatures above the optimal range for Bd are more likely to serve as an environmental refuge from the pathogen, and amphibians at high altitudes are at reduced risk of Bd-related declines because Bd is less common in the environment.

Tables and Figures

Table 4.1. Percentage of Gastrotheca pseustes tadpoles infected with Bd at the localities sampled in this study. Locality Month Altitude (m) N % infected Monay Bioparque April 2594 15 7 Monay Bioparque May 2615 15 0 Monay Bioparque October 2624 15 7 Monay Bioparque October 2624 15 20 Mazan November 3090 15 27 Mazan February 3090 15 40 Mazan February 3100 10 30 Mazan May 3131 15 0 Llaviucu April 3142 15 7 San Joaquin November 3325 15 0 Control Quinoas May 3462 11 0 Control Quinoas November 3624 15 0 Control Quinoas February 3624 10 0 Control Quinoas October 3628 15 0 Chulo February 3731 5 40 Totoras February 3760 10 0 Estrellas Cocha April 3763 10 0 Estrellas Cocha April 3773 10 0 Patul October 3775 15 7 Patul October 3775 10 10 Chusalongo April 3779 10 0 Estrellas Cocha April 3782 10 40 Luspa March 3795 25 16 Jigeno March 3870 15 0 Cajas South October 3887 15 7 Laguna Toreadora November 3922 1 0 Laguna Toreadora February 3922 10 0 Laguna Toreadora May 3922 1 0 Laguna Toreadora October 3931 15 0 Patul February 3932 15 13 Lagunas de Pato April 3947 10 0 Lagunas de Pato April 3978 10 0 Lagunas de Pato April 3980 15 7 Cajas South March 4039 15 0 Patul October 4055 4 0 Tres Cruces March 4122 10 0 Tres Cruces March 4191 6 0 Loma de Caja February 4196 10 10 Loma de Caja November 4212 20 0

Table 4.2. Aquatic and terrestrial temperatures ( C) from select tadpole collection pools ranging from 2500-4200 masl.

Altitude (masl) Tem eratures C ) N Mean stdev Min Q1 Med Q3 Max Range 2500 (water) 1465 16.48 0.24 15.66 1.33 16.52 16.62 17.1 1.43 3000 (water) 1465 12.57 0.71 10.94 12.01 12. 13.17 14.4 3.47 4000 (water) 1465 11.27 1.11 7.48 10.65 11.33 12.01 14.4 6.94 4200 (water) 1465 7.68 3.74 1.22 5.45 6.57 9.01 27.4 26.15

3000 (ground) 1465 10.94 1.27 6.67 10.26 10.94 11.72 15.4 8.7 4200 (ground) 1465 6.8 1.87 2.1 5.55 6.57 8.03 13.9 11.76

______

Figure 4.1. Correlates of Bd prevalence in Gastrotheca pseustes tadpoles with A) altitude, B) time of year, and C) water temperature C ). 82

Figure 4.2. Temperature profiles of select sites in Cajas National Park. A) Aquatic pool water temperatures along an altitudinal gradient (2500, 3000, 4000, 4200 masl). Bd growth is ecomes im aired elo a out 0 C line shaded area). ) Seasonal stream water temperature and average rainfall (Instituto Nacional de Meterologia y Hidrologia, Ecuador (INAMHI)) at 3000 masl. C) Seasonal profile of stream water temperatures from March –May 2011 at select pools from 2500, 3000, 4000, and 4200 masl.

Figure 4.3. Bd prevalence and tadpole growth and development. A) Graph of Bd prevalence plotted against Gosner (1960) developmental stages (B) Bd prevalence in three categories of tadpole body condition ((weight/length)*100): 0-1.99 (n=256), 2.00- 3.99 (n=143), and 4.00-5.99 (n=26) and (C) Body condition of Bd positive tadpoles by altitude. 84

Chapter 5: Conservation Status of the Cajas Green Harlequin frog, Atelopus exiguus

INTRODUCTION Amphibian populations worldwide have declined dramatically in the past 30 years (IUCN, Blaustein and Johnson 2010, DiRosa et al. 2007, Lips et al. 2005, Mendelson et al. 2004, Pounds et al. 2006, Wake and Vredenburg 2008, Kiesecker 2011). Estimates vary, but one third to one half of the orld’s 6000 am hi ian s ecies are threatened ith extinction (Zippel and Mendelson III 2008, Kiesecker 2011). This severe decline in amphibian populations is possibly the most severe decline of any vertebrate group in recent history (La Marca et al. 2005). 168 amphibian species have gone extinct, and 43% of amphibian species (2469) have populations that are declining (Kiesecker 2011). Declines and s ecies’ disa earances have een most dramatic in tro ical montane habitats in the New World where species have small geographic ranges (Wake and Vredenburg 2008). Population declines are widespread in Latin America, including declines in 30 genera and 9 families in 13 countries (Young et al. 2001). Ecuador boasts the third highest amphibian diversity worldwide with 518 species of which 214 are endemic (Ron et al. 2012). The Ecuadorian Andes have lower diversity than adjacent lowlands, but endemism is as high as 52% (Ron et al. 2012). A conservative estimate of 25 Ecuadorian amphibian species had experienced declines by 2000 (Ron 2000) and by 2008 that number was increased to 141 species with as many as 25 species extinct (Zippel and Mendelson III 2008). The genus with the most species affected is Atelopus (Ron 2000, Lampo et al. 2012).

The genus Atelopus (family Bufonidae) contains many species which are characterized by local endemism, so many harlequin frogs are particularly vulnerable to extinction (La Marca et al. 2005). The genus occurs from sea level to 4000 masl across a geographic range from northern Costa Rica along the Andes south into central Bolivia and eastward toward the Amazon basin and into the Guianas (Lotters 2007). Harlequin frogs are diurnal, aposematic in coloration, and contain potent skin toxins. Males are territorial and aggregate along streams to breed during the dry season (Lotters 2007). About two- thirds of the 110 species of Atelopus endemic to central and South America are believed to be extinct (Kiesecker 2011, Pounds et al. 2006). 42 of the 52 Atelopus populations with sufficient data have been reduced by half or more and only 10 species are known to have stable populations (La Marca et al. 2005). Many records of last observations are from the 980’s, and 30 s ecies have disa eared from kno n localities and have not been seen since 2000 (La Marca et al. 2005). All Atelopus species restricted to habitat above 1000 masl have declined and 75% have disappeared (La Marca et al. 2005). In Venezuela, there are 10 endemic harlequin frogs of which one is extinct, 8 have not been observed since their decline in the 980’s, and one A. cruciger) persists but remains critically endangered (Lampo et al. 2012). In Ecuador, Ron et al. (2003) conducted surveys in 1999-2001 of A. ignescens populations surveyed previously from 1967 and 1981 and documented to be extremely common, but extensive search efforts were unable to locate any individuals of this species.

A exiguus may be one of only two high- Andean Atelopus species remaining in the wild (La Marca et al. 2005). A. exiguus has been documented in the sub-paramo and paramo of Azuay province, Ecuador (Toral et al. 2010). These localities and the most recent observations of A. exiguus include Mazan (2011), Dos Chorreras (2008) and Quimsacocha (2005), Llaviuco (2000), and Laguna Luspa (1979). A. exiguus status was 86

re orted as “declinin ” in 2005 La Marca et al. 2005) and in 2010 was listed as “criticall endan ered” Toral et al. 2010). Exploration in 2010 and 2011 found A. exiguus at only one of these locations.

Widespread amphibian population declines are attributed to a variety of factors acting synergistically on many species in different habitats (Ron et al. 2003). The climate-linked epidemic hypothesis suggests that large scale warming is a key factor in the disappearance of amphibians (Pounds et al. 2006). Years that have been unusually warm have marked declines and have been associated with last observations of populations in many locations (Blaustein and Dobson 2006, Pounds et al. 2006, Pounds et al. 2008). Another general trend is that declines have been associated with aquatic, and more specifically, stream-breeding amphibians (Kilpatrick et al. 2009, Kriger et al. 2007, Bustamante et al. 2005). Finally, high elevation species (above 1000 masl) in tropical regions have been impacted more severely than lower elevation species (La Marca et al. 2005, Young et al. 2001, Pounds et al. 2006, Lips et al. 2008). Specific threats that have been implicated in amphibian declines include: habitat destruction (deforestation, agriculture, construction of dams), pollution and pesticide drift, introduced predators (trout), climate change, increased UV-B radiation, and the amphibian disease chytridiomycosis (Toral et al. 2010, Zippel and Mendelson III 2008, Young et al. 2001, Wake et al. 2008, Ron et al. 2003).

Chytridiomycosis is an amphibian fungal disease which has been implicated in global amphibian declines (Kiesecker 2011, Blaustein and Johnson 2010). It is caused by the pathogen Batrachochytrium dendrobatidis (Bd), and has likely spread by global commerce (Kiesecker 2011). It spread quickly through amphibian populations in Latin America (25-282 km/yr) the Sierra Nevada Mountains, USA (700 m/yr) and Australia 87

(17 km/yr) impacting upland amphibian populations in a relatively short period of time (Lips et al. 2008, Vredenburg et al. 2010, Laurance et al. 1996). Upon introduction, the novel pathogen has caused severe declines and in some cases localized extirpations (Morgan et al. 2007, James et al. 2009). However, mortality rates range from 0-100% and there is differential susceptibility among species, populations, and individuals (Rosenblum et al. 2010). Chytridiomycosis impacts populations that are already impacted by other threats (Wake et al. 2008). Some populations, including a harlequin frog population of A. cruciger, have recovered and persist with endemic infections (Blaustein et al. 2010, Kriger and Hero 2006, Retallick et al. 2004, Lampo et al. 2012). There is some evidence that selection pressure alters an amphibians population by favoring individuals with stronger immune defenses against Bd which may include antimicrobial skin peptides, mucosal antibodies, complement factors, and the involvement of cytokine receptors (Richmond et al. 2009).

The history of chytridiomycosis in Ecuador suggests that it arrived during or before 1980. Lips et al. (2008) tested 32 museum specimens which were collected in Ecuador before 1980 and none had Bd infections. Bd was first documented in Ecuador in 1980 when it was detected on a museum specimen of Atelopus bombolochus from Canar province (Ron et al. 2003). Examination of additional museum specimens collected from 3100-4000 masl in Carchi and Azuay provinces found infections in A. bombolochus (1991), A. ignescens (1989, 1992, 1993), Gastrotheca pseustes (1998, 1999), Hyla psarolaima (1989) and Telmatobius niger (1989). Field samples of G. pseustes were collected in 1998-1999 from Azuay province, Ecuador and tested positive for Bd (Merino-Viteri 2001). Additional G. pseustes samples were collected in 2010-2011 from tadpoles and sample analysis identified widespread Bd in Cajas National Park. Bd infected tadpoles ranging from 2500- 4200 masl at a prevalence rate of 8% infected (Chapter 4). 88

In addition to the presence of Bd in Atelopus species mentioned above, Bd is suspected to be involved in large scale declines, extirpations, and extinctions in the genus. In cases where Bd swabs were collected from declining Atelopus populations, Bd has been identified within the population (La Marca et al. 2005). These include A. zeteki (Lips et al. 2003) and A. cruciger (Bonnaccorso and Guayasamin 2003). However, many species disappeared before samples could be collected to determine the presence / absence of Bd. In the case of A. exiguus, no samples were collected at the time of decline but a museum specimen of A. exiguus collected in February 1978 (likely prior to the arrival of Bd) was negative for Bd (Merino-Viteri 2002).

Trout have been introduced for sport fishing worldwide (Jonsson 2006). Most species grow optimally around 17 C and spawn at 12 C (Jonsson 2006), making montane streams and ponds ideal habitat. When introduced, they have negative impacts on native fish and amphibians (Jonsson 2006). There have been no studies of the impacts of trout on Atelopus, but they have likely contributed to population declines in habitats where they coexist (Ron et al. 2003). However, at some Andean localities, Atelopus have coexisted with trout for as long as three decades before noticeable declines occurred (La Marca et al. 2005).

Climate chan e could im act s ecies directl reducin the area of a s ecies’ fundamental niche or indirectly by interacting with other risk factors. Pounds et al. (2006) argued that large scale warming is a key factor in the disappearance of amphibians. The climate-linked epidemic hypothesis proposes that amphibian declines occur in unusually warm years (Pounds et al. 2006). In Ecuador, 1987 was unusually warm and dry, and A. ignescens was last observed the following year (Ron et al. 2003). From 1987-1989 the last recorded observations were made of 5 additional Atelopus species in Ecuador (Ron et 89

al. 2003). Temperatures at high altitudes are increasing, and in the Ecuadorian Andes this increase may be as much as 2 C, or four times the global average in increased temperatures over the past century (Ron et al. 2003). A second hypothesis, the chytrid thermal optimum hypothesis, proposes that cloud cover increases night time temperatures while also decreasing daytime temperatures. This ultimately reduces daily temperature variation (Pounds et al. 2006). Less temperature variation and less exposure to high temperatures prevent amphibians from basking to induce a fever which could clear disease (Richards-Zawacki 2009).

Rates of declines and extinctions among harlequin frogs are the highest among amphibians worldwide. Here, we examine the status of a small but persisting population of the harlequin frog, A. exiguus. Risk factors for this population include isolation from other populations of A. exiguus, increased risk of chytridiomycosis and predation by trout due to its stream association, high altitude distribution, vulnerability of the genus to Bd. Here, we consider the natural history of the species, potential threats that are limiting population growth, and assess changes in the demographics of the population based on 25 years of research.

MATERIALS AND METHODS This paper is a compilation of studies conducted on the same population of Atelopus exiguus over the past 25 years (1986-2010) and is aimed at increasing our understanding of the natural history and p1opulation status of the species. These studies, the years they were conducted, and the methods employed are included in Table 5.1.

Species Description Atelopus exiguus is endemic to the Cordillera Occidental of Ecuador, and inhabits paramo and subparamo ecosystems adjacent to rivers and streams. Ecuador hosts 110 species of harlequin frogs, but A. exiguus is one of only two persisting species inhabiting the Ecuadorian high Andes (the other is A. nanay). A. exiguus is small (males 21.1- 27.1mm, females 28.7-35.4mm) but robust. It has entirely black eyes, has a uniformly green dorsum with yellow spinules, and a ventrum that is yellow anteriorly and pale orange posteriorly (Coloma et al. 2000). Historically, it has inhabited elevations ranging from 3150-3850 masl in Azuay province of southern Ecuador where annual mean precipitation is 100-200 cm and monthly temperature averages range from is 7-12 C. Its call, typical of species of Atelopus inhabiting low to middle elevations, is weak, high pitched, and pulsed. Note rate remains consistent through the call (Coloma et al. 2000). Among other highland Atelopus species, it is common for males to remain in territories close to the river while females seek refuge in surrounding upland habitat for most of the year and return to streams to breed. Amplectant pairs remain together for weeks before spawning, and the female (but not the male) feeds during this time (Peters 1973).

Site Description The river locality flows through Andean cloud forest in a biological reserve from 3000 to 3400 masl. The river begins in a nearby national park, and is part of a protected drainage system which supplies water to the city of Cuenca. The river locality contributes 20% to the Tomebamba River, one of the major rivers which flows through Cuenca. A. exiguus inhabits a short section of the river corridor and surrounding upland habitat at 3100 masl. The population is concentrated at a section where the remains of an old rock slide make up the northern bank of the river and provide suitable refuge. The dominant vegetation is siksalis (Cortadeiras nitida), a dense, tufted grass with cutting edges. Annual 91

temperatures range from 10-20 C. Average annual precipitation is 100 cm, and most of the rain comes between July and September while December-February are typically the driest months.

Land Use History From 1976-1983, the Andean forest on the land which now comprises the biological reserve was logged. In 1985, the land was acquired by the government and protected as a park but also used for tourism and recreation. From 1983-1995, tourists visited the park and sport fishing was a popular activity. Two species of trout inhabit the river locality: Onchocynchus mykiss and Salmo trutta. They were first introduced to the region in the 9 0’s as a Peace Cor initiative to rovide recreation and nutrition to Andean people. In the 980’s the land was protected as a park but open to tourism and the main attraction was sport fishing. It is likely that trout were common, but that populations were suppressed by the impact of fishing. The park was given increased protection in 1995 when it was declared a biological reserve and closed to tourism. At this time the impact of fishing was reduced although not eliminated, as evidence of trespassing fishermen has been documented as recently as 2010. By removing the impact of fishing, trout populations have gone unchecked and likely increased.

Temperature Collection From 2010-2011, we deployed two waterproof temperature data loggers (Onset Temperature) in A. exiguus habitat. The first was deployed in the river where they breed, the second in microhabitat where adults and juveniles forage and seek refuge. The river data logger was tied to tree roots and rocks and was submerged in a small cave that the river flows through. The microhabitat data logger was tied to a rock and buried in leaf litter six horizontal meters and two vertical meters from the river. Temperature was 92

recorded hourly and the data analyzed to determine maximum and minimum temperatures, daily fluctuations, and averages.

Bd Swab Samples Skin swab samples were collected from frogs encountered in the surveys conducted between 2009 and 2011 to test for the presence of the fungal pathogen, Batrachochytrium dendrobatidis (Bd). Frogs were captured in individual plastic bags to prevent contamination (Mendez 2008) and released immediately after data and skin swabs were taken. Swabs were collected from the drink patch, thighs, and webbing between the toes of adult and juvenile A. exiguus (technique adapted from Hyatt et al. 2007). Swabs were air dried, placed in a small labeled vial, and stored in a 4 C freezer prior to processing. Samples were delivered to the amphibian laboratory at Pontifica Uniersidad Catolica del Ecuador in Quito for processing. All samples were processed using qualitative PCR to detect the presence of Bd (Hyatt et al. 2007, Annis et al. 2004). Conventional PCR, which measures end-point products of PCR and can detect at a minimum of 10 zoospores or 10 of DNA, used the rimers d a 5’-CAGTGTGCCATATGTCACG-3’) and d2a 5’- CATGGTTCATATCGTCCAG-3’), each 300 . Am lification reactions ere carried out in 25 µl which included 1µl of each primer, 0.9 mM MgCl2, 1 X Taq buffer, and 0.2 nM of each dNTP and 0.8 units of Platinum, and Taq polymerase. The amplification conditions incudes an initial denaturation at 93°C for 10 minutes followed by 30 cycles of 45 seconds at 93°C, 45 seconds at 60°C, 1 minute at 72°C and a final extension of 10 minutes at 72°C. 10µl of the reaction were then run on 1.2% agarose to visualize the bands (Annis et al. 2004).

RESULTS Description of Species Most Atelopus exiguus encountered along the Mazan river valley matched previous descriptions of the species. Some juveniles had a black pelvic spot (Figure 5.1), but this was not observed in adults. Among both juveniles and adults, we observed dorsal skin patterns that were mottled with dark green and the more characteristic light green (Figure 5.1). Several juveniles displaying this pattern were observed by Read (1987), and in 2010-11 and we confirmed that some individuals retain this pattern as adults. Gravid females had slightly translucent bellies through which eggs were occasionally observed (Figure 5.1). In 1986, it was noted that all frogs had white pads on their feet and none were black. In 2010, approximately 50% of the population, both juveniles and adults, had feet marked with black (Figure 5.2). The shift in foot color in the population may indicate connectivity to another population or expansion of a rare allele following a population bottleneck. Frog size was consistent in all of the studies. In 1986 mean male snout-vent length (SVL) was 25.2 ±1.3 mm (n=5) and mean female SVL was 34.4 ±1.3 mm (n=5). In 2010, mean male SVL was 24.1 ±1.0 mm, tibia-fibia length (TF) 9.8 ±0.5 mm and weight 2.1 ±0.5 g (n=5). Mean female SVL was 33.3 ±1.0 mm, TF 12.4 ± 0.3 mm and weight 4.1 ± 0.1 g (n=4).

Natural history and habitat characteristics Atelopus exiguus is a diurnal, conspicuous anuran which was historically found in abundant populations alongside rivers and tributary streams (Figure 5.3). Despite the presence of adjacent secondary forest habitat, frogs were most commonly encountered in open canopy areas including the rocky riverbanks and a maintained trail. Frogs were found hiding either in caves, beneath rocks, or leaf litter, and were commonly associated ith siksalis lants Fi ure 5.3). In the 980’s, it was common to find the frogs walking 94

on the surface, but in more recent studies frogs were rarely encountered above ground. Refugia typically had little light, high humidity (92-100%), and were within six meters of the river. A. exiguus displays slow and measured movement, and adults walk instead of jump. Males establish territories in crevices beside running seepages and call chiefly in the morning and particularly on warm days following heavy rains. When the population was abundant, it was not unusual to hear many males calling at the same time. The number of calling males decreased in 1988, and a few calls were heard in 1994. Since then, no calls have been observed. In the 980’s, am lectant airs ere encountered between July and September (dry season), and spent females were common in the middle of September. Strands of eggs were encountered attached to the bottom of stones in slowly flowing waters, and tadpoles were found in considerable numbers in small tributaries. Tadpoles are black with iridescent blue or white stripes and a ventral sucker that they use to adhere to rocks. Juveniles and newly metamorphosed frogs were found in September and their main refuge was in piles of loose stones along the Mazan river. However, different timing of reproductive behavior has been observed in more recent years (2009-2011). Gravid females were encountered several months later (March, April, and May) and juveniles were found most commonly in February and March.

The Mazan river temperatures collected once daily in 1987 ranged from 8.7 C to 10.5 C, while river temperatures (collected hourly) in 2010-2011 averaged 11.2 C and ranged from 7.3 C to 15.2 C (Figure 5.4). Data collected in 2010 had a broader range due to continuous sampling, but it is interesting to note how much warmer the river was in 2010-2011. Air temperatures at Mazan collected in 1987 at 10:00 ranged from 10-25 C (mean 14.4 C) and at 20:00 dropped to 4.5-13 C (mean 9.9 C) throughout one year, and in 2010 average daily refugia temperature ranged from 6.1 to 15.4C (mean 10.7 C) (Figure 5.4). Frogs were seldom encountered above ground on relatively cool, rainy days. 95

In days with 75-100% cloud cover, frogs were more commonly encountered in refuge sites.

Changes in the population In the 980’s, and resuma l in revious decades, A. exiguus was common throughout a known river valley above 2800 masl, and was found in tributaries to the river locality and above the Andean forest tree line in paramo habitat. The population of Atelopus exiguus at the river locality has notably declined from 1987 to 2010 with the most dramatic decline occurring between 1988 and 1994. Population declines were first noted in 1988 when Read noted that the number of calling males had decreased. In 1994, Toral had only 33 encounters during 10 months of research (encounter rate 0.08 frogs/ search hour). Since 1995, it is difficult to assess changes in the population because it has remained so small. In 2004, the encounter rate was 0.06, and was at its lowest in 2006 at 0.02. Since then, encounter rates have increased slightly to 0.04 and 0.09 in 2009 and 2010, respectively. However, population estimates remain dangerously low at an estimated 20 frogs in 2009 and 18 in 2010 (Schnabel mark recapture population estimate). In 1987, the species was most a undant at the localit “ edre al” where there remain si ns of re roduction. In the 980’s, calls, amplexed pairs, eggs, tadpoles, and ne l metamor hosed fro s ere recorded here. In the 990’s calls, juveniles, and an amplexed pair were recorded at the site. From 2004 to 2010 the only evidence of successful reproduction has been a few juveniles. Also, in 2009-2010 gravid females were encountered in March, April, and May. These females were carrying eggs well past the typical breeding season, and this may suggest that they were unable to find a suitable adult male

Threats to the population Studies on this A. exiguus population have identified several potential threats, including invasive species, disease, and climate change.

Disease: Batrachochytrium dendrobatidis has been documented in the river locality area in close proximity to the persisting population of A. exiguus. Swabs collected from A. exiguus in 2009 -2010 did not detect the presence of Bd among this species, but in 2010 and 2011 it was detected in Gastrotheca pseustes in emergent, riverine pools approximately 500 m do nstream of the “pedre al” site discussed (Chapter 4). G. pseustes is a pond breeding species, but adults were observed beneath rocks and vegetation in the same area utilized by A. exiguus. Earlier studies did not collect samples to test for Bd in this population of A. exiguus. Records of the fungal pathogen have been documented as early as 1989 in the nearby national park approximately 10 km away (Merino-Viteri et al. 2001, but it has never been documented in A. exiguus.

Invasive species: We observed trout in the river adjacent to rock piles where we found juvenile, adult, and gravid female A. exiguus.

Climate: 1991 and 1992 were particularly hot and dry years in the nearby city of Cuenca, Ecuador.

DISCUSSION This population of Atelopus exiguus has declined markedly since 1988 and population estimates suggest that it is in eminent danger of extinction. To date, the cause of decline is unknown but several threats are suspected: climate change, non-native trout, and the amphibian disease chytridiomycosis. The years 1991-1992 were particularly hot and dry 97

years in the region. The timing of these hot and dry years fits the period when A. exiguus experienced dramatic declines. Regional peaks in temperature in Panama, Costa Rica, and Puerto Rico have been associated with the disappearance of tropical frogs from pristine habitat in the years that followed (Pounds and Coloma 2008). We documented the presence of trout in the river. Trout impact amphibian populations through direct predation and competition for resources. In California, the mountain yellow legged frog (Lithobates muscosa) has been extirpated from alpine pools, but when trout were removed, amphibians quickly rebounded in population size (Vredenburg et al. 2007). Another study in California found that trout compete with amphibians for food resources. Insect emergence was 20 times higher in fishless ponds than in those inhabited by trout (Finlay and Vredenburg 2007). In Idaho, introduced trout inhabited the majority of permanently inundated pools, and fishless habitats were too shallow to provide suitable breeding and overwintering sites for long toed salamanders (Ambystoma macrodactylum) and Columbia spotted frogs (Lithobates luteiventris) (Pilliod and Peterson 2001). Trout are voracious predators and may be preying on A. exiguus eggs, tadpoles, and adults. If this is the case, persisting individuals may be the result of breeding activity that occurred in areas lacking trout such as small, semi-permanent streams. However, trout coexisted with A. exiguus for at least 10 years and perhaps as many as 20 years before declines were observed. We suggest that future research directly measure trout predation on A. exiguus at each lifestage, and that in-situ conservation efforts focus on the exclusion of trout from critical A. exiguus breeding habitat.

Bd has not been detected among this population of A. exiguus; however, its presence may have gone undetected and the fungus may have impacted the population in the past. There are two possible scenarios for the interaction between Bd and A. exiguus at this locality. First, the population may never have been exposed to Bd and remains vulnerable 98

to potential impacts, and second, the population has been exposed to Bd and the present population is at least partially resistant to the pathogen. Atelopus and other species e erienced dramatic declines in the 980’s and recovered o ulations ersist ith endemic Bd infections (Lampo et al. 2012, Retallick et al. 2004). It is likely that this population of A. exiguus has been exposed to Bd and impacted by the fungus. The timing of A. exiguus population declines corresponds to the time when Bd was first detected in surrounding areas and the timing of widespread amphibian declines in the region. Bd has been detected recently (2010-2011) in Gastrotheca pseustes, a species which shares upland refugia with A. exiguus. The refugia are characterized by high humidity and temperatures just below the ideal temperature range for Bd, so it is likely that Bd would be able to persist in the environment. Atelopus is a particularly susceptible genus to Bd (La Marca 2005), and it is likely that infection of the A. exiguus population would produce significant mortality. This potentially created a bottleneck effect that produced a population more resistant to Bd. The recent finding of a small population with no evidence of Bd is consistent with this scenario. A. exiguus is the only Andean harlequin frog which has been thoroughly tested for Bd where Bd has not been detected. This could be the result of the limited sample size of animals tested, but given the history of Bd in the area, it probably is more likely evidence of evolved resistance to the pathogen. The population of A. exiguus along the Mazan river therefore provides hopeful evidence that at least some members of this genus may be capable of surviving Bd infestations and avoid extinction. Also, A. exiguus is unique in that it persists without endemic infections. Future work is necessary to determine the susceptibility of individuals from this population to Bd.

In 1987, most Mazan specimens had white markings on their feet and none were marked with black. In 1984 Barnett noted that specimens from Cajas NP had black feet (personal 99

communication). In 2010, half of the frogs we encountered in the Mazan population had feet marked with black. This may suggest that there was gene flow between the two populations in recent years. Recent observations have found no other populations in the Mazan drainage, but it remains possible that individuals may move between localized populations. The neighboring valley of Llaviucu is the closest locality to Mazan where A. exiguus has been observed recently (2000). However, our search efforts there did not result in encounters with the species. More time should be invested in scouting for persisting remnant populations in neighboring valleys.

CONCLUSION A. exiguus has persisted two decades since the Mazan population experienced dramatic declines between 1988-1994. However, the population size remains dangerously low. No evidence was found to implicate chytridiomycosis in A. exiguus declines, although the pattern of decline fits the temporal pattern observed in other Bd-induced declines in the region. No Bd was detected in the 41 swabs collected between 2009-2011, but Bd occurs in the general area and it is possible that the existing population has evolved resistance to the fungus. Trout were present in the Mazan river in large enough populations to support recreational fishing for 10-30 years before A. exiguus populations declined. However, it is possible that the establishment of trout in the area poses a threat to the small surviving population. Climate change is a potential factor behind declines in the A. exiguus population, especially given that declines occurred in proximity to significantly hot and dry years in 1991-1992. Despite many threats, a reproducing population of A. exiguus continues to persist in the biological reserve, and this situation provides hope for a genus that has experienced such widespread declines and extinctions.

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Tables and Figures

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Table 5.1. Population parameters from surveys conducted 1986-2010 including: lead scientist, survey method, number of Atelopus exiguus encountered, estimated population size, encounter frequency, population status, and co-occurring species. Year Lead Scientist Survey Number Estimated Population Encounter Frequency Co-occurring species Methods Encountered Population Size Status (per search hour) 1986- M. Read Call Abundant Abundant Stable >1.0 Hyloxalus vertebralis, H. 1987 surveys anthracinus, Pristimantis riveti, P. cryophilus, Telmatobius niger, Centrolenella buckleyi, Gastrotheca pseustes, G. litonedis 1988 M. Read Call Abundant Abundant Declining >1.0 Hyloxalus vertebralis, H. surveys anthracinus, Pristimantis riveti, P. cryophilus, Telmatobius niger, Centrolenella buckleyi, Gastrotheca pseustes, G.litonedis 1994- E. Toral Timed VES 33 Threatened 0.08 Hyloxalus anthracinus, 1995 Pristimantis riveti, P. cryophilus, Gastrotheca pseustes 2004- E. Toral Timed VES 23 Threatened 0.06 None documented 2005 2006 E. Toral Timed VES 7 Threatened 0.02 None documented 2009- G. Maldonado Timed VES 14 20 Threatened 0.04 Pristimantis riveti, P. 2010 cryophilus. Gastrotheca pseustes 2010- C. Korfel Timed VES 13 18 Threatened 0.09 Pristimantis riveti, P. 2011 cryophilus, Gastrotheca pseustes

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Figure 5.2. A. Atelopus exiguus with white feet, B. A.exiguus with black feet.

Figure 5.3. Characteristic habitat where Atelopus exiguus are found. A. River B. Siksalis plant.

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Figure 5.4. Daily average temperatures of Atelopus exiguus refuge site and riverine aquatic habitat.

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Conclusion: Insights to Environmental Factors Influencing Impacts of Bd on Global Amphibian Populations

OVERVIEW This dissertation presented several studies analyzing the temperature and habitat correlates of Bd dynamics in both temperate and tropical montane amphibian communities. Results of these studies have increased our understanding of the interactions between host and pathogen in natural environments. Previous laboratory work suggested that Bd is temperature-limited; however, little research has been done to investigate the suggested limitations in wild amphibian host populations. The juxtaposition of temperate and tropical work on this particular host- pathogen relationship is important to gain understanding of how the fungus is adapted to a broad variety of climactic conditions, and how differences in these conditions may favor or reduce survival rates in infected amphibians. Previous studies suggest that Bd infections are contributing to large-scale amphibian declines in tropical environments, but that temperate amphibian populations remain stable despite Bd infections. Work described in this dissertation investigating the persistence of a critically endangered harlequin frog (Atelopus exiguus), belonging to a highly susceptible genus and inhabiting the tropical montane environment to which Bd is well adapted, will help guide conservation efforts for amphibian species that are susceptible to Bd infections and inhabit areas where climactic conditions favor Bd virulence.

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RATIONALE A study of the seasonal and microhabitat impacts on host – pathogen dynamics in temperate central Ohio was pursued to further our understanding of how Bd population growth and infectivity respond to seasonal cues and differ between habitat types in a temperate environment. Initial work determined the taxonomic and geographic distribution of Bd in Ohio. This initial work indicated that Bd was widespread both taxonomically and geographically, and suggested that samples collected from two common species, Lithobates clamitans and L. catesbeianus which occur in a variety of aquatic, semi-aquatic, and terrestrial habitats, would provide important information addressing my questions about Bd distribution and rates of infection among amphibian hosts in different microhabitats and different seasons. Amphibians in temperate environments have not experienced notable declines due to Bd infections but widespread amphibian declines attributed to Bd have occurred in tropical montane regions. To develop a more global understanding of Bd dynamics, the work in a temperate Ohio environment was complemented by a study of the dynamics between host and pathogen in tropical, montane Ecuador. An altitudinal gradient from 2500 – 4200 masl was selected to explore the impacts of a variety of climactic conditions on the interaction between Bd and Gastrotheca pseustes, an endangered but locally common amphibian species. Finally, Bd presence was investigated in a small but persisting population of the critically endangered Andean harlequin frog, Atelopus exiguus. This genus is famous for the widespread declines and extinctions occurring in recent decades and presumably as a result of the arrival of Bd. Together, these studies provide information on the influence of climatic conditions on Bd disease dynamics in wild amphibian populations and insights applicable to the conservation of at- risk amphibian species.

Bd DYNAMICS IN A TEMPERATE ENVIRONMENT AND IMPLICATIONS FOR AMPHIBIAN CONSERVATION Work in temperate Ohio determined that infection prevalence and intensity have different relationships with environmental temperature. High infection prevalence was characteristic of cooler environmental temperatures, including cool seasons (spring, autumn) and cool, forested stream habitats. High infection intensity was observed more

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commonly in association with warmer environmental temperatures such as warm seasons (summer) and emergent wetlands with full sun exposure. This work adds two important pieces of information to our understanding of the influence of temperature on dynamics between amphibians and disease and provides insight to the ecological context of disease. Among temperate amphibians in Ohio, high Bd prevalence occurred at a mean temperature of about 16 C while high infection intensity occurred at a mean temperature of around 25 C (Figure 1). The environmental mediation of the dynamic interaction between amphibian resistance to chytridiomycosis and Bd virulence may be explained as follows. In springtime, Bd produces large numbers of zoospores slowly (Woodhams et al. 2008), amphibian immune systems are recovering from a compromised state associated with cool temperatures, and springtime breeders congregate at pools where mating activity increases skin-to-skin contact. During the summer, Bd reproduces more quickly but warm temperatures decrease zoospore survivability, favoring re-infection of the same frog compared to infection of a new host (Woodhams et al. 2008). Amphibian immune systems are generally more competent at warmer temperatures, and behaviors such as basking may easily elevate skin temperatures above the lethal threshold for Bd. During this time of high re-infectivity, amphibians with robust immune systems should be able to clear infections whereas those individuals with weaker immune function (due to parasite loads, other diseases, or individual variation in immune function) may develop more intense infections. The infection threshold at which species succumb to the disease varies, but this study provides insight into when die-offs are most likely to occur in seasonal environments. In summary, Bd population growth and distribution is high, and may be facilitated by amphibian hosts, during cool spring months, but infectivity and lethality among susceptible amphibians may be higher during warm summer months. Amphibian conservationists should be wary of the effects of climate change and extended periods of warm temperatures on temperate amphibian populations.

Bd DYNAMICS IN A HIGH ALTITUDE TROPICAL ENVIRONMENT AND IMPLICATIONS FOR AMPHIBIAN CONSERVATION In contrast to my findings in temperate Ohio environments, work in tropical, montane Ecuador found that Bd prevalence was low (8%) but the fungus was widespread from

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mid (2500 masl) to high altitude (4200 masl) sites where mean aquatic temperatures range from 9-23 C. There was no difference in Bd prevalence over the full range of elevations. This finding was unexpected, as laboratory studies suggest that the temperatures at the higher altitude sites were at the low end of suitable temperatures for the fungus (Woodhams et al. 2008). However, studies on other amphibian species occurring at lower elevations (0-2600 masl, Brem et al. 2008 and Puschendorf et al. 2006) found an altitudinal gradient in which infection prevalence increases with altitude and generally occurs at much higher prevalence than the 8% identified here in G. pseustes. The low prevalence among G. pseustes tadpoles at my study sites does not indicate that the species is at risk of population declines, but this species may put more susceptible species (i.e., species with a lower tolerance threshold for Bd infections) at risk by serving as a vector throughout its widespread geographic and altitudinal distribution.

Another significant result from this work is the finding that infected tadpoles had poorer body condition compared to their uninfected conspecifics, a relationship that was consistent from 2500-4200 masl. Although infection intensity was not calculated as the relative number of zoospores collected by a standard technique swab sample, reduced body condition in tadpoles serves as a measure of the impact the pathogen has on its host and as an indicator of the severity of an infection. Therefore, it appears that infections were not more severe at lower elevations, as we would predict based on warmer temperatures that favor Bd zoospores to re-infect their host. Bd persists in the Ecuadorian Andes where mean aquatic temperatures range from 9-23 C, and infected tadpoles are affected equally over this gradient (Figure 6.1). Bd continues to affect amphibians in wild populations experiencing cool temperatures that were suggested in laboratory studies to be at the low end of suitable temperatures for the fungus (Woodhams et al. 2008). The decrease in body condition of infected tadpoles is an important finding because the effects of chytridiomycosis on tadpoles are poorly understood.

Atelopus exiguus, a critically endangered harlequin frog with a historical distribution of 3150 to 3850 masl in the Ecuadorian Andes, lives in an altitudinal range much higher than most harlequin frogs. One known population suffered dramatic declines at the same

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time as Bd arrived to the region. These declines are representative of declines experienced within this genus throughout its distribution in Latin America. Bd was not detected among the A. exiguus population despite intensive sampling and its detection in a sympatric population of G. pseustes. Perhaps the current population of A. exiguus is resistant to Bd infections as a result of selection on the population since the arrival of Bd. Alternatively, the population may have suffered declines as the result of an entirely different impact. However, given the vulnerability of this genus to Bd, and the presence of the fungus in the environment, the former appears more likely. The persistence of this population in an environment where Bd exists provides much needed hope for harlequin frogs and other tropical, montane amphibian species. Perhaps a low environmental prevalence as indicated by samples collected from G. pseustes tadpoles reduces the risk of infection of A. exiguus compared to harlequin frogs occurring at lower elevations. If this is the case, then high altitude may, in fact, provide an environmental refuge from the impacts of Bd to some amphibian species despite its presence in the environment.

A SYNTHESIS OF STUDY FINDINGS Dynamics between amphibians and Bd in the environment are affected by temperature, and temperature varies both spatially and temporally. Warmer temperatures may have a more profound impact on the spread and virulence of Bd than cool temperatures experienced by amphibians (see Figure 1). In temperate Ohio, high intensity infections were observed during warm conditions. This trend did not apply to the altitudinal gradient studied in the tropics; however, the temperature at which high intensity infections occurred in Ohio (25 ̊C) is warmer than the mean temperature at 2500 masl in the Ecuadorian Andes. Applying findings from the temperate study to our tropical montane study suggests that amphibian Bd infection intensities may be higher at lower elevations (as supported by the findings of Brem et al. 2008, Puschendorf et al. 2006). Thus, amphibians at lower elevations may be at increased risk of declines or extinctions due to the temperature regime. The high Bd prevalence among Ohio amphibian populations observed during cool conditions (spring mean temperature 16 C, Bd prevalence 89%) was much higher than in similar temperature conditions in tropical, montane Ecuador (mean temperature about 16 C at 2500 masl; Bd prevalence 8%).

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Springtime aquatic temperatures in Ohio fluctuated widely each day (as much as 12 ̊C daily in emergent wetlands), whereas aquatic temperatures were fairly constant in Ecuador (varying as little as 3.5 C seasonally at 3000 masl). High Bd prevalence during springtime in Ohio may be facilitated by temperature variation and the struggle of amphibians to thermally acclimate. However, Bd prevalence was also higher among autumn samples than summer samples when temperatures were also cooler but more constant. Constant temperatures in the Andes, more similar to autumn temperatures in Ohio, may favor well-acclimated amphibians and more effective immune function, resulting in lower infection prevalence. The population of A. exiguus persisting at 3100 masl (mean temperature 11 C) contrasts with the Bd-driven extirpation of many lower elevation harlequin frogs, suggesting that at high elevations and cooler temperatures, amphibians may have the advantage over Bd compared to amphibians distributed from 0- 2500 masl. In this dissertation, I have focused on the possible impacts of both temperature and temperature variation on Bd/host relationships in both temperate and tropical regions. However, interactions between host, pathogen, and the environment are likely complex and probably involve a synergy of many factors, such as species-specific susceptibility, moisture gradients, stress, and amphibian species community diversity. A full understanding of amphibian-Bd dynamics will require additional studies exploring how the interactions between these many factors affect the abundance and virulence of Bd.

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Tables and Figures

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Figure 6.1. Summary of temperature effects on Bd and amphibian–Bd infections based on laboratory and field based studies. Contributions from the research described in this dissertation are in red.

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