Batrachochytrium Dendrobatidis) Infection in Amphibians in the Rincon Mountains and Tucson, Arizona

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Dynamics of Chytrid Fungus (Batrachochytrium Dendrobatidis) Infection in Amphibians in the Rincon Mountains and Tucson, Arizona

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Authors Ratzlaff, Kristina M.

Publisher The University of Arizona.

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Link to Item http://hdl.handle.net/10150/228467 DYNAMICS OF CHYTRID FUNGUS (BATRACHOCHYTRIUM DENDROBATIDIS)

INFECTION IN AMPHIBIANS IN THE RINCON MOUNTAINS AND TUCSON,

ARIZONA

Kristina Ratzlaff

A Thesis Submitted to the Faculty of the

SCHOOL OF NATURAL RESOURCES

In Partial Fulfillment of the Requirements For the Degree of

MASTER OF SCIENCE

WITH A MAJOR IN WILDLIFE AND FISHERIES SCIENCE

In the Graduate College

THE UNIVERSITY OF ARIZONA

2012

STATEMENT BY AUTHOR

This thesis has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.

Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the copyright holder.

SIGNED: Kristina Marie Ratzlaff

APPROVAL BY THESIS DIRECTOR

This thesis has been approved on the date shown below:

Cecil R . Schwalbe 5/9/2012 CECIL R. SCHWALBE Date Thesis Advisor Assistant Professor School of Natural Resources & the Environment

Philip C. Rosen 5/9/2012 PHILIP C. ROSEN Date Academic Associate School of Natural Resources & the Environment

Don E. Swann 5/9/2012 DON E. SWANN Date Biologist Saguaro National Park

William J Matter 5/10/2012 WILLIAM J. MATTER Date Professor School of Natural Resources & the Environment 3

ACKNOWLEDGEMENTS

First I would like to thank my major advisor, Cecil Schwalbe, for his help and support throughout this process – we had our ups and downs, and came through it in the end! Thanks also to my other committee members: Don Swann, for reminding me to think about the big picture and for being an awesome mentor, mediator, and friend; Phil Rosen, for challenging me to think about different statistics and ways to interpret my data; Bill Matter, for coming onto my committee in the 12th hour to allow me to graduate in time. I had four excellent field assistants, and I’m still friends with each of them. Laura Tennant helped explore unknown territory and figure out how to catch and process frogs without losing them. Beau Barber and Maggie Blais: you were both awesome, despite some hot days and scary (not really) encounters with mountain lions, bears, and coatis. Samantha Barnett had the challenge of helping me finish my field work in four weeks instead of five due to the fire – yet you never complained and trucked along with me the entire time. Thank you all! There were times when I didn’t have a field assistant, and during these times numerous field volunteers helped me out: Tiffany Alvarez, Kim Baker, Shauna Barnett, Erika Bassaraba, Sam Birk, Dennis Caldwell, Matt Christensen, Sabrina DeGeest, Kim Diamond, Aubree Duplessis, Bethany Hontz, Justin Kolb, Kim Kwasny, Kara O’Brien, Johnny Ortiz, JD Paes, Chuck Perger, Adrien Pesque, Chris Pruden, Russ Solsky, Bryan Starrett, Phillip Switzer, Michael Ward, Bruce Weise, Erin Zylstra. All laboratory work was done at the Human Origins Genotyping Laboratory, which provided state of the art equipment and help whenever necessary. Tara Luckau and Oliver Hymen were invaluable with the qPCR up and running – I’m not sure the lab work would have been finished without you! Thanks also go to Taylor Edwards, Jon Galina - Mehlman, Hans-Werner Herrmann, Vangie Rodriguez, Ryan Sprissler, and everyone else. Additional support for my thesis was provided by Dana Backer, Kevin Bonine, Dennis Caldwell, Karen Hancock, Dr. Jim Jarchow, Jaide Miller, Brent Sigafus, and Jason Welborn. Meg Weesner and Becky MacEwan at Saguaro National Park both helped with the permits required for my project. Pond owners were gracious in allowing me on their property whenever I asked: Berghausen, Baker, Caruso, Cebedilla Estates, Crosby, Davison, Delaney, Fraiser, Hernbrode, Olias, Reifshneider, Rosco, Schwalbe, and Tanque Verde Guest Ranch. . Funding for this project came from a USGS Park Oriented Biological Support grant, Human Origins Genotyping Laboratory and USGS Amphibian Research and Monitoring Initiative. Above all, my family - Mom, Dad, Aimee, and Ben - was essential to the success of this project. Whenever I just could not find someone else to go into the field with me, one of you came along. Ben braved allergic reactions to riparian areas; Aimee sacrificed nights and spring break days; Mom and Dad were willing to drive into town at night for yet another bullfrog catching endeavor. Whenever I wanted to give up, vent, or do some meaningless crying, you encouraged me to keep on going. Thank you for your continued love and support. 4

DEDICATION

To my mom and dad, because in the end, I’m not sure I would have finished without you.

TABLE OF CONTENTS

LIST OF TABLES ……………………………………………………………………... 7 LIST OF FIGURES …………………………………………………………………… 8 ABSTRACT ……………………………………………………………………………. 10

CHAPTER 1 - PATTERNS OF CHYTRID FUNGUS (BATRACHOCHYTRIUM DENDROBATIDIS) INFECTION IN AMPHIBIANS OF THE RINCON MOUNTAINS AND TUCSON BASIN, ARIZONA ………………………………………………...…. 11 Abstract ……...………………………………………………………...…………… 11 Introduction …………………………………………………………………...……. 13 Methods …………………………………………………………………………... 22 Study Sites……………………………………………………………………… 22 Rincon Mountains: Hyla arenicolor and Rana yavapaiensis ……….…….. 22 Chimenea Canyon ...... ……………………………… 23 Tanque Verde Drainage …………………….……………………….… 24 Wildhorse Canyon …………………………………….………………. 25 Urban and Suburban Ponds: Rana catesbeiana and Rana yavapaiensis … 25 Sample Collection ……...…………………………...……………………….. 26 Real Time Quantitative PCR ....…………………………………………...…. 29 Data Analysis ……...……………………………………………………….... 30 Results .…………………………………………………………………..………. 33 Species …………………………………………………………………….… 33 Location ……………………………………………………………………... 33 Elevation …………………………………………………………………….. 35 Season ..……………………………………………………………………… 36 Life Stage ……………………………………………………………………. 37 Water ……………………………………………………………………..…... 38 Discussion ……………………………………………………………….….…….. 39 Differences in Bd among species ……………………………………….….… 39 Differences among locations …………..………………………………..……. 41 Elevation ………………………………………………………………...…… 52 Season ……………………………………………………………………….. 53 Life Stage ……………………………………………………………………. 55 Water …………………………………………………………………………. 55 Management Implications ………………………………………………………... 58 Works Cited .……………………………………………………………..……….. 82

CHAPTER 2 – ITRACONAZOLE IS NOT EFFECTIVE IN REDUCING WINTER MORTALITY OF WILD LOWLAND LEOPARD FROGS (RANA YAVAPAIENSIS) FROM CHYTRIDIOMYCOSIS ……………………………………………………….. 92 Abstract ……...……………………………………………………………………... 92 Introduction ……………………………………………………………………….... 93 Methods ………………………………………………………………………….… 95 Study Sites ……………………………………………………………………... 95 Field Work ……………………………………………………………………. . 95 6

TABLE OF CONTENTS - Continued

Treatment ………………………………………………………………………. 96 Post-Treatment ………………………………………………………………… 97 Results .…………………………………………………………………...…..…….. 98 Pre-Treatment …………………………………………………….……………. 98 Treatment ………………………………………………………….…………… 98 Post-Treatment ………………………………………………………………… 99 Long-Term Captivity Frogs ……………………………………………………100 Discussion .…………………………………………………………………..……. 101 Works Cited .………………………………………………………………….…... 107

CHAPTER 3: NO DIFFERENCE BETWEEN BATRACHOCHYTRIUM DENDROBATIDIS ZOOSPORE COUNTS IN FROGS FOUND RECENTLY DEAD, AFTER BEING FROZEN, AND THEN THAWED …………………………………. 111 Abstract ……...……………………………………………………………….…… 111 Introduction ……………………………………………………………………...... 112 Methods ……………………………………………………………………….….. 114 Results….. ………………………………………………………………………… 116 Discussion .……………………………………………………………………..…. 117 Works Cited .…………………………………………………………………….... 121

REFERENCES …………………………………………………………………………123

APPENDIX A – ALL FROG SAMPLES COLLECTED AT PONDS FROM THE RINCON MOUNTAINS AND TUCSON BASIN ………………………….………… 135

APPENDIX B – ALL WATER FILTER SAMPLES COLLECTED AT PONDS FROM THE RINCON MOUNTAINS AND TUCSON BASIN ……………………………… 178

APPENDIX C – LOCATIONS WHERE BOTH FROGS WERE SWABBED AND WATER FILTRATION SAMPLES WERE COLLECTED .…………………………. 183

APPENDIX D – ADDITIONAL FIGURES FOR CHAPTER 1 ….………………….. 188

APPENDIX E – STATISTICAL TABLES FOR CHAPTER 1……………….………. 201

APPENDIX F – BACKYARD POND INFORMATION …………….……………….. 229

LIST OF TABLES

Table 1.1 Patterns of Batrachochytrium dendrobatidis infection among species and locations ………………………………………………………………………………. 60

Table 1.2 Chi square values and probabilities from generalized linear models to see which variables of interest were correlated with prevalence and infection intensity of Batrachochytrium dendrobatidis from Hyla arenicolor and Rana yavapaiensis at the Rincon Mountains and Rana catesbeiana from the Tucson Basin …………………… 62

Table 1.3 Patterns of Batrachochytrium dendrobatidis infection across species, elevations, and locations …………………………………………………………….... 63

Table 1.4 Patterns of Batrachochytrium dendrobatidis across species, seasons, and locations……………………………………………………………………………...... 65

Table 1.5 Patterns of Batrachochytrium dendrobatidis infection across species, life stages, and locations …………………………………………………………………... 67

Table 1.6 Presence of Batrachochytrium dendrobatidis (+/-) in water filter samples and frog samples from the same location ……………………………………………… 69

Table 2.1 Live caught Rana yavapaiensis that presented signs of chytridiomycosis and died within a day of capture ……………………………………………………… 104

Table 2.2 Infection intensity of Batrachochytrium dendrobatidis from Rana yavapaiensis before, during, and after first treatment with itraconazole ……………… 104

Table 2.3 Infection intensity of Rana yavapaiensis treated for Batrachochytrium dendrobatidis with itraconozole, released into home pond, and recaptured at a later date …………………………………………………………………………………………. 105

Table 2.4 Infection intensity of Batrachochytrium dendrobatidis from Rana yavapaiensis kept in captivity throughout the winter ……………………………..…... 105

Table 3.1 Batrachochytrium dendrobatidis zoospore counts for each dead frog collected when first found (fresh), while frozen, and after thawing …………………… 119

LIST OF FIGURES

Figure 1.1 Distribution of three canyons in Saguaro National Park, Rincon Mountain District (Arizona, USA) where Hyla arenicolor and Rana yavapaiensis were tested for Batrachochytrium dendrobatidis …………………………………………………….. 70

Figure 1.2 Ponds across the Tucson basin (Arizona, USA) where Rana catesbeiana were tested for Batrachochytrium dendrobatidis ……………………………………… 71

Figure 1.3 Backyard ponds across the Tucson Basin (Arizona, USA) where Rana yavapaiensis were tested for Batrachochytrium dendrobatidis ……………………….. 72

Figure 1.4 Prevalence (a) and intensity (b) of Batrachochytrium dendrobatidis infection across species, all life stages, locations, seasons, and elevations combined … 73

Figure 1.5 Prevalence (a & c) and intensity (b & d) of Batrachochytrium dendrobatidis from Hyla arenicolor (top) and Rana yavapaiensis (bottom) across locations; all life stages, elevations and seasons combined ……………………………………………… 74

Figure 1.6 Prevalence (a) and intensity (b) of Batrachochytrium dendrobatidis from Rana catesbeiana across ponds throughout the Tucson Basin, Arizona, USA ……… 75

Figure 1.7 Prevalence of Batrachochytrium dendrobatidis from Rana yavapaiensis across ponds throughout the Tucson Basin, Arizona, USA ……………………………. 76

Figure 1.8 Intensity of Batrachochytrium dendrobatidis from Rana yavapaiensis across ponds throughout the Tucson Basin, Arizona, USA …………………………… 77

Figure 1.9 Prevalence and infection intensity of Batrachochytrium dendrobatidis from Hyla arenicolor across elevational gradients in Chimenea Canyon (a-b), Tanque Verde Canyon (c-d), and Wildhorse Canyon (e-f), all life stages and seasons combined …… 78

Figure 1.10 Prevalence (a) and infection intensity (b) of Batrachochytrium dendrobatidis from Rana yavapaiensis across elevational gradients in Chimenea Canyon, Tanque Verde Canyon, and Wildhorse Canyon, all life stages and seasons combined … 79

Figure 1.11 Prevalence (a) and intensity (b) of Batrachochytrium dendrobatidis infection of Rana yavapaiensis and Hyla arenicolor, with each data point representing average values where the two species co-exist in the same elevational gradient in the same canyon …………………………………………………………………………….…. 80

LIST OF FIGURES – Continued

Figure 1.12 Batrachochytrium dendrobatidis zoospore counts per liter of water versus average zoospore counts from Hyla arenicolor and Rana yavapaiensis from pools in three canyons at the Rincon Mountains and R. catesbeiana and R. yavapaiensis from ponds throughout the Tucson Basin, Arizona ……………………………………………… 81

Figure 2.1 Average infection intensity of Batrachochytrium dendtobatidis from Rana yavapaiensis before, during, and after treatment with itraconazole …………….. 106

Figure 3.1 Average number of Batrachochytrium dendrobatidis zoospores at each stage of swabbing dead Rana yavapaiensis (fresh, frozen, and thawed) ……………… 120

ABSTRACT

The chytrid fungus Batrachochytrium dendrobatidis (Bd) has been implicated in amphibian declines around the world, including the southwestern United States. I studied patterns of Bd infection in Hyla arenicolor, Rana catesbeiana, and R. yavapaiensis in the

Rincon Mountains and Tucson Basin in Arizona. Bd prevalence and infection intensity

were location dependent in all species and R. yavapaiensis may be a reservoir of Bd for H.

arenicolor, where they co-occurred. Treatment of a backyard population of R.

yavapaiensis with itraconazole did not reduce winter frog mortality due to Bd. The lethal

Bd infection threshold of this population was between 59,847 and 4,237,330 zoospores.

Zoospore loads from swabs of freshly dead frogs did not differ significantly from those

taken from those same frogs following freezing and thawing. Thus, important information

regarding infection intensity and probable cause of death can be gathered from frogs

collected by others and frozen until convenient to process.

CHAPTER 1 - PATTERNS OF CHYTRID FUNGUS (BATRACHOCHYTRIUM DENDROBATIDIS) INFECTION IN AMPHIBIANS OF THE RINCON MOUNTAINS AND TUCSON BASIN, ARIZONA

Abstract

The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has been implicated in amphibian declines around the world, including in the southwestern United

States. While amphibians of southern Arizona were known to test positive for Bd, little was known about how, or if, these amphibians were affected by this disease. I swabbed lowland leopard frogs (Rana yavapaiensis) and canyon tree frogs (Hyla arenicolor) from streams in the Rincon Mountains and used quantitative PCR to determine patterns of Bd prevalence and infection intensity in both species, in different life stages, at different locations, and across elevations and seasons. I also studied patterns of Bd infection in

American bullfrogs (R. catesbeiana) and R. yavapaiensis from ponds across the Tucson

Basin neighboring the Rincon Mountains. I filtered water at each location to see if Bd would be detected in the water when frogs at that location also tested positive for Bd. In

the Rincon Mountains, prevalence and infection intensity (averaged across all sites) was higher for R. yavapaiensis than H. arenicolor. In the Tucson Basin, mean prevalence and infection intensity was higher for R. yavapaiensis than R. catesbeiana.

Prevalence and infection intensity of Bd in H. arenicolor were associated with

location and season, as well as the interactions of location and elevation and location and

season. Prevalence of Bd was also associated with the interaction between location and

life stage. 12

Prevalence and infection intensity of Bd in R. yavapaiensis were associated with

location and the interaction between location and elevation. Infection intensity of Bd was

correlated with elevation. Neither prevalence nor infection intensity of Bd in R. yavapaiensis from the Rincon Mountains were associated with life stage, season, or the interaction between location and life stage and season.

I found some evidence that R. yavapaiensis from the Rincon Mountains caused the prevalence and infection intensity of Bd in H. arenicolor to be greater, where the species co-occurred. Patterns of Bd infection in H. arenicolor were temperature dependent (increasing with elevation) when R. yavapaiensis were not present, suggesting

that R. yavapaiensis may be a reservoir of Bd for H. arenicolor.

Prevalence and infection intensity of Bd in R. catesbeiana differed between

locations, but was not associated with life stage. Prevalence and infection intensity of Bd

in R. yavapaiensis from the Tucson Basin also differed between locations.

Bd results from water filtrates were inconclusive.

Introduction

Amphibians are experiencing declines and extinctions worldwide (Stuart et al.

2004). While habitat loss and over-harvesting are the major contributors, disease has emerged as an additional cause of declines and extinctions, especially those that occur in pristine habitats (Stuart et al. 2004, Wake and Vredenburg 2008, Collins 2010).

Specifically, the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has been implicated in global amphibian declines, and is believed to have caused the decline or extinction of at least 200 amphibian species (Skerratt et al. 2007).

Bd was first described in frogs from Australia and Panama (Berger et al. 1998) and was identified as a previously undescribed fungal pathogen of amphibians (Longcore et al. 1999). Retrospective studies provide evidence that Bd was the cause of the declines of several amphibian species, decades before its 1998 discovery (Lips et al. 2003a,

Retallick et al. 2004, Hale et al. 2006). Bd zoospores infect and grow in amphibian keratin cells in structures called zoosporangia (Berger et al. 2005a). Zoosporangia development coincides with maturation of amphibian keratin cells, and when a keratin cell is ready to be shed, zoosporangia discharge their zoospores into the water to reinfect the host and infect new hosts (Berger et al. 2005a). Bd zoospores are waterborne, and die when desiccated (Johnson et al. 2003).

Bd infection is localized to where keratin is located: tadpoles only have keratin in their mouthparts, whereas metamorphosed and adult frogs have keratin in their skin at the bottoms of their hands and feet and within the ventral abdomen (Marantelli et al. 2004). 14

Most species are not affected by Bd as tadpoles, but some experience high rates of Bd

related mortality post-metamorphosis (Lamirande and Nichols 2002, Muths et al. 2003,

Marantelli et al. 2004, Rachowicz and Vredenburg 2004, Woodhams and Alford 2005).

In a Bd-positive captive population of great barred frogs (Mixophyes fasciolatus), it was

found that rapid redistribution of Bd at metamorphosis from mouthparts to keratinized skin caused all individuals (n > 500) to die from their infection (Marantelli et al. 2004).

Recently metamorphosed poison dart frogs (Dendrobates tinctorius) were also more susceptible to high Bd infection loads then adults (Lamirande and Nichols 2002).

Bd causes the disease chytridiomycosis, which is thought to occur by causing an imbalance in osmotic homeostasis through the inhibition of electrolyte transport across an amphibian’s skin, causing a decrease in sodium and potassium, resulting in cardiac arrest and death (Voyles et al. 2007). There is some evidence that higher Bd levels also lead to dehydration (Voyles et al. 2012).

Bd grows and reproduces best from 17-25oC, can survive freezing, and dies when exposed to high temperatures for extended periods of time (e.g., 37oC for 4 hours)

(Longcore et al. 1999, Johnson et al. 2003, Piotrowski et al. 2004). Many studies found a

higher prevalence of infection in the winter and spring than in the summer (Aplin and

Kirkpatrick 2000; Berger et al. 2004; Kriger and Hero 2007a; McDonald et al. 2005;

Ouellet et al. 2005; Pearl et al. 2007; Retallick et al. 2004; Rothermel et al. 2008;

Woodhams and Alford 2005), due to these seasons being near the optimal temperature for

Bd. Despite the high winter prevalence, some frog populations appeared to persist with

stable infections of Bd (McDonald et al. 2005; Retallick et al. 2004; but see Green et al. 15

2002). Large numbers of dead or dying frogs, thought to be due to chytridiomycosis,

have also been found in the winter (Berger et al. 2004; Bradley et al. 2002).

Frog populations at elevations with optimal temperatures for Bd generally

experience a higher Bd prevalence and are at more risk to decline due to Bd (Burrowes et

al. 2004, Brem and Lips 2008). Prevalence and infection intensity of Bd in frogs in

Brazil was highest at lentic breeding habitats at high elevations (1600m versus 0-1000m,

Gründler et al. 2012. Infected Cascades frogs (Rana cascadae) in Oregon were more

prevalent at high elevations in the early season (cooler months) than lower elevations at

all other seasons (2400m versus 730-2000m, Piovia-Scott et al. 2011).

Species that are more aquatic experience more frequent Bd-related declines than

terrestrial species (Lips et al. 2003b, Kriger and Hero 2007b), though some aquatic

species, like American bullfrogs (R. catesbeiana) and African clawed frogs (Xenopus

spp.), are resistant to Bd (Daszak et al. 2004, Garner et al. 2006, Kielgast et al. 2010) due

to a strong anti-Bd response of their antimicrobial peptides (Rollins-Smith et al. 2002,

Ramsey et al. 2010). As a result, R. catesbeiana and Xenopus spp. are considered

reservoirs of Bd and vectors of the fungus to naïve populations of other species (Weldon

et al. 2004, Garner et al. 2006, Bai et al. 2010). Pacific chorus frogs (Pseudacris regilla)

have recently been implicated as reservoirs of Bd in the Sierra Nevada of California, with

Bd-positive individuals carrying infection loads up to ten times greater than those found lethal for the sympatric mountain yellow-legged frog (R. sierra and R. muscosa), even at

sites where mountain yellow-legged frogs were extirpated (Reeder et al. 2012). Other

potential reservoirs of Bd include pond water, certain aquatic insects, waterfowl, and fish, 16

and it is suggested that any of these reservoirs (or something else) could spread Bd from one location to another (Collins et al. 2005, Johnson and Speare 2005, Garmyn et al.

2012).

Much debate revolves around whether Bd is a recently introduced disease (the novel pathogen hypothesis) or is endemic to populations and has increased in virulence and pathogenicity due to changes in the environment (the endemic pathogen hypothesis,

Fisher et al. 2009). Genotyping of Bd strains has revealed little difference between strains worldwide and Bd has not been detected in frog specimens in North America prior to 1961, supporting the novel pathogen hypothesis (Morehouse et al. 2003, Ouellet et al.

2005, Morgan et al. 2007). Bd is thought to spread to different countries through international trade (Daszak and Cunningham 2003, Mazzoni et al. 2003, Bai et al. 2010).

The origin of Bd has been hypothesized to be in Africa with initial spread from the

Xenopus spp. trade and further spread from the exportation of R. catesbeiana from North

America (Weldon et al. 2004, Garner et al. 2006, Fisher and Garner 2007).

Lowland leopard frog (R. yavapaiensis) declines have been recorded in the

American Southwest (Sredl et al. 1997), and some of these declines have been attributed to chytridiomycosis (Bradley et al. 2002, Sredl 2005). Conservation of R. yavapaiensis is a major goal of the National Park Service, National Forest Service, and Arizona Game and Fish Department due to the apparent vulnerability of this species and the implication of Bd-related declines of R. yavapaiensis. Bd has been documented in R. yavapaiensis from across the state, including the Rincon Mountains, where Bd-positive dead individuals have been found in winter on several occasions since 2000 (Saguaro National 17

Park, unpublished data). Rana yavapaiensis in the Rincon Mountains co-exist with canyon tree frogs (Hyla arenicolor) at lower elevations (≤ 1372m), with only H. arenicolor occurring higher. Rana yavapaiensis tadpoles take three to nine months to metamorphose (Sredl 2005) and can overwinter (Collins and Lewis 1979). Individuals that metamorphose in the spring can breed as early as the fall of the same year (personal observation)

Hyla arenicolor in Arizona have tested positive for Bd, but are not known to be negatively affected by this disease (Sredl and Caldwell 2000, Baker 2010), possibly because of its more terrestrial nature and affinity for high temperatures on rocks, presumably enabling it to “bake” off the disease. Hyla arenicolor throughout the Santa

Catalina Mountains in Arizona only tested positive for Bd at higher elevations (1341-

2286m versus 863-1265m), and over time, certain individuals were found to have cleared themselves of their infection (Baker 2010), likely due to the warmer temperatures at lower elevations and the ability of this species to withstand high temperatures.

Non-native R. catesbeiana are found in pools across the Tucson Basin west of the

Rincon Mountains but rarely come into contact with the native frogs (Saguaro National

Park, unpublished data). However, R. catesbeiana do occur in the Tanque Verde drainage northwest of the Rincon Mountains and are negatively affecting backyard pond populations of R. yavapaiensis as predators (personal communication with D. Caldwell) and, potentially, as carriers of Bd (Farrer et al. 2011).

The DNA collected on frog swabs in this study was extracted and analyzed with real time quantitative polymerase chain reaction (qPCR). This is a lab technique which 18

was used to determine if Bd DNA was present on the swabs as well as report,

quantitatively, how infected the individual was relative to others in the study. The

extracted DNA of the unknown frog swabs was run against standards of known amounts

of Bd zoospores (e.g., 1, 10, 100, 1000). The amount of DNA in the standards vial was

considered the “genomic equivalent” as the numbers of zoospores that were initially

counted out (Boyle et al. 2004, Reeder et al. 2012). These standards were what frog swab

samples were compared against, with the resulting qPCR data being reported as

“zoospore equivalents” (Boyle et al. 2004, Hyatt et al. 2007) with the assumption that the

amount of DNA present on that sample was the same amount as that many zoospores

would contain. In this study I use “zoospores” instead of “zoospore equivalents.”

If all frogs in a particular study are swabbed in the same standardized manner,

infection intensities of individuals and/or assemblages (species, populations, life stages, etc.) can be compared relative to each other (Kriger et al. 2007a). In some cases, lethal

levels of zoospores are known for a specific species (e.g., 10,000 zoospores in the

mountain yellow legged frog, Briggs et al. 2010, Vredenburg et al. 2010, Kinney et al.

2011), allowing further inferences to be made on what level of infection a frog has (low,

intermediate, or high). It is important to note that low “zoospore equivalent” amounts

reported by the qPCR could be a result of poor lab work or low amounts of Bd DNA

present on the frog’s skin (Smith 2007). However, in this study I believe sufficient

caution and care was taken while swabbing and in the laboratory to obtain reliable

results. 19

Swabbing the frog’s skin removes sloughing skin containing Bd zoospores

(Kriger et al. 2006), while the severity of Bd infection depends on how many

zoosporangia are infecting a frog’s skin. Kriger et al. (2007a) found a direct relationship

between the number of zoospores detected in qPCR from a swab and the number of

zoosporangia detected in the skin of that same frog using histology, showing that the

more zoospores are detected using qPCR, the more intense the Bd infection.

The lethal load of Bd zoospores in R. catesbeiana is unknown. In one study, R.

catesbeiana were inoculated with up to ten million zoospores a day for four weeks, and

no individuals showed signs of chytridiomycosis or died (Dazak et al. 2004). Bd lethal

loads in R. yavapaiensis are suggested to be above 60,000 zoospores (see chapter 2 of

this document). One H. arenicolor, collected “dead or dying” in Montrose Canyon was

declared Bd positive through histological inspection (Bradley et al. 2002), but there was

no mention in the same paper if this frog had chytridiomycosis or died of other causes

while it was infected with Bd. Of over 400 H. arenicolor tested in a previous study

(Baker 2010), and the 870 tested in this study, no individuals had visible signs of

chytridiomycosis, despite at least one individual with a Bd-infection intensity of over a million zoospores (this study), indicating that the lethal load of Bd in H. arenicolor is unknown.

The goals of this study were to determine the relationship between Bd and H.

arenicolor, R. yavapaiensis, and R. catesbeiana in the Rincon Mountains and

surrounding Tucson Basin, and to establish the baseline distribution of these three frogs

and Bd for measuring any changes in the future. 20

This study had seven main objectives:

1. Determine Bd prevalence and infection intensity in H. arenicolor, R. catesbeiana,

and R. yavapaiensis. I expected H. arenicolor to have the lowest prevalence and

infection intensity of Bd and R. yavapaiensis to have the highest prevalence and

intensity, because of H. arenicolor’s likelihood of clearing Bd through basking and

R. yavapaiensis’ likelihood of extended contact with the waterborne zoospores of

Bd.

2. Determine Bd prevalence and infection intensity across locations for those three

species in the Rincon Mountains and across the Tucson Basin. I expected

locations in the Rincon Mountains with a southern aspect to have a lower

prevalence and intensity due to higher solar radiation and locations with a northern

aspect to have a higher prevalence. These predictions are based on the optimal

temperature range for Bd growth (17-25oC, Longcore et al. 1999, Johnson et al.

2003, Piotrowski et al. 2004).

3. Determine Bd prevalence and infection intensity in H. arenicolor across elevations

in the Rincon Mountains. Based on work done in the Santa Catalinas (Baker

2010), and the optimal temperatures of Bd, I expected prevalence and infection

intensity to be lowest at the warmer, lower elevations and increase at cooler, higher

elevations.

4. Determine Bd prevalence and infection intensity in H. arenicolor and R.

yavapaiensis across seasons. I expected prevalence and infection intensity to be

lowest in the hot summer, highest in the cold winter, and intermediate in the fall 21

and spring due to similar findings in other studies (e.g., Alpin and Kirkpatrick

2000; Berger et al. 2004; Kriger and Hero 2007a).

5. Determine Bd prevalence and infection intensity across life stages. Based on

studies on life stages of other amphibian species with Bd, I expected prevalence

and intensity to be highest in recently metamorphosed individuals, as this is the

stage when many amphibian species are most susceptible to chytridiomycosis.

6. Determine if Bd can be detected in the water bodies of this system and how the

detection rate correlates with prevalence and infection intensity of Bd in

amphibians at the same bodies of water. I expected areas with a high prevalence

and infection intensity in amphibians to be more likely to have water samples test

positive for Bd, due to the greater numbers of zoospores in the system (Carey et al.

2006).

7. Determine if there are any interspecific dynamics, specifically between H.

arenicolor and R. yavapaiensis, which would be important determinants of

chytridiomycosis in R. yavapaiensis. I was initially concerned that H. arenicolor

might act as a reservoir of Bd in the Rincon Mountains (like P. regilla and

mountain yellow-legged frogs in the Sierra Nevada).

Methods

Study Sites

Rincon Mountains: Hyla arenicolor and Rana yavapaiensis

I studied H. arenicolor and R. yavapaiensis in three canyons in the Rincon

Mountains, Pima County, Arizona, USA (Figure 1.1). Elevations in the canyons ranged from 920 to 2429 meters. Canyons had year round water in tinajas (bedrock pools) and plunge pools (scour pools with sandy bottoms and vegetation). Flowing water only occurred after adequate summer monsoon rainstorms or when winter storms provided enough snow for snow melt in the spring, but flows only lasted for a month or two at most. All canyons contained riparian vegetation in the drainage with non-riparian vegetation on the surrounding slopes.

From 1994 to 2004 the Sonoran desert scrub of the Rincon Mountains (~900m elevation) had an average high of 37.8oC in July, with average maximum temperatures

above 30oC from May through September and below 20oC in December and January

(based on data at the University of Arizona, the closest climate monitoring station to the

study areas, Powell et al. 2006). Conifer forest at ~2400m elevation had an average high

temperature of 29.2oC in July, with average maximum temperatures between 20-29oC

from May to October, and below 20oC the rest of the year (Powell et al. 2006). Average 23

yearly precipitation in Sonoran desert scrub was 28.5cm, whereas that in conifer forest

was 69.1cm (from Manning Camp climate station, Powell et al. 2006).

Rana yavapaiensis were associated with tinajas and plunge pools in the Rincon

Mountains (Wallace et al. 2010). In this study, H. arenicolor were typically only found

at pools that were bordered on at least one side by a rock wall or large boulder, on which

frogs were observed sitting and basking much of the day; the presence of these rock walls

or boulders was a better indicator of H. arenicolor presence than vegetation type. Rana

yavapaiensis were common in plunge pools with clear water and vegetation or rock

crevices near the water’s edge for a quick escape, though individuals were found in

tinajas in areas where abundance of frogs was high or where plunge pools were scarce.

Chimenea Canyon

Chimenea is a south-facing canyon that extends from 900-2429m elevation.

Frogs at lower elevations (Sonoran desert scrub, 900-1200m) were found at pools amid

desert cotton (Gossypium thurberi), rushes (Juncus spp.), willows (Salix spp.) and stands of cattails (Typha domingensis). One or two trees were usually found at plunge pools, including Fremont cottonwood (Populus fremontii), Arizona sycamore (Plantanus wrightii), and velvet ash (Fraxinus velutina. Frogs at intermediate elevations (oak savannah to pine-oak woodland, 1200-2000m) were found at pools surrounded by deer grass (Muhlenbergia rigens), oak trees (Quercus spp.), shin dagger (Agave schottii), velvet ash, and Arizona walnut (Juglans major). No pools were found from 1550-1600m 24

or from 1800-2000m. The majority of pools at high elevations (conifer forest, 2000-

2500m) were tinajas with no vegetation. The plunge pools there were surrounded by canyon grape (Vitis arizonica) and oaks, with ponderosa pine (P. ponderosa) and southwestern white pine (P. strobiformis) as the dominant trees.

Tanque Verde Drainage

Joaquin Canyon on the north side of the Rincon Mountains feeds into Tanque

Verde Creek at 1200m. Throughout the rest of this paper Joaquin Canyon and Tanque

Verde Creek are considered Tanque Verde Canyon. Tanque Verde Canyon extends from

900-2100m; the upper 600m of this canyon’s elevational range was narrow and did not have tinajas or plunge pools for frogs. The majority of the pools in the canyon were plunge pools with large boulders. Frogs at lower elevations (Sonoran desert scrub, 900-

1200m) were found at pools bordered by rough cockleburs (Xanthium strumarium),

Bermuda grass (Cynodon dactylon), with a few oaks, Fremont cottonwood, and Arizona sycamore scattered throughout the canyon. No pools were found from 1100-1200m.

Frogs at middle elevations (oak savannah, 1200-1500m) were found at pools surrounded by oaks, desert cotton, and rushes.

Wildhorse Canyon

Wildhorse Canyon is a west-facing drainage that extends from 900-2000m

elevation, though the upper 200m is narrow and does not have tinajas or plunge pools for

frogs. Plunge pools are more abundant from 900-1000m with tinajas consisting of the

majority of pools above this point. Pools at lower elevations (Sonoran desert scrub, 900-

1200m) were surrounded by stands of cattails, rough cockleburs, Bermuda grass, willows

(Salix sp.) and fountain grass (Pennisetum setaceum). Few trees were found near pools at

lower elevations. No pools were found from 1280-1500m. Frogs at higher elevations

(oak-juniper woodland, 1200-2000m) were found at pools surrounded by oaks and

junipers (Juniperus spp.).

Urban and Suburban Ponds: Rana catesbeiana and Rana yavapaiensis

I studied R. catesbeiana from seven ponds across the Tucson Basin, Pima County

(Figure 1.2). Ponds at Kennedy and Columbus parks were near the Santa Cruz River.

Both had stands of cattails along part of the pond’s shorelines. Ponds at Campus

Agriculture Center and Ft. Lowell Park were near the Rillito River. Ft. Lowell Pond had

dense stands of cattails and the ponds at Campus Agriculture Center were surrounded by

American bulrush (Schoenoplectus americanus) and whorled marsh pennywort

(Hydrocotle verticillata). Crosby’s pond, Cebadilla Lake, and a pond at Tanque Verde

Guest Ranch were along Tanque Verde Creek and had stands of cattails and Bermuda 26

grass along the edges. Crosby’s pond and that at Tanque Verde Guest Ranch were

smaller and had more tree cover than Cebadilla Lake.

I studied R. yavapaiensis from 12 ponds across the Tucson Basin, Pima County

(Figure 1.3). All were man-made ponds at private residences that were stocked with eggs

or tadpoles from other backyard ponds or from the wild. To preserve the privacy of these

households, no names or specific locations of the ponds will be given. Two ponds were

near Kennedy Park, five were in the Notch Neighborhood between Tanque Verde Creek

and Saguaro National Park, two were near the intersection of Houghton Road and Old

Spanish Trail, two more were near the intersection of Old Spanish Trail and Escalante

Road. Rana yavapaiensis were also caught and swabbed at the Campus Agricultural

Center.

Sample Collection

All frogs were swabbed with sterilized Whatman OmniSwabs and assigned a

unique identification code to each sample. I swabbed frogs using a standardized swabbing protocol resulting in a total of 31 strokes (five on each calf, thigh, and the ventral surface of the abdomen, and three on the bottom of each hind foot). All swabs were placed into individual vials with lysis buffer for future extraction and analysis. 27

I swabbed adult and metamorph H. arenicolor and R. yavapaiensis from fall 2008 through summer 2010 from the three canyons in the Rincon Mountains. Metamorphs included all frogs estimated to have metamorphosed in the past 30 days: these included

H. arenicolor with snout-vent-length (SVL: length from tip of nose to vent) less than

~30mm and R. yavapaiensis with SVL less than ~50mm. I considered all other individuals to be adults. I swabbed adult and metamorph H. arenicolor and R. yavapaiensis each season across their entire elevational range in each target drainage. I found H. arenicolor from 920-2429m in Chimenea Canyon, 920-1523m in Tanque Verde

Canyon, and 920-1825m in Wildhorse Canyon, and R. yavapaiensis from 920-1372m elevation in each canyon. I based seasons on the unique climate of southern Arizona: fall

= September-November; winter = December-February; spring = March-May; summer =

June-August. Hyla arenicolor were not swabbed above 1372m in the winter or spring, because this species is not usually available for capture at higher elevations in these seasons (Baker 2010).

To test tadpoles for Bd I excised tadpole mouthparts and collected 10 tadpoles of each species per canyon per season, but only when more than 1000 tadpoles of that species were present in the canyon. I euthanized tadpoles with an overdose of MS-222

(Webb et al. 2005) prior to cutting off mouthparts for Bd testing. 28

I visited ponds with R. catesbeiana or R. yavapaiensis throughout the Tucson

Basin three to five times from spring 2009 through summer 2010. I sampled

representatives of each life stage (tadpoles, metamorphs, and adults) at each pond when

each was present. All R. catesbeiana that had an SVL less than 70mm were considered

metamorphs.

I used a separate pair of non-latex gloves to handle each frog. For each

individual, I recorded weight (g), SVL (mm), age (adult, metamorph, or tadpole), sex

(male, female, or unknown), and water temperature (10cm deep, degrees Celsius) of

nearest water. I also counted the number of trombiculid mites (Hannemania sp.) on each

frog.

I collected water filtration samples from each frog pond and from every 150m of elevation in the three Rincon Mountain study drainages. I collected samples using methods described by Kirstein et al. (2007), except that I filtered 120ml of water, broke open the plastic filter casing, and placed the filter in a 5ml tube filled with 200ul of lysis buffer.

I disinfected all measuring instruments, frog bags, and footwear every 150m of elevation, between each drainage, and between each Tucson Basin pond using at least one of the methods outlined in Speare et al. (2004), including complete drying for over three hours, soaking in 10% bleach for ten minutes, or using a hot wash at 60oC for more than five minutes.

All work for this project was approved by the Institutional Animal Care and Use

Committee at the University of Arizona (#07-020 and #08-135), Arizona Game and Fish 29

Commission (#SP584012), Saguaro National Park (# SAGU-2008-SCI-0002), and

Coronado National Forest (written permission from D.A. Bennett).

Real Time Quantitative PCR

I extracted all swabs, tadpole samples, and filtration samples and analyzed them with real-time quantitative PCR using the methods described in Boyle et al. (2004) with

the following modifications: each sample was run in a 10ul reaction with 5uL of 2X

Taqman MM, 0.5uL of each primer (each diluted to 20uM), 0.25uL of the probe, 1.25uL of H2O, and 2.5uL of DNA. I also used an internal positive control in one of each of the triplicate wells, with 0.60uL 10X IPC mix and 0.12 50x IPC DNA (Applied Biosystems)

(Hyatt et al. 2007).

I estimated zoospore loads for swabs and tadpole samples by taking an average of the triplicates of each sample and adjusting this number to account for the fraction of the original extract used in the qPCR (Briggs et al. 2010). I reported zoospore counts for

water filtration samples as zoospores per liter by taking an average of the triplicates of

each sample (reported by the qPCR) and adjusting this number to account for the fraction

of the extract from the filter used in the qPCR as well as the amount of water filtered.

Data Analysis

I defined prevalence values as the percentage of frogs that tested positive for Bd

for any given assemblage. Infection intensity values were only reported from the frogs

that tested positive for Bd and are reported as zoospores.

To determine the differences in prevalence and infection intensity of Bd across

species, I compared Bd infection between H. arenicolor and R. yavapaiensis from the

Rincon Mountains and R. catesbeiana from the Tucson Basin (all locations and life

stages combined in each species) using generalized linear models (GLMs) with indicator

variables for each species. I then looked at patterns of Bd infection of each species individually:

To determine if location, elevation, season, or life stage was associated with Bd prevalence and infection intensity, I analyzed patterns of Bd infection among locations, elevations, seasons, and life stages for H. arenicolor and R. yavapaiensis separately using

GLMs. The objective of the GLM analyses was to determine the contribution of each

fixed effect (location, elevation, season, life stage, and interactions between location and

the other three fixed effects) to prevalence or intensity of infection of Bd of each species

after adjusting for covariates (mite load, sex, snout-vent-length, water temperature, and

year).

To further determine if there was a correlation between Bd and elevation, I

calculated the prevalence and mean intensity of Bd from H. arenicolor and R.

yavapaiensis (separately) for each 120-m elevational segment where each species was 31

found and compared these values to elevation. During preliminary analysis of H.

arenicolor elevation data, I detected a pattern of prevalence and infection intensity being

high at lower elevations, decreasing, and then increasing after a certain elevation was

reached. For this reason, I fit a 2nd degree polynomial regression line for Chimenea and

Wildhorse Canyon elevation data. I fit a linear regression line for Tanque Verde Canyon

elevation data due to H. arenicolor not being found as high in elevation in this canyon as

in the other two canyons, and thus not having as many data points to fit a regression line

(i.e., four points). I also used simple linear regression to test if the prevalence or

infection intensity of Bd in R. yavapaiensis was correlated with prevalence or infection

intensity of Bd in H. arenicolor, where they co-occurred in the same elevational segment.

I analyzed patterns of Bd infection (prevalence and intensity) between locations and life stages for R. catesbeiana also using GLMs, with the objective to determine the contribution of each fixed effect (location and life stage) to prevalence and intensity of infection (separately) after adjusting for covariates (sex, SVL, water temperature, and year). Rana catesbeiena individuals of each life stage were not collected from each pond, so an interaction between life stage and location was not included in the model.

Elevational variation between sites was limited, and seasonal sampling was not pursued in this study, so elevation and season were not included in the model either.

I analyzed patterns of Bd infection between locations in R. yavapaiensis from backyard ponds using GLMS, with the objective to determine the contribution of location to Bd prevalence and intensity of infection after adjusting for covariates (life stage, sex, snout-vent-length, water temperature, year). Associations between prevalence or 32

intensity of Bd and elevation, seasonality, and life stage were not pursued with R. yavapaiensis from backyard ponds, so these variables were not included in the model.

All infection intensity data were log10-transformed to meet assumptions of normality. I chose distribution and link functions by choosing those that gave the lowest

AIC values (data not shown); in all cases the binomial distribution with a logit link function had the lowest AIC values for the prevalence data, and the normal distribution with an identity link function had the lowest AIC value for the intensity data.

My final objective was to determine if infection intensity of Bd of frogs at a pool

(H. arenicolor and R. yavapaiensis together from the Rincon Mountains or R. catesbeiana from the Tucson Basin) was correlated with the number of zoospores found in a water filtration sample taken at that same pool. I averaged the Bd infection load from all frogs found at a specific pool (H. arenicolor and R. yavapaiensis together from the Rincon Mountains or R. catesbeiana from the Tucson Basin) and used linear regression to compare this amount to the to the Bd zoospore count per liter of water filtered at that same pool.

I used Jmp 9.0.0 (SAS Institute Inc.) for all analyses.

I calculated odds and multiplicative differences between variables (Appendix E), but report means (and 95% confidence intervals) in Tables 1.1 and 1.3-5 for simplicity.

Results

Species

I swabbed 870 H. arenicolor (821 frogs, 49 tadpoles) and 358 R. yavapaiensis

(320, 38) from three canyons in the Rincon Mountains from fall 2008 through summer

2010, 320 R. catesbeiana (213, 107) from seven ponds in the Tucson Basin from spring

2009 through summer 2010, and 204 R. yavapaiensis (192, 12) from 12 ponds in the

Tucson Basin from fall 2008 through summer 2010. Hyla arenicolor and R. yavapaiensis

from all surveyed canyons and R. catesbeiana from all surveyed ponds were infected

with Bd, with different levels of prevalence and infection intensity (Figure 1.4, Table

1.1). Prevalence and mean intensity of Bd infections (all life stages, drainages, and elevations combined) were highest in R. yavapaiensis, intermediate in R. catesbeiana,

and lowest in H. arenicolor (Table 1.1).

Location

For H. arenicolor, Bd prevalence and infection intensity were highly associated with location, as well as the interactions between location and life stage, elevation, and season, after accounting for SVL, sex, mite load, life stage, elevation, water temperature, season, and year (Table 1.2, Figure 1.5a-b). For R. yavapaiensis in the Rincon

Mountains, Bd prevalence and infection intensity were associated with location and the 34

interaction between location and elevation after accounting for snout-vent-length, sex, mite load, life stage, elevation, water temperature, season, and year (Table 1.2, Figure

1.5c-d).

Prevalence and intensity in H. arenicolor were lowest at Wildhorse Canyon and moderate-low in Chimenea and Tanque Verde Canyons (Table 1.1). Prevalence and intensity in R. yavapaiensis were also lowest at Wildhorse Canyon. Prevalence in R. yavapaiensis was high at Chimenea and Tanque Verde Canyons, but intensity was moderate at Chimenea Canyon and high at Tanque Verde Canyon (Table 1.1).

For R. catesbeiana, Bd prevalence and infection intensity were associated with location and varied depending on the pond R. catesbeiana were sampled at, after accounting for snout-vent-length, sex, life stage, water temperature, and year (Table 1.2,

Figure 1.6). The lowest mean prevalence occurred in frogs at Columbus Park and

Cebadilla Lake (4 and 6%, respectively); those at Crosby Pond had the highest prevalence (94%). Rana catesbeiana at Cebadilla Lake had the lowest mean infection intensity (mean 16 zoospores); those at Campus Agriculture Farm had the highest (5,955 zoospores, Table 1.1).

Prevalence and mean infection intensity of Bd were higher in R. yavapaiensis from backyard ponds than in R. yavapaiensis from the Rincon Mountains. For R. yavapaiensis in the backyard ponds, Bd prevalence and infection intensity were associated with location (prevalence X2 = 94.57, p <0.0001; intensity X2 = 69.54,

p<0.0001), after accounting for life stage, SVL, sex, water temperature, and year (Figures

1.7-8). No frogs tested positive at house # 1 (near Kennedy Park) or 11 (near Old 35

Spanish Trail and Escalante) and 13% and 17% of frogs tested positive at houses 2 (near

Kennedy Park) and 12 (near Old Spanish Trail and Escalante), respectively (Table 1.1).

At the other eight backyard ponds, 75-100% of R. yavapaiensis tested positive for Bd.

Frogs at houses 2 and 12 also had the lowest infection intensities (mean 3 and 14 zoospores, respectively); those at Campus Agricultural Farm had the highest (144,941 zoospores, Table 1.1).

Elevation

There was a strong association in H. arenicolor between Bd prevalence and infection intensity and the interaction of elevation and location, after accounting for snout-vent-length, sex, mite load, life stage, water temperature, season, and year (Table

1.2). Bd prevalence and infection intensity in H. arenicolor at Chimenea Canyon decreased with elevation from 920-1523m and increased from 1524-2429m (Figure 1.9a- b, Table 1.3). Prevalence and infection intensity decreased at Tanque Verde Canyon from

920-1523m (Figure 1.9c-d, Table 1.3). Prevalence increased from 920-1825m at

Wildhorse Canyon, and intensity decreased slightly from 920-1221m and increased from

1222-1825m (Figure 1.9e-f, Table 1.3).

Bd positive Rana yavapaiensis from Wildhorse Canyon were only found between

920-1070m (Figure 1.10a, Table 1.3). I found and swabbed one R. yavapaiensis adult in

Chimenea Canyon between 1524-1674m. This frog tested positive for Bd and had an 36

infection load of 1049 zoospores, but was not included in the analyses because it was the

only R. yavapaiensis detected in that elevation range throughout the study.

Where they co-occurred, there was a positive correlation between Bd prevalence

and infection intensity from R. yavapaiensis and H. arenicolor (Figure 1.11). For every

10% increase in prevalence of Bd from R. yavapaiensis within a 120-m elevational

segment, prevalence of Bd from H. arenicolor from that same elevational segment

increased 3% (H. arenicolor prevalence = 0.057 + 0.302*R.yavapaiensis prevalence).

For every 10x increase in the zoospore load from R. yavapaiensis within a 120-m

elevational segment, the zoospore load of H. arenicolor from that same elevational

0.27x segment increased 10 [log10(H. arenicolor infection intensity) = 1.153 + log10(R.

yavapaiensis infection intensity)]. Linear correlation coefficients for both relationships

(Figure 1.11) were significant at P < 0.04.

Season

Prevalence and infection intensity of Bd in H. arenicolor varied between seasons after accounting for SVL, sex, elevation, mite load, life stage, water temperature, season, and year (Table 1.2). Prevalence was highest in the winter and spring and lowest in the summer and fall (Table 1.4). Mean zoospore loads were highest in the spring, less high in summer and winter, and were lowest in the fall (Table 1.4).

There was a strong relationship between Bd prevalence and infection intensity in

H. arenicolor and the interaction between season and location. The highest prevalence of 37

Bd was 83% at Wildhorse Canyon in winter and 96% at Chimenea Canyon in spring, whereas H. arenicolor from Tanque Verde and Wildhorse Canyons in summer and

Chimenea Canyon in fall all had a prevalence of less than 10% (Table 1.4).

Moderate infection loads (compared to those in R. yavapaiensis, ~300-1000 zoospores) were found in H. arenicolor at Chimenea Canyon in spring and summer and at Tanque Verde Canyon in spring and fall, whereas low infection loads (compared to R. yavapaiensis, ~ ten zoospores) were found in H. arenicolor at Wildhorse in spring and summer.

In R. yavapaiensis there was no relationship between season and Bd prevalence or infection intensity.

Life Stage

Bd prevalence and infection intensity in H. arenicolor varied with the interaction between life stage and location, after accounting for SVL, sex, mite load, elevation, water temperature, season, and year (Table 1.5). Hyla arenicolor tadpoles tested positive for

Bd at Tanque Verde Canyon, but all those from Chimenea and Wildhorse canyons tested negative (Table 1.5). Metamorphosed H. arenicolor in Wildhorse Canyon had a higher prevalence and lower infection intensity than metamorphs and adults in Chimenea or

Tanque Verde canyons (Table 1.5).

For R. catesbeiana and R. yavapaiensis, there was no evidence of a relationship between Bd prevalence or infection intensity and life stage. 38

Water

There were too few locations that had both frogs and water samples testing positive for Bd to make any strong correlations between the zoospore counts of frogs and water (Figure 1.12, Table 1.6). However, out of the ponds that had both positive frogs and water samples, 82% occurred when the mean zoospore count of frogs was greater than 4,800 zoospores (9 out of 11 ponds, Figure 1.12). Eight of the ponds that had both positive frogs and water samples were ponds where only R. yavapaiensis were sampled: three of these ponds were in Chimenea Canyon and had H. arenicolor present that were not swabbed, and the other five were backyard ponds where only R. yavapaiensis were swabbed (Appendix C).

Discussion

Differences in Bd among species

Differences in prevalence and infection intensity of Bd in amphibian species found at the same site have been reported from several locations, including Queensland,

Australia, and California and Tennessee, USA (McDonald et al. 2005, Padgett-Flohr and

Hopkins 2009, Venesky et al. 2011). These differences have been attributed to

differences in skin immune defenses (Rollins-Smith et al. 2011); life history traits, such

as tadpole behavior (Venesky et al. 2001); and dependence on water (Kriger and Hero

2007b).

A previous study of Bd in R. yavapaiensis from different locations in Arizona

found an overall prevalence similar to the one in this study (43% vs. 49%, Shlaepfer et al.

2007). Another study found a mean infection intensity of Bd to be lower than ours (276

vs. 2,461 zoospores, Savage et al. 2011). The high prevalence and infection intensity of

Bd in R. yavapaiensis could be due to the aquatic nature of this species. Aquatic frogs

have been found to be more affected by Bd than terrestrial frogs in Panama and Australia

(Lips 1999, Rowley and Alford 2007). Epidemics of chytridiomycosis in R. yavapaiensis have also been recorded in several populations across Arizona (Sredl et al. 2003, Sredl

2005), indicating that this species can be negatively affected by this disease. Recent evidence also suggests that R. yavapaiensis with certain peptide encoding genes that 40

initiate acquired immunity may be able to survive Bd infection better than R. yavapaiensis without those genes (Savage and Zamudio 2011).

Rana yavapaiensis from backyard ponds may have had a higher prevalence and infection intensity of Bd than those individuals from the Rincon Mountains because backyard ponds may be at different temperatures then in the wild; more significantly, the water isn’t refreshed in the backyard ponds and these ponds are often the only source of water for what are, at times, high density populations. Therefore, backyard pond water would likely have a higher zoospore count and frogs there would be more likely to be in close proximity that might lead to infection transfer by direct contact.

While R. catesbeiana are also aquatic, prevalence and infection intensity of Bd in this species was less than that of R. yavapaiensis, though still higher than that in H. arenicolor. Overall prevalence of Bd in R. catesbeiana frogs and tadpoles in this study was similar to but slightly higher than prevalence measured in Virginia (41% vs. 35%,

Pullen et al. 2010), and more than double the overall prevalence found in another dataset on bullfrogs from Arizona (18%, Shlaepfer et al. 2007). Infection intensity of Bd in R. catesbeiana tends to be reported on a site-by-site basis and will be discussed below.

Rana catesbeiana have antimicrobial peptides that provide resistance against Bd (Rollins-

Smith et al. 2002). This species is relatively unaffected by Bd, acts as a reservoir for Bd, and is one of the main frog species implicated in spreading Bd to naïve amphibian populations across the world (Garner et al. 2006, Fisher and Garner 2007, Goka et al.

2009, Schloegel et al. 2010, Farrer et al. 2011). 41

The prevalence of Bd in H. arenicolor from this study (23%) was very similar to

that found in another study (21%, Baker 2010). This is the first report of Bd infection

intensities in H. arenicolor. The low prevalence and infection intensity of Bd in H. arenicolor was likely due to the more terrestrial nature of this species and its propensity

to achieve high body temperatures by basking. Individuals bask on rocks surrounding

pools or streams, and often only visit the water once a day to get their hydration needs for

the day (Snyder and Hammerson 1993). Because of this, H. arenicolor individuals would

come into contact with Bd zoospores less often than the two ranid species, and thus

would be less likely to get infected and reinfected by zoospores. There is some evidence

that wild H. arenicolor have also cleared themselves of Bd infection (Baker 2010),

presumably through basking at temperatures high enough to kill the fungus. When

Pseudacris triseriata were incubated at 32oC and Litoria chloris at 37oC, Bd was cleared

from all infected frogs (Woodhams et al. 2003, Retallick and Miera 2007). The rocks that

H. arenicolor bask on can reach temperatures of at least 36.8oC at lower elevations

(900m) with cloacal temperatures up to 34.2oC (Baker 2010), both of which are temperatures that would, if sustained for long enough, allow Bd to be cleared from H.

arenicolor individuals as well.

Differences among locations

Prevalence of Bd in H. arenicolor, R. yavapaiensis, and R. catesbeiana was found to be location dependent in several studies (Baker 2010, Bai et al. 2010, Savage et al. 42

2011, Shlaepfer et al. 2007). Infection intensity of Bd in R. yavapaiensis and R.

catesbeiana has also been found to be location dependent (Bai et al. 2010, Savage et al.

2011). Differences between locations in Bd prevalence in H. arenicolor have been

attributed to temperature variation between locations (Baker 2010), whereas those in R.

yavapaiensis have been attributed to immunogenetic differences between populations

(Savage and Zamudio 2011). No hypotheses were given on why there were differences

between locations in Bd prevalence and infection intensities in R. catesbeiana (Bai et al.

2010). Location dependence in Bd prevalence and infection intensity in H. arenicolor, R.

yavapaiensis, and R. catesbeiana in this study could be due to a variety of factors,

including:

a. Temperature differences among the locations;

b. Local populations are at different stages of Bd infection;

c. Differences in innate immune responses to Bd among populations;

d. Differences among localities in Bd strains of varying virulence;

e. Other, yet unrecognized, factor(s).

a. Temperature differences

For H. arenicolor and R. yavapaiensis in the Rincon Mountains, based on aspect

alone, pools at Chimenea (a south-facing drainage) would be expected to be hotter than

pools at the same elevations at north-facing Tanque Verde (Burnett et al. 2008). Because

Bd does better in cooler and wetter locations (Drew et al. 2006, Kriger et al. 2007b), 43

prevalence and infection intensity of Bd would be expected to be highest at Tanque Verde

Canyon, intermediate at Wildhorse Canyon, and lowest at Chimenea Canyon. Data from

this study do not support this hypothesis, however.

For R. catesbeiana and R. yavapaiensis in pools throughout the Tucson Basin,

aspect would not likely play a major role in pool temperature, since terrain is nearly flat.

Pools did have different levels of shade available, but these levels were not quantified.

Temperature data-loggers were not used in this study, but could be employed in the

future to document temperatures at various pools in the Tucson Basin to explore

differences in Bd prevalence and infection intensity found in this study among

populations of R. catesbeiana and R. yavapaiensis.

b. Populations at different stages of Bd infection

Different stages of Bd infection have been described in studies on mountain

yellow-legged frogs (R. muscosa and R. sierrae) in the Sierra Nevada, California. In one

study, abundances of these species were relatively high and constant until Bd arrived, at

which point the population declined rapidly in two to four years (Vredenburg et al. 2010).

Briggs et al. (2010) used Bd data from R. sierrae to design a model to describe a potential

pattern of Bd introduction into naïve populations:

1. Bd arrives into an uninfected frog population; prevalence and infection

intensity of Bd in susceptible individuals increase rapidly. 44

2. Buildup of infection intensity occurs rapidly in dense frog populations and

under conditions that promote continual reinfection (e.g., small volumes of

non flowing water).

3. Extirpation of the population is possible if the Bd load on all individuals

reaches a certain threshold.

4. If some individuals survive the initial epidemic then persistence of the disease

in the population in a new stable state is possible, with infection intensity

levels being relatively low.

Numerous R. yavapaiensis populations in the Rincon Mountains have been

monitored by Saguaro National Park since 1996, including Chimenea and Wildhorse

Canyons (Swann et al., in prep). Data from this monitoring effort suggest that abundance

and occupancy of R. yavapaiensis depended primarily on water availability, with the

main causes of decline being drought and sedimentation of pools. It is uncertain when Bd

first arrived in any of the canyons of this study, but Bd-positive R. yavapaiensis have been found in Chimenea Canyon since 2000 and Wildhorse Canyon since 2008 (Saguaro

National Park, unpublished data) with no Bd-related declines reported in either canyon since monitoring began. Bd-positive R. yavapaiensis have been found in Tanque Verde

Canyon since 2000 (Dennis Caldwell, unpublished data), and the abundance of R. yavapaiensis was relatively high (compared to other populations in the Rincon

Mountains) from spring/summer 2006 to spring/summer 2009 before numbers plummeted in 2010, with the population extirpation suspected as of 2011 (Savage et al.

2011, Swann et al. in prep). Compared to other drainages in the Rincon Mountains 45

during this time period, Tanque Verde had high water availability from 2009 to 2010

(Swann et al. in prep), suggesting that drought and sedimentation were not causes of the

decline. High Bd prevalence and infection intensity in frogs from Tanque Verde (Savage

et al. 2011, this study) indicate that the R. yavapaiensis population may have been

extirpated by chytridiomycosis. The ten year delay between the first recorded Bd- positive frog and the decline of frogs in Tanque Verde does not appear to fit the Briggs model, unless (a) it took >10 years for infection thresholds in R. yavapaiensis to reach a level to promote declines; (b) some other factor (like a different Bd strain or a new or

temporarily varying environmental stressor) caused Bd virulence to increase, (c) natural

fluctuation in frog abundance produced high densities conducive to Bd transmission prior

to the decline, or (d) the frogs declined due to a factor unrelated to Bd. One potential

stressor may have been the large amount of sediment upstream of Tanque Verde Creek

after the fires in 2003, which depressed the number of frogs in its feeder tributaries

(Parker 2006, Wallace et al. 2006). However, the true causes of this population decline,

and possible extirpation, remain unknown.

If the Briggs et al. model applies to R. yavapaiensis in Chimenea or Wildhorse

Canyons, then Bd would have to have been introduced into the Rincon Mountains, caused

population declines, and reached a stable state (stage 4) prior to 1996. The low levels of

Bd prevalence and infection intensity in frogs in Wildhorse Canyon corresponds with the

Briggs et al. model; however, the high levels of prevalence and infection intensity of Bd

in frogs in Chimenea suggest that some other factor may also be at play (e.g., virulence of

Bd strains). 46

Hyla arenicolor populations showed no signs of Bd-related declines in any of the canyons throughout the sampling period of this study, even in areas and times of high prevalence, such as at high elevations, in colder seasons, and in Chimenea and Tanque

Verde Canyon where they co-exist with R. yavapaiensis. The mean infection intensity of

Bd in H. arenicolor never approached the levels found in R. yavapaiensis at Chimenea or

Tanque Verde Canyons. There are no accounts of Bd-related die-offs or declines of this species in Arizona (Bradley et al. 2002, Baker 2010), and they may rarely, if ever, experience fatal Bd infection intensities.

This is the first study to report Bd results in R. catesbeiana from around the

Tucson Basin. Rana catesbeiana can travel at least 11 km from their home pond (Suhre

2010) and the majority of the ponds in this study were within less than this distance via drainage channel from at least one other pond with bullfrogs. Thus, it is possible that R. catesbeiana share genes and pathogens widely among populations in the Tucson area and might be expected to be at the same Briggs et al. model stage. However, Bd prevalence and intensity in the Tucson Basin in R. catesbeiana were highly variable among locations, suggesting that other factors (like environmental factors, population density, or non-Bd health factors) modulate Bd infection.

Perhaps most notable was the difference in infection patterns between Cebadilla

Lake and Crosby pond, which were about 2.5 km apart and both located near Tanque

Verde Creek. Rana catesbeiana at Cebadilla Lake had the lowest prevalence and infection intensity levels, whereas Crosby pond had some of the highest. Considering the vagility of R. catesbeiana, it seems unlikely that the differences in infection patterns 47

between these two ponds were due to different Briggs et al. stages of infection or that

different Bd strains uniquely infected each site. Other differences between the ponds

were more likely involved, such as temperature or population density. Crosby pond had

more tree cover and was, on average, 14.3oC cooler than Cebadilla Lake. Perhaps Bd at

Crosby pond reproduced more in those cooler temperatures, resulting in more infected frogs with higher zoospore loads.

Rana yavapaiensis populations near Kennedy Park and Campus Agricultural

Center were established using eggs and tadpoles from a single Bd-free captive source. In both cases, Bd likely arrived with bullfrogs. The other nine ponds were stocked with eggs or tadpoles from the Rincon Mountains. Bd could have entered these ponds any number of ways, including from water that was brought with the eggs/tadpoles from their original location; from infected bullfrogs that invaded the pond; from cross-contamination via fish, plants, nets, or any type of material brought from one infected drainage or pond to another; from infected aquatic insects or birds; from the wind; or from any other source of bringing zoospores in water to the backyard pond.

It is difficult to assign a stage of infection to frog populations in the 12 backyard ponds, due to unknowns about Bd arrival. Similarly, Bd strains to which their gene pools may have been exposed prior to and post establishment in Tucson yards are unknown.

c. Population level innate immune system differences

Different R. muscosa populations from California had differences in the

proportion of individuals with anti-Bd skin bacteria: populations containing higher

proportions persisted with Bd longer than populations with lower proportions (Lam et al.

2010). Different populations of the salamander species Hemidactylium scutatum and

Plethodon cinereus also had unique antifungal bacteria species on their skin (Lauer et al.

2007). Bd infection survival in different R. yavapaiensis populations across Arizona was

correlated with the presence of certain MHC class IIB genotypes, which code for certain

immune responses (Savage and Zamudio 2011). Populations with one specific

immunogenetic genotype not only survived Bd infection but had a lower prevalence and

lower infection intensities than populations lacking that genotype (Savage and Zamudio

2011), suggesting that populations that survive Bd infection may have a genetically based

resistance against the disease, whereas populations that decline or become extirpated lack

resistance or have resistant alleles at low frequency.

A population genetics study of R. yavapaiensis throughout the Rincon Mountains

reported that populations of frogs from each drainage were genetically unique with

limited recent mixing between populations (Goldberg et al. 2004) so the possibility that

populations from Chimenea, Tanque Verde, and Wildhorse Canyons have different

peptides, anti-Bd bacteria, or immunogenetic genotypes cannot be ruled out even though

they are close geographically. Neither anti-Bd skin bacteria nor immunogenetic

genotypes have been studied in R. yavapaiensis from the Rincon Mountains. However, 49

two of the backyard R. yavapaiensis ponds (near Old Spanish Trail and Houghton Road)

were stocked with eggs and tadpoles from Wildhorse and Tanque Verde. If populations

of frogs from Wildhorse had unique anti-Bd skin bacteria or genotypes that allowed

individuals to survive Bd infection, I would expect the frogs from Wildhorse tadpoles at

these backyard ponds to have survived. In both backyard ponds, R. yavapaiensis

experienced significant die-offs in the winters after the introduced tadpoles

metamorphosed, with only overwintering tadpoles keeping the populations going. This

provides anecdotal evidence that population level innate immune system differences are

probably not causing the differences in Bd prevalence and infection intensity between R.

yavapaiensis from Wildhorse and Tanque Verde.

Frogs from backyard ponds stocked only with tadpoles from Chimenea Canyon

(two houses near Escalante and Old Spanish Trail) and frogs from ponds near Kennedy

Park (stocked with tadpoles from a Bd-free captive population with a mixed origin from

eggs in the San Pedro River, Tumacacori Mountains, and Pajarito Mountains) either had

not tested Bd-positive yet or had a low prevalence and infection intensity, so a population level innate system response from these individuals cannot be ruled out.

Considering the vagility of R. catesbeiana (and other ranids) in valley environments, it seems somewhat less likely that the differences in infection patterns between ponds across the Tucson Basin were due to genetics or population specific anti-

Bd bacteria and instead may have been due to other differences between ponds.

However, this cannot be ruled out in this study.

d. Virulence differences

Strains of Bd around the world have been shown to differ in their virulence

(Berger et al. 2005b, Fisher et al. 2009), and variation between Bd strains has been found

among populations of R. sierrae from the same geographical area (Morgan et al. 2007).

Bd genotype has been found to be an important determinant of whether an amphibian

community can coexist with Bd or not (Farrer et al. 2011). Genotyping of Bd strains

from around the world has strongly supported the hypothesis that the international trade

of amphibians has caused endemic strains of Bd to come into contact with other strains

and recombine to form hypervirulent strains. These closely related hypervirulent strains

are a worldwide panzootic group that is causing amphibian declines and extinctions

(Farrer et al. 2011). Multiple strains of the panzootic group with varying virulence can

be found in a single locality (Farrer et al. 2011) and could be present in the Rincon

Mountains. If variation in strain virulence accounts for differences in infection found in

this study, Bd in Tanque Verde Canyon had the highest virulence, and Bd in Wildhorse

Canyon had the least virulent strain or strains. The declines of frogs in the backyard

ponds from Old Spanish Trail and Houghton might then be explained by predicted

dominance of the virulent strain (Farrer et al. 2011) even if strains of varying virulence

were introduced from Wildhorse and Tanque Verde into the backyard ponds. Backyard

ponds that came into contact with only a less virulent strain would thus be more likely to

coexist with Bd. 51

Once again, the ability of R. catesbeiana to travel long distances makes it unlikely

that differences in Bd prevalence and infection intensity among the neighborhood ponds

is due to different strains of Bd infecting each pond. Instead, what is more likely is that

there is one strain infecting all the bullfrog ponds and that the differences in prevalence

and infection intensity are due to something else.

e. Other Factors

Without more data about these locations, it is impossible to know what is causing

the location-based differences in prevalence and infection intensity of Bd between

drainages in the Rincon Mountains, backyard ponds, and bullfrog ponds. Bd prevalence

and infection intensity at a given location may be related to two or more of the factors

discussed above. In addition, it is also possible that location based differences are due to

as yet unidentified or unmeasured variables, including numerous environmental factors,

or that the differences are random. The prevalence of Bd in the environment and rate at

which an individual frog would get infected when Bd was introduced to a specific pool

would also affect the prevalence and infection intensity of Bd at a location. It cannot be

ruled out that the differences between locations are random, based on small sample size,

or due to differences among frogs within seasons.

Elevation

While the natural history of H. arenicolor indicates that this species would likely

not be affected by Bd at lower elevations, one might expect the prevalence and intensity of Bd to increase with elevation due to the lapse rates of air temperatures with increasing

elevation, rates which can vary from 0.6 – 3.4oC per 100m depending on moisture

content of the air and nearness to the earth (Lowry 1967). Prevalence of Bd in H.

arenicolor increased with elevation in the Santa Catalina Mountains, a mountain range adjoining the Rincon Mountains (Baker 2010). Higher elevations might often be at the

optimal growing temperatures of Bd, and H. arenicolor may then be unable to regularly

achieve temperatures lethal to Bd. Studies in Latin America and Iberian Peninsula found

frogs were more affected by Bd at higher elevations than frogs at lower elevations

(Young et al. 2001, Walker et al. 2010).

Baker (2010) found prevalence of Bd in H. arenicolor to increase with elevation

in several drainages in which R. yavapaiensis is presumed to be extirpated (Lazaroff et al.

2006); no H. arenicolor at elevations where R. yavapaiensis were traditionally found

tested positive for Bd (Lazaroff et al. 2006, Baker 2010). The long term persistence of

Bd in H. arenicolor in the absence of infected ranid populations has never been

determined, and it remains unknown if high elevation persistence of Bd in these hylid

frogs is a permanent condition.

Where R. yavapaiensis and H. arenicolor coexisted, prevalence and intensity in

H. arenicolor correlated positively and significantly with that of R. yavapaiensis: 53

prevalence and infection intensity of both species were high in Chimenea and Tanque

Verde Canyon and low in Wildhorse Canyon. The greater the prevalence and infection

intensity of R. yavapaiensis were, the higher were the prevalence and infection intensity

of H. arenicolor.

Where the two species coexisted, Bd prevalence and intensity in H. arenicolor

decreased as elevation increased. At the lowest elevations, there were many more

infected R. yavapaiensis present to shed zoospores in the water. As elevation increased,

there were fewer and fewer R. yavapaiensis to shed zoospores, and at a certain elevation

(around 1370m) only H. arenicolor were found. Prevalence and infection intensity in H. arenicolor were lowest at this elevation, and then increased with elevation. These findings suggest that infection rates in H. arenicolor at low elevations are governed by transmission from the Bd reservoir in R. yavapaiensis, whereas H. arenicolor at high elevation are governed by lower temperatures restricting the frog’s ability to achieve high enough temperatures to kill Bd.

Season

There was a higher Bd prevalence in H. arenicolor in winter and spring than in summer and fall, and a higher Bd infection intensity in spring than in all other seasons.

This increased prevalence could be due to the water being cooler in winter and spring and closer to optimal growth temperature for Bd (Longcore et al. 1999, Johnson et al. 2003,

Piotrowski et al. 2004). Several other studies have reported seasonal patterns of Bd 54

prevalence, with the highest prevalence occurring in the coldest seasons (Berger et al.

2004, Retallick et al. 2004, McDonald et al. 2005, Kriger and Hero 2007a, Kinney et al.

2011). A study of H. arenicolor from the Rincon Mountains and Santa Catalina

Mountains also found that the highest prevalence of Bd occurred early in the year, when water was cooler, though overall prevalence was highest in the winter and decreased throughout the spring (Baker 2010).

Although prevalence in this study was location dependent, differences among locations did not change the expected pattern: high prevalence was observed in canyons in winter and spring and a low prevalence in summer and fall. Infection intensity patterns loosely followed this expected pattern: moderate levels of infection in spring and fall, although not winter. The winter findings may be due to low sample sizes of H. arenicolor; these frogs apparently brumate in winter (Hammerson 1999, Baker 2010) and do not emerge until early spring when it is warmer, thus resulting in a small sample size.

Whereas Bd prevalence and infection intensity overall were higher in Chimenea and Tanque Verde, when the data were broken into seasons, Tanque Verde and

Wildhorse had a higher Bd prevalence and infection intensity in fall than Chimenea, and

H. arenicolor from Wildhorse were the only individuals to test positive for Bd in winter.

The reason for fall differences in H. arenicolor remains unclear, but the high winter Bd prevalence in Wildhorse is likely the result of few frogs observed in the winter and the possibility that the only frogs observed were infected and trying to clear the disease through basking.

Life Stage

Bd positive H. arenicolor tadpoles were only found in Tanque Verde Canyon, which may be a result of sampling bias: groups of tadpoles were usually sampled from the same pools, and there is a possibility that Bd present in Chimenea and Wildhorse

Canyons had not yet infected tadpoles at the times of sampling. Hyla arenicolor

metamorphs from Wildhorse had higher prevalence and lower infection intensity than

metamorphs and adults from the other two canyons. One study reported high mortality of

the frog Mixophyes fasciolatus with heavy infection loads of Bd sporangia within 30 days

of metamorphosis (Marantelli et al. 2004). Hyla arenicolor metamorphs could also be

following the same pattern of having a higher Bd prevalence soon after metamorphosis

than when a tadpole or an adult. Why these metamorphs would have a high prevalence

but low intensity, when individuals from the other canyons had a low prevalence and

high intensity, is uncertain, but could be due to sample size: tadpole and metamorph

sample sizes were much lower than for adults in each drainage sampled.

Water

The finding of Bd-positive water samples at sites with only negative frog samples

may be explained by sample limitations for the frogs. Infected tadpoles can also be a

source of zoospores in the water (Rachowicz and Vredenburg 2004), so zoospores in the

water could have been from infected tadpoles or frogs that were not sampled. A non- 56

amphibian reservoir of Bd may also be a source of zoospores; Bd has been found to reside in feathers, sediment, crayfish, fish, and some species of aquatic insects (Collins et al. 2005, Johnson and Speare 2005). The viability of the zoospores collected in the water filter was also unknown, as the qPCR only detects Bd DNA and does not determine if the filter collected live zoospores or simply Bd DNA in the water.

Almost half of the locations studied had Bd-negative water samples but Bd-

positive frogs. This indicates that water filters should not be the only samples collected

to determine if Bd is present at a location.

Of the ponds that had both positive frogs and water samples, 82% occurred when the mean zoospore count of frogs was greater than 4,800 zoospores (9/11), suggesting that there may be a threshold effect, where water samples are more likely to test positive for Bd after infection loads are greater than 4,800 zoospores. However, ten sites (10/18) with mean Bd infection intensity over 4,800 zoospores had water samples test negative, indicating that this threshold value may not be as reliable as first thought (Appendix C).

Most of the ponds that had both positive frogs and water samples (73%, or 8/11) were ponds where only R. yavapaiensis were sampled, giving the indication that ponds with R. yavapaiensis only are more likely to have a water sample test positive for Bd.

Three of these ponds, however, were in an area where H. arenicolor were present but not

sampled too. The other five ponds were backyard ponds with only R. yavapaiensis

present. The water in these ponds is not refreshed, and is often the only source of water

for what can be high density populations. Consequently, the water in these ponds would

likely have a higher zoospore count, and so the water in backyard ponds may be more 57

likely to test positive, not solely because of the presence of R. yavapaiensis, but because of the water dynamics as well. Seven other backyard ponds with only R. yavapaiensis present had positive frogs but negative water samples, once again supporting the conclusion that water filters should not be the only samples collected to determine if Bd is present at a location, even in areas where the chances of getting a positive water sample may be high.

Management Implications

More research is needed to identify causes underlying location-based differences

in Bd prevalence and infection intensity in all three species: H. arenicolor, R. yavapaiensis, and R. catesbeiana. It is of particular importance to identify Bd strains in the Rincon Mountains and wider Tucson region, and test for virulence differences if multiple strains are present. Major variation in virulence would provide several important guideposts for modes of reintroduction and even field sampling and disinfection protocols.

In the Rincon Mountains, and more generally for native Southwestern ranid frog populations experiencing Bd-related declines, it will be potentially extremely useful to catalog and understand anti-microbial, and particularly anti-Bd, properties of bacteria or other microbial flora living on frog skin in ranid populations. This holds promise that inoculation trials could be attempted to preserve or restore populations. It would also be important to know if any of the populations of R. yavapaiensis in the study region, and especially at Saguaro National Park, had immunogenetic variation supporting resistance to and coexisting with Bd. Resistant stock, or at least resistant alleles, could then be selected for potential reintroduction establishment programs.

Active management of amphibian chytridiomycosis in the Rincon Mountains, and elsewhere in nature, does not seem plausible at present: this study suggests that more first must be learned about location-based differences in Bd prevalence, infection intensity, and morbidity. This study highlights the complexity of Bd infection in this system, as 59

well as the widespread, multi-species, entrenched nature of the infection. What is certain is that Bd is here to stay, and any management actions need to take into account the futility of trying to get rid of Bd and instead consider what can be done to allow frog populations to persist in the face of this disease.

Table 1.1 Patterns of Batrachochytrium dendrobatidis infection among species and locations Prevalence 95% Mean 95% CI CI House Mean # Range of Species Location #a # Positive Total Prevalence Lower Upper Zoospores Lower Upper Zoospores Hyla Rincon Mountains 197 870 0.23 0.20 0.26 274 172 436 (1 – 1,021,184) arenicolor Rana Tucson Basin 129 317 0.41 0.36 0.47 849 522 1,381 (1 - 201,684) catesbeiana Rana Rincon Mountains 175 358 0.49 0.44 0.54 2,461 1,447 4,185 (1 – 1,526,741) yavapaiensis Rana Tucson Basin 118 203 0.58 0.51 0.64 6,273 2,993 13,145 (1-11,498,309) yavapaiensis Hyla Chimenea 91 383 0.24 0.20 0.28 392 191 803 (1 – 1,021,184) arenicolor Tanque Verde 67 263 0.25 0.21 0.31 354 159 789 (1 – 42,971) Wildhorse 39 224 0.17 0.13 0.23 77 31 190 (2 – 77,317) Rana Chimenea 100 123 0.81 0.74 0.87 889 454 1,741 (1 – 667,675) yavapaiensis Tanque Verde 67 73 0.92 0.83 0.96 19,691 10,231 37,900 (3 – 1,526,741) Wildhorse 8 162 0.05 0.03 0.09 22 5 111 (5 – 1,017)

Table 1.1 (continued) Patterns of Batrachochytrium dendrobatidis infection among species and locations Prevalence 95% Mean 95% CI CI House # Mean # Range of Species Location #a Positive Total Prevalence Lower Upper Zoospores Lower Upper Zoospores Rana Near Kennedy Park 1 0 5 0.00 yavapaiensis Near Kennedy Park 2 3 24 0.13 0.04 0.31 3 1 38 (1 - 7) Campus Ag Farm 3 12 16 0.75 0.51 0.90 144,941 63,059 333,143 (3,155 - 590,210) Notch Neighborhood 4 1 1 1.00 581 Notch Neighborhood 5 14 18 0.78 0.55 0.91 185 13 2,105 (2 - 1,006,288) Notch Neighborhood 6 13 13 1.00 0.77 1.00 12,469 3,445 45,125 (136 - 1,482,726) Notch Neighborhood 7 1 1 1.00 1,761 Notch Neighborhood 8 1 1 1.00 38 Houghton and Old Spanish Trail 9 29 29 1.00 0.88 1.00 21,994 6,438 75,131 (14 - 4,140,300) Houghton and Old Spanish Trail 10 35 36 0.97 0.86 1.00 30,167 10,880 83,645 (24 - 11,498,309) Old Spanish Trail and Escalante 11 0 6 0.00 Old Spanish Trail and Escalante 12 9 53 0.17 0.09 0.29 14 5 39 (3 - 134) Rana Campus Ag. Farm 23 36 0.64 0.49 0.79 5,955 1,561 22,714 (12 - 201,684) catesbeiana Cebedilla Lake 3 47 0.06 0.02 0.17 16 5 45 (10 - 21) Columbus Park 2 52 0.04 0.01 0.13 5 (1 - 25) Crosby Pond 77 82 0.94 0.87 0.97 862 505 1,472 (8 - 155,996) Ft. Lowell Park 11 25 0.44 0.27 0.63 312 74 1,310 (14 - 19,444) Kennedy Park 2 15 0.13 0.04 0.38 20 (2 - 190) TVGR 11 60 0.18 0.11 0.30 838 93 7,529 (3 – 24,254) a) Corresponds to locations indicated in Figure 1.3.

61 Table 1.2 Chi-square values and probabilities from generalized linear models to see which variables of interest were correlated with prevalence and infection intensity of Batrachochytrium dendrobatidis from Hyla arenicolor and Rana yavapaiensis at the Rincon Mountains and Rana catesbeiana from the Tucson Basin. Asterisks indicate significance (p ≤0.05). Hyla arenicolor Rana yavapaiensis Rana catesbeiana

Prevalence Intensity Prevalence Intensity Prevalence Intensity Elevation 0.01 (0.9206) 0.02 (0.8771) 0.36 (0.5488) 13.11 (0.0003) * Life Stage 1.73 (0.1886) 1.31 (0.2529) 0.96 (0.3281) 0.43 (0.5122) 1.42 (0.2336) 2.23 (0.1355) Location 26.83 (<0.0001) * 8.57 (0.0138) * 23.62 (<0.0001) * 33.44 (<0.0001) * 107.31 (<0.0001) * 32.28 (<0.0001) * Season 24.09 (<0.0001) * 11.26 (0.0036) * 1.89 (0.3882) 6.35 (0.0957) Life Stage*Location 6.58 (0.0372) * 5.86 (0.0534) 1.11 (0.5738) 1.67 (0.4330) Elevation*Location 57.17 (<0.0001) * 20.88 (<0.0001) * 6.20 (0.0451) * 10.98 (0.0041) * Season*Location 131.27 (<0.0001) * 56.45 (<0.0001) * 4.05 (0.3990) 2.68 (0.1018)

62 Table 1.3 Patterns of Batrachochytrium dendrobatidis infection across species, elevations, and locations. Elevation # Prevalence 95% CI Mean # Mean 95% CI Species (m) Location Positive Total Prevalence Lower Upper Zoospores Lower Upper Range of Zoospores Hyla arenicolor 920-1070 Chimenea 23 56 0.41 0.29 0.54 121 32 466 (1 – 41,615) 1071-1221 Chimenea 12 30 0.40 0.25 0.58 723 59 8,861 (1 – 1,021,184) 1222-1372 Chimenea 4 49 0.08 0.03 0.19 92 9 1,002 (13 - 460) 1373-1523 Chimenea 0 4 0.00 1524-1674 Chimenea 3 61 0.05 0.02 0.13 25 0 9,598 (3 - 335) 1675-1825 Chimenea 4 33 0.12 0.05 0.27 203 14 2,927 (22 – 1,009) 1826-1976 Chimenea 1977-2127 Chimenea 23 71 0.32 0.23 0.44 529 125 2,243 (1 – 64,607) 2128-2278 Chimenea 17 40 0.43 0.29 0.58 593 114 3,098 (1 – 31,434) 2279-2429 Chimenea 5 28 0.18 0.08 0.36 28,831 75 11,127,792 (7 – 969,050) 920-1070 Tanque Verde 37 105 0.35 0.27 0.45 547 191 1,566 (2 – 42,971) 1071-1221 Tanque Verde 19 52 0.37 0.25 0.50 588 106 3,258 (1 – 40,605) 1222-1372 Tanque Verde 2 70 0.03 0.01 0.10 50 3 816 (40 - 62) 1373-1523 Tanque Verde 4 17 0.24 0.10 0.47 26 7 92 (12 - 58) 920-1070 Wildhorse 14 105 0.13 0.08 0.21 13 5 33 (2 - 303) 1071-1221 Wildhorse 1 22 0.05 0.01 0.22 10 1222-1372 Wildhorse 2 43 0.05 0.01 0.15 9 (4 - 25) 1373-1523 Wildhorse 3 10 0.30 0.11 0.60 32 6 183 (19 - 71) 1524-1674 Wildhorse 10 10 1.00 0.72 1.00 328 44 2,444 (13 – 77,317) 1675-1825 Wildhorse 9 13 0.69 0.42 0.87 620 63 6,133 (11 – 18,576)

63 Table 1.3 (continued) Patterns of Batrachochytrium dendrobatidis infection across species, elevations, and locations. Elevation # Prevalence 95% CI Mean # Mean 95% CI Species (m) Location Positive Total Prevalence Lower Upper Zoospores Lower Upper Range of Zoospores Rana 920-1070 Chimenea 83 106 0.78 0.70 0.85 581 270 1,249 (1 – 667,675) yavapaiensis 1071-1221 Chimenea 12 12 1.00 0.76 1.00 14,242 6,246 32,477 (922 – 70,749) 1222-1372 Chimenea 4 4 1.00 0.51 1.00 1,247 106 14,679 (202 – 8,901) 920-1070 Tanque Verde 55 59 0.93 0.84 0.97 16,216 7,861 33,449 (3 – 1,526,740) 1071-1221 Tanque Verde 10 10 1.00 0.72 1.00 27,012 4,517 161,517 (73 – 249,092) 1222-1372 Tanque Verde 2 4 0.50 0.15 0.85 846,081 (486,913 – 1,470,188) 920-1070 Wildhorse 8 152 0.05 0.03 0.10 22 5 111 (5 – 1,017) 1071-1221 Wildhorse 0 8 0.00 1222-1372 Wildhorse 0 2 0.00

64 Table 1.4 Patterns of Batrachochytrium dendrobatidis across species, seasons, and locations. # Prevalence 95% CI Mean # Mean 95% CI Species Season Location Positive Total Prevalence Lower Upper Zoospores Lower Upper Range Zoospores Hyla Fall All 85 411 0.21 0.17 0.25 27 8 91 (1 – 34,729) arenicolor Winter All 5 9 0.56 0.27 0.81 72 16 327 (12 - 303) Spring All 43 104 0.41 0.32 0.51 414 128 1,336 (2 – 20,224) Summer All 64 346 0.18 0.15 0.23 297 119 740 (1 – 969,050) Hyla Fall Chimenea 14 189 0.07 0.04 0.12 13 4 39 (1 - 916) arenicolor Fall Tanque Verde 45 111 0.41 0.32 0.50 884 367 2,130 (1 – 42,971) Fall Wildhorse 26 111 0.23 0.17 0.32 163 49 545 (2 – 77,317) Winter Chimenea

Winter Tanque Verde 0 3 0.00 Winter Wildhorse 5 6 0.83 0.44 0.97 72 16 327 (12 - 303) Spring Chimenea 25 26 0.96 0.81 0.99 973 312 3,035 (13 – 1,021,184) Spring Tanque Verde 14 43 0.33 0.20 0.47 328 18 5,832 (4 – 20,224) Spring Wildhorse 4 35 0.11 0.05 0.26 4 1 18 (2 - 15) Summer Chimenea 52 168 0.31 0.24 0.38 626 231 1,696 (1 – 969,050) Summer Tanque Verde 8 106 0.08 0.04 0.14 12 2 78 (2 – 1,031) Summer Wildhorse 4 72 0.06 0.02 0.13 10 3 38 (4 - 25)

65 Table 1.4 (continued) Patterns of Batrachochytrium dendrobatidis across species, seasons, and locations. # Prevalence 95% CI Mean # Mean 95% CI Species Season Location Positive Total Prevalence Lower Upper Zoospores Lower Upper Range Zoospores Rana Fall All 101 178 0.57 0.49 0.64 418 144 1,219 (1 – 122,059) yavapaiensis Winter All 7 7 1.00 0.65 1.00 6,166 199 191,045 (41 – 667,675) Spring All 1 24 0.04 0.01 0.20 Summer All 66 149 0.44 0.37 0.52 1,427 660 3,087 (1 – 1,470,188) Rana Fall Chimenea 59 63 0.94 0.85 0.98 1,137 457 2,827 (1 – 307,885) yavapaiensis Fall Tanque Verde 37 40 0.93 0.80 0.97 36,879 15,011 90,603 (3 – 1,526,740) Fall Wildhorse 5 75 0.07 0.03 0.15 7 4 11 (5 - 13) Winter Chimenea 5 5 1.00 0.57 1.00 37,146 1,593 866,255 (891 – 667,675) Winter Tanque Verde

Winter Wildhorse 2 2 1.00 0.34 1.00 69 (41 - 116) Spring Chimenea 1 1 1.00 118,313 Spring Tanque Verde 0 1 1.00 Spring Wildhorse 0 22 0.00 Summer Chimenea 35 54 0.65 0.51 0.76 295 112 775 (1 – 238,867) Summer Tanque Verde 30 32 0.94 0.80 0.98 9,082 3,569 23,109 (76 – 1,470,188) Summer Wildhorse 1 63 0.02 0.00 0.08 1,017

66 Table 1.5 Patterns of Batrachochytrium dendrobatidis infection across species, life stages, and locations. Prevalence 95% Life # CI Mean # Mean 95% CI Range of Species Stage Location Positive Total Prevalence Lower Upper Zoospores Lower Upper Zoospores Hyla Tadpoles All 4 49 0.08 0.03 0.19 21 0 1,671 (3 – 1,031) arenicolor Juveniles All 13 67 0.19 0.12 0.30 220 24 2,013 (2 – 1,021,184) Adults All 180 754 0.24 0.21 0.27 294 181 478 (1 – 969,050) Rana Tadpoles All 30 107 0.28 0.20 0.37 7,180 2,749 18,754 (10 – 201,684) catesbeiana Juveniles All 33 93 0.35 0.27 0.46 415 155 1,109 (2 – 155,996) Adults All 68 120 0.57 0.48 0.65 496 266 926 (1 – 46,428) Rana Tadpoles All 9 38 0.24 0.13 0.39 5,163 846 31,527 (448 – 238,867) yavapaiensis Juveniles All 60 96 0.63 0.53 0.72 885 402 1,950 (1 – 119,933) Adults All 106 224 0.47 0.41 0.54 4,120 1,983 8,563 (1 – 1,526,741)

67 Table 1.5 (Continued) Patterns of Batrachochytrium dendrobatidis infection across species, life stages, and locations. Prevalence 95% Life # CI Mean # Mean 95% CI Range of Species Stage Location Positive Total Prevalence Lower Upper Zoospores Lower Upper Zoospores Hyla Tadpoles Chimenea 0 10 0.00 0 arenicolor

Tadpoles Tanque Verde 4 18 0.22 0.09 0.45 21 0 1,671 (3 – 1,031) Tadpoles Wildhorse 0 21 0.00 0

Juveniles Chimenea 4 33 0.12 0.05 0.27 1,424 1 2,237,593 (21 – 1,021,184)

Juveniles Tanque Verde 3 25 0.12 0.04 0.30 2,282 1 6,370,859 (58 – 17,895)

Juveniles Wildhorse 6 9 0.67 0.35 0.88 20 4 94 (2 - 104)

Adults Chimenea 87 340 0.26 0.21 0.30 369 178 763 (1 – 969,050)

Adults Tanque Verde 60 220 0.27 0.22 0.34 390 168 905 (1 – 42,971)

Adults Wildhorse 33 194 0.17 0.12 0.23 98 35 276 (2 – 77,317) Rana Tadpoles Chimenea 2 10 0.20 0.06 0.51 16,520 (1,143 – 238,867) yavapaiensis

Tadpoles Tanque Verde 6 8 0.75 0.41 0.93 4,593 447 47,161 (448 – 43,221) Tadpoles Wildhorse 1 20 0.05 0.01 0.24 1,017

Juveniles Chimenea 35 46 0.76 0.62 0.86 328 127 847 (1 – 118,313) Juveniles Tanque Verde 22 22 1.00 0.85 1.00 7,243 2,612 20,085 (76 – 119,933)

Juveniles Wildhorse 3 28 0.11 0.04 0.27 19 0 1,098 (5 - 116)

Adults Chimenea 63 67 0.94 0.86 0.98 1,411 572 3,478 (1 – 667,675)

Adults Tanque Verde 39 43 0.91 0.78 0.96 43,307 17,940 104,544 (3 – 1,526,741)

Adults Wildhorse 4 114 0.04 0.01 0.09 10 2 46 (5 - 41)

68 Table 1.6 Presence of Batrachochytrium dendrobatidis (+/-) in water filter samples and frog samples from the same location. # Positive Frogs Water (n=115) % Positive + + 11 9.56 - + 6 5.22 + - 51 44.35 - - 47 40.87

69 70

Figure 1.1 Distribution of three canyons in Saguaro National Park, Rincon Mountain District (Arizona, USA) where Hyla arenicolor and Rana yavapaiensis were tested for Batrachochytrium dendrobatidis.

Figure 1.2 Ponds across the Tucson Basin (Arizona, USA) where Rana catesbeiana were tested for Batrachochytrium dendrobatidis. Location names with total number of individuals (tadpoles, metamorphs, and adults combined) are: (1) Kennedy Park (n=15); (2) Columbus Park (n=52); (3) Campus Agriculture Farm (n=36); (4) Ft. Lowell Park (n=25); (5) Crosby pond (n=82); (6) Tanque Verde Guest Ranch (n=60); (7) Cebadilla Estates Lake (n=47).

Figure 1.3 Backyard ponds across the Tucson Basin (Arizona, USA) where Rana yavapaiensis were tested for Batrachochytrium dendrobatidis. Location names are: (1-2) near Kennedy Park; (3) Campus Agricultural Center; (4-8) Notch Neighborhood; (9-10) Houghton and Old Spanish Trail; (11-12) Old Spanish Trail and Escalante.

(a) 1

0.8

0.6

0.4

0.2 Infection Prevalence (%) Prevalence Infection

(b) 100000

10000

1000

100

10 (no. of zoospores) Infection Intensity Infection Intensity

1 HYAR RACA RAYA (Rincon Mts) RAYA (Tucson Basin)

Figure 1.4 Prevalence (a) and intensity (b) of Batrachochytrium dendrobatidis infection across species, all life stages, locations, seasons, and elevations combined. HYAR = Hyla arenicolor, RACA = Rana catesbeiana, RAYA = Rana yavapaiensis. Mean and 95% confidence intervals. 73 (a) 1 (b) Hyla arenicolor 100,000 0.8 10,000

0.6 1,000

0.4 100 (no. of zoospores) Infection Intensity Infection Intensity 0.2 10 Infection Prevalence (%) Prevalence Infection

0 1 Chimenea Tanque Verde Wildhorse Chimenea Tanque Verde Wildhorse

0.6 1,000

0.4 100 (no. of zoospores) Infection Intensity Infection Intensity 0.2 10 Infection Prevalence (%) Prevalence Infection 0 1 Chimenea Tanque Verde Wildhorse Chimenea Tanque Verde Wildhorse

(a) (b) 100,000,000,000,000 10,000,000,000,000 1 1,000,000,000,000 100,000,000,000 10,000,000,000 0.8 1,000,000,000 100,000,000 0.6 10,000,000 1,000,000 0.4 100,000 (no. of zoospores) Infection Intensity Infection Intensity 10,000

Infection Prevalence (%) Prevalence Infection 0.2 1,000 100 10 0 1

Figure 1.6 Prevalence (a) and intensity (b) of Batrachochytrium dendrobatidis from Rana catesbeiana across ponds throughout the Tucson Basin, Arizona, USA. TVGR = Tanque Verde Guest Ranch. Mean and 95% confidence intervals.

75 1.00

0.80

0.60

0.40

0.20 Infection Prevalence (%) Prevalence Infection 0.00

Figure 1.7 Prevalence of Batrachochytrium dendrobatidis from Rana yavapaiensis across ponds throughout the Tucson Basin, Arizona, USA. Numbers correspond to locations on Figure 1.3. Only one frog was swabbed at location 4, 7, and 8. Means and 95% confidence intervals. 76 1,000,000

100,000

10,000

1,000

100 (no. of zoospores) Infection Intensity Infection Intensity 10

Figure 1.8 Intensity of Batrachochytrium dendrobatidis from Rana yavapaiensis across ponds throughout the Tucson Basin, Arizona, USA. Numbers correspond to locations on Figure 1.3. Only one frog was swabbed at location 4, 7, and 8. Means and 95% confidence intervals.

77 78

(a) (b) 1.00 Chimenea Canyon 0.80 R² = 0.7463 R² = 0.4218 6 0.60 4 0.40 0.20 2 0.00 0 Infection Intensity Infection Intensity Infection Prevalence (%) Prevalence Infection [Log10(no. of [Log10(no. zoospores)]

(c) (d) 1.00 Tanque Verde Canyon 0.80 6 R² = 0.8614 0.60 R² = 0.3245 4 0.40 2 0.20 0.00 0 Infection Intensity Infection Intensity Infection Prevalence (%) Prevalence Infection [Log10(no. of [Log10(no. zoospores)]

(e) (f) 1.00 Wildhorse Canyon 6 0.80 R² = 0.9350 0.60 4 0.40 2 0.20 R² = 0.6915 Infection Intensity Infection Prevalence (%) Prevalence Infection 0.00 [Log10(no. of [Log10(no. zoospores)] 0

Elevation Range (m) Elevation Range (m)

0.80 10000

0.60 1000

0.40 100 (no. of zoospores) Infection Intensity

Infection Prevalence (%) Prevalence Infection 0.20 10

0.00 1 920-1070 1071-1221 1222-1372 920-1070 1071-1221 1222-1372 Elevation Range (m) Elevation Range (m) Figure 1.10 Prevalence (a) and infection intensity (b) of Batrachochytrium dendrobatidis from Rana yavapaiensis across elevational gradients in Chimenea Canyon, Tanque Verde Canyon, and Wildhorse Canyon, all life stages and seasons combined. Mean and 95% confidence intervals.

79 (a) (b) 1.00 Chimenea 4 Tanque Verde R² = 0.5497 0.80 Wildhorse 3

0.60 R² = 0.5231 2 0.40

0.20 oflog10(no. zoospores+1) Hyla arenicolor Infection Intensity Infection Intensity arenicolorHyla 0.00 0 0.00 0.20 0.40 0.60 0.80 1.00 0 2 4 6 Hyla arenicolor Infection Prevalence (%) Prevalence Infection arenicolor Hyla Rana yavapaiensis Infection Prevalence (%) Rana yavapaiensis Infection Intensity log10(no. of zoospores+1)

6 HYAR HYAR and RAYA 5 RACA RAYA 4

2 Log10(zoospores +1) Log10(zoospores Water Zoospore Count Zoospore Water 1

0 0 1 2 3 4 5 6 7 Frog Zoospore Count Log10(zoospores +1)

Works Cited

Aplin, K., and P. Kirkpatrick. 2000. Chytridiomycosis in southwest Australia: historical sampling documents the date of introduction, rates of spread and seasonal epidemiology, and sheds new light on chytrid ecology. In Getting the Jump! on Amphibian Disease: Conference and Workshop Compendium, Cairns, Australia.

Bai, C. M.,T. W. J. Garner, and Y. M. Li. 2010. First evidence of Batrachochytrium dendrobatidis in China: discovery of chytridiomycosis in introduced American bullfrogs and native amphibians in the Yunnan Province, China. EcoHealth, 7, 127–134.

Baker, K. D. 2010. Disease and natural history of canyon treefrogs (Hyla arenicolor). University of Arizona, Tucson Arizona.

Berger, L., A. D. Hyatt, R. Speare, and J. E. Longcore. 2005a. Life cycle stages of the amphibian chytrid Batrachochytrium dendrobatidis. Diseases of Aquatic Organisms 68:51-63.

Berger, L., G. Marantelli, L. F. Skerratt, and R. Speare. 2005b. Virulence of the amphibian chytrid fungus, Batrachochytrium dendrobatidis, varies with the strain. Diseases of Aquatic Organisms 68:47-50.

Berger, L., R. Speare, H. Hines, G. Marantelli, A. D. Hyatt, K. R. McDonald, L. F. Skerratt, V. Olsen, J. M. Clarke, G. Gillespie, M. Mahony, N. Sheppard, C. Williams, and M. Tyler. 2004. Effect of season and temperature on mortality in amphibians due to chytridiomycosis. Australian Veterinary Journal 82:31-36.

Berger, L., R. Speare, P. Daszak, D. E. Green, A. A. Cunningham, C. L. Goggin, R. Slocombe, M. A. Ragan, A. H. Hyatt, K. R. McDonald, H. B. Hines, K. R. Lips, G. Marantelli, and H. Parkes. 1998. Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America. Proceedings of the National Academy of Science of the United States of America 95:9031-9036.

Boyle, D. G., D. B. Boyle, V. Olsen, J. A. T. Morgan, and A. D. Hyatt. 2004. Rapid quantitative detection of chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples using real-time Taqman PCR assay. Diseases of Aquatic Organisms 60:141-148.

Bradley, G. A., P. C. Rosen, M. J. Sredl, T. R. Jones, and J. E. Longcore. 2002. Chytridiomycosis in native Arizona frogs. Journal of Wildlife Diseases 38:206- 212.

Briggs, C. J., R. A. Knapp, and V. T. Vredenburg. 2010. Enzootic and epizootic dynamics of the chytrid fungal pathogen of amphibians. Proceedings of the National Academy of Sciences of the United States of America 107:9695-9700.

Burnett, B. N., G. A. Meyer, and L. D. McFadden. 2008. Aspect-related microclimatic influences on slope forms and processes, northeastern Arizona. Journal of Geophysical Research-Earth Surface 113:18.

Burrowes, P. A., R. L. Joglar, and D. E. Green. 2004. Potential causes for amphibian declines in Puerto Rico. Herpetologica 60: 141-154.

Carey, C., J. E. Bruzgul, L. J. Livo, M. L. Walling, K. A. Kuehl, B. F. Dixon, A. P. Pessier, R. A. Alford, and K. B. Rodgers. 2006. Experimental exposures of boreal toads (Bufo boreas) to a pathogenic chytrid fungus (Batrachochytrium dendrobatidis). EcoHealth 3: 5-21.

Collins, J. P. 2010. Amphibian decline and extinction: What we know and what we need to learn. Diseases of Aquatic Organisms 92:93-99.

Collins, J. P., and M. A. Lewis. 1979. Overwintering tadpoles and breeding season variation in the Rana pipiens complex in Arizona. Southwestern Naturalist 24: 371-373.

Collins, J. P., V. Miera, E. W. Davidson, and R. W. Retallick. 2005. Host-pathogen community ecology in frogs. Arizona Game and Fish Department Heritage Grant, Project #I03006.

Daszak, P., A. Strieby, A. A. Cunningham, J. E. Longcore, C. C. Brown, and D. Porter. 2004. Experimental evidence that the bullfrog (Rana catesbeiana) is a potential carrier of chytridiomycosis, an emerging fungal disease of amphibians. Herpetological Journal 14:201-207.

Daszak, P., and A. A. Cunningham. 2003. Anthropogenic change, biodiversity loss, and a new agenda for emerging diseases. Journal of Parasitology 89:S37-S41.

Drew, A., E. J. Allen, and L. J. S. Allen. 2006. Analysis of climatic and geographic factors affecting the presence of chytridiomycosis in Australia. Diseases of Aquatic Organisms 68:245-250.

Farrer, R.A., L. A. Weinert, J. Bielby, T. W. J. Garner, F. Balloux, F. Clare, J. Bosch, A. A. Cunningham, C. Weldon, L. H. du Preez, L. Anderson, S. L. K. Pond, R. Shahar-Golan, D. A. Henk, and M. C. Fisher. 2011. Multiple emergences of genetically diverse amphibian-infecting chytrids include a globalized hypervirulent recombinant lineage. Proceedings of the National Academy of Sciences of the United States of America 108: 18732-18736.

Fisher, M. C., and T. W. J. Garner. 2007. The relationship between the emergence of Batrachochytrium dendrobatidis, the international trade in amphibians and introduced amphibian species. Fungal Biology Reviews 21:2-9.

Fisher, M. C., J. Bosch, Z. Yin, D. A. Stead, J. Walker, L. Selway, A. J. P. Brown, L. A. Walker, N. A. R. Gow, J. E. Stajich, and T. W. J. Garner. 2009. Proteomic and phenotypic profiling of the amphibian pathogen Batrachochytrium dendrobatidis shows that genotype is linked to virulence. Molecular Ecology 18:415-429.

Fisher, M. C., T. W. J. Garner, and S. F. Walker. 2009. Global emergence of Batrachochytrium dendrobatidis and amphibian chytridiomycosis in space, time, and host. Annual Review of Microbiology 63:291-310.

Garmyn A, P. Van Rooij, F. Pasmans, T. Hellebuyck, W. Van Den Broeck, F. Haesebouck, and A. Martel. 2012. Waterfowl: potential environmental reservoirs of the chytrid fungus Batrachochytrium dendrobatidis. PLoS ONE 7: e35038.

Garner, T. W. J., M. W. Perkins, P. Govindarajulu, D. Seglie, S. Walker, A. A. Cunningham, and M. C. Fisher. 2006. The emerging pathogen Batrachochytrium dendrobatidis globally infects introduced populations of the North American bullfrog, Rana catesbeiana. Biology Letters 2:455-459.

Goka, K., J. Yokoyama, Y. Une, T. Kuroki, K. Suzuki, M. Nakahara, A. Kobayashi, S. Inaba, T. Mizutani, and A. D. Hyatt. 2009. Amphibian chytridiomycosis in Japan: distribution, haplotypes and possible route of entry into Japan. Molecular Ecology 18:4757-4774.

Goldberg, C., D. E. Swann, and J. E. Wallace. 2004. Genetic Structure of Lowland Leopard Frog (Rana yavapaiensis) Populations in and near Saguaro National Park, Arizona. Final Report to Western National Parks Association (Grant 03-11) and Saguaro National Park, Tucson, Arizona.

Green, D. E., K. A. Converse, and A. K. Schrader. 2002. Epizootiology of sixty-four amphibian morbidity and mortality events in the USA, 1996-2001. Annals of the New York Academy of Science 969: 323-339.

Gründler, M.C., L. F. Toledo, G. Parra-Olea, C. F.B. Haddad, L. O. M. Giasson, R. J. Sawaya, C. P. A. Prado, O. G. S. Araujo, F. J. Zara, C. F. Centeno, and K. R. Zamudio. 2012. Interaction between breeding habitat and elevation affects prevalence but not infection intensity of Batrachochytrium dendrobatidis in Brazilian anuran assemblages. Diseases of Aquatic Organisms 97: 173-184.

Hale, S. F., P. C. Rosen, J. L. Jarchow, and G. A. Bradley. 2006. Effects of the chytrid fungus on the Tarahumara frog (Rana tarahumarae) in Arizona and Sonora, Mexico. Pages 407-411 in G. J. Gottfried, B. S. Gebow, L. G. Eskew, andC. B. Edminster, editors. Connecting mountain islands and desert seas: biodiversity and management of the Madrean Archipelago II. U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station, Fort Collins, CO.

Hammerson, G. A. 1999. Amphibians and reptiles in Colorado. University Press of Colorado. Colorado Division of Wildlife, Niwot, Colorado.

Hyatt, A. D., D. G. Boyle, V. Olsen, D. B. Boyle, L. Berger, D. Obendorf, A. Dalton, K. Kriger, M. Hero, H. Hines, R. Phillott, R. Campbell, G. Marantelli, F. Gleason, and A. Colling. 2007. Diagnostic assays and sampling protocols for the detection of Batrachochytrium dendrobatidis. Diseases of Aquatic Organisms 73:175-192.

Johnson, M., and R. Speare. 2005. Possible modes of dissemination of the amphibian chytrid Batrachochytrium dendrobatidis in the environment. Diseases of Aquatic Organisms 65:181-186.

Johnson, M., L. Berger, L. Phillips, and R. Speare. 2003. Fungicidal effects of chemical disinfectants, UV light, desiccation and heat on the amphibian chytrid, Batrachochytrium dendrobatidis. Diseases of Aquatic Organisms 57`:255-260.

Kielgast, J., D. Rodder, M. Veith, and S. Lotters. 2010. Widespread occurrence of the amphibian chytrid fungus in Kenya. Animal Conservation 13:36-43.

Kinney, V. C., J. L. Heemeyer, A. P. Pessier, and M. J. Lannoo. 2011. Seasonal pattern of Batrachochytrium dendrobatidis infection and mortality in Lithobates areolatus: affirmation of Vredenburg's "10,000 zoospore rule". Plos One 6:10.

Kriger, K. M., and J. M. Hero. 2007a. Large-scale seasonal variation in the prevalence and severity of chytridiomycosis. Journal of Zoology 271:352-359.

Kriger, K. M., and J. M. Hero. 2007b. The chytrid fungus Batrachochytrium dendrobatidis is non-randomly distributed across amphibian breeding habitats. Diversity and Distributions 13:781-788.

Kriger, K. M., K. J. Ashton, H. B. Hines, and J. M. Hero. 2007a. On the biological relevance of a single Batrachochytrium dendrobatidis zoospore: a reply to Smith (2007). Diseases of Aquatic Organisms 73: 257-260.

Kriger, K. M., F. Pereoglou, and J.-M. Hero. 2007b. Latitudinal variation in the prevalence and intensity of chytrid (Batrachochytrium dendrobatidis) infection in Eastern Australia. Conservation Biology 21:1280-1290.

Lam, B. A., J. B. Walke, V. T. Vredenburg, and R. N. Harris. 2010. Proportion of individuals with anti-Batrachochytrium dendrobatidis skin bacteria is associated with population persistence in the frog Rana muscosa. Biological Conservation 143:529-531.

Lamirande, E. W., and D. K. Nichols. 2002. Effects of host age on susceptibility to cutaneous chytridiomycosis in blue-and-yellow poison dart frogs (Dendrobates tinctorius). Pages 3-13 in Proceedings of Proceedings of the Sixth International Symposium on the Pathology of Reptiles and Amphibians.3-13.

Lauer, A., M. A. Simon, J. L. Banning, B. A. Lam, and R. N. Harris. 2007. Diversity of cutaneous bacteria with antifungal activity isolated from female four-toed salamanders. The ISME Journal 2:145-157.

Lazaroff, D. W., P. C. Rosen, and C. H. Lowe, Jr. 2006. Amphibians, reptiles, and their habitats at Sabino Canyon. University of Arizona Press, Tucson, Arizona.

Lips, K. R. 1999. Mass mortality and population declines of anurans at an upland site in Western Panama. Conservation Biology 13:117-125.

Lips, K. R., D. E. Green, and R. Papendick. 2003a. Chytridiomycosis in wild frogs from Southern Costa Rica. Journal of Herpetology 37:215-218.

Lips, K. R., J. D. Reeve, and L. R. Witters. 2003b. Ecological traits predicting amphibian population declines in Central America. Conservation Biology 17:1078-1088.

Longcore, J. E., A. P. Pessier, and D. K. Nichols. 1999. Batrachochytrium dendrobatidis gen. et sp. nov., a chytrid pathogenic to amphibians. Mycologia 91:219-227.

Lowry, W. P. 1967. Weather and life: an introduction to biometeorology. Academic Press, New York and London.

Marantelli, G., L. Berger, R. Speare, and L. Keegan. 2004. Changes in distribution of Batrachochytrium dendrobatidis and keratin during tadpole development leading to high mortality after metamorphosis. Pacific Conservation Biology 10:173-179.

Mazzoni, R., A. C. Cunningham, P. Daszak, A. Apolo, E. Perdomo, and G. Speranza. 2003. Emerging pathogen of wild amphibians in frogs (Rana catesbiana) farmed for international trade. Emerging Infectious Diseases 9:995-998.

McDonald, K. R., D. Méndez, R. Müller, A. B. Freeman, and R. Speare. 2005. Decline in the prevalence of chytridiomycosis in upland frog populations in North Queensland, Australia. Pacific Conservation Biology 11:114-120.

Morehouse, E. A., T. Y. James, A. R. D. Ganley, R. Vilgaly, L. Berger, P. J. Murphy, and J. E. Longcore. 2003. Multilocus sequence typing suggests the chytrid pathogen of amphibians is a recently emerged clone. Molecular Ecology 12:395-403.

Morgan, J. A. T., V. T. Vredenburg, L. J. Rachowicz, R. A. Knapp, M. J. Stice, T. Tunstall, R. E. Bingham, J. M. Parker, J. E. Longcore, C. Moritz, C. J. Briggs, and J. W. Taylor. 2007. Population genetics of the frog-killing fungus Batrachochytrium dendrobatidis. Proceedings of the National Academy of Science of USA 104:13845-13850.

Muths, E., P. S. Corn, A. P. Pessier, and D. E. Green. 2003. Evidence for disease related amphibian decline in Colorado. Biological Conservation 110:357-365.

Ouellet M, I. Mikaelian, B. D. Pauli, J. Rodrigue, and D. M. Green. 2005. Historical evidence of widespread chytrid infection in North American amphibian populations. Conservation Biology 19: 1431–1440.

Padgett-Flohr, G. E. , and R. L. Hopkins. 2009. Batrachochytrium dendrobatidis, a novel pathogen approaching endemism in central California. Diseases of Aquatic Organisms 83: 1-9.

Parker, J.T.C. 2006. Post-wildfire sedimentation in Saguaro National Park, Rincon Mountain District, and effects on lowland leopard frog habitat. US Geological Survey Scientific Investigations Report 2006-5235.

Parris, M. J., and T. O. Cornelius. 2004. Fungal pathogen causes competitive and developmental stress in larval amphibian communities. Ecology 85:3385-3395.

Pearl, C.A., E. L. Bull, D. E. Green, J. Bowerman,M. J. Adams, A. Hyatt, and W. H. Wente. 2007. Occurrence of the amphibian pathogen Batrachochytrium dendrobatidis in the Pacific Northwest. Journal of Herpetology 41: 145-149.

Piotrowski, J. S., S. L. Annis, and J. F. Longcore. 2004. Physiology of Batrachochytrium dendrobatidis, a chytrid pathogen of amphibians. Mycologia 96:9-15.

Piovia-Scott, J., K. L. Pope, S. P. Lawler, E. M. Cole, and J. E. Foley. 2011. Factors related to the distribution and prevalence of the fungal pathogen Batrachochytrium dendrobatidis in Rana cascadae and other amphibians in the Klamath Mountains. Biological Conservation 144: 2913-2921.

Powell, B. F., W. L. Halvorson, and C. A. Schmidt, editors. 2006. Vascular plant and vertebrate inventory of Saguaro National Park, Rincon Mountain District. OFR 2006-1075. U.S. Geological Survey, Southwest Biological Science Center, Sonoran Desert Research Station, University of Arizona, Tucson, AZ.

Pullen, K.D., A.M. Best, and J. L. Ware. 2010. Amphibian pathogen Batrachochytrium dendrobatidis prevalence is correlated with season and not urbanization in central Virginia. Diseases of Aquatic Organisms 91: 9-16.

Rachowicz, L. J., and V. T. Vredenburg. 2004. Transmission of Batrachochytrium dendrobatidis within and between amphibian life stages. Diseases of Aquatic Organisms 61:75-83.

Ramsey, J. P., L. K. Reinert, L. K. Harper, D. C. Woodhams, and L. A. Rollins-Smith. 2010. Immune defenses against Batrachochytrium dendrobatidis, a fungus linked to global amphibian declines, in the South African clawed frog, Xenopus laevis. Infection and Immunity 78:3981-3992.

Reeder N. M. M., A. P. Pessier, and V. T. Vredenburg. 2012. A reservoir species for the emerging amphibian pathogen Batrachochytrium dendrobatidis thrives in a landscape decimated by disease. PLoS ONE 7: e33567.

Retallick, R. W. R., and V. Miera. 2007. Strain differences in the amphibian chytrid Batrachochytrium dendrobatidis and non-permanent, sub-lethal effects of infection. Diseases of Aquatic Organisms 75:201-207.

Retallick, R. W. R., H. McCallum, and R. Speare. 2004. Endemic infection of the amphibian chytrid fungus in a frog community post-decline. Plos Biology 2:1965- 1971.

Rollins-Smith, L. A., C. Carey, J. Longcore, J. K. Doersam, A. Boutte, J. E. Bruzgal, and J. M. Conlon. 2002. Activity of antimicrobial skin peptides from ranid frogs against Batrachochytrium dendrobatidis, the chytrid fungus associated with global amphibian declines. Developmental and Comparative Immunology 26:471- 479.

Rothermel, B. B., S. C. Walls, J. C. Mitchell, C. K. Dodd, L. K. Irwin, D. E. Green, V. M. Vazquez, J. W. Petranka, and D. J. Stevenson. 2008. Widespread occurrence 89

of the amphibian chytrid fungus Batrachochytrium dendrobatidis in the southeastern USA. Diseases of Aquatic Organisms 82: 3-18.

Rowley, J., and R. A. Alford. 2007. Behaviour of Australian rainforest stream frogs may affect the transmission of chytridiomycosis. Diseases of Aquatic Organisms 77:1- 9.

Savage, A. E. and K. R. Zamudio. 2011. MHC genotypes associate with resistance to a frog-killing fungus. Proceedings of the National Academy of Sciences of the United States of America 108: 16705-16710.

Savage, A. E., M. J. Sredl, and K. R. Zamudio. Disease dynamics vary spatially and temporally in a North American amphibian. Biological Conservation 144: 1910- 1915.

Schloegel, L. M., C. M. Ferreira, T. Y. James, M. Hipolito, J. E. Longcore, A. D. Hyatt, M. Yabsley, A. Martins, R. Mazzoni, A. J. Davies, and P. Daszak. 2010. The North American bullfrog as a reservoir for the spread of Batrachochytrium dendrobatidis in Brazil. Animal Conservation 13:53-61.

Shlaepfer, M. A., M. J. Sredl, P. C. Rosen, and M. J. Ryan. 2007. High prevalence of Batrachochytrium dendrobatidis in wild populations of lowland leopard frogs Rana yavapaiensis in Arizona. EcoHealth 4: 421-427.

Skerratt, L. F., L. Berger, R. Speare, S. Cashins, K. R. McDonald, A. D. Phillott, H. B. Hines, and N. Kenyon. 2007. Spread of chytridiomycosis has caused the rapid global decline and extinction of frogs. EcoHealth 4:125-134.

Smith, K. G. 2007. Use of quantitative PCR assay for amphibian chytrid detection: comment on Kriger et al. (2006a,b). Diseases of Aquatic Organisms 73: 253-255.

Snyder, G. K., and G. A. Hammerson. 1993. Interrelationships between water economy and thermoregulation in the canyon tree-frog Hyla arenicolor Journal of Arid Environments 25:321-329.

Speare, R., L. Berger, L. F. Skerratt, R. Alford, D. Mendez, S. Cashins, N. Kenyon, K. Hauselberger, and J. Rowley. 2004. Hygiene protocol for handling amphibians in field studies. in http://www.jcu.edu.au/school/phtm/PHTM/frogs/field- hygiene.doc.

Sredl, M. J. 2005. Rana yavapaiensis Platz and Frost, 1984. in M. J. Lannoo, editor. Amphibian Declines: The Conservation Status of United States Species. University of California Press, Berkeley, California.

Sredl, M. J., and D. J. Caldwell. 2000. Update on chytrid surveys of frogs from Arizona. Sonoran Herpetologist. Tucson Herpetological Newsletter 13: 1.

Sredl, M. J., J. P. Collins, and J. M. Howland. 1997. Mark-recapture of Arizona leopard frogs. Pages 1-20 in M. J. Sredl, editor. Ranid frog conservation and management. Arizona Game and Fish Department,Nongame and Endangered Wildlife Program Technical Report 121.

Sredl, M. J., K. J. Field, and A. M. Peterson. 2003. Understanding and mitigating effects of chytrid fungus to amphibian populations in Arizona. Technical Report 208, Nongame Branch, Wildlife Management Division, Arizona Game and Fish Department.

Stuart, S., N. , J. S. Chanson, N. A. Cox, B. E. Young, A. S. L. Rodrigues, D. L. Fischman, and R. W. Waller. 2004. Status and Trends of Amphibian Declines and Extinctions Worldwide. Science 306:1783-1786.

Suhre, D. O. 2010. Dispersal and demography of the American bullfrog in a semi-arid grassland. University of Arizona, Tucson, Arizona.

Swann, D. E., K. M. Ratzlaff, and M. K. Ward. In prep. Lowland leopard frogs in the Rincon Mountains, Saguaro National Park, Arizona: a 15 year study. Saguaro National Park Technical Report, Tucson, Arizona.

Venesky, M. D., J. L. Kerby, A. Storfer, and M. J. Parris. 2011. Can differences in host behavior drive patterns of disease prevalence in tadpoles? PLoS ONE 6: e24991.

Voyles J., V. T. Vredenburg, T. S. Tunstall, J. M. Parker, C. J. Briggs and E. B. Rosenblum. 2012. Pathophysiology in mountain yellow-legged frogs (Rana muscosa) during a chytridiomycosis outbreak. PLoS ONE 7: e35374.

Voyles, J., L. Berger, S. Young, R. Speare, R. Webb, J. Warner, D. Rudd, R. Campbell, and L. F. Skerratt. 2007. Electrolyte depletion and osmotic imbalance in amphibians with chytridiomycosis. Diseases of Aquatic Organisms 77:113-118.

Vredenburg, V. T., R. A. Knapp, T. S. Tunstall, and C. J. Briggs. 2010. Dynamics of an emerging disease drive large-scale amphibian population extinctions. Proceedings of the National Academy of Sciences of the United States of America 107:9689- 9694.

Wake, D. B., and V. T. Vredenburg. 2008. Are we in the midst of the sixth mass extinction? A view from the world of amphibians. Proceedings of the National Academy of Sciences of the United States of America 105:11466-11473.

Walker, S. F., J. Bosch, V. Gomez, T. W. J. Garner, A. A. Cunningham, D. S. Schmeller, M. Ninyerola, D. A. Henk, C. Ginestet, C. P. Arthur, and M. C. Fisher. 2010. Factors driving pathogenicity vs. prevalence of amphibian panzootic chytridiomycosis in Iberia. Ecology Letters 13:372-382.

Wallace, J. E., R. J. Steidl, and D. E. Swann. 2010. Habitat characteristics of lowland leopard frogs in mountain canyons of Southeastern Arizona. Journal of Wildlife Management 74:808-815.

Wallace, J.E., D.E. Swann, and R.J. Steidl. 2006. Effects of wildland fire on lowland leopard frogs and their habitat at Saguaro National Park. Final report to Western National Parks Association and Saguaro National Park, Tucson, Arizona.

Webb, R., L. Berger, D. Mendez, and R. Speare. 2005. MS-222 (tricaine methane sulfonate) does not kill the amphibian chytrid fungus Batrachochytrium dendrobatidis. Diseases of Aquatic Organisms 68:89-90.

Weldon, C., L. H. du Preez, A. D. Hyatt, R. Muller, and R. Speare. 2004. Origin of the amphibian chytrid fungus. Emerging Infectious Diseases 10:2100-2105.

Woodhams, D. C., and R. A. Alford. 2005. Ecology of chytridiomycosis in rainforest stream frog assemblages of tropical Queensland. Conservation Biology 19:1449- 1459.

Woodhams, D. C., R. A. Alford, and G. Marantelli. 2003. Emerging disease of amphibians cured by elevated body temperature. Diseases of Aquatic Organisms 55:65-67.

Young, B. E., K. R. Lips, J. K. Reaser, R. Ibanez, A. W. Salas, J. R. Cedeno, L. A. Coloma, S. R. Ron, E. L. Marca, J. A. Meyer, A. Munoz, F. Bolanos, G. Chaves, and D. Romo. 2001. Population declines and priorities for amphibian conservation in Latin America. Conservation Biology 15:1213-1223.

CHAPTER 2 – ITRACONAZOLE IS NOT EFFECTIVE IN REDUCING WINTER MORTALITY OF WILD LOWLAND LEOPARD FROGS (RANA YAVAPAIENSIS) FROM CHYTRIDIOMYCOSIS

Abstract

The decline of amphibians worldwide has been attributed in part to the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd). When Bd enters a naïve amphibian population it can cause most, if not all, of the population to die. This was the case in a backyard population of lowland leopard frogs, Rana yavapaiensis, in Tucson, Arizona, in winter 2007 and 2008. In an attempt to prevent a third Bd-related die-off in 2009, I treated over 50% of the population with itraconazole to a zero detectable zoospore infection load before returning them to their home pond. Despite this treatment, no frogs survived the winter, and all dead individuals tested positive for Bd. Zoospore counts of frogs that died soon after capture suggested that the lethal Bd infection threshold of R. yavapaiensis was between 59,847 and 4,237,330 zoospores. Of six frogs kept in captivity, only one survived to the spring, despite repeated treatments with itraconazole.

One of the dead captive frogs was found to have died of a secondary infection due to a compromised immune system, likely because of stress of having the Bd infection and being in captivity. This study found that treatment of Bd in R. yavapaiensis with itraconazole was not successful in reducing winter mortality of these frogs due to Bd.

Introduction

Amphibians have been experiencing worldwide declines in the 21st century with

current extinction rates estimated to be 200 times greater than natural prehistoric rates

(McCallum 2007). One of the proximate causes of amphibian declines is the amphibian

chytrid fungus Batrachochytrium dendrobatidis (Bd), which has been found in over 350 amphibians and has been implicated in the decline of at least 200 species (Skerratt et al.

2007, Fisher et al. 2009, Collins 2010). Bd infects the keratinized skin of amphibians and disrupts electrolyte intake of skin, causing the often fatal disease chytridiomycosis

(Daszak et al. 1999, Berger et al. 2004, Voyles et al. 2007). This fungus has been implicated in declines of amphibians in Arizona, especially ranid frogs (Bradley et al.

2002, Hale et al. 2006, Schlaepfer et al. 2007).

Saguaro National Park and Arizona Game and Fish Department have collaborated with neighbors of the park to create backyard ponds stocked with eggs and tadpoles of lowland leopard frogs (Rana yavapaiensis) from the park. The aim of this project was to

better ensure that genetic stocks of frogs from those drainages were preserved, while at

the same time creating supplemental populations of R. yavapaiensis for potential

reintroduction into drainages from which the species had disappeared. Since their

creation (2005 through 2009), some of the ponds in the project experienced 100% mortality of R. yavapaiensis at least once due to predation and disease.

Our study focuses on two pond complexes that have had little observed predation and a high level of winter mortality of R. yavapaiensis thought to be caused by Bd. The

frogs in these ponds experienced 100% mortality in winter 2007 and near 100% mortality 94

in winter 2008 (one surviving adult was observed the following spring), with the

population persisting solely from overwintering tadpoles, which metamorphosed in

spring and summer. From 2007-2008 all R. yavapaiensis sampled from these ponds

tested positive for Bd (n=16; Ratzlaff, unpublished data).

One treatment regime reported to clear Bd infections in several amphibian species

involves soaking the infected frog in an antifungal solution (itraconazole; Marantelli et al.

2004, Forzan et al. 2008, Une et al. 2008). The goal of this study was to determine if

treating wild frogs with itraconazole until they cleared the fungus, and then releasing

them back into their habitat, would increase their chances of surviving the cold season.

This project was conducted under Arizona Game and Fish Department scientific collections permit (#SP584012) and approved University of Arizona Animal Care and

Use Protocols (# 07-020 and #08-135).

Methods

Study Sites

The leopard frogs in this study were from two small pond complexes (about 30 meters apart) at neighboring houses approximately five kilometers west of the Rincon

Mountain District of Saguaro National Park. Pond owners frequently observed frogs traveling between the pond complexes; thus, I considered the frogs in the two complexes to be one population. Both pond complexes consisted of two backyard ponds, constructed for the frogs, linked by a flowing stream containing low waterfalls, with cattails and rock nooks as the main sources of cover. One complex included a 2.0 x 1.5 x

0.5m deep upper pond and a 2.0 x 1.5 x 0.3m deep lower pond; the other complex contained a 0.75 x 1.0 x 0.3m deep upper pond and a 2.0 x 1.5 x 0.6m deep lower pond.

Field Work

I captured 29 frogs (out of 40 detected) for two days and four nights in October

2009 by hand, dip netting, and live trapping. I measured, weighed, swabbed, photographed, and assigned a unique identification number to each frog. I swabbed all frogs using a standardized swabbing protocol resulting in a total of 31 strokes (5 on each calf, thigh, and the ventral surface of the abdomen, and 3 on the bottom of each hind foot); I handled each frog with a separate pair of non-latex gloves. I placed all swabs into individual vials containing lysis buffer for future extraction and analysis. I also collected water filtration samples from each pond using methods described by Kirshtein et al. 96

(2007), except after I filtered 120 ml of water I broke open the plastic filter casing and

placed the filter in a 5-ml tube containing 200 ul of lysis buffer.

Treatment

Frogs were housed at the Central Animal Facility at the University of Arizona. I

treated each frog with 0.01% itraconazole in Amphibian’s Ringer’s Solution. The

itraconazole solution consisted of ten mg of itraconazole in one ml of 50% water and

50% methyl cellulose, with ascorbic acid added to adjust pH. The day of the treatment, I diluted one ml of the itraconazole solution in 100 ml of Amphibian Ringer's Solution for each frog. I soaked frogs in this solution for five minutes a day for 11 days. While each frog was soaking, I cleaned its cage with a 10% bleach solution and thoroughly rinsed it

before I returned the frog after treatment. I handled each frog with a separate pair of

non-latex gloves.

I swabbed each frog three times: immediately after capture, day six of treatment,

and the day after its last treatment. I extracted DNA at the Human Origins Genotyping

Laboratory, Arizona Research Laboratories, University of Arizona, and ran a polymerase

chain reaction (PCR) and used agarose-gel electrophoresis for the presence of Bd DNA to

determine if frogs were still infected. I returned frogs back to their pond if they tested

negative for Bd after their last treatment. Frogs that tested positive for Bd began a second

round of treatment and I kept them in captivity over the winter. After three of these frogs

died without signs of chytridiomycosis, I submitted the third dead frog to the University

of Arizona Veterinary Diagnostic Laboratory for necropsy. 97

Post-Treatment

I monitored the ponds once a week to look for any live, sick or dead frogs. I swabbed dead frogs that were not too decayed for Bd analysis. I compared dead frogs with a visible spot pattern to my database to determine if they were one of the itraconazole-treated frogs.

I re-ran all samples in summer 2010 with real time quantitative polymerase chain reaction (qPCR) using the methods described in Boyle et al. (2004) with some modifications: I ran each sample in ten-uL reactions with five uL of 2X Taqman MM, 0.5 uL of each primer (each diluted to 20uM), 0.25 uL of the probe, and 2.5 uL of DNA. I used an internal positive control in one of each of the triplicate wells, with 0.60 uL 10X

IPC mix and 0.12 50x IPC DNA (Applied Biosystems; Hyatt et al. 2007). I estimated zoospore counts for frog samples by taking an average of the triplicates of each sample and adjusting this number to account for the fraction of the original extract used in the qPCR (Briggs et al. 2010). I reported zoospore counts for water filtration samples as zoospores per liter by taking an average of the triplicates of each sample and adjusting this number to account for the fraction of the extract from the filter used in the qPCR as well as the amount of water filtered.

The number of zoospores found using qPCR is often reported as “genomic equivalents” or “zoospore equivalents” (Boyle et al. 2004, Hyatt et al. 2007, Reeder et al.

2012). In this study I use “zoospores” instead of “zoospore equivalents.”

Results

Pre-Treatment

Five of the twenty-nine frogs that were caught died prior to treatment, with four

of those displaying signs of chytridiomycosis (lethargy, no righting reflex, red thighs,

poor muscle contraction; Daszak et al. 1999, Mazzoni et al. 2003). Infection loads in

these individuals ranged from 59,847 to four million zoospores (Table 2.1).

Water filtration samples from both pond complexes were positive, with zoospore

loads of 442 zoospores per liter filtered in one pond complex and 90 zoospores per liter

filtered in the other pond complex.

Treatment

Two of the 24 R. yavapaiensis that began treatment died from non-disease-related causes. Of the 22 frogs that completed treatment, 16 tested negative with the PCR agarose-gel method and were released back into their home ponds in November 2009

(Table 2.2). The remaining six began a second round of treatment. Quantitative PCR is favored over traditional PCR because qPCR is more sensitive to detecting Bd and reports the infection intensity relative to other frogs swabbed the same way. When I later used

qPCR to reanalyze the same samples, one of the 15 released frogs tested positive with an infection load of seven zoospores.

On average, the infection load of Bd in R. yavapaiensis decreased by 7818

zoospores after being treated with itraconazole (one sided p-value >0.0001, from a 99

matched pairs t-test, t-ratio = 14.31, df = 21, 95 % CI = 9368 to 6901 zoospores; Figure

2.1). The infection load decreased by an average 7650 zoospores by the sixth day of

treatment (95% CI = 7684 to 7806 zoospores) and an additional 168 zoospores (95% CI =

152 to 214 zoospores) between the sixth day and the day after finishing treatment (day

12).

Post Treatment

I found 12 dead R. yavapaiensis in the ponds from November 2009 to March

2010. Three were confirmed recaptures of frogs that had been treated, five were too

decayed to identify, and four were frogs that had not been treated. In addition, I found a

decayed piece of skin, but could not determine its species.

I detected four live frogs (at least 2 individuals, maximum of 4) over three

occasions from November 2009 to January 2010. I saw the last live individual on

January 9, 2010, and did not detect frogs again until late July 2010 following metamorphosis of overwintered tadpoles.

There were four confirmed “recaptures” (three dead and one live) of frogs that had been treated, but only the live frog and one of the dead frogs were in good enough condition to be swabbed (Table 2.3). The recaptured live frog had almost 100 times more zoospores than when it was first captured, prior to treatment.

100

Long-Term Captivity Frogs

I kept six frogs in captivity, which underwent a second round of treatment with

itraconazole. Two of these frogs died due to cage malfunctions and three died with no

obvious signs of chytridiomycosis. One of these frogs was diagnosed through necropsy

to have died of an infection likely reflecting a weakened immune system (Table 2.4).

One frog after two series of treatments had an infection load over 25 times greater than

when it was first captured. Two other treated frogs also tested positive after two series of treatments, though both had an infection load at least 17 times less than when first captured. I returned the remaining live frog to its home pond on March 9, 2010; its fate is unknown.

101

Discussion

The infection load of individuals with signs of chytridiomycosis that died soon

after capture gives an indication of the lethal infection threshold of Bd in R. yavapaiensis.

Lethal infection thresholds of other amphibian species have been estimated at ten million to one hundred million zoosporangia in Bufo boreas (analyzed with histology, Carey et al. 2006), 32 genomic equivalents in Litoria aurea (qPCR, Stockwell et al. 2010) and

10,000 zoospores in R. muscosa (qPCR, Vredenburg et al. 2010). Though I only have a sample size of four, the suggested infection threshold for R. yavapaiensis is between

59,000 and four million zoospores - at least with my swabbing method, brand of swab,

and from my population (Savage and Zamudio 2011). Several other amphibian studies

have reported a high winter prevalence of Bd infection as well as Bd related winter die-

offs (Bradley et al. 2002, Berger et al. 2004, Retallick et al. 2004, McDonald et al. 2005,

Kriger and Hero 2007, Pearl et al. 2007, Rothermel et al. 2008). Frog immune systems

may be weakened in cold weather, with decreased, if not undetectable, concentrations of

antimicrobial peptides in the skin (Mattute et al. 2000, Rollins-Smith et al. 2002).

Amphibian antimicrobial peptides are part of an innate immune response against Bd infection (Woodhams et al. 2007), and the winter die-offs I observed could be caused in part by such a seasonal decline in antimicrobial peptides.

Itraconazole baths have been used to treat amphibians for Bd, with some success

(Forzan et al. 2008, Une et al. 2008, Berger et al. 2010). The Bd treatment in this study caused infection intensity to decrease over time, but 32% (7/22) of the treated frogs still had low levels of infection at the end of the first 11 day treatment regimen. A longer 102

soaking and treatment period, as Une et al. (2008) practiced, might solve this problem.

Rana yavapaiensis in this study tended to have a nervous temperament, and long-term captivity appeared to stress the frogs, despite limited contact with humans (~30 minutes a day). Heat is the only well supported treatment of Bd (Berger et al. 2010) with treatments occurring for two to five day periods (Woodams et al. 2003, Retallick and Miera 2007). I did not use heat to treat chytridiomycosis, and suggest that heat should be tried as a treatment of Bd in wild R. yavapaiensis to attempt to decrease the amount of time these frogs stay in captivity and maximize clearance rates.

Treatment of wild populations of amphibians has been recommended to decrease the impact of chytridiomycosis until host resistance develops (Berger et al. 2010). It was my hope that eliminating Bd infection, for at least a critical pre-winter period of time, would help the frogs survive the disease when the cold weather set in, but this was not the case. Despite treatment, no adult R. yavapaiensis from the two pond complexes survived the winter. This was likely partially due to the fact that it was impossible to clear Bd from the environment prior to release; Bd was found in water samples and likely helped maintain any infection in untreated frogs and tadpoles. Zoospores in the water were likely sufficient to re-infect the treated frogs after release, and cause their deaths.

Throughout my monitoring of the pond complex post-treatment, only 12 frogs were found dead even though all of the frogs in the pond (>30 individuals) apparently died during the winter. Homeowners in our study were looking for any obvious dead frogs and I visited the ponds once a week after release, and yet the death of over 50% of the pond complex’s amphibian inhabitants went undetected. This illustrates how 103

difficult it is to find dead or dying frogs, even in a closely monitored small aquatic system. If one is interested in determining if amphibian declines in an area could be due to Bd, this highlights the need for intensive monitoring to positively identify chytridiomycosis impacts even when impacts are severe, and highlights the likelihood of failing to observe even population-level mortality events.

Rana yavapaiensis kept in long term captivity fared little better than their released counterparts, with only one surviving to be released in the following spring. Infection load fluctuated in the captive frogs, with some swabs testing negative one month and positive the next, and frogs never presenting signs of chytridiomycosis. Cause of death of two captive frogs was unknown, but necropsy of a third dead frog (University of

Arizona Veterinary Diagnostic Laboratory, Reference Case 10-590) provided some insight into what the causes could have been. This frog was suspected to have died of an overwhelming opportunistic Pseudomonas infection, secondary to debilitation from Bd infection, seasonal changes in immune system, and/or stress of captivity, causing the frog to die from a bacterium that is ubiquitous in the environment (personal communication with Dr. J. Jarchow, Zapata et al. 1992, Morgan and Tromborg 2007).

104

Table 2.1 Wild caught Rana yavapaiensis that presented signs of chytridiomycosis and died within a day of capture. M = metamorph, A = adult. Snout-Vent Infection Life Weight Length Intensity (no. of Stage (g) (mm) zoospores) M 4.0 29 59,847 M 3.0 26 260,961 M 15.5 36 2,580,090 A 18.0 61 4,237,330

Table 2.2 Infection intensity of Batrachochytrium dendrobatidis from Rana yavapaiensis before, during, and after first treatment with itraconazole. Zoospore loads from qPCR and results from traditional PCR gel are given; note that frog SAGU00566 tested negative via the gel but had a very low zoospore count with qPCR. # Zoospores at # Zoospores at # Zoospores End of First Frog ID Initial Capture Mid-Treatment Treatment Gel Result SAGU00656 337,831 810 0 Negative SAGU01170 189,247 2,309 0 Negative SAGU01188 54,606 172 0 Negative SAGU01184 32,427 121 0 Negative SAGU01180 21,158 3,401 0 Negative SAGU01176 13,797 13 0 Negative SAGU01416 12,541 217 0 Negative SAGU01173 10,866 1,601 0 Negative SAGU01363 9,774 600 10 Positive SAGU01187 8,832 26 0 Negative SAGU01182 8,479 41 19 Positive SAGU01163 5,656 373 136 Positive SAGU01175 5,393 8 0 Negative SAGU01353 4,867 912 0 Negative SAGU01168 3,703 36 0 Negative SAGU00566 3,629 62,202 7 Negative SAGU01166 3,182 3 0 Negative SAGU01189 2,802 25 0 Negative SAGU01185 2,798 8,799 19 Positive SAGU01181 1,669 59 6 Positive SAGU01178 1,257 66 65 Positive SAGU01179 129 0 0 Negative

Table 2.3 Infection intensity of Rana yavapaiensis treated for Batrachochytrium dendrobatidis with itraconazole, released into home pond, and recaptured at a later date. # # Zoospores # Zoospores Zoospores When Date When Date When Frog ID Captured Released Released Recaptured Recaptured State When Recaptured SAGU01188 54,606 2-Nov-09 0 9-Jan-10 3,248 Dead SAGU01189 2,802 2-Nov-09 0 9-Jan-10 260,463 Alive, active, good righting reflex SAGU01168 3,703 2-Nov-09 0 19-Dec-09 ---- Dead - too decayed to swab SAGU01353 4,867 2-Nov-09 0 19-Dec-09 ---- Dead - too decayed to swab

Table 2.4 Infection intensity of Batrachochytrium dendrobatidis from Rana yavapaiensis kept in captivity throughout the winter. Frogs that died due to cage malfunctions are not shown. Frog ID Date Location SAGU01163 SAGU01181 SAGU01363 SAGU01182 16-Oct-09 Initial Capture 5,656 1,669 9,774 8,479 22-Oct-09 Mid-Treatment 373 59 600 41 28-Oct-09 End of 1st Treatment 136 6 10 19 2-Nov-09 2nd Treatment Begins 13-Nov-09 2nd Treatment Ends 30-Nov-09 Re-swab Frogs 13 0 0 43 2-Jan-10 Re-swab Frogs 154,765 289 0 0 3-Feb-10 Re-swab Frogs Found Dead 0 0 10 7-Feb-10 3rd Treatment Begins Found Dead Found Dead 18-Feb-10 3rd Treatment Ends 16-Mar-10 Released 0 105 106

100000

10000

1000

100 (no. of of (no. zoospores) Infection Intensity Intensity Infection 10

1 First Caught Mid Treatment End of 1st Treatment

Figure 2.1 Average infection intensity of Batrachochytrium dendtobatidis from Rana yavapaiensis before, during, and after treatment with itraconazole. Error bars represent 95% confidence intervals. 107

Works Cited

Berger, L., R. Speare, A. Pessier, J. Voyles, and L. F. Skerratt. 2010. Treatment of chytridiomycosis requires urgent clinical trials. Diseases of Aquatic Organisms 92:165-174.

Bradley, G. A., P. C. Rosen, M. J. Sredl, T. R. Jones, and J. E. Longcore. 2002. Chytridiomycosis in native Arizona frogs. Journal of Wildlife Diseases 38:206- 212.

Collins, J. P. 2010. Amphibian decline and extinction: What we know and what we need to learn. Diseases of Aquatic Organisms 92:93-99.

Daszak, P., L. Berger, A. A. Cunningham, A. D. Hyatt, D. E. Green, and R. Speare. 1999. Emerging infectious diseases and amphibian population declines. Emerging Infectious Diseases 5:735-748.

Forzan, M. J., H. Gunn, and P. Scott. 2008. Chytridiomycosis in an aquarium collection of frogs: diagnosis, treatment, and control. Journal of Zoo and Wildlife Medicine 39:406-411. 108

Hale, S. F., P. C. Rosen, J. L. Jarchow, and G. A. Bradley. 2006. Effects of the chytrid fungus on the Tarahumara frog (Rana tarahumarae) in Arizona and Sonora, Mexico. Pages 407-411 in G. J. Gottfried, B. S. Gebow, L. G. Eskew, andC. B. Edminster, editors. Connecting mountain islands and desert seas: biodiversity and management of the Madrean Archipelago II. U.S. Department of Agriculture, Forest Service,Rocky Mountain Research Station, Fort Collins, Colorado.

Kirshtein, J. D., C. W. Anderson, J. S. Wood, J. E. Longcore, and M. A. Voytek. 2007. Quantitative PCR detection of Batrachochytrium dendrobatidis DNA from sediments and water. Diseases of Aquatic Organisms 77:11-15.

Kriger, K. M., and J. M. Hero. 2007. Large-scale seasonal variation in the prevalence and severity of chytridiomycosis. Journal of Zoology 271:352-359.

Mattute, B., K. B. Storey, F. C. Knoop, and J. M. Conlon. 2000. Induction of synthesis of an antimicrobial peptide in the skin of the freeze-tolerant frog, Rana sylvatica, in response to environmental stimuli. FEBS Letters 24209:1-4.

McCallum, M. L. 2007. Amphibian decline or extinction? Current declines dwarf background extinction rate. Journal of Herpetology 41:483-491.

Morgan, K. N., and C. T. Tromborg. 2007. Sources of stress in captivity. Applied Animal Behaviour Science 102:262-302.

109

Pearl, C. A., E. L. Bull, D. E. Green, J. Bowerman, M. J. Adams, A. Hyatt, and W. H. Wente. 2007. Occurrence of the amphibian pathogen Batrachochytrium dendrobatidis in the Pacific Northwest. Journal of Herpetology 41:145-149.

Reeder, N. M. M., A. P. Pessier, and V. T. Vredenburg. A reservoir species for the emerging amphibian pathogen Batrachochytrium dendrobatidis thrives in a landscape decimated by disease. PLoS ONE 7:e33567.

Retallick, R. W. R., H. McCallum, and R. Speare. 2004. Endemic infection of the amphibian chytrid fungus in a frog community post-decline. Plos Biology 2:1965- 1971.

Rollins-Smith, L. A., L. K. Reinert, V. Miera, and J. M. Conlon. 2002. Antimicrobial peptide defenses of the Tarahumara frog, Rana tarahumarae. Biochemical and Biophysical Research Communications 297:361-367.

Rothermel, B. B., S. C. Walls, J. C. Mitchell, C. K. Dodd, L. K. Irwin, D. E. Green, V. M. Vazquez, J. W. Petranka, and D. J. Stevenson. 2008. Widespread occurrence of the amphibian chytrid fungus Batrachochytrium dendrobatidis in the southeastern USA. Diseases of Aquatic Organisms 82:3-18.

Savage, A. E., and K. R. Zamudio. MHC genotypes associate with resistance to a frog- killing fungus. PNAS 108: 16705-16710.

Schlaepfer, M. A., M. J. Sredl, P. C. Rosen, and M. J. Ryan. 2007. High prevalence of Batrachochytrium dendrobatidis in wild populations of lowland leopard frogs Rana yavapaiensis in Arizona. EcoHealth 4:421-427.

Stockwell, M. P., J. Clulow, and M. J. Mahony. 2010. Host species determines whether infection load increases beyond disease-causing thresholds following exposure to the amphibian chytrid fungus. Animal Conservation 13:62-71.

Une, Y., S. Kadekaru, K. Tamukai, K. Goka, and T. Kuroki. 2008. First report of spontaneous chytridiomycosis in frogs in Asia. Diseases of Aquatic Organisms 82:157-160.

110

Woodams, D. C., R. A. Alford, and G. Marantelli. 2003. Emerging disease of amphibians cured by elevated body temperature. Diseases of Aquatic Organisms 55:65-67.

Woodhams, D. C., K. Ardipradja, R. A. Alford, G. Marantelli, L. K. Reinert, and L. A. Rollins-Smith. 2007. Resistance to chytridiomycosis varies among amphibian species and is correlated with skin peptide defenses. Animal Conservation 10:409-417.

Zapata, A. G., A. Varas, and M. Torroba. 1992. Seasonal variations in the immune system of lower vertebrates. Immunology Today 13:142-147. 111 CHAPTER 3: NO DIFFERENCE BETWEEN BATRACHOCHYTRIUM DENDROBATIDIS ZOOSPORE COUNTS IN FROGS FOUND RECENTLY DEAD, AFTER BEING FROZEN, AND THEN THAWED

Abstract

Amphibians around the world are in decline, in part due to infection by the chytrid fungus, Batrachochytrium dendrobatidis (Bd). When large numbers of frogs are found dead, it is important to determine if those deaths were due to Bd, especially if Bd had not yet been documented in that area. Diagnosis of death due to Bd infection

(chytridiomycosis) can be difficult if dead frogs are collected and stored before they can be tested for Bd or if proper histological techniques or equipment are not used. To address this issue, I looked at the reliability of freezing recently dead frogs and swabbing them for Bd DNA after thawing. I found that the number of Bd zoospores detected on a swab from a recently dead frog did not differ from the number detected on a swab from that same animal after the frog had been frozen and thawed. Results of this study indicate that researchers can reliably use and report zoospore loads from frogs collected and frozen by others, as well as use these zoospore loads to help determine if the frogs might have died of chytridiomycosis or not.

112 Introduction

The chytrid fungus Batrachocytrium dendrobatidis (Bd) has been implicated in amphibian declines and extinctions across the world (Kilpatrick et al. 2010). Knowing the prevalence and intensity of Bd infections is important in diagnosing and understanding

the dynamics of chytridiomycosis in declining amphibian populations. It is important to

investigate the cause of death of amphibians, especially when there is a major die-off

(Lips et al. 2006), the species is endangered (Schoegel et al. 2005, Barrionuevo and

Mangione 2006), or the previous disease status of the population is unknown (Herrera et

al. 2005). Histology on dead frogs is useful because it can confirm the presence of Bd

and provide a direct measure of disease severity; real-time quantitative polymerase chain reaction (qPCR) provides a proxy level of severity through an estimate of the Bd zoospore load of an individual (Kriger et al. 2007, Kilpatrick et al. 2010).

Processing dead frogs for Bd analysis requires skill, knowledge, and access to chemicals and equipment (Berger and Speare 2002). Postmortem decomposition of dead frogs occurs quickly, especially at high temperatures, so rapid collection and preservation of specimens is necessary to maximize chances of identifying cause(s) of death (Berger and Speare 2002). Non-biologists who opportunistically find dead frogs are often willing to collect and freeze them for researchers, but are usually unable to keep Bd-detection equipment or learn swabbing techniques. If infection prevalence or load are similar in frozen and fresh, dead frogs, then valuable information about Bd in a population may be efficiently captured, which would otherwise be lost. The purpose of this study was to test whether numbers of zoospores from recently dead frogs using quantitative PCR 113 would be similar to zoospore counts of the same frogs that were frozen and swabbed at a later date.

114 Methods

Samples in this study came from an experimental R. yavapaisensis population in two backyard ponds approximately 3 miles west of Saguaro National Park, Rincon

Mountain District, near Tucson, Arizona. Both ponds had frogs from two drainages from the park. After introduction of tadpoles during fall-spring of 2006-7, both ponds saw die- offs of juvenile and adult frogs each winter, resulting in few (one frog in 2009) to no survivors (2008 and 2010) the following spring. The next years’ cohorts of frogs were thus from tadpoles produced the preceding year, which survived the winter and metamorphosed in spring and summer.

I searched for and collected dead frogs three times in December 2008 after pond owners reported seeing at least one dead frog in their pond. I swabbed all dead frogs

(n=12) with sterilized Whatman OmniSwabs and assigned each a unique identification code. I swabbed all frogs using a standardized swabbing protocol resulting in a total of

31 strokes: (5 on each calf, thigh, and the ventral surface of the abdomen, and 3 on the bottom of each hind foot). I placed all swabs into individual vials containing lysis buffer for future extraction and analysis.

Two to four weeks after initial freezing, I removed all frogs from the freezer and immediately re-swabbed them. I then allowed frogs to thaw (so that at least the skin was thawed) and swabbed them a final time.

I extracted DNA from the swabs and analyzed it with qPCR using methods described in Boyle et al. (2004) with the following modifications: I ran each sample in

10-uL reactions with 5 uL of 2X Taqman MM, 0.5 uL of each primer (each diluted to

20uM), 0.25 uL of the probe, and 2.5 uL of DNA. I ran an internal positive control in 115 one of each of the triplicate wells, with 0.60 uL 10X IPC mix and 0.12 50x IPC DNA

(Applied Biosystems; Hyatt et al. 2007). I estimated zoospore counts by taking an average of the triplicates of each sample and adjusting this number to account for the fraction of the original extract volume used in the PCR (Briggs et al. 2010).

116 Results

The number of Bd zoospores detected on swabs from recently dead frogs did not differ from the number detected on swabs from those same animals after the frogs had been frozen and thawed (two-sided p-value =0.52, matched paired t-test, t = -0.66, df =

11). Swabs from those recently dead R. yavapaiensis contained significantly more zoospores than those from the same animals while frozen (one-sided p > 0.0145, paired t- test, t = 2.51, df = 11; Figure 3.1), on average 784,019 zoospores more (95% CI = 96,559 to 1,471,478 zoospores; Table 3.1). In all cases, frogs tested positive for Bd when they were dead, frozen, or thawed.

117 Discussion

Many scientists have analyzed dead frogs using histology and determined cause

of death to be chytridiomycosis (Berger et al. 1998, Berger et al. 2005b, Herrera et al.

2005, Barrionuevo and Mangione 2006, Lips et al. 2006, Rothermel et al. 2008), at times

after ruling out other factors, such as other diseases, habitat loss, or predation (Bosch and

Martinez-Solano 2006). The similarity of zoospore loads of freshly dead frogs and

those same frogs after being frozen and thawed demonstrates that researchers can gather

useful information regarding zoospore loads from frogs collected and frozen. If the

infection threshold of a species has already been estimated (e.g., Carey et al. 2006), then

a reasonable assumption could be made that once an individual frog’s zoospore count

reaches that threshold, it is in danger of succumbing to chytridiomycosis under the right

stressors, such as cold weather, pollution, or even being captured and swabbed (Hale et

al. 2006). In my study 83% (10/12) of the dead frogs had initial infection loads measured

at above the suggested lethal load of Bd in R. yavapaiensis (59,000 zoospores, Chapter 2

of this document), as did 11 of 12 of these frogs after freezing and thawing; all 12 frogs

had at least one measurement (fresh or thawed) over 35,000. These infection loads, along

with the history of amphibian die offs in the study ponds and the winter-only die-offs in these frog populations (Chapter 2), caused me to conclude that at least 10, and likely all

12 individuals probably died from chytridiomycosis.

Though the difference was not statistically significant (Table 3.1), recently dead frogs had an average 186,431 zoospores less than those same frogs frozen then thawed.

A possible explanation for this unexpected result could be that the third swabbing (of the thawed frog) might have broken through the skin and picked up additional zoospores 118 from the thalli inside the skin, in addition to or instead of the zoospores on the skin

(Berger et al. 2005a). This could result in a much higher zoospore count than when the

frog was first found dead. To clarify this, it will be necessary to swab larger samples,

taken from single mortality events, before, after, and both before and after freezing and

thawing.

Since all frogs tested positive for Bd in all three instances (fresh dead, frozen, and

thawed), if a researcher was simply testing for prevalence of Bd, any of the swabbing

methods may be sufficient. However, the lower zoospore counts from still-frozen frogs

indicate that swabbing is less effective on a frozen surface. It is preferable to test freshly dead specimens or defrost the specimen’s skin before swabbing for Bd testing in frogs.

Being able to determine Bd presence, and an estimate of infection load from frozen frog specimens collected freshly dead and analyzed when convenient, will facilitate valuable insights into the disease process in frog populations, particularly in situations such as parks, refuges, and assurance populations where volunteers or staff are often present to observe, collect, and freeze moribund or freshly dead frogs. This technique might also be used to stockpile frozen specimens for a season or, likely, periods of a year or perhaps longer, although additional testing should be done to confirm

this for frozen storage longer than the 2-4 weeks used in this study.

119 Table 3.1 Batrachochytrium dendrobatidis zoospore counts for each dead frog collected when first found (fresh), while frozen, and after thawing. Frog # Fresh Frozen Thawed 1 4,207 108,494 1,344,476 2 35,629 607 3,719 3 302,967 402,920 59,606 4 452,744 378,363 1,165,416 5 637,572 652,942 1,668,071 6 919,309 311,985 857,336 7 1,080,001 150,078 1,680,116 8 1,311,276 448,744 105,592 9 1,366,733 627,196 2,127,350 10 1,411,645 495,582 1,500,731 11 2,031,708 285,019 3,228,540 12 4,237,330 520,964 2,287,341 Average 1,149,260 365,241 1,335,691 Std Error 331,770 58,752 282,644 95% CI - Lower 419,038 235,929 713,596 95% CI - Upper 1,879,482 494,554 1,957,786

120

Figure 3.1 Average number of Batrachochytrium dendrobatidis zoospores at each stage of swabbing dead Rana yavapaiensis (fresh, frozen, and thawed). Error bars represent 95% confidence intervals.

121 Works Cited

Barrionuevo, S., and S. Mangione. 2006. Chytridiomycosis in two species of Telmatobius (Anura : Leptodactylidae) from Argentina. Diseases of Aquatic Organisms 73:171-174.

Berger, L., A. D. Hyatt, R. Speare, and J. E. Longcore. 2005a. Life cycle stages of the amphibian chytrid Batrachochytrium dendrobatidis. Diseases of Aquatic Organisms 68:51-63.

Berger, L., and B. J. Speare. 2002. What to do with dead or ill frogs. Australian Amphibian Disease Group http://www.jcu.edu.au/school/phtm/PHTM/frogs/pmfrog.htm.

Berger, L., R. Speare, and L. Skerratt. 2005b. Distribution of Batrachochytrium dendrobatidis and pathology in the skin of green tree frogs (Litoria caerulea) with severe chytridiomycosis. Diseases of Aquatic Organisms 68:65-70.

Bosch, J., and I. Martinez-Solano. 2006. Chytrid fungus infection related to unusual mortalities of Salamandra salamandra and Bufo bufo in the Penalara Natural Park, Spain. Oryx 40:84-89.

Hale, S. F., P. C. Rosen, J. L. Jarchow, and G. A. Bradley. 2006. Effects of the chytrid fungus on the Tarahumara frog (Rana tarahumarae) in Arizona and Sonora, Mexico. Pages 407-411 in G. J. Gottfried, B. S. Gebow, L. G. Eskew, andC. B. Edminster, editors. Connecting mountain islands and desert seas: biodiversity and 122 management of the Madrean Archipelago II. U.S. Department of Agriculture, Forest Service,Rocky Mountain Research Station, Fort Collins, Colorado.

Herrera, R. A., M. M. Steciow, and G. S. Natale. 2005. Chytrid fungus parasitizing the wild amphibian Leptodactylus ocellatus (Anura: Leptodactylidae) in Argentina. Diseases of Aquatic Organisms 64:247-252.

Kilpatrick, A. M., C. J. Briggs, and P. Daszak. 2010. The ecology and impact of chytridiomycosis: an emerging disease of amphibians. Trends in Ecology & Evolution 25:109-118.

Kriger, K. M., K. J. Ashton, H. B. Hines, and J. M. Hero. 2007. On the biological relevance of a single Batrachochytrium dendrobatidis zoospore: a reply to Smith (2007). Diseases of Aquatic Organisms 73:257-260.

Lips, K. R., F. Brem, R. Brenes, J. D. Reeve, R. A. Alford, J. Voyles, C. Carey, L. Livo, A. P. Pessier, and J. P. Collins. 2006. Emerging infectious disease and the loss of biodiversity in a Neotropical amphibian community. Proceedings of the National Academy of Science of USA 102:3165-3170.

Lips, K. R., D. E. Green, and R. Papendick. 2003. Chytridiomycosis in wild frogs from Southern Costa Rica. Journal of Herpetology 37:215-218.

Schoegel, L. M., J. M. Hero, L. Berger, R. Speare, K. McDonald, and P. Daszak. 2005. The decline of the sharp-snouted day frog (Taudactylus acutirostris): the first documented case of extinction by infection in a free-ranging wildlife species? EcoHealth 3:35-40.

123

REFERENCES

Baker, K. D. 2010. Disease and natural history of canyon treefrogs (Hyla arenicolor). University of Arizona, Tucson Arizona.

Barrionuevo, S., and S. Mangione. 2006. Chytridiomycosis in two species of Telmatobius (Anura : Leptodactylidae) from Argentina. Diseases of Aquatic Organisms 73:171-174.

Berger, L., A. D. Hyatt, R. Speare, and J. E. Longcore. 2005. Life cycle stages of the amphibian chytrid Batrachochytrium dendrobatidis. Diseases of Aquatic Organisms 68:51-63.

Berger, L., R. Speare, and L. Skerratt. 2005. Distribution of Batrachochytrium dendrobatidis and pathology in the skin of green tree frogs (Litoria caerulea) with severe chytridiomycosis. Diseases of Aquatic Organisms 68:65-70.

Berger, L., and B. J. Speare. 2002. What to do with dead or ill frogs. Australian Amphibian Disease Group http://www.jcu.edu.au/school/phtm/PHTM/frogs/pmfrog.htm.

Berger, L., G. Marantelli, L. F. Skerratt, and R. Speare. 2005. Virulence of the amphibian chytrid fungus, Batrachochytrium dendrobatidis, varies with the strain. Diseases of Aquatic Organisms 68:47-50.

Berger, L., R. Speare, A. Pessier, J. Voyles, and L. F. Skerratt. 2010. Treatment of chytridiomycosis requires urgent clinical trials. Diseases of Aquatic Organisms 92:165-174.

Bosch, J., and I. Martinez-Solano. 2006. Chytrid fungus infection related to unusual mortalities of Salamandra salamandra and Bufo bufo in the Penalara Natural Park, Spain. Oryx 40:84-89.

Bradley, G. A., P. C. Rosen, M. J. Sredl, T. R. Jones, and J. E. Longcore. 2002. Chytridiomycosis in native Arizona frogs. Journal of Wildlife Diseases 38:206- 212.

Burrowes, P. A., R. L. Joglar, and D. E. Green. 2004. Potential causes for amphibian declines in Puerto Rico. Herpetologica 60: 141-154.

Collins, J. P. 2010. Amphibian decline and extinction: What we know and what we need to learn. Diseases of Aquatic Organisms 92:93-99.

Collins, J. P., and M. A. Lewis. 1979. Overwintering tadpoles and breeding season variation in the Rana pipiens complex in Arizona. Southwestern Naturalist 24: 371-373.

125

Collins, J. P., V. Miera, E. W. Davidson, and R. W. Retallick. 2005. Host-pathogen community ecology in frogs. Arizona Game and Fish Department Heritage Grant, Project #I03006.

Daszak, P., and A. A. Cunningham. 2003. Anthropogenic change, biodiversity loss, and a new agenda for emerging diseases. Journal of Parasitology 89:S37-S41.

Daszak, P., L. Berger, A. A. Cunningham, A. D. Hyatt, D. E. Green, and R. Speare. 1999. Emerging infectious diseases and amphibian population declines. Emerging Infectious Diseases 5:735-748.

Drew, A., E. J. Allen, and L. J. S. Allen. 2006. Analysis of climatic and geographic factors affecting the presence of chytridiomycosis in Australia. Diseases of Aquatic Organisms 68:245-250.

Forzan, M. J., H. Gunn, and P. Scott. 2008. Chytridiomycosis in an aquarium collection of frogs: diagnosis, treatment, and control. Journal of Zoo and Wildlife Medicine 39:406-411.

126

Goldberg, C., D. E. Swann, and J. E. Wallace. 2004. Genetic structure of lowland leopard frog (Rana yavapaiensis) populations in and near Saguaro National Park, Arizona. Final Report to Western National Parks Association (Grant 03-11) and Saguaro National Park, Tucson, Arizona.

Hale, S. F., P. C. Rosen, J. L. Jarchow, and G. A. Bradley. 2006. Effects of the chytrid fungus on the Tarahumara frog (Rana tarahumarae) in Arizona and Sonora, Mexico. Pages 407-411 in G. J. Gottfried, B. S. Gebow, L. G. Eskew, andC. B. Edminster, editors. Connecting mountain islands and desert seas: biodiversity and management of the Madrean Archipelago II. U.S. Department of Agriculture, Forest Service,Rocky Mountain Research Station, Fort Collins, Colorado.

Hammerson, G. A. 1999. Amphibians and reptiles in Colorado. University Press of Colorado. Colorado Division of Wildlife, Niwot, Colorado.

127

Johnson, M., and R. Speare. 2005. Possible modes of dissemination of the amphibian chytrid Batrachochytrium dendrobatidis in the environment. Diseases of Aquatic Organisms 65:181-186.

Kielgast, J., D. Rodder, M. Veith, and S. Lotters. 2010. Widespread occurrence of the amphibian chytrid fungus in Kenya. Animal Conservation 13:36-43.

Kilpatrick, A. M., C. J. Briggs, and P. Daszak. 2010. The ecology and impact of chytridiomycosis: an emerging disease of amphibians. Trends in Ecology & Evolution 25:109-118.

Kriger, K. M., and J. M. Hero. 2007. Large-scale seasonal variation in the prevalence and severity of chytridiomycosis. Journal of Zoology 271:352-359.

Kriger, K. M., and J. M. Hero. 2007b. The chytrid fungus Batrachochytrium dendrobatidis is non-randomly distributed across amphibian breeding habitats. Diversity and Distributions 13:781-788.

128

Lazaroff, D. W., P. C. Rosen, and C. H. Lowe, Jr. 2006. Amphibians, reptiles, and their habitats at Sabino Canyon. University of Arizona Press, Tucson, Arizona.

Lips, K. R. 1999. Mass mortality and population declines of anurans at an upland site in Western Panama. Conservation Biology 13:117-125.

Lips, K. R., D. E. Green, and R. Papendick. 2003. Chytridiomycosis in wild frogs from Southern Costa Rica. Journal of Herpetology 37:215-218.

Lips, K. R., J. D. Reeve, and L. R. Witters. 2003. Ecological traits predicting amphibian population declines in Central America. Conservation Biology 17:1078-1088.

Longcore, J. E., A. P. Pessier, and D. K. Nichols. 1999. Batrachochytrium dendrobatidis gen. et sp. nov., a chytrid pathogenic to amphibians. Mycologia 91:219-227.

Lowry, W. P. 1967. Weather and life: an introduction to biometeorology. Academic Press, New York and London.

McCallum, M. L. 2007. Amphibian decline or extinction? Current declines dwarf background extinction rate. Journal of Herpetology 41:483-491.

Morgan, K. N., and C. T. Tromborg. 2007. Sources of stress in captivity. Applied Animal Behaviour Science 102:262-302.

Muths, E., P. S. Corn, A. P. Pessier, and D. E. Green. 2003. Evidence for disease related amphibian decline in Colorado. Biological Conservation 110:357-365.

Padgett-Flohr, G. E. , and R. L. Hopkins. 2009. Batrachochytrium dendrobatidis, a novel pathogen approaching endemism in central California. Diseases of Aquatic Organisms 83: 1-9.

Parris, M. J., and T. O. Cornelius. 2004. Fungal pathogen causes competitive and developmental stress in larval amphibian communities. Ecology 85:3385-3395.

130

Pearl, C. A., E. L. Bull, D. E. Green, J. Bowerman, M. J. Adams, A. Hyatt, and W. H. Wente. 2007. Occurrence of the amphibian pathogen Batrachochytrium dendrobatidis in the Pacific Northwest. Journal of Herpetology 41:145-149.

Piotrowski, J. S., S. L. Annis, and J. F. Longcore. 2004. Physiology of Batrachochytrium dendrobatidis, a chytrid pathogen of amphibians. Mycologia 96:9-15.

Rachowicz, L. J., and V. T. Vredenburg. 2004. Transmission of Batrachochytrium dendrobatidis within and between amphibian life stages. Diseases of Aquatic Organisms 61:75-83.

Retallick, R. W. R., H. McCallum, and R. Speare. 2004. Endemic infection of the amphibian chytrid fungus in a frog community post-decline. Plos Biology 2:1965- 1971.

Rollins-Smith, L. A., C. Carey, J. Longcore, J. K. Doersam, A. Boutte, J. E. Bruzgal, and J. M. Conlon. 2002. Activity of antimicrobial skin peptides from ranid frogs 131

against Batrachochytrium dendrobatidis, the chytrid fungus associated with global amphibian declines. Developmental and Comparative Immunology 26:471- 479.

Rowley, J., and R. A. Alford. 2007. Behaviour of Australian rainforest stream frogs may affect the transmission of chytridiomycosis. Diseases of Aquatic Organisms 77:1- 9.

Savage, A. E., M. J. Sredl, and K. R. Zamudio. Disease dynamics vary spatially and temporally in a North American amphibian. Biological Conservation 144: 1910- 1915.

132

Smith, K. G. 2007. Use of quantitative PCR assay for amphibian chytrid detection: comment on Kriger et al. (2006a,b). Diseases of Aquatic Organisms 73: 253-255.

Snyder, G. K., and G. A. Hammerson. 1993. Interrelationships between water economy and thermoregulation in the canyon tree-frog Hyla arenicolor. Journal of Arid Environments 25:321-329.

Sredl, M. J., and D. J. Caldwell. 2000. Update on chytrid surveys of frogs from Arizona. Sonoran Herpetologist. Tucson Herpetological Newsletter 13: 1.

Suhre, D. O. 2010. Dispersal and demography of the American bullfrog in a semi-arid grassland. University of Arizona, Tucson, Arizona.

133

Une, Y., S. Kadekaru, K. Tamukai, K. Goka, and T. Kuroki. 2008. First report of spontaneous chytridiomycosis in frogs in Asia. Diseases of Aquatic Organisms 82:157-160.

Venesky, M. D., J. L. Kerby, A. Storfer, and M. J. Parris. 2011. Can differences in host behavior drive patterns of disease prevalence in tadpoles? PLoS ONE 6: e24991.

Wallace, J. E., R. J. Steidl, and D. E. Swann. 2010. Habitat Characteristics of Lowland Leopard Frogs in Mountain Canyons of Southeastern Arizona. Journal of Wildlife Management 74:808-815.

Weldon, C., L. H. du Preez, A. D. Hyatt, R. Muller, and R. Speare. 2004. Origin of the amphibian chytrid fungus. Emerging Infectious Diseases 10:2100-2105.

Woodhams, D. C., and R. A. Alford. 2005. Ecology of chytridiomycosis in rainforest stream frog assemblages of tropical Queensland. Conservation Biology 19:1449- 1459.

Woodhams, D. C., R. A. Alford, and G. Marantelli. 2003. Emerging disease of amphibians cured by elevated body temperature. Diseases of Aquatic Organisms 55:65-67.

Zapata, A. G., A. Varas, and M. Torroba. 1992. Seasonal variations in the immune system of lower vertebrates. Immunology Today 13:142-147. APPENDIX A: ALL FROG SAMPLES COLLECTED AT PONDS FROM THE RINCON MOUNTAINS AND TUCSON BASIN

Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU00379 4-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 542038 3563509 7916 A 59 F 4.6 35 25 SAGU00352 4-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 542038 3563509 7916 A 59 M 2.5 38 30 SAGU00364 4-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 542038 3563509 7916 A 59 M 3.5 36 50 SAGU00377 4-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 541847 3562692 7398 A 59 M 3.0 38 80 SAGU00369 4-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 542036 3562871 7516 A 59 M 2.5 32 30 SAGU00368 4-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 542036 3562871 7516 A 59 M 4.0 33 200 SAGU00363 4-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 541982 3562836 7476 A 58 M 4.0 36 200 SAGU00380 4-Oct-08 HYAR POSITIVE 7 2008 Fall Chimenea 542036 3562871 7516 A 59 M 3.0 34 60 SAGU00372 4-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 541847 3562692 7398 A 59 F 6.5 45 80 SAGU00355 4-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 542036 3562871 7516 A 59 F 10.5 49 100 SAGU00375 4-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 542038 3563509 7916 A 59 M 10.4 52 200 SAGU00356 4-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 542038 3563509 7916 A 59 M 16.6 57 300 SAGU00362 4-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 542038 3563509 7916 A 59 M 12.6 53 300 SAGU00371 4-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 542038 3563509 7916 A 59 M 9.0 49 25 SAGU00374 4-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 542038 3563509 7916 A 59 M 9.0 47 150 SAGU00367 4-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 542038 3563509 7916 A 59 M 9.6 57 130 SAGU00354 4-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 542038 3563509 7916 A 59 M 8.6 47 100 SAGU00358 4-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 542047 3562890 7580 A 60 M 10.0 47 100 SAGU00378 4-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 541846 3562670 7388 A 59 M 9.5 48 30 SAGU00373 4-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 541846 3562670 7388 A 59 M 9.0 46 100 SAGU00359 4-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 541846 3562670 7388 A 59 M 13.5 56 50 SAGU00376 4-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 541846 3562670 7388 A 59 M 13.0 53 150 SAGU00361 4-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 541846 3562670 7388 A 59 M 10.0 50 200 SAGU00351 4-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 542036 3562871 7516 A 59 M 15.0 54 30 SAGU00366 4-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 541982 3562836 7476 A 58 M 10.0 47 125 SAGU00370 4-Oct-08 HYAR POSITIVE 28 2008 Fall Chimenea 541846 3562670 7388 A 59 M 7.0 47 25 SAGU00324 5-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 540726 3562100 6509 A 62 F 2.0 36 50 SAGU00353 5-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 541566 3562376 7088 A 58 F 3.5 37 50 SAGU00340 5-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 540726 3562100 6509 A 62 M 2.5 36 100 SAGU00360 5-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 541502 3562340 7048 A 58 F 10.0 49 100 SAGU00322 5-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 540867 3562148 6653 A 60 F 9.5 50 80 SAGU00346 5-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 540840 3562138 6601 A 60 F 10.0 48 60 SAGU00328 5-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 540726 3562100 6509 A 62 F 10.0 45 120 SAGU00336 5-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 540726 3562100 6509 A 62 F 6.0 41 100 SAGU00325 5-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 540726 3562100 6509 A 62 F 3.0 38 80 SAGU00356 5-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 541566 3562376 7088 A 58 M 14.0 54 40 SAGU00347 5-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 541146 3562271 6745 A 61 M 6.5 41 150

SAGU00342 5-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 541146 3562271 6745 A 61 M 7.0 44 100 135 SAGU00337 5-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 540887 3562166 6669 A 60 M 11.5 49 200 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU00345 5-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 540887 3562166 6669 A 60 M 12.0 51 80 SAGU00323 5-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 540867 3562148 6653 A 60 M 9.5 46 180 SAGU00338 5-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 540867 3562148 6653 A 60 M 12.5 50 60 SAGU00333 5-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 540867 3562148 6653 A 60 M 11.0 50 150 SAGU00327 5-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 540840 3562138 6601 A 60 M 8.5 47 100 SAGU00329 5-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 540801 3562135 6562 A 61 M 12.0 51 150 SAGU00348 5-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 540726 3562100 6509 A 62 M 7.0 45 120 SAGU00321 5-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 540887 3562166 6669 A 60 M 11.5 51 300 SAGU00330 5-Oct-08 HYAR POSITIVE 7 2008 Fall Chimenea 540867 3562148 6653 A 60 M 11.0 51 200 SAGU00331 5-Oct-08 HYAR POSITIVE 8 2008 Fall Chimenea 540867 3562148 6653 A 60 M 15.0 54 100 SAGU00357 5-Oct-08 HYAR POSITIVE 8 2008 Fall Chimenea 541502 3562340 7048 A 58 F 6.5 44 50 SAGU00344 5-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 540764 3562110 6561 J 63 U 0.7 20 2 SAGU00207 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 539173 3561445 5571 A 67 F 2.8 34 150 SAGU00262 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 539173 3561445 5571 A 67 F 2.6 36 30 SAGU00266 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 539173 3561445 5571 A 67 F 2.0 36 80 SAGU00210 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 538696 3561245 5461 A 72 F 3.0 34 100 SAGU00261 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 538635 3561128 5380 A 71 F 2.0 38 100 SAGU00250 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 539173 3561445 5571 A 67 F 3.0 35 80 SAGU00242 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 538696 3561245 5461 A 72 F 13.5 55 100 SAGU00247 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 538635 3561128 5380 A 71 F 11.0 56 200 SAGU00201 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 538635 3561128 5380 A 71 F 7.0 46 200 SAGU00206 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 538635 3561128 5380 A 71 F 5.0 48 200 SAGU00205 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 538635 3561128 5380 A 71 F 9.0 48 250 SAGU00248 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 538696 3561245 5461 A 72 M 10.5 47 150 SAGU00263 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 538696 3561245 5461 A 72 M 7.0 44 150 SAGU00241 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 538696 3561245 5461 A 72 M 7.0 41 200 SAGU00208 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 538635 3561128 5380 A 71 M 6.5 45 100 SAGU00264 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 538635 3561128 5380 A 71 M 8.0 49 80 SAGU00268 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 539173 3561445 5571 A 67 M 8.8 48 400 SAGU00203 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 539173 3561445 5571 J 67 U 1.4 27 100 SAGU00244 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 539173 3561445 5571 J 67 U 0.9 29 8 SAGU00265 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 538833 3561372 5459 J 72 F 2.3 34 100 SAGU00245 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 538696 3561245 5461 J 72 F 1.0 24 10 SAGU00204 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 538635 3561128 5380 J 71 F 2.0 36 100 SAGU00243 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 538833 3561372 5459 J 72 M 2.1 30 150 SAGU00270 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 538833 3561372 5459 J 72 M 1.0 30 100 SAGU00202 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 538635 3561128 5380 J 71 M 2.5 31 100 SAGU00246 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 538833 3561372 5459 J 72 U 1.7 29 80 SAGU00209 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 538833 3561372 5459 J 72 U 2.2 31 100

SAGU00249 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 539173 3561445 5571 J 67 U 0.7 23 8 136 SAGU00267 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 538696 3561245 5461 J 72 U 0.9 26 100 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU00269 6-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 539173 3561445 5571 J 67 U 0.8 22 25? SAGU00109 10-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 537142 3558827 4080 A 73 F 2.8 32 50 SAGU00106 10-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 537142 3558827 4080 A 73 F 2.5 32 25 SAGU00226 10-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 537142 3558827 4080 A 73 F 1.5 27 40 SAGU00214 10-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 537084 3559946 4420 A 79 F 1.5 30 40 SAGU00104 10-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 537010 3559466 4196 A 76 F 1.5 27 10 SAGU00197 10-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 537010 3559466 4196 A 76 F 1.2 24 25 SAGU00200 10-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 537084 3559946 4420 A 79 F 2.0 36 25 SAGU00191 10-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 537010 3559466 4196 A 76 M 2.0 32 15 SAGU00198 10-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 537084 3559946 4420 A 79 M 2.8 38 80 SAGU00101 10-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 537142 3558827 4080 A 73 F 4.0 36 80 SAGU00212 10-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 537142 3558827 4080 A 73 F 3.5 36 40 SAGU00195 10-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 537142 3558827 4080 A 73 F 3.8 34 40 SAGU00222 10-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 537142 3558827 4080 A 73 F 8.0 45 40 SAGU00227 10-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 537010 3559466 4196 A 76 M 6.5 43 15 SAGU00218 10-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 537084 3559946 4420 A 79 M 5.3 44 80 SAGU00215 10-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 537084 3559946 4420 A 79 M 9.0 47 150 SAGU00192 10-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 537010 3559466 4196 J 76 U 1.3 26 40 SAGU00224 10-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 537142 3558827 4080 J 73 U 1.0 21 8 SAGU00211 10-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 537010 3559466 4196 J 76 U 1.2 25 20 SAGU00223 10-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 537084 3559946 4420 J 79 U 0.7 21 80 U58 12-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 536909 3557519 3414 A 72 F 6.3 38 10 U52 12-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 536909 3557519 3414 A 70 M 7.7 41 5 U56 12-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 536909 3557519 3414 A 70 M 8.4 43 23 U17 12-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 536909 3557519 3414 A 70 F 14.2 52 25 U50 12-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 536909 3557519 3414 A 70 F 14.5 51 25 U53 12-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 536909 3557519 3414 A 70 F 9.4 44 50 U54 12-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 536909 3557519 3414 A 70 F 9.1 44 4 U13 12-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 536909 3557519 3414 A 72 M 10.2 45 35 U18 12-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 536909 3557519 3414 A 70 M 8.5 46 40 U20 12-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 536909 3557519 3414 A 72 M 7.2 41 35 U49 12-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 536909 3557519 3414 A 70 M 9.5 43 15 U55 12-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 536909 3557519 3414 A 70 M 7.4 42 20 U61 12-Oct-08 HYAR POSITIVE 1 2008 Fall Chimenea 536909 3557519 3414 A 72 F 11.3 50 30 U59 12-Oct-08 HYAR POSITIVE 4 2008 Fall Chimenea 536909 3557519 3414 A 72 F 8.1 44 24 U48 12-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 536909 3557519 3414 J 70 U 5.3 35 8 U57 12-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 536909 3557519 3414 J 70 U 4.9 36 18 U60 12-Oct-08 HYAR NEGATIVE 0 2008 Fall Chimenea 536909 3557519 3414 J 72 U 4.3 33 5 U51 12-Oct-08 HYAR POSITIVE 21 2008 Fall Chimenea 536909 3557519 3414 J 70 U 6.8 39 15

SAGU00050 16-Mar-09 HYAR POSITIVE 41 2009 Spring Chimenea 536906 3557511 3394 A 61 U 3.5 33 5 137 SAGU00045 16-Mar-09 HYAR POSITIVE 274 2009 Spring Chimenea 536906 3557511 3394 A 61 F 3.0 32 3 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU00052 16-Mar-09 HYAR POSITIVE 1,276 2009 Spring Chimenea 537042 3557863 3518 A 65 F 3.5 31 8 SAGU00049 16-Mar-09 HYAR POSITIVE 1,403 2009 Spring Chimenea 537042 3557863 3518 A 65 F 2.5 31 2 SAGU00046 16-Mar-09 HYAR NEGATIVE 0 2009 Spring Chimenea 536924 3557689 3452 A 70 M 5.0 38 0 SAGU00060 16-Mar-09 HYAR POSITIVE 13 2009 Spring Chimenea 536906 3557511 3394 A 61 M 5.5 41 10 SAGU00038 16-Mar-09 HYAR POSITIVE 15 2009 Spring Chimenea 536906 3557511 3394 A 61 M 5.5 35 20 SAGU00042 16-Mar-09 HYAR POSITIVE 20 2009 Spring Chimenea 536906 3557511 3394 A 61 M 4.8 34 2 SAGU00040 16-Mar-09 HYAR POSITIVE 38 2009 Spring Chimenea 537042 3557863 3518 A 65 M 7.0 38 20 SAGU00055 16-Mar-09 HYAR POSITIVE 54 2009 Spring Chimenea 537042 3557863 3518 A 65 F 4.0 35 10 SAGU00034 16-Mar-09 HYAR POSITIVE 430 2009 Spring Chimenea 536906 3557511 3394 A 61 M 5.5 40 5 SAGU00047 16-Mar-09 HYAR POSITIVE 513 2009 Spring Chimenea 536924 3557689 3452 A 70 M 3.8 38 15 SAGU00035 16-Mar-09 HYAR POSITIVE 761 2009 Spring Chimenea 536906 3557511 3394 A 61 M 5.0 36 0 SAGU00048 16-Mar-09 HYAR POSITIVE 1,156 2009 Spring Chimenea 537042 3557863 3518 A 65 M 8.0 42 5 SAGU00039 16-Mar-09 HYAR POSITIVE 1,536 2009 Spring Chimenea 536924 3557689 3452 A 70 F 3.0 31 0 SAGU00031 16-Mar-09 HYAR POSITIVE 1,824 2009 Spring Chimenea 536906 3557511 3394 A 61 F 9.5 45 2 SAGU00044 16-Mar-09 HYAR POSITIVE 2,053 2009 Spring Chimenea 536924 3557689 3452 A 70 M 7.0 41 25 SAGU00037 16-Mar-09 HYAR POSITIVE 8,585 2009 Spring Chimenea 536906 3557511 3394 A 61 M 8.0 42 15 SAGU00057 16-Mar-09 HYAR POSITIVE 9,590 2009 Spring Chimenea 537042 3557863 3518 A 65 M 4.8 38 1 SAGU00056 16-Mar-09 HYAR POSITIVE 14,264 2009 Spring Chimenea 537042 3557863 3518 A 65 M 6.0 47 2 SAGU00043 16-Mar-09 HYAR POSITIVE 14,714 2009 Spring Chimenea 537042 3557863 3518 A 65 F 7.0 38 10 SAGU00053 16-Mar-09 HYAR POSITIVE 20,407 2009 Spring Chimenea 536924 3557689 3452 A 70 M 5.0 38 3 SAGU00032 16-Mar-09 HYAR POSITIVE 289 2009 Spring Chimenea 536906 3557511 3394 J 61 M 2.5 25 1 SAGU00041 16-Mar-09 HYAR POSITIVE 667 2009 Spring Chimenea 536924 3557689 3452 J 70 F 2.5 26 0 SAGU00036 16-Mar-09 HYAR POSITIVE 1,021,184 2009 Spring Chimenea 537042 3557863 3518 J 65 M 1.5 25 1 SAGU00872 2-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 541168 3562279 6797 A 70 F 3.5 32 50 SAGU00887 2-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 540665 3562048 6529 A 78 F 4.5 42 80 SAGU00900 2-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 542039 3563518 7924 A 68 F 7.0 47 20 SAGU00874 2-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 541168 3562279 6797 A 70 M 5.0 40 70 SAGU00886 2-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 542039 3563518 7924 A 68 M 7.0 44 30 SAGU00889 2-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 542039 3563518 7924 A 68 M 9.0 46 20 SAGU00894 2-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 542039 3563518 7924 A 68 M 7.0 46 60 SAGU00965 2-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 542039 3563518 7924 A 68 M 6.0 41 150 SAGU00893 2-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 542039 3563518 7924 A 68 M 7.5 44 5 SAGU00891 2-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 542039 3563518 7924 A 68 M 7.5 41 150 SAGU00989 2-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 542039 3563518 7924 A 68 M 7.0 43 60 SAGU00890 2-Jun-09 HYAR POSITIVE 1 2009 Summer Chimenea 541853 3562637 7380 A 58 M 7.0 40 150 SAGU00896 2-Jun-09 HYAR POSITIVE 6 2009 Summer Chimenea 541379 3562369 6942 A 70 F 7.0 46 30 SAGU00877 2-Jun-09 HYAR POSITIVE 9 2009 Summer Chimenea 540665 3562048 6529 A 78 M 7.0 44 8 SAGU00897 2-Jun-09 HYAR POSITIVE 15 2009 Summer Chimenea 541379 3562369 6942 A 70 F 7.0 45 60 SAGU00883 2-Jun-09 HYAR POSITIVE 18 2009 Summer Chimenea 541853 3562637 7380 A 58 M 6.5 42 50

SAGU00899 2-Jun-09 HYAR POSITIVE 65 2009 Summer Chimenea 540665 3562048 6529 A 78 M 6.0 42 40 138 SAGU00875 2-Jun-09 HYAR POSITIVE 82 2009 Summer Chimenea 541728 3562513 7223 A 61 F 9.0 48 10 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU00880 2-Jun-09 HYAR POSITIVE 91 2009 Summer Chimenea 541379 3562369 6942 A 70 F 8.5 45 50 SAGU00873 2-Jun-09 HYAR POSITIVE 356 2009 Summer Chimenea 540665 3562048 6529 A 78 M 6.0 41 20 SAGU00879 2-Jun-09 HYAR POSITIVE 367 2009 Summer Chimenea 541728 3562513 7223 A 61 M 4.5 42 30 SAGU00876 2-Jun-09 HYAR POSITIVE 1,785 2009 Summer Chimenea 541755 3562519 7252 A 60 M 7.0 45 10 SAGU00885 2-Jun-09 HYAR POSITIVE 4,047 2009 Summer Chimenea 541379 3562369 6942 A 70 F 8.0 45 100 SAGU00898 2-Jun-09 HYAR POSITIVE 5,246 2009 Summer Chimenea 540665 3562048 6529 A 78 M 3.5 35 100 SAGU00888 2-Jun-09 HYAR POSITIVE 5,468 2009 Summer Chimenea 541379 3562369 6942 A 70 M 4.5 42 25 SAGU00892 2-Jun-09 HYAR POSITIVE 8,163 2009 Summer Chimenea 541853 3562637 7380 A 58 F 7.0 42 100 SAGU00878 2-Jun-09 HYAR POSITIVE 8,855 2009 Summer Chimenea 540665 3562048 6529 A 78 F 8.0 47 20 SAGU00882 2-Jun-09 HYAR POSITIVE 13,744 2009 Summer Chimenea 540665 3562048 6529 A 78 M 4.5 37 40 SAGU00871 2-Jun-09 HYAR POSITIVE 21,154 2009 Summer Chimenea 541354 3562350 6922 A 76 F 10.0 48 40 SAGU00884 2-Jun-09 HYAR POSITIVE 25,076 2009 Summer Chimenea 541728 3562513 7223 A 61 F 13.5 52 50 SAGU00881 2-Jun-09 HYAR POSITIVE 31,434 2009 Summer Chimenea 541853 3562637 7380 A 58 F 10.5 50 40 SAGU00895 2-Jun-09 HYAR POSITIVE 61,735 2009 Summer Chimenea 540665 3562048 6529 A 78 M 6.0 42 60 SAGU01021 3-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 539753 3561629 5929 A 62 F 2.0 32 10 SAGU00985 3-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 539422 3561588 5826 A 73 F 2.0 29 40 SAGU00972 3-Jun-09 HYAR POSITIVE 335 2009 Summer Chimenea 538799 3561352 5479 A 72 F 2.0 34 40 SAGU00986 3-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 539422 3561588 5826 A 73 F 10.0 46 50 SAGU00964 3-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 538799 3561352 5479 A 72 F 10.0 47 100 SAGU00969 3-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 538799 3561352 5479 A 72 F 8.0 46 20 SAGU00977 3-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 538799 3561352 5479 A 72 F 7.5 44 150 SAGU00988 3-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 539422 3561588 5826 A 73 M 4.5 42 25 SAGU00990 3-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 538799 3561352 5479 A 72 M 4.5 37 30 SAGU00982 3-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 538799 3561352 5479 A 72 M 7.0 43 100 SAGU00968 3-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 538799 3561352 5479 A 72 M 4.0 36 40 SAGU00971 3-Jun-09 HYAR POSITIVE 3 2009 Summer Chimenea 538834 3561379 5482 A 81 M 4.5 37 70 SAGU00979 3-Jun-09 HYAR POSITIVE 22 2009 Summer Chimenea 539422 3561588 5826 A 73 F 9.0 47 50 SAGU01032 3-Jun-09 HYAR POSITIVE 154 2009 Summer Chimenea 539753 3561629 5929 A 62 M 6.0 43 10 SAGU01030 3-Jun-09 HYAR POSITIVE 503 2009 Summer Chimenea 539701 3561649 5916 A 63 F 11.0 53 3 SAGU01031 3-Jun-09 HYAR POSITIVE 1,009 2009 Summer Chimenea 539753 3561629 5929 A 62 F 9.0 46 50 SAGU00980 8-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 536919 3557546 3443 A 79 F 6.0 41 5 SAGU01011 8-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 536919 3557546 3443 A 79 F 7.0 41 10 SAGU00991 8-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 536919 3557546 3443 A 79 M 5.0 37 20 SAGU01005 8-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 536919 3557546 3443 A 79 M 4.0 35 25 SAGU00966 8-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 536919 3557546 3443 A 79 M 5.0 35 15 SAGU01006 8-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 536919 3557546 3443 A 79 M 3.5 36 15 SAGU00973 8-Jun-09 HYAR POSITIVE 4 2009 Summer Chimenea 536919 3557546 3443 A 79 F 3.0 33 50 SAGU01003 8-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 536919 3557546 3443 J 79 U 0.5 22 1 SAGU01014 9-Jun-09 HYAR POSITIVE 5 2009 Summer Chimenea 537038 3557851 3546 A 76 M 2.5 32 80

SAGU01020 9-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537038 3557851 3546 A 76 F 4.0 39 40 139 SAGU01002 9-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537038 3557851 3546 A 76 F 5.0 40 80 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU01010 9-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537038 3557851 3546 A 76 M 4.5 38 200 SAGU00987 9-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537038 3557851 3546 A 76 M 3.5 35 80 SAGU01004 9-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537038 3557851 3546 A 76 M 3.5 34 50 SAGU01008 9-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537038 3557851 3546 A 76 M 3.5 34 150 SAGU01019 9-Jun-09 HYAR POSITIVE 1 2009 Summer Chimenea 537038 3557851 3546 A 76 M 4.0 37 15 SAGU00587 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537579 3560322 5010 A 70 F 2.0 33 60 SAGU00573 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537148 3558639 3978 A 80 F 1.5 30 60 SAGU00571 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537148 3558639 3978 A 80 M 3.0 32 200 SAGU00581 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537148 3558639 3978 A 80 M 2.5 34 300 SAGU00600 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537579 3560322 5010 A 70 F 6.0 42 80 SAGU00583 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537579 3560322 5010 A 70 F 7.5 45 30 SAGU00584 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537579 3560322 5010 A 70 F 6.0 42 120 SAGU00577 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537579 3560322 5010 A 70 F 7.5 44 50 SAGU00575 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537089 3559933 4407 A 82 F 5.0 39 20 SAGU00572 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537089 3559933 4407 A 82 F 6.0 39 40 SAGU00574 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537148 3558639 3978 A 80 F 3.0 36 50 SAGU00585 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537579 3560322 5010 A 70 M 5.0 39 100 SAGU00576 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537579 3560322 5010 A 70 M 6.0 40 200 SAGU00591 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537579 3560322 5010 A 70 M 3.0 36 120 SAGU00594 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537089 3559933 4407 A 82 M 5.0 39 100 SAGU00599 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537089 3559933 4407 A 82 M 5.0 38 200 SAGU00595 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537089 3559933 4407 A 82 M 5.0 40 200 SAGU00580 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537089 3559933 4407 A 82 M 4.0 36 150 SAGU00597 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537089 3559933 4407 A 82 M 4.0 36 50 SAGU00589 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537089 3559933 4407 A 82 M 3.5 32 30 SAGU00579 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537148 3558639 3978 A 80 M 4.0 38 100 SAGU00598 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537148 3558639 3978 A 80 M 3.0 35 50 SAGU00593 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537148 3558639 3978 A 80 M 4.0 37 60 SAGU00590 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537148 3558639 3978 A 80 M 3.0 34 100 SAGU00946 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537148 3558639 3978 T 80 U SAGU00938 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537148 3558639 3978 T 80 U SAGU00956 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537148 3558639 3978 T 80 U SAGU00936 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537148 3558639 3978 T 80 U SAGU00959 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537148 3558639 3978 T 80 U SAGU00947 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537148 3558639 3978 T 80 U SAGU00931 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537148 3558639 3978 T 80 U SAGU00937 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537148 3558639 3978 T 80 U SAGU00941 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537148 3558639 3978 T 80 U SAGU00933 15-Jun-09 HYAR NEGATIVE 0 2009 Summer Chimenea 537148 3558639 3978 T 80 U

SAGU01075 30-Sep-09 HYAR NEGATIVE 0 2009 Fall Chimenea 541854 3562651 7367 A 62 F 2.5 21 12 140 SAGU01055 30-Sep-09 HYAR NEGATIVE 0 2009 Fall Chimenea 541752 3562516 7308 A 62 F 3.5 31 40 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU00922 30-Sep-09 HYAR NEGATIVE 0 2009 Fall Chimenea 541382 3562404 6969 A 67 F 3.0 29 50 SAGU01069 30-Sep-09 HYAR NEGATIVE 0 2009 Fall Chimenea 541382 3562404 6969 A 67 F 3.5 28 20 SAGU01059 30-Sep-09 HYAR NEGATIVE 0 2009 Fall Chimenea 541854 3562651 7367 A 62 F 12.0 42 80 SAGU01076 30-Sep-09 HYAR NEGATIVE 0 2009 Fall Chimenea 541854 3562651 7367 A 62 F 10.5 46 80 SAGU01062 30-Sep-09 HYAR NEGATIVE 0 2009 Fall Chimenea 541752 3562516 7308 A 62 F 11.0 48 200 SAGU01068 30-Sep-09 HYAR NEGATIVE 0 2009 Fall Chimenea 541382 3562404 6969 A 67 F 14.0 51 10 SAGU01067 30-Sep-09 HYAR NEGATIVE 0 2009 Fall Chimenea 541253 3562294 6856 A 74 F 6.0 38 100 SAGU01060 30-Sep-09 HYAR NEGATIVE 0 2009 Fall Chimenea 540730 3562090 6554 A 72 F 17.0 57 80 SAGU01074 30-Sep-09 HYAR NEGATIVE 0 2009 Fall Chimenea 540730 3562090 6554 A 72 F 6.0 41 60 SAGU01071 30-Sep-09 HYAR NEGATIVE 0 2009 Fall Chimenea 540730 3562090 6554 A 72 F 10.0 48 100 SAGU01079 30-Sep-09 HYAR NEGATIVE 0 2009 Fall Chimenea 540730 3562090 6554 A 72 F 8.0 42 40 SAGU01077 30-Sep-09 HYAR NEGATIVE 0 2009 Fall Chimenea 540730 3562090 6554 A 72 F 15.0 51 50 SAGU01078 30-Sep-09 HYAR NEGATIVE 0 2009 Fall Chimenea 541854 3562651 7367 A 62 M 14.0 50 50 SAGU00909 30-Sep-09 HYAR NEGATIVE 0 2009 Fall Chimenea 541752 3562516 7308 A 62 M 12.0 49 200 SAGU00912 30-Sep-09 HYAR NEGATIVE 0 2009 Fall Chimenea 541752 3562516 7308 A 62 M 9.5 46 100 SAGU01072 30-Sep-09 HYAR NEGATIVE 0 2009 Fall Chimenea 541382 3562404 6969 A 67 M 9.0 42 50 SAGU01063 30-Sep-09 HYAR NEGATIVE 0 2009 Fall Chimenea 541382 3562404 6969 A 67 M 9.0 44 60 SAGU01065 30-Sep-09 HYAR NEGATIVE 0 2009 Fall Chimenea 541382 3562404 6969 A 67 M 9.0 44 50 SAGU01052 30-Sep-09 HYAR NEGATIVE 0 2009 Fall Chimenea 540730 3562090 6554 A 72 M 9.5 44 40 SAGU01054 30-Sep-09 HYAR NEGATIVE 0 2009 Fall Chimenea 540730 3562090 6554 A 72 M 10.0 46 40 SAGU01070 30-Sep-09 HYAR POSITIVE 629 2009 Fall Chimenea 541253 3562294 6856 A 74 M 10.5 45 60 SAGU01058 30-Sep-09 HYAR NEGATIVE 0 2009 Fall Chimenea 540730 3562090 6554 J 72 F 1.0 23 30 SAGU01422 1-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 539429 3561593 5845 A 69 F 4.0 32 50 SAGU01080 1-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 539429 3561593 5845 A 69 F 3.5 34 30 SAGU01066 1-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 539429 3561593 5845 A 69 M 2.5 25 40 SAGU01414 1-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 539429 3561593 5845 A 69 F 14.0 57 40 SAGU01056 1-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 539429 3561593 5845 A 69 F 8.0 45 50 SAGU01419 1-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 538635 3561111 5389 A 80 F 4.5 36 30 SAGU01426 1-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 538635 3561111 5389 A 80 F 6.0 41 30 SAGU01417 1-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 538635 3561111 5389 A 80 F 10.0 46 50 SAGU01430 1-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 538635 3561111 5389 A 80 F 5.0 35 30 SAGU01424 1-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 539429 3561593 5845 A 69 F 12.5 45 150 SAGU01436 1-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 539429 3561593 5845 A 69 M 5.0 36 80 SAGU01438 1-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 538635 3561111 5389 A 80 M 6.0 38 30 SAGU01051 1-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 538635 3561111 5389 A 80 M 8.0 44 30 SAGU01427 1-Oct-09 HYAR POSITIVE 16 2009 Fall Chimenea 538635 3561111 5389 A 80 F 8.0 41 60 SAGU01413 1-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 538635 3561111 5389 J 80 U 2.0 22 20 SAGU01435 2-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 537596 3560329 5028 A 70 U 2.5 25 25 SAGU01421 2-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 537596 3560329 5028 A 70 F 11.0 46 60

SAGU01423 2-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 537596 3560329 5028 A 70 F 10.0 46 40 141 SAGU01434 2-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 537596 3560329 5028 A 70 F 8.0 44 80 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU01433 2-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 537417 3560235 4827 A 70 F 6.5 40 60 SAGU01437 2-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 537417 3560235 4827 A 70 F 6.0 38 80 SAGU01420 2-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 537081 3559959 4495 A 79 F 4.0 33 40 SAGU01429 2-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 537081 3559959 4495 A 79 F 3.5 35 20 SAGU01432 2-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 537081 3559959 4495 A 79 F 7.0 38 30 SAGU01425 2-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 537325 3560193 4687 A 78 M 6.0 40 50 SAGU01431 2-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 537081 3559959 4495 A 79 M 5.5 40 50 SAGU01428 2-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 537325 3560193 4687 J 78 U 1.0 17 10 SAGU01418 2-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 537081 3559959 4495 J 79 U 1.0 17 0 SAGU01057 2-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 537081 3559959 4495 J 79 U 1.0 22 10 SAGU01415 2-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 537081 3559959 4495 J 79 U 1.0 21 3 SAGU00907 2-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 537081 3559959 4495 J 79 U 1.0 17 3 SAGU00830 5-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 537040 3557865 3640 A 72 F 3.0 25 30 SAGU01102 5-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 537026 3557772 3522 A F 6.0 40 80 SAGU00761 5-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 537040 3557865 3640 A 72 M 6.5 39 30 SAGU01119 5-Oct-09 HYAR POSITIVE 2 2009 Fall Chimenea 536936 3557673 3473 A 80 M 5.0 36 40 SAGU01111 5-Oct-09 HYAR POSITIVE 3 2009 Fall Chimenea 536936 3557673 3473 A 80 F 7.0 41 30 SAGU00781 5-Oct-09 HYAR POSITIVE 916 2009 Fall Chimenea 537221 3558462 3838 A F 6.0 38 50 SAGU01125 10-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 536909 3557519 3414 A 70 F 7.0 40 20 SAGU00633 10-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 536909 3557519 3414 A 70 F 6.5 41 40 SAGU01101 10-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 536909 3557519 3414 A 70 M 6.0 39 25 SAGU01380 10-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 536909 3557519 3414 A 70 M 6.0 39 15 SAGU00640 10-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 536909 3557519 3414 A 70 M 5.0 38 15 SAGU01108 10-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 536909 3557519 3414 A 70 M 5.5 38 20 SAGU01112 10-Oct-09 HYAR NEGATIVE 0 2009 Fall Chimenea 536909 3557519 3414 A 70 M 6.0 38 30 SAGU00827 10-Oct-09 HYAR POSITIVE 7 2009 Fall Chimenea 536909 3557519 3414 A 70 M 6.0 37 20 SAGU01158 16-Mar-10 HYAR POSITIVE 41,615 2010 Spring Chimenea 536913 3557511 3432 A 40 F 13.5 44 50 SAGU02010 10-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 536901 3557513 3432 A 78 F 10.5 46 60 SAGU01578 10-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 536901 3557513 3432 A 78 F 11.5 45 200 SAGU02059 10-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 537036 3557802 3513 A 79 F 10.5 46 200 SAGU02082 10-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 536901 3557513 3432 A 78 M 8.5 45 40 SAGU02093 10-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 536901 3557513 3432 A 78 M 9.5 45 20 SAGU01672 15-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 541737 3562509 7234 A 64 F 6.0 34 5 SAGU01990 15-Jun-10 HYAR POSITIVE 1 2010 Summer Chimenea 541167 3562217 6838 A 68 M 4.5 35 60 SAGU01699 15-Jun-10 HYAR POSITIVE 449 2010 Summer Chimenea 541737 3562509 7234 A 60 F 4.5 31 40 SAGU01966 15-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 541860 3562691 7400 A 52 F 9.0 45 3 SAGU01916 15-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 541737 3562509 7234 A 62 F 9.5 46 10 SAGU01861 15-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 541309 3562316 6908 A 70 F 6.5 37 15 SAGU01894 15-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 541167 3562217 6838 A 68 F 8.0 42 25

SAGU01827 15-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 541167 3562217 6838 A 68 F 9.0 47 8 142 SAGU01575 15-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 540719 3562104 6542 A 76 F 9.5 44 25 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU01877 15-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 540719 3562104 6542 A 76 F 7.0 36 25 SAGU01740 15-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 540719 3562104 6542 A 76 F 11.0 47 40 SAGU01806 15-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 541412 3562384 6923 A 78 M 11.0 46 30 SAGU01769 15-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 540719 3562104 6542 A 76 M 9.5 43 50 SAGU01793 15-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 540719 3562104 6542 A 76 M 9.0 40 80 SAGU01993 15-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 540719 3562104 6542 A 76 M 7.0 37 60 SAGU01777 15-Jun-10 HYAR POSITIVE 63 2010 Summer Chimenea 541860 3562691 7400 A 52 M 10.5 40 30 SAGU01850 15-Jun-10 HYAR POSITIVE 134 2010 Summer Chimenea 540719 3562104 6542 A 76 F 10.5 45 80 SAGU01772 15-Jun-10 HYAR POSITIVE 247 2010 Summer Chimenea 541737 3562509 7234 A 63 M 9.0 43 40 SAGU01765 15-Jun-10 HYAR POSITIVE 443 2010 Summer Chimenea 541309 3562316 6908 A 70 M 8.5 42 30 SAGU01673 15-Jun-10 HYAR POSITIVE 1,406 2010 Summer Chimenea 541309 3562316 6908 A 70 M 6.5 38 30 SAGU01949 15-Jun-10 HYAR POSITIVE 2,146 2010 Summer Chimenea 541309 3562316 6908 A 70 M 9.5 44 60 SAGU01836 15-Jun-10 HYAR POSITIVE 2,797 2010 Summer Chimenea 541847 3562694 7375 A 56 M 9.5 47 60 SAGU01618 15-Jun-10 HYAR POSITIVE 6,488 2010 Summer Chimenea 541737 3562509 7234 A 61 M 9.0 44 60 SAGU01801 15-Jun-10 HYAR POSITIVE 23,762 2010 Summer Chimenea 541847 3562694 7375 A 56 M 10.0 42 100 SAGU01676 15-Jun-10 HYAR POSITIVE 30,611 2010 Summer Chimenea 541309 3562316 6908 A 70 M 5.5 37 15 SAGU01757 15-Jun-10 HYAR POSITIVE 31,071 2010 Summer Chimenea 541847 3562694 7375 A 56 M 9.5 42 80 SAGU01695 15-Jun-10 HYAR POSITIVE 64,607 2010 Summer Chimenea 541309 3562316 6908 A 70 M 8.5 40 60 SAGU01979 16-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 539379 3561509 5738 A 73 F 3.5 30 30 SAGU02012 16-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 539379 3561509 5738 A 73 F 3.5 32 50 SAGU01998 16-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 539379 3561509 5738 A 73 F 10.5 47 100 SAGU01986 16-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 539379 3561509 5738 A 73 F 6.5 37 30 SAGU01961 16-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 539379 3561509 5738 A 73 F 11.0 45 100 SAGU02045 16-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 539379 3561509 5738 A 73 F 5.5 35 40 SAGU01968 16-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 539379 3561509 5738 A 73 F 10.5 47 50 SAGU01957 16-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 538638 3561124 5384 A 73 F 6.0 40 20 SAGU01987 16-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 538638 3561124 5384 A 73 F 7.0 36 30 SAGU01967 16-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 538638 3561124 5384 A 73 F 7.5 42 25 SAGU01971 16-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 538638 3561124 5384 A 73 F 7.5 40 50 SAGU02028 16-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 539379 3561509 5738 A 73 M 7.5 36 50 SAGU01984 16-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 539379 3561509 5738 A 73 M 8.5 43 40 SAGU02003 16-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 538638 3561124 5384 A 73 M 6.5 41 80 SAGU02051 16-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 538638 3561124 5384 A 73 M 6.0 35 50 SAGU01985 16-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 538638 3561124 5384 A 73 M 8.0 39 15 SAGU01999 16-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 538638 3561124 5384 A 73 M 7.5 38 50 SAGU01958 16-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 538638 3561124 5384 A 73 M 6.5 40 40 SAGU02004 16-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 538638 3561124 5384 A 73 M 5.5 35 20 SAGU02002 16-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 538638 3561124 5384 A 73 M 7.5 42 30 SAGU02015 16-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 538638 3561124 5384 A 73 M 7.0 36 20

SAGU02077 16-Jun-10 HYAR POSITIVE 70,780 2010 Summer Chimenea 542040 3563520 7926 A 61 M 9.5 47 10 143 SAGU01996 16-Jun-10 HYAR POSITIVE 84,225 2010 Summer Chimenea 542040 3563520 7926 A 61 M 10.0 45 20 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU01582 16-Jun-10 HYAR POSITIVE 501,170 2010 Summer Chimenea 542040 3563520 7926 A 61 M 10.5 48 15 SAGU02000 16-Jun-10 HYAR POSITIVE 969,050 2010 Summer Chimenea 542040 3563520 7926 A 61 M 8.5 44 20 SAGU01843 17-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 537090 3559920 4461 A 80 F 9.0 43 30 SAGU01835 17-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 537090 3559920 4461 A 80 F 12.0 44 50 SAGU01891 17-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 537090 3559920 4461 A 80 F 10.5 43 50 SAGU01896 17-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 537090 3559920 4461 A 80 F 11.5 44 50 SAGU02023 17-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 537090 3559920 4461 A 80 M 7.5 35 25 SAGU01805 17-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 537090 3559920 4461 A 80 M 7.5 38 20 SAGU02027 17-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 537090 3559920 4461 A 80 M 8.0 40 30 SAGU01962 17-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 537090 3559920 4461 A 80 M 8.0 39 50 SAGU01884 17-Jun-10 HYAR NEGATIVE 0 2010 Summer Chimenea 537090 3559920 4461 A 80 M 8.5 37 50 SAGU02044 17-Jun-10 HYAR POSITIVE 13 2010 Summer Chimenea 537090 3559920 4461 A 80 M 10.0 45 30 SAGU01808 17-Jun-10 HYAR POSITIVE 84 2010 Summer Chimenea 537090 3559920 4461 A 80 M 8.5 45 12 SAGU01708 17-Jun-10 HYAR POSITIVE 149 2010 Summer Chimenea 537090 3559920 4461 A 80 F 8.5 41 30 SAGU01764 17-Jun-10 HYAR POSITIVE 460 2010 Summer Chimenea 537090 3559920 4461 A 80 F 11.5 49 50 SAGU00108 8-Oct-08 HYAR NEGATIVE 0 2008 Fall Tanque Verde 538110 3569124 4004 A 69 F 2.7 33 50 SAGU00219 8-Oct-08 HYAR NEGATIVE 0 2008 Fall Tanque Verde 538110 3569124 4004 A 69 F 2.6 34 40 SAGU00117 8-Oct-08 HYAR NEGATIVE 0 2008 Fall Tanque Verde 538110 3569124 4004 A 69 F 3.1 35 60 SAGU00116 8-Oct-08 HYAR NEGATIVE 0 2008 Fall Tanque Verde 538110 3569124 4004 A 69 F 2.1 33 40 SAGU00341 8-Oct-08 HYAR NEGATIVE 0 2008 Fall Tanque Verde 538884 3568673 4213 A 80 F 2.0 30 30 SAGU00339 8-Oct-08 HYAR NEGATIVE 0 2008 Fall Tanque Verde 538110 3569124 4004 A 69 F 2.9 32 80 SAGU00279 8-Oct-08 HYAR NEGATIVE 0 2008 Fall Tanque Verde 538110 3569124 4004 A 69 F 1.9 31 50 SAGU00196 8-Oct-08 HYAR NEGATIVE 0 2008 Fall Tanque Verde 538884 3568673 4213 A 80 F 2.0 29 40 SAGU00291 8-Oct-08 HYAR NEGATIVE 0 2008 Fall Tanque Verde 538884 3568673 4213 A 80 M 2.3 33 25 SAGU00220 8-Oct-08 HYAR NEGATIVE 0 2008 Fall Tanque Verde 538884 3568673 4213 A 80 M 3.0 36 25 SAGU00225 8-Oct-08 HYAR NEGATIVE 0 2008 Fall Tanque Verde 538110 3569124 4004 A 69 M 2.9 33 60 SAGU00199 8-Oct-08 HYAR NEGATIVE 0 2008 Fall Tanque Verde 538110 3569124 4004 A 69 F 3.2 36 30 SAGU00349 8-Oct-08 HYAR NEGATIVE 0 2008 Fall Tanque Verde 538884 3568673 4213 A 80 M 4.3 36 70 SAGU00334 8-Oct-08 HYAR NEGATIVE 0 2008 Fall Tanque Verde 538110 3569124 4004 A 69 M 2.9 35 80 SAGU00335 8-Oct-08 HYAR NEGATIVE 0 2008 Fall Tanque Verde 538110 3569124 4004 J 69 F 1.6 28 60 SAGU00118 8-Oct-08 HYAR NEGATIVE 0 2008 Fall Tanque Verde 538884 3568673 4213 J 80 U 0.6 1 2 SAGU00405 17-Oct-08 HYAR POSITIVE 433 2008 Fall Tanque Verde 534131 3569561 3403 A 76 F 1.0 32 20 SAGU00276 17-Oct-08 HYAR POSITIVE 653 2008 Fall Tanque Verde 534131 3569561 3403 A 76 F 1.0 28 4 SAGU00137 17-Oct-08 HYAR POSITIVE 3,290 2008 Fall Tanque Verde 533044 3568954 3262 A 63 F 2.0 36 30 SAGU00439 17-Oct-08 HYAR POSITIVE 9,097 2008 Fall Tanque Verde 533044 3568954 3262 A 63 M 1.0 30 3 SAGU00412 17-Oct-08 HYAR POSITIVE 15,986 2008 Fall Tanque Verde 534131 3569561 3403 A 76 M 1.0 28 20 SAGU00317 17-Oct-08 HYAR POSITIVE 20,807 2008 Fall Tanque Verde 534131 3569561 3403 A 76 M 1.5 31 5 SAGU00113 17-Oct-08 HYAR POSITIVE 531 2008 Fall Tanque Verde 533044 3568954 3262 A 63 M 3.5 36 20 SAGU00428 17-Oct-08 HYAR POSITIVE 608 2008 Fall Tanque Verde 533044 3568954 3262 A 63 F 7.0 44 15

SAGU00282 17-Oct-08 HYAR POSITIVE 675 2008 Fall Tanque Verde 533044 3568954 3262 A 63 F 4.0 41 15 144 SAGU00446 17-Oct-08 HYAR POSITIVE 1,403 2008 Fall Tanque Verde 533044 3568954 3262 A 63 F 7.5 44 20 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU00417 17-Oct-08 HYAR POSITIVE 2,625 2008 Fall Tanque Verde 533044 3568954 3262 A 63 M 3.5 41 50 SAGU00483 17-Oct-08 HYAR POSITIVE 4,863 2008 Fall Tanque Verde 533044 3568954 3262 A 63 F 7.5 45 80 SAGU00487 17-Oct-08 HYAR POSITIVE 6,580 2008 Fall Tanque Verde 534131 3569561 3403 A 76 F 6.0 45 40 SAGU00121 17-Oct-08 HYAR POSITIVE 36,511 2008 Fall Tanque Verde 534131 3569561 3403 A 76 F 9.0 47 40 SAGU00231 18-Oct-08 HYAR POSITIVE 1,641 2008 Fall Tanque Verde 534870 3569546 3477 A 68 F 3.0 39 20 SAGU00216 18-Oct-08 HYAR POSITIVE 1,251 2008 Fall Tanque Verde 534870 3569546 3477 A 68 M 4.0 33 15 SAGU00125 18-Oct-08 HYAR POSITIVE 2,168 2008 Fall Tanque Verde 535466 3569792 3565 A 72 F 6.0 42 20 SAGU00140 18-Oct-08 HYAR POSITIVE 2,952 2008 Fall Tanque Verde 535466 3569792 3565 A 72 M 4.0 37 50 SAGU00452 18-Oct-08 HYAR POSITIVE 3,173 2008 Fall Tanque Verde 535466 3569792 3565 A 72 F 9.0 47 40 SAGU00414 18-Oct-08 HYAR POSITIVE 4,302 2008 Fall Tanque Verde 534870 3569546 3477 A 68 M 3.0 37 80 SAGU00141 18-Oct-08 HYAR POSITIVE 6,947 2008 Fall Tanque Verde 535466 3569792 3565 A 72 F 8.0 50 80 SAGU00484 18-Oct-08 HYAR POSITIVE 7,540 2008 Fall Tanque Verde 535466 3569792 3565 A 72 F 5.0 36 30 SAGU00419 18-Oct-08 HYAR POSITIVE 8,581 2008 Fall Tanque Verde 534870 3569546 3477 A 68 F 5.0 42 20 SAGU00235 18-Oct-08 HYAR POSITIVE 10,121 2008 Fall Tanque Verde 534870 3569546 3477 A 68 F 6.0 47 25 SAGU00273 18-Oct-08 HYAR POSITIVE 11,046 2008 Fall Tanque Verde 535466 3569792 3565 A 72 F 9.0 49 40 SAGU00499 18-Oct-08 HYAR POSITIVE 12,568 2008 Fall Tanque Verde 535466 3569792 3565 A 72 M 5.0 36 30 SAGU00490 18-Oct-08 HYAR POSITIVE 40,605 2008 Fall Tanque Verde 535466 3569792 3565 A 72 F 7.5 46 40 SAGU00399 18-Oct-08 HYAR POSITIVE 42,971 2008 Fall Tanque Verde 534870 3569546 3477 A 68 M 5.5 41 20 SAGU00131 18-Oct-08 HYAR POSITIVE 11,530 2008 Fall Tanque Verde 534870 3569546 3477 J 68 U 0.7 22 3 SAGU00122 18-Oct-08 HYAR POSITIVE 17,895 2008 Fall Tanque Verde 534870 3569546 3477 J 68 U 1.0 29 8 SAGU00022 3-Nov-08 HYAR NEGATIVE 0 2008 Fall Tanque Verde 540576 3567704 4997 A 63 M 1.0 25 80 SAGU00071 3-Nov-08 HYAR POSITIVE 12 2008 Fall Tanque Verde 539979 3568602 4551 A 70 F 1.5 26 40 SAGU00073 3-Nov-08 HYAR NEGATIVE 0 2008 Fall Tanque Verde 539979 3568602 4551 A 70 F 3.0 35 30 SAGU00063 3-Nov-08 HYAR NEGATIVE 0 2008 Fall Tanque Verde 540576 3567704 4997 A 63 F 2.2 30 200 SAGU00017 3-Nov-08 HYAR NEGATIVE 0 2008 Fall Tanque Verde 539979 3568602 4551 A 70 M 2.5 32 25 SAGU00062 3-Nov-08 HYAR NEGATIVE 0 2008 Fall Tanque Verde 540576 3567704 4997 A 63 M 5.0 40 300 SAGU00030 3-Nov-08 HYAR NEGATIVE 0 2008 Fall Tanque Verde 540576 3567704 4997 A 63 M 4.0 36 300 SAGU00005 3-Nov-08 HYAR NEGATIVE 0 2008 Fall Tanque Verde 540576 3567704 4997 A 63 M 3.5 36 200 SAGU00066 3-Nov-08 HYAR POSITIVE 14 2008 Fall Tanque Verde 539979 3568602 4551 A 70 M 4.5 50 100 SAGU00086 3-Nov-08 HYAR POSITIVE 46 2008 Fall Tanque Verde 539979 3568602 4551 A 70 M 8.5 44 100 SAGU00079 3-Nov-08 HYAR NEGATIVE 0 2008 Fall Tanque Verde 539979 3568602 4551 J 70 F 1.5 30 12 SAGU00024 3-Nov-08 HYAR NEGATIVE 0 2008 Fall Tanque Verde 540576 3567704 4997 J 63 F 0.7 27 80 SAGU00085 3-Nov-08 HYAR NEGATIVE 0 2008 Fall Tanque Verde 540576 3567704 4997 J 63 F 0.7 24 50 SAGU00003 3-Nov-08 HYAR NEGATIVE 0 2008 Fall Tanque Verde 540576 3567704 4997 J 63 F 0.5 22 100 SAGU00072 3-Nov-08 HYAR NEGATIVE 0 2008 Fall Tanque Verde 539979 3568602 4551 J 70 U 0.5 21 12 SAGU00076 3-Nov-08 HYAR POSITIVE 58 2008 Fall Tanque Verde 539979 3568602 4551 J 70 M 1.5 26 20 SAGU00285 24-Jan-09 HYAR NEGATIVE 0 2008 Winter Tanque Verde 533683 3569276 3415 J 56 U 1.1 24 0 SAGU00077 24-Jan-09 HYAR NEGATIVE 0 2008 Winter Tanque Verde 533479 3569218 3332 J 59 U 2.3 30 3 SAGU00289 24-Jan-09 HYAR NEGATIVE 0 2008 Winter Tanque Verde 533485 3569150 3344 J 59 U 1.4 26 4

SAGU00298 17-Mar-09 HYAR NEGATIVE 0 2009 Spring Tanque Verde 534245 3569483 3394 A 65 F 2.0 29 5 145 SAGU00496 17-Mar-09 HYAR NEGATIVE 0 2009 Spring Tanque Verde 533117 3569084 3287 A 72 F 2.0 29 15 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU00316 17-Mar-09 HYAR NEGATIVE 0 2009 Spring Tanque Verde 534245 3569483 3394 A 65 M 3.0 32 0 SAGU00054 17-Mar-09 HYAR NEGATIVE 0 2009 Spring Tanque Verde 534245 3569483 3394 A 65 M 3.5 33 0 SAGU00188 17-Mar-09 HYAR NEGATIVE 0 2009 Spring Tanque Verde 533507 3569181 3507 A 72 M 3.5 34 0 SAGU00310 17-Mar-09 HYAR POSITIVE 828 2009 Spring Tanque Verde 533117 3569084 3287 A 72 F 3.0 31 23 SAGU00059 17-Mar-09 HYAR NEGATIVE 0 2009 Spring Tanque Verde 534245 3569483 3394 A 65 F 4.0 34 7 SAGU00495 17-Mar-09 HYAR NEGATIVE 0 2009 Spring Tanque Verde 533507 3569181 3507 A 72 F 6.0 39 2 SAGU00128 17-Mar-09 HYAR NEGATIVE 0 2009 Spring Tanque Verde 533117 3569084 3287 A 72 F 9.0 48 10 SAGU00234 17-Mar-09 HYAR NEGATIVE 0 2009 Spring Tanque Verde 533117 3569084 3287 A 72 F 4.0 35 0 SAGU00305 17-Mar-09 HYAR NEGATIVE 0 2009 Spring Tanque Verde 533117 3569084 3287 A 72 F 6.0 39 0 SAGU00306 17-Mar-09 HYAR NEGATIVE 0 2009 Spring Tanque Verde 533117 3569084 3287 A 72 F 6.0 41 0 SAGU00058 17-Mar-09 HYAR NEGATIVE 0 2009 Spring Tanque Verde 534245 3569483 3394 A 65 M 3.0 31 6 SAGU00051 17-Mar-09 HYAR NEGATIVE 0 2009 Spring Tanque Verde 533507 3569181 3507 A 72 M 5.0 39 5 SAGU00238 17-Mar-09 HYAR NEGATIVE 0 2009 Spring Tanque Verde 533507 3569181 3507 A 72 M 5.0 40 15 SAGU00299 17-Mar-09 HYAR NEGATIVE 0 2009 Spring Tanque Verde 533507 3569181 3507 A 72 M 4.0 38 0 SAGU00318 17-Mar-09 HYAR NEGATIVE 0 2009 Spring Tanque Verde 533507 3569181 3507 A 72 M 3.0 34 3 SAGU00136 17-Mar-09 HYAR NEGATIVE 0 2009 Spring Tanque Verde 533117 3569084 3287 A 72 M 4.8 36 1 SAGU00260 17-Mar-09 HYAR NEGATIVE 0 2009 Spring Tanque Verde 533117 3569084 3287 A 72 M 4.0 35 7 SAGU00494 17-Mar-09 HYAR NEGATIVE 0 2009 Spring Tanque Verde 533117 3569084 3287 A 72 M 4.5 39 15 SAGU00169 17-Mar-09 HYAR POSITIVE 4 2009 Spring Tanque Verde 534245 3569483 3394 A 65 F 6.0 37 4 SAGU00296 17-Mar-09 HYAR POSITIVE 8 2009 Spring Tanque Verde 533507 3569181 3507 A 72 F 5.0 38 30 SAGU00138 17-Mar-09 HYAR POSITIVE 13 2009 Spring Tanque Verde 534245 3569483 3394 A 65 F 6.0 40 1 SAGU00277 17-Mar-09 HYAR POSITIVE 14 2009 Spring Tanque Verde 534245 3569483 3394 A 65 F 3.5 36 10 SAGU00278 17-Mar-09 HYAR POSITIVE 18 2009 Spring Tanque Verde 533117 3569084 3287 A 72 M 4.0 36 0 SAGU00139 17-Mar-09 HYAR POSITIVE 41 2009 Spring Tanque Verde 533507 3569181 3507 A 72 F 4.2 35 15 SAGU00186 17-Mar-09 HYAR POSITIVE 877 2009 Spring Tanque Verde 534245 3569483 3394 A 65 F 3.5 37 0 SAGU00142 17-Mar-09 HYAR POSITIVE 16,524 2009 Spring Tanque Verde 533507 3569181 3507 A 72 M 6.2 42 25 SAGU00311 17-Mar-09 HYAR POSITIVE 18,585 2009 Spring Tanque Verde 534245 3569483 3394 A 65 M 4.1 37 6 SAGU00129 17-Mar-09 HYAR POSITIVE 20,224 2009 Spring Tanque Verde 533507 3569181 3507 A 72 M 4.0 36 50 SAGU00845 16-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 532906 3564020 3214 A 73 F 7.5 42 50 SAGU00846 16-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 532906 3564020 3214 A 73 F 4.5 37 15 SAGU00849 16-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 532906 3564020 3214 A 73 M 5.0 39 40 SAGU00857 16-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 532906 3564020 3214 A 73 M 5.5 40 40 SAGU00862 16-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 532906 3564020 3214 A 73 M 4.5 37 30 SAGU00864 16-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 532906 3564020 3214 A 73 M 3.5 35 25 SAGU00859 16-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 532906 3564020 3214 A 73 M 4.5 37 30 SAGU00852 16-Jun-09 HYAR POSITIVE 99 2009 Summer Tanque Verde 532906 3564020 3214 A 73 F 4.5 39 80 SAGU00850 17-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 534672 3569573 3484 A 76 F 3.0 34 50 SAGU00803 17-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 534672 3569573 3484 A 76 F 5.5 39 30 SAGU00866 17-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 534672 3569573 3484 A 76 M 4.0 38 50

SAGU00863 17-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 534672 3569573 3484 A 76 M 4.5 38 30 146 SAGU00785 17-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 534672 3569573 3484 A 76 M 4.5 37 30 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU00805 17-Jun-09 HYAR POSITIVE 2 2009 Summer Tanque Verde 534672 3569573 3484 A 76 M 4.5 34 40 SAGU00800 17-Jun-09 HYAR POSITIVE 3 2009 Summer Tanque Verde 534672 3569573 3484 A 76 M 4.5 38 30 SAGU00795 17-Jun-09 HYAR POSITIVE 4 2009 Summer Tanque Verde 534672 3569573 3484 A 76 F 6.0 43 50 SAGU00523 17-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 534672 3569573 3484 T 76 U SAGU00515 17-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 534672 3569573 3484 T 76 U SAGU00528 17-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 534672 3569573 3484 T 76 U SAGU00507 17-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 534672 3569573 3484 T 76 U SAGU00520 17-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 534672 3569573 3484 T 76 U SAGU00526 17-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 534672 3569573 3484 T 76 U SAGU00505 17-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 534672 3569573 3484 T 76 U SAGU00524 17-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 534672 3569573 3484 T 76 U SAGU00502 17-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 534672 3569573 3484 T 76 U SAGU00529 17-Jun-09 HYAR POSITIVE 1,031 2009 Summer Tanque Verde 534672 3569573 3484 T 76 U SAGU00601 22-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 538116 3569138 4026 A 89 F 3.5 34 60 SAGU00627 22-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 538116 3569138 4026 A 89 F 5.5 38 10 SAGU00820 22-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 539677 3568593 4450 A 88 F 5.0 37 15 SAGU00838 22-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 539677 3568593 4450 A 88 F 3.5 35 40 SAGU00818 22-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 539677 3568593 4450 A 88 F 6.0 38 50 SAGU00831 22-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 539677 3568593 4450 A 88 F 7.5 41 20 SAGU00812 22-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 539677 3568593 4450 A 88 F 6.5 42 40 SAGU00629 22-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 538116 3569138 4026 A 89 M 5.0 35 30 SAGU00621 22-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 538116 3569138 4026 A 89 M 4.5 36 15 SAGU00614 22-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 538116 3569138 4026 A 89 M 7.0 42 60 SAGU00611 22-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 538116 3569138 4026 A 89 M 5.0 36 100 SAGU00623 22-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 538116 3569138 4026 A 89 M 5.5 42 60 SAGU00617 22-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 538116 3569138 4026 A 89 M 5.0 38 50 SAGU00834 22-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 539677 3568593 4450 A 88 M 5.0 35 60 SAGU00836 22-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 539677 3568593 4450 A 88 M 3.5 31 30 SAGU00828 22-Jun-09 HYAR NEGATIVE 0 2009 Summer Tanque Verde 539677 3568593 4450 A 88 M 5.0 38 30 SAGU01053 6-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 538106 3569121 4012 A 75 F 6.5 41 40 SAGU01379 6-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 538106 3569121 4012 A 75 F 7.0 40 30 SAGU01412 6-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 538106 3569121 4012 A 75 F 6.5 37 80 SAGU01265 6-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 538106 3569121 4012 A 75 M 6.5 37 15 SAGU00798 6-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 538106 3569121 4012 A 75 M 7.0 37 30 SAGU01271 6-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 538106 3569121 4012 A 75 M 6.0 40 80 SAGU00068 6-Oct-09 HYAR POSITIVE 40 2009 Fall Tanque Verde 538106 3569121 4012 A 75 M 7.0 40 15 SAGU01375 6-Oct-09 HYAR POSITIVE 62 2009 Fall Tanque Verde 538106 3569121 4012 A 75 M 7.0 36 50 SAGU01259 6-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 538106 3569121 4012 J 75 U 1.0 21 20 SAGU01064 6-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 538106 3569121 4012 J 75 U 2.0 26 40

SAGU01129 7-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 540587 3568351 4756 A 76 F 2.0 23 50 147 SAGU01376 7-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 539688 3568600 4462 A 70 F 7.0 42 100 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU00839 7-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 539688 3568600 4462 A 70 F 4.0 34 40 SAGU01273 7-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 539688 3568600 4462 A 70 F 9.0 43 70 SAGU00811 7-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 539752 3568633 4458 A 70 F 7.0 38 100 SAGU01261 7-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 539688 3568600 4462 A 70 M 7.5 41 50 SAGU01377 7-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 539688 3568600 4462 A 70 M 7.0 41 40 SAGU00762 7-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 539688 3568600 4462 A 70 M 6.5 39 40 SAGU00768 7-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 539610 3568625 4451 A 67 M 7.0 39 100 SAGU01399 7-Apr-10 HYAR NEGATIVE 0 2010 Spring Tanque Verde 533400 3569043 3349 A 53 F 14.5 47 30 SAGU00917 7-Apr-10 HYAR NEGATIVE 0 2010 Spring Tanque Verde 533481 3569091 3357 A 60 F 17.5 46 100 SAGU00901 7-Apr-10 HYAR NEGATIVE 0 2010 Spring Tanque Verde 533481 3569091 3357 A 60 F 11.5 41 70 SAGU01388 7-Apr-10 HYAR NEGATIVE 0 2010 Spring Tanque Verde 534664 3569488 3566 A 62 F 15.5 43 7 SAGU01386 7-Apr-10 HYAR NEGATIVE 0 2010 Spring Tanque Verde 533149 3569093 3317 A 51 M 13.5 40 30 SAGU01316 7-Apr-10 HYAR NEGATIVE 0 2010 Spring Tanque Verde 533149 3569093 3317 A 51 M 10.5 37 20 SAGU01295 7-Apr-10 HYAR NEGATIVE 0 2010 Spring Tanque Verde 533481 3569091 3357 A 60 M 11.5 37 25 SAGU01396 7-Apr-10 HYAR NEGATIVE 0 2010 Spring Tanque Verde 533481 3569091 3357 A 60 M 12.5 41 30 SAGU00916 7-Apr-10 HYAR NEGATIVE 0 2010 Spring Tanque Verde 533481 3569091 3357 A 60 M 13.5 40 10 SAGU01407 7-Apr-10 HYAR NEGATIVE 0 2010 Spring Tanque Verde 534518 3569419 3418 A 62 M 10.5 38 40 SAGU00914 7-Apr-10 HYAR POSITIVE 4 2010 Spring Tanque Verde 533149 3569093 3317 A 51 M 12.5 44 50 SAGU01393 7-Apr-10 HYAR POSITIVE 14 2010 Spring Tanque Verde 533481 3569091 3357 A 60 M 12.5 40 45 SAGU01642 8-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 532920 3569019 3360 A 70 F 9.5 42 40 SAGU01629 8-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 532920 3569019 3360 A 70 F 8.5 44 60 SAGU01563 8-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 533172 3569103 3321 A 75 F 9.5 43 80 SAGU01846 8-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 534679 3569564 3439 A 80 F 11.5 48 60 SAGU01794 8-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 534885 3569544 3485 A 82 F 11.0 48 20 SAGU01636 8-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 532920 3569019 3360 A 70 M 5.5 38 30 SAGU01620 8-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 532920 3569019 3360 A 70 M 6.5 37 60 SAGU01649 8-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 532920 3569019 3360 A 70 M 6.5 37 30 SAGU01633 8-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 532920 3569019 3360 A 70 M 6.5 40 30 SAGU01596 8-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 532920 3569019 3360 A 70 M 7.5 45 30 SAGU01648 8-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 532920 3569019 3360 A 70 M 7.0 39 30 SAGU01651 8-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 533056 3568957 3244 A 69 M 6.5 38 25 SAGU01572 8-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 533172 3569103 3321 A 75 M 7.5 38 30 SAGU01847 8-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 533172 3569103 3321 A 75 M 8.0 42 25 SAGU01892 8-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 533172 3569103 3321 A 75 M 6.5 39 80 SAGU01619 8-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 533502 3569133 3350 A 72 M 8.0 41 150 SAGU01568 8-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 534679 3569564 3439 A 80 M 7.5 39 150 SAGU01904 8-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 534679 3569564 3439 A 80 M 9.5 42 100 SAGU00636 20-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 533160 3569115 3317 A 76 F 3.0 30 10 SAGU01247 20-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 533160 3569115 3317 A 76 F 3.0 27 20

SAGU01444 20-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 532924 3569016 3234 A 75 F 7.0 39 30 148 SAGU01448 20-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 532924 3569016 3234 A 75 F 4.0 36 40 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU01467 20-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 532924 3569016 3234 A 75 F 6.0 37 20 SAGU01468 20-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 532924 3569016 3234 A 75 F 6.0 37 30 SAGU01441 20-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 532924 3569016 3234 A 75 F 4.0 33 40 SAGU01453 20-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 532924 3569016 3234 A 75 M 5.0 37 40 SAGU01469 20-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 532924 3569016 3234 A 75 M 5.0 37 30 SAGU01456 20-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 532924 3569016 3234 A 75 M 6.0 37 30 SAGU01470 20-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 532924 3569016 3234 A 75 M 7.0 41 30 SAGU01269 20-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 533160 3569115 3317 A 76 M 7.0 38 40 SAGU01250 20-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 533160 3569115 3317 A 76 M 8.0 42 100 SAGU01087 20-Oct-09 HYAR POSITIVE 3 2009 Fall Tanque Verde 533160 3569115 3317 A 76 F 7.0 40 20 SAGU01450 20-Oct-09 HYAR POSITIVE 155 2009 Fall Tanque Verde 532924 3569016 3234 A 75 M 4.0 34 20 SAGU01192 26-Oct-09 HYAR POSITIVE 50 2009 Fall Tanque Verde 534652 3569600 3508 A 63 F 3.0 27 40 SAGU01198 26-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 534652 3569600 3508 A 63 F 4.0 31 80 SAGU01197 26-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 534652 3569600 3508 A 63 F 7.0 41 60 SAGU01210 26-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 534652 3569600 3508 A 63 F 8.5 42 100 SAGU01199 26-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 534652 3569600 3508 A 63 F 7.5 39 120 SAGU01205 26-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 534652 3569600 3508 A 63 M 6.0 35 150 SAGU01202 26-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 534652 3569600 3508 A 63 M 6.0 38 150 SAGU01204 26-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 534652 3569600 3508 A 63 M 6.5 40 200 SAGU01206 26-Oct-09 HYAR NEGATIVE 0 2009 Fall Tanque Verde 534652 3569600 3508 A 63 M 7.0 38 50 SAGU01191 26-Oct-09 HYAR POSITIVE 1 2009 Fall Tanque Verde 534652 3569600 3508 A 63 F 7.0 39 80 SAGU01211 26-Oct-09 HYAR POSITIVE 4 2009 Fall Tanque Verde 534652 3569600 3508 A 63 F 7.5 42 50 SAGU01194 26-Oct-09 HYAR POSITIVE 16 2009 Fall Tanque Verde 534652 3569600 3508 A 63 F 6.5 40 50 SAGU01220 26-Oct-09 HYAR POSITIVE 20 2009 Fall Tanque Verde 534652 3569600 3508 A 63 F 7.5 43 40 SAGU01208 26-Oct-09 HYAR POSITIVE 32 2009 Fall Tanque Verde 534652 3569600 3508 A 63 M 6.0 38 100 SAGU01218 26-Oct-09 HYAR POSITIVE 34,729 2009 Fall Tanque Verde 534652 3569600 3508 A 63 M 7.0 41 60 SAGU01706 1-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538110 3569116 3983 A 68 M 11.0 41 30 SAGU01868 2-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538111 3569116 3983 A 68 F 12.0 41 10 SAGU01922 2-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538111 3569116 3983 A 68 F 13.0 43 25 SAGU01720 2-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538111 3569116 3983 A 68 F 13.0 42 25 SAGU01865 2-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538111 3569116 3983 A 68 F 12.0 43 25 SAGU01709 2-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538111 3569116 3983 A 68 F 9.0 45 20 SAGU01743 2-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538111 3569116 3983 A 68 F 9.0 43 30 SAGU01751 2-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538111 3569116 3983 A 68 M 11.0 44 50 SAGU01787 2-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538111 3569116 3983 A 68 M 9.5 39 20 SAGU01726 2-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538111 3569116 3983 A 68 M 11.0 41 40 SAGU01690 2-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538111 3569116 3983 A 68 M 6.5 38 20 SAGU01837 2-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538111 3569116 3983 A 68 M 8.0 41 40 SAGU01921 2-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538111 3569116 3983 A 68 M 7.0 38 20

SAGU01905 2-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538111 3569116 3983 A 68 M 6.0 38 30 149 SAGU01684 2-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538111 3569116 3983 A 68 M 7.0 41 35 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU01692 2-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538111 3569116 3983 A 68 M 6.5 33 20 SAGU01780 2-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538111 3569116 3983 A 68 M 6.0 39 20 SAGU02017 2-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538111 3569116 3983 A 68 M 8.0 42 40 SAGU01622 2-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538111 3569116 3983 T 68 U SAGU01567 2-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538111 3569116 3983 T 68 U SAGU01613 2-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538111 3569116 3983 T 68 U SAGU01610 2-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538111 3569116 3983 T 68 U SAGU01564 2-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538111 3569116 3983 T 68 U SAGU01607 2-Jun-10 HYAR POSITIVE 3 2010 Summer Tanque Verde 538111 3569116 3983 T 68 U SAGU01654 2-Jun-10 HYAR POSITIVE 3 2010 Summer Tanque Verde 538111 3569116 3983 T 68 U SAGU01645 2-Jun-10 HYAR POSITIVE 21 2010 Summer Tanque Verde 538111 3569116 3983 T 68 U SAGU01869 3-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 539107 3568579 4310 A 70 F 10.0 46 30 SAGU01863 3-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 539628 3568599 4439 A 78 F 10.0 45 25 SAGU01784 3-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 539748 3568634 4499 A 79 F 10.0 48 40 SAGU01735 3-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 539107 3568579 4310 A 70 M 8.0 42 30 SAGU01697 3-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 539107 3568579 4310 A 70 M 6.5 40 25 SAGU01759 3-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 539628 3568599 4439 A 78 M 8.0 42 40 SAGU01887 3-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 539628 3568599 4439 A 78 M 6.5 41 40 SAGU01704 3-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 539697 3568599 4484 A 84 M 6.5 38 40 SAGU01694 3-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 539748 3568634 4499 A 79 M 10.5 44 30 SAGU01785 3-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 539748 3568634 4499 A 79 M 7.5 39 30 SAGU01574 3-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538911 3568543 4275 J 90 U 24.0 2 12 SAGU01779 3-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538911 3568543 4275 J 90 U 20.0 1 2 SAGU01760 3-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538911 3568543 4275 J 90 U 24.0 1 12 SAGU01833 3-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538911 3568543 4275 J 90 U 24.0 2 10 SAGU01681 3-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538911 3568543 4275 J 90 U 23.0 1 12 SAGU01711 3-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538911 3568543 4275 J 90 U 21.0 1 2 SAGU01935 3-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538911 3568543 4275 J 90 U 21.0 1 3 SAGU01820 3-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538911 3568543 4275 J 90 U 22.0 1 6 SAGU01888 3-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538911 3568543 4275 J 90 U 23.0 2 8 SAGU01977 3-Jun-10 HYAR NEGATIVE 0 2010 Summer Tanque Verde 538911 3568543 4275 J 90 U 22.0 2 8 SAGU00492 1-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 530140 3563430 3309 A 85 F 4.8 38 35 SAGU00127 1-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 530140 3563430 3309 A 85 F 3.7 36 25 SAGU00498 1-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 530140 3563430 3309 A 85 F 2.1 35 15 SAGU00148 1-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 530140 3563430 3309 A 85 F 2.3 40 20 SAGU00124 1-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 530140 3563430 3309 A 85 F 2.0 31 25 SAGU00491 1-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 530140 3563430 3309 A 85 M 4.1 38 50 SAGU00256 1-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 530140 3563430 3309 A 85 F 5.4 45 50 SAGU00144 1-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 530140 3563430 3309 A 85 F 6.6 51 25

SAGU00133 1-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 530140 3563430 3309 A 85 M 5.8 43 25 150 SAGU00185 1-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 530140 3563430 3309 A 85 M 6.2 53 100 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU00150 1-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 530140 3563430 3309 A 85 M 4.4 41 40 SAGU00132 1-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 530140 3563430 3309 J 85 F 2.0 29 20 SAGU00254 1-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 530140 3563430 3309 J 85 U 0.3 25 20 SAGU00149 1-Oct-08 HYAR POSITIVE 2 2008 Fall Wildhorse 530140 3563430 3309 J 85 U 0.5 22 4 SAGU00181 1-Oct-08 HYAR POSITIVE 6 2008 Fall Wildhorse 530140 3563430 3309 J 85 U 2.3 31 12 HYAR00192 11-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 529465 3564079 3033 A 78 F 7.1 41 16 HYAR00193 11-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 529465 3564079 3033 A 78 F 6.3 40 15 HYAR00194 11-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 529465 3564079 3033 A 78 F 6.5 41 20 U04 11-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 529465 3564079 3033 A 78 F 6.9 39 40 U45 11-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 529465 3564079 3033 A 78 F 7.5 41 10 HYAR00196 11-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 529465 3564079 3033 A 78 M 7.9 42 20 U46 11-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 529465 3564079 3033 A 78 M 8.1 42 25 U47 11-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 529465 3564079 3033 A 78 U 5.4 39 40 HYAR00195 11-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 529465 3564079 3033 A 78 F 13.7 51 45 HYAR00197 11-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 529465 3564079 3033 A 78 F 13.4 52 25 HYAR00199 11-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 529465 3564079 3033 A 78 F 12.2 50 30 HYAR00200 11-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 529465 3564079 3033 A 78 F 9.0 44 25 U07 11-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 529465 3564079 3033 A 78 F 10.7 50 17 HYAR00198 11-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 529465 3564079 3033 A 78 M 10.5 46 15 U43 11-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 529465 3564079 3033 A 78 M 10.7 46 90 U44 11-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 529465 3564079 3033 A 78 M 9.0 46 5 SAGU00430 20-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 533644 3563060 5599 A 57 M 2.0 30 200 SAGU00308 20-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 533169 3563636 4907 A 61 M 1.5 29 150 SAGU00479 20-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 533169 3563636 4907 A 61 M 1.5 30 200 SAGU00485 20-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 533169 3563636 4907 A 61 M 2.5 32 150 SAGU00438 20-Oct-08 HYAR POSITIVE 12 2008 Fall Wildhorse 533644 3563060 5599 A 57 M 2.0 30 80 SAGU00436 20-Oct-08 HYAR POSITIVE 23 2008 Fall Wildhorse 533281 3563576 4992 A 60 F 1.5 28 150 SAGU00385 20-Oct-08 HYAR POSITIVE 68 2008 Fall Wildhorse 533644 3563060 5599 A 57 M 1.5 28 100 SAGU00422 20-Oct-08 HYAR POSITIVE 152 2008 Fall Wildhorse 533655 3563047 5646 A 60 M 1.5 30 150 SAGU00307 20-Oct-08 HYAR POSITIVE 3,681 2008 Fall Wildhorse 533644 3563060 5599 A 57 F 1.7 28 200 SAGU00477 20-Oct-08 HYAR POSITIVE 16,835 2008 Fall Wildhorse 533655 3563047 5646 A 60 M 2.0 32 120 SAGU00407 20-Oct-08 HYAR POSITIVE 18,576 2008 Fall Wildhorse 533655 3563047 5646 A 60 F 2.0 30 130 SAGU00236 20-Oct-08 HYAR POSITIVE 77,317 2008 Fall Wildhorse 534187 3564189 5132 A 59 F 2.4 31 200 SAGU00281 20-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 533169 3563636 4907 A 61 F 8.5 47 300 SAGU00384 20-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 533169 3563636 4907 A 61 M 3.5 37 200 SAGU00381 20-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 533169 3563636 4907 A 61 M 2.5 32 200 SAGU00473 20-Oct-08 HYAR POSITIVE 11 2008 Fall Wildhorse 533644 3563060 5599 A 57 M 5.0 38 250 SAGU00392 20-Oct-08 HYAR POSITIVE 13 2008 Fall Wildhorse 534187 3564189 5132 A 59 M 2.5 35 100 SAGU00398 20-Oct-08 HYAR POSITIVE 14 2008 Fall Wildhorse 534187 3564189 5132 A 59 F 8.5 45 300

SAGU00415 20-Oct-08 HYAR POSITIVE 59 2008 Fall Wildhorse 534187 3564189 5132 A 59 F 7.5 45 300 151 SAGU00466 20-Oct-08 HYAR POSITIVE 728 2008 Fall Wildhorse 534187 3564189 5132 A 59 F 8.0 48 250 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU00500 20-Oct-08 HYAR POSITIVE 1,015 2008 Fall Wildhorse 534187 3564189 5132 A 59 M 7.0 44 300 SAGU00390 20-Oct-08 HYAR POSITIVE 1,193 2008 Fall Wildhorse 533655 3563047 5646 A 60 F 3.5 39 200 SAGU00294 20-Oct-08 HYAR POSITIVE 1,957 2008 Fall Wildhorse 534187 3564189 5132 A 59 F 3.0 34 200 SAGU00421 20-Oct-08 HYAR POSITIVE 3,655 2008 Fall Wildhorse 534187 3564189 5132 A 59 M 9.5 50 300 SAGU00453 20-Oct-08 HYAR POSITIVE 7,411 2008 Fall Wildhorse 533655 3563047 5646 A 60 M 3.5 36 150 SAGU00382 20-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 533281 3563576 4992 J 60 U 0.7 28 150 SAGU00315 20-Oct-08 HYAR POSITIVE 19 2008 Fall Wildhorse 533281 3563576 4992 J 60 M 1.0 25 200 SAGU00397 20-Oct-08 HYAR POSITIVE 33 2008 Fall Wildhorse 534187 3564189 5132 J 59 M 1.0 27 80 SAGU00481 20-Oct-08 HYAR POSITIVE 71 2008 Fall Wildhorse 533281 3563576 4992 J 60 U 0.5 23 60 SAGU00429 20-Oct-08 HYAR POSITIVE 104 2008 Fall Wildhorse 534187 3564189 5132 J 59 U 0.6 29 50 SAGU00413 22-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 531316 3563358 4074 A 63 F 1.5 37 30 SAGU00445 22-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 531316 3563358 4074 A 63 M 3.0 35 150 SAGU00449 22-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 531316 3563358 4074 A 63 M 1.0 27 30 SAGU00403 22-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 530644 3563348 3518 A 59 F 9.5 47 50 SAGU00432 22-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 530644 3563348 3518 A 59 F 3.5 38 80 SAGU00287 22-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 530644 3563348 3518 A 59 F 5.5 42 100 SAGU00389 22-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 530644 3563348 3518 A 59 F 3.0 36 80 SAGU00402 22-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 531316 3563358 4074 A 63 F 3.5 39 40 SAGU00476 22-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 531316 3563358 4074 A 63 F 3.5 38 200 SAGU00395 22-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 531316 3563358 4074 A 63 M 2.5 38 100 SAGU00441 22-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 531316 3563358 4074 A 63 M 3.5 38 50 SAGU00463 22-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 531316 3563358 4074 A 63 M 3.5 35 30 SAGU00497 22-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 530644 3563348 3518 A 59 M 5.5 41 40 SAGU00284 22-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 530644 3563348 3518 A 59 M 7.0 44 80 SAGU00304 22-Oct-08 HYAR NEGATIVE 0 2008 Fall Wildhorse 530644 3563348 3518 A 59 M 7.5 47 80 SAGU00010 5-Feb-09 HYAR NEGATIVE 0 2008 Winter Wildhorse 529057 3564298 2872 A 59 M 6.3 39 20 SAGU00011 5-Feb-09 HYAR POSITIVE 12 2008 Winter Wildhorse 529057 3564298 2872 A 59 F 10.0 45 10 SAGU00313 5-Feb-09 HYAR POSITIVE 47 2008 Winter Wildhorse 529454 3564074 2979 A 59 F 4.0 38 30 SAGU00029 5-Feb-09 HYAR POSITIVE 84 2008 Winter Wildhorse 529454 3564074 2979 A 59 F 4.5 38 20 SAGU00013 5-Feb-09 HYAR POSITIVE 137 2008 Winter Wildhorse 528876 3564508 2803 A 59 M 33 20 SAGU00020 5-Feb-09 HYAR POSITIVE 303 2008 Winter Wildhorse 529454 3564074 2979 A 59 M 7.5 44 15 SAGU00514 20-Mar-09 HYAR NEGATIVE 0 2009 Spring Wildhorse 529882 3563699 3181 A 71 F 2.2 31 10 SAGU00509 20-Mar-09 HYAR NEGATIVE 0 2009 Spring Wildhorse 529882 3563699 3181 A 71 F 1.8 27 10 SAGU00512 20-Mar-09 HYAR NEGATIVE 0 2009 Spring Wildhorse 529882 3563699 3181 A 71 F 3.0 31 6 SAGU00506 20-Mar-09 HYAR NEGATIVE 0 2009 Spring Wildhorse 529882 3563699 3181 A 71 F 2.2 29 8 SAGU00501 20-Mar-09 HYAR NEGATIVE 0 2009 Spring Wildhorse 529882 3563699 3181 A 71 F 5.5 41 6 SAGU00518 20-Mar-09 HYAR NEGATIVE 0 2009 Spring Wildhorse 529882 3563699 3181 A 71 F 4.0 33 6 SAGU00516 20-Mar-09 HYAR NEGATIVE 0 2009 Spring Wildhorse 529882 3563699 3181 A 71 F 4.0 34 20 SAGU00531 20-Mar-09 HYAR NEGATIVE 0 2009 Spring Wildhorse 529475 3564059 3055 A 72 F 3.0 33 5

SAGU00544 20-Mar-09 HYAR NEGATIVE 0 2009 Spring Wildhorse 529475 3564059 3055 A 72 F 5.5 38 0 152 SAGU00521 20-Mar-09 HYAR NEGATIVE 0 2009 Spring Wildhorse 529882 3563699 3181 A 71 M 8.0 42 200 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU00503 20-Mar-09 HYAR NEGATIVE 0 2009 Spring Wildhorse 529882 3563699 3181 A 71 M 7.0 41 20 SAGU00548 20-Mar-09 HYAR NEGATIVE 0 2009 Spring Wildhorse 529475 3564059 3055 A 72 M 7.0 39 20 SAGU00557 20-Mar-09 HYAR NEGATIVE 0 2009 Spring Wildhorse 529475 3564059 3055 A 72 M 6.0 38 10 SAGU00532 20-Mar-09 HYAR NEGATIVE 0 2009 Spring Wildhorse 529475 3564059 3055 A 72 M 9.0 43 50 SAGU00536 20-Mar-09 HYAR NEGATIVE 0 2009 Spring Wildhorse 529475 3564059 3055 A 72 M 7.0 41 15 SAGU00552 20-Mar-09 HYAR NEGATIVE 0 2009 Spring Wildhorse 529475 3564059 3055 A 72 M 7.0 42 30 SAGU00533 20-Mar-09 HYAR NEGATIVE 0 2009 Spring Wildhorse 529475 3564059 3055 A 72 M 7.0 43 10 SAGU00535 20-Mar-09 HYAR NEGATIVE 0 2009 Spring Wildhorse 529475 3564059 3055 A 72 M 7.0 41 40 SAGU00534 20-Mar-09 HYAR NEGATIVE 0 2009 Spring Wildhorse 529475 3564059 3055 A 72 M 7.0 40 20 SAGU00550 20-Mar-09 HYAR POSITIVE 2 2009 Spring Wildhorse 529475 3564059 3055 A 72 F 4.0 33 10 SAGU00560 4-Apr-09 HYAR NEGATIVE 0 2009 Spring Wildhorse 530186 3563472 3307 A 63 F 2.0 30 40 SAGU00098 4-Apr-09 HYAR NEGATIVE 0 2009 Spring Wildhorse 530186 3563472 3307 A 63 F 6.0 43 30 SAGU00480 4-Apr-09 HYAR NEGATIVE 0 2009 Spring Wildhorse 530186 3563472 3307 A 63 F 6.0 43 70 SAGU00394 4-Apr-09 HYAR NEGATIVE 0 2009 Spring Wildhorse 530186 3563472 3307 A 63 F 5.0 39 50 SAGU00302 4-Apr-09 HYAR NEGATIVE 0 2009 Spring Wildhorse 530186 3563472 3307 A 63 F 5.0 40 10 SAGU00545 4-Apr-09 HYAR NEGATIVE 0 2009 Spring Wildhorse 530186 3563472 3307 A 63 F 4.5 34 8 SAGU00097 4-Apr-09 HYAR NEGATIVE 0 2009 Spring Wildhorse 530186 3563472 3307 A 63 F 3.0 33 5 SAGU00406 4-Apr-09 HYAR NEGATIVE 0 2009 Spring Wildhorse 530186 3563472 3307 A 63 F 3.0 34 6 SAGU00540 4-Apr-09 HYAR NEGATIVE 0 2009 Spring Wildhorse 530186 3563472 3307 A 63 M 3.5 36 12 SAGU00554 4-Apr-09 HYAR NEGATIVE 0 2009 Spring Wildhorse 530186 3563472 3307 A 63 M 5.5 40 25 SAGU00682 28-May-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 531259 3563340 4036 A 80 F 2.0 29 15 SAGU00668 28-May-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 531259 3563340 4036 A 80 F 7.0 42 8 SAGU00671 28-May-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 531259 3563340 4036 A 80 F 6.0 39 15 SAGU00689 28-May-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 531259 3563340 4036 A 80 F 5.0 39 20 SAGU00687 28-May-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 531259 3563340 4036 A 80 M 4.5 39 40 SAGU00666 28-May-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 531259 3563340 4036 A 80 M 4.0 38 60 SAGU00661 28-May-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 531259 3563340 4036 A 80 M 4.0 34 100 SAGU00684 28-May-09 HYAR POSITIVE 4 2009 Summer Wildhorse 531259 3563340 4036 A 80 M 4.0 37 100 SAGU00677 29-May-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 530219 3563481 3354 A 81 F 9.0 45 20 SAGU00688 29-May-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 530219 3563481 3354 A 81 F 4.0 37 30 SAGU00674 29-May-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 530219 3563481 3354 A 81 F 7.0 41 12 SAGU00679 29-May-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 530219 3563481 3354 A 81 M 5.0 38 15 SAGU00669 29-May-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 530219 3563481 3354 A 81 M 5.0 37 15 SAGU00680 29-May-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 530219 3563481 3354 A 81 M 5.0 38 3 SAGU00681 29-May-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 530219 3563481 3354 A 81 M 4.5 39 30 SAGU00673 29-May-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 530219 3563481 3354 A 81 M 4.0 38 25 SAGU00709 11-Jun-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 529465 3564079 3033 A 79 F 4.5 37 2 SAGU00696 11-Jun-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 529465 3564079 3033 A 79 F 3.5 34 15 SAGU00707 11-Jun-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 529465 3564079 3033 A 79 F 9.0 49 15

SAGU00704 11-Jun-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 529465 3564079 3033 A 79 F 6.0 43 25 153 SAGU00069 11-Jun-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 529465 3564079 3033 A 79 M 4.0 34 5 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU00716 11-Jun-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 529465 3564079 3033 A 79 M 4.0 36 25 SAGU00309 11-Jun-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 529465 3564079 3033 A 79 M 3.5 35 30 SAGU00703 11-Jun-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 529465 3564079 3033 A 79 M 6.0 41 30 SAGU00511 26-Jun-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 529477 3564004 3074 T 92 U SAGU00705 26-Jun-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 529477 3564004 3074 T 92 U SAGU00953 26-Jun-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 529477 3564004 3074 T 92 U SAGU00948 26-Jun-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 529477 3564004 3074 T 92 U SAGU00252 26-Jun-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 529477 3564004 3074 T 92 U SAGU00782 26-Jun-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 529477 3564004 3074 T 92 U SAGU00143 26-Jun-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 529477 3564004 3074 T 92 U SAGU00504 26-Jun-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 529477 3564004 3074 T 92 U SAGU00940 26-Jun-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 529477 3564004 3074 T 92 U SAGU00797 26-Jun-09 HYAR NEGATIVE 0 2009 Summer Wildhorse 529477 3564004 3074 T 92 U SAGU01097 12-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 533623 3563055 5593 A 60 F 5.0 38 30 SAGU00650 12-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 533623 3563055 5593 A 60 F 4.5 36 20 SAGU01083 12-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 533623 3563055 5593 A 60 M 6.0 38 20 SAGU01325 14-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 529459 3564060 3061 A 72 F 4.0 37 30 SAGU01340 14-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 529459 3564060 3061 A 72 F 7.5 45 20 SAGU01242 19-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 531210 3563331 4030 A 73 U 2.5 25 15 SAGU01126 19-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 531292 3563346 4065 A 70 F 8.0 44 30 SAGU01411 19-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 531292 3563346 4065 A 70 F 9.0 45 100 SAGU01230 19-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 531210 3563331 4030 A 73 F 9.0 45 80 SAGU00653 19-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 530565 3563366 3542 A 71 F 10.0 45 20 SAGU01358 19-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 530565 3563366 3542 A 71 F 5.0 34 15 SAGU01109 19-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 530565 3563366 3542 A 71 F 6.5 41 20 SAGU01235 19-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 530103 3563460 3281 A 75 F 8.0 45 10 SAGU01233 19-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 530103 3563460 3281 A 75 F 6.0 40 10 SAGU01234 19-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 530103 3563460 3281 A 75 F 7.0 42 8 SAGU01221 19-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 530103 3563460 3281 A 75 F 6.5 40 5 SAGU01090 19-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 531292 3563346 4065 A 70 M 5.5 36 80 SAGU00654 19-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 531292 3563346 4065 A 70 M 5.5 36 50 SAGU00631 19-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 531292 3563346 4065 A 70 M 7.0 39 20 SAGU00563 19-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 531292 3563346 4065 A 70 M 6.0 37 80 SAGU00658 19-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 531292 3563346 4065 A 70 M 6.0 38 25 SAGU01236 19-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 531210 3563331 4030 A 73 M 4.0 34 10 SAGU01241 19-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 531210 3563331 4030 A 73 M 5.0 35 40 SAGU01245 19-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 531210 3563331 4030 A 73 M 6.0 40 80 SAGU00659 19-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 530565 3563366 3542 A 71 M 5.0 36 15 SAGU01361 19-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 530565 3563366 3542 A 71 M 5.5 37 30

SAGU01190 19-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 530565 3563366 3542 A 71 M 4.5 34 5 154 SAGU01117 19-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 530565 3563366 3542 A 71 M 6.5 36 35 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU01086 19-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 530565 3563366 3542 A 71 M 4.0 37 20 SAGU01098 19-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 530565 3563366 3542 A 71 M 5.0 35 20 SAGU01246 19-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 530103 3563460 3281 A 75 M 4.0 34 10 SAGU01227 19-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 530103 3563460 3281 A 75 M 5.0 34 10 SAGU01232 19-Oct-09 HYAR NEGATIVE 0 2009 Fall Wildhorse 530103 3563460 3281 A 75 M 5.0 37 15 SAGU00647 19-Oct-09 HYAR POSITIVE 3 2009 Fall Wildhorse 530437 3563350 3417 A F 6.5 38 20 SAGU01244 19-Oct-09 HYAR POSITIVE 12 2009 Fall Wildhorse 530103 3563460 3281 A 75 M 5.0 36 10 SAGU01140 18-Mar-10 HYAR NEGATIVE 0 2010 Spring Wildhorse 530142 3563434 3315 A M 9.5 38 4 SAGU01150 18-Mar-10 HYAR NEGATIVE 0 2010 Spring Wildhorse 530220 3563480 3349 A M 9.0 40 10 SAGU01148 18-Mar-10 HYAR POSITIVE 3 2010 Spring Wildhorse 529463 3564076 3017 A 50 M 8.5 41 25 SAGU01154 18-Mar-10 HYAR POSITIVE 5 2010 Spring Wildhorse 530142 3563434 3315 A M 11.5 42 7 SAGU01144 18-Mar-10 HYAR POSITIVE 15 2010 Spring Wildhorse 530142 3563434 3315 A M 10.5 42 15 SAGU01839 29-May-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 530103 3563478 3270 A 64 F 14.0 50 100 SAGU01758 29-May-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 530103 3563478 3270 A 64 F 13.0 48 60 SAGU01855 29-May-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 530103 3563478 3270 A 64 F 16.5 50 70 SAGU01897 29-May-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 530103 3563478 3270 A 64 M 8.5 42 50 SAGU01770 29-May-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 530103 3563478 3270 A 64 M 7.5 40 60 SAGU01946 29-May-10 HYAR POSITIVE 13 2010 Summer Wildhorse 529796 3563840 3161 A 81 F 8.5 49 200 SAGU01671 29-May-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 529887 3563720 3169 T 70 U SAGU01919 29-May-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 529887 3563720 3169 T 70 U SAGU01889 29-May-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 529887 3563720 3169 T 70 U SAGU01844 29-May-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 529887 3563720 3169 T 70 U SAGU01688 29-May-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 529887 3563720 3169 T 70 U SAGU01859 29-May-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 529887 3563720 3169 T 70 U SAGU01712 29-May-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 529887 3563720 3169 T 70 U SAGU01878 29-May-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 529887 3563720 3169 T 70 U SAGU01812 29-May-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 529887 3563720 3169 T 70 U SAGU01700 29-May-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 529887 3563720 3169 T 70 U SAGU01791 29-May-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 529887 3563720 3169 T 70 U SAGU01901 1-Jun-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 531683 3563343 4202 A 69 F 11.0 45 35 SAGU01816 1-Jun-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 531683 3563343 4202 A 69 F 11.0 45 25 SAGU01886 1-Jun-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 531683 3563343 4202 A 69 F 10.0 46 25 SAGU01938 1-Jun-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 531003 3563335 3831 A 81 F 9.5 42 30 SAGU01755 1-Jun-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 531003 3563335 3831 A 81 F 10.5 45 20 SAGU01799 1-Jun-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 531003 3563335 3831 A 81 F 9.5 44 30 SAGU01931 1-Jun-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 531683 3563343 4202 A 69 M 7.5 39 50 SAGU01790 1-Jun-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 531683 3563343 4202 A 69 M 9.5 44 50 SAGU01851 1-Jun-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 531683 3563343 4202 A 69 M 8.0 40 25 SAGU01974 1-Jun-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 531683 3563343 4202 A 69 M 7.5 41 20

SAGU01832 1-Jun-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 531683 3563343 4202 A 69 M 7.0 40 40 155 SAGU01761 1-Jun-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 531683 3563343 4202 A 69 M 7.0 43 30 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU01893 1-Jun-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 531683 3563343 4202 A 69 M 7.0 35 25 SAGU02022 1-Jun-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 531683 3563343 4202 A 69 M 7.0 41 30 SAGU01741 1-Jun-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 531683 3563343 4202 A 69 M 7.5 42 30 SAGU01775 1-Jun-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 531683 3563343 4202 A 69 M 7.0 42 25 SAGU01698 1-Jun-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 531683 3563343 4202 A 69 M 5.5 40 25 SAGU02026 1-Jun-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 531003 3563335 3831 A 81 M 7.0 39 80 SAGU01800 1-Jun-10 HYAR NEGATIVE 0 2010 Summer Wildhorse 531003 3563335 3831 A 81 M 6.5 41 100 SAGU01825 1-Jun-10 HYAR POSITIVE 11 2010 Summer Wildhorse 531106 3563320 3948 A 78 M 6.5 38 70 SAGU01679 1-Jun-10 HYAR POSITIVE 25 2010 Summer Wildhorse 531683 3563343 4202 A 69 M 6.5 38 30 SAGU00470 10-Jun-09 RACA POSITIVE 18 2009 Summer Campus Ag Farm 505852 3571359 2323 A U SAGU00945 14-Jun-09 RACA POSITIVE 1,240 2009 Summer Campus Ag Farm 505852 3571359 2323 T U SAGU00958 14-Jun-09 RACA POSITIVE 10,899 2009 Summer Campus Ag Farm 505852 3571359 2323 T U SAGU00949 14-Jun-09 RACA POSITIVE 13,394 2009 Summer Campus Ag Farm 505852 3571359 2323 T U SAGU00957 14-Jun-09 RACA POSITIVE 17,849 2009 Summer Campus Ag Farm 505852 3571359 2323 T U SAGU00950 14-Jun-09 RACA POSITIVE 24,927 2009 Summer Campus Ag Farm 505852 3571359 2323 T U SAGU00960 14-Jun-09 RACA POSITIVE 30,758 2009 Summer Campus Ag Farm 505852 3571359 2323 T U SAGU00934 14-Jun-09 RACA POSITIVE 45,873 2009 Summer Campus Ag Farm 505852 3571359 2323 T U SAGU00943 14-Jun-09 RACA POSITIVE 71,891 2009 Summer Campus Ag Farm 505852 3571359 2323 T U SAGU00939 14-Jun-09 RACA POSITIVE 86,330 2009 Summer Campus Ag Farm 505852 3571359 2323 T U SAGU00954 14-Jun-09 RACA POSITIVE 128,618 2009 Summer Campus Ag Farm 505852 3571359 2323 T U CHY02841 17-Oct-09 RACA POSITIVE 12 2009 Fall Campus Ag Farm J U CHY02850 17-Oct-09 RACA POSITIVE 41 2009 Fall Campus Ag Farm J U SAGU01822 6-Jun-10 RACA NEGATIVE 0 2010 Summer Campus Ag Farm 505852 3571359 2323 J 79 U 31.5 70 0 SAGU01571 6-Jun-10 RACA POSITIVE 28 2010 Summer Campus Ag Farm 505852 3571359 2323 T U SAGU01612 6-Jun-10 RACA POSITIVE 160 2010 Summer Campus Ag Farm 505852 3571359 2323 T U SAGU02087 6-Jun-10 RACA POSITIVE 3,388 2010 Summer Campus Ag Farm 505852 3571359 2323 T U SAGU01604 6-Jun-10 RACA POSITIVE 4,027 2010 Summer Campus Ag Farm 505852 3571359 2323 T U SAGU01615 6-Jun-10 RACA POSITIVE 5,614 2010 Summer Campus Ag Farm 505852 3571359 2323 T U SAGU02098 6-Jun-10 RACA POSITIVE 33,990 2010 Summer Campus Ag Farm 505852 3571359 2323 T U SAGU01644 6-Jun-10 RACA POSITIVE 37,718 2010 Summer Campus Ag Farm 505852 3571359 2323 T U SAGU01631 6-Jun-10 RACA POSITIVE 75,451 2010 Summer Campus Ag Farm 505852 3571359 2323 T U SAGU01573 6-Jun-10 RACA POSITIVE 128,441 2010 Summer Campus Ag Farm 505852 3571359 2323 T U SAGU02090 6-Jun-10 RACA POSITIVE 201,684 2010 Summer Campus Ag Farm 505852 3571359 2323 T U SAGU01285 13-Oct-09 RACA NEGATIVE 0 2009 Fall Campus Ag Farm 505852 3571359 2323 T 75 U SAGU01100 13-Oct-09 RACA NEGATIVE 0 2009 Fall Campus Ag Farm 505852 3571359 2323 T 75 U SAGU01287 13-Oct-09 RACA NEGATIVE 0 2009 Fall Campus Ag Farm 505852 3571359 2323 T 75 U SAGU01281 13-Oct-09 RACA NEGATIVE 0 2009 Fall Campus Ag Farm 505852 3571359 2323 T 75 U SAGU01346 13-Oct-09 RACA NEGATIVE 0 2009 Fall Campus Ag Farm 505852 3571359 2323 T 75 U SAGU01352 13-Oct-09 RACA NEGATIVE 0 2009 Fall Campus Ag Farm 505852 3571359 2323 T 75 U

SAGU01282 13-Oct-09 RACA NEGATIVE 0 2009 Fall Campus Ag Farm 505852 3571359 2323 T 75 U 156 SAGU01335 13-Oct-09 RACA NEGATIVE 0 2009 Fall Campus Ag Farm 505852 3571359 2323 T 75 U Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU01351 13-Oct-09 RACA NEGATIVE 0 2009 Fall Campus Ag Farm 505852 3571359 2323 T 75 U SAGU01365 13-Oct-09 RACA NEGATIVE 0 2009 Fall Campus Ag Farm 505852 3571359 2323 T 75 U SAGU01369 13-Oct-09 RACA NEGATIVE 0 2009 Fall Campus Ag Farm 505852 3571359 2323 T 75 U SAGU01330 15-Oct-09 RACA NEGATIVE 0 2009 Fall Cebedilla 528955 3567668 2691 A 71 F 10.0 53 0 SAGU00637 15-Oct-09 RACA NEGATIVE 0 2009 Fall Cebedilla 528955 3567668 2691 A 71 F 7.0 51 0 SAGU01327 15-Oct-09 RACA NEGATIVE 0 2009 Fall Cebedilla 528955 3567668 2691 A 71 M 35.0 76 0 SAGU00642 15-Oct-09 RACA NEGATIVE 0 2009 Fall Cebedilla 528955 3567668 2691 A 71 F 210.0 128 0 SAGU01349 15-Oct-09 RACA NEGATIVE 0 2009 Fall Cebedilla 528955 3567668 2691 A 71 F 263.0 147 0 SAGU01343 15-Oct-09 RACA NEGATIVE 0 2009 Fall Cebedilla 528955 3567668 2691 A 71 M 316.0 161 0 SAGU01336 15-Oct-09 RACA NEGATIVE 0 2009 Fall Cebedilla 528955 3567668 2691 J 71 U 7.0 41 0 SAGU01342 15-Oct-09 RACA NEGATIVE 0 2009 Fall Cebedilla 528955 3567668 2691 J 71 U 7.0 40 0 SAGU01345 15-Oct-09 RACA NEGATIVE 0 2009 Fall Cebedilla 528955 3567668 2691 J 71 U 5.0 36 0 SAGU01324 15-Oct-09 RACA NEGATIVE 0 2009 Fall Cebedilla 528955 3567668 2691 J 71 U 7.0 44 0 SAGU01095 30-Oct-09 RACA POSITIVE 21 2009 Fall Cebedilla 528955 3567668 2691 A 60 F 16.5 50 0 SAGU01362 30-Oct-09 RACA NEGATIVE 0 2009 Fall Cebedilla 528955 3567668 2691 J 60 U 10.0 43 0 SAGU00651 30-Oct-09 RACA POSITIVE 18 2009 Fall Cebedilla 528955 3567668 2691 J 60 U 10.0 44 0 SAGU01151 19-Mar-10 RACA NEGATIVE 0 2010 Spring Cebedilla 528943 3567760 2718 A 50 F 240.0 125 0 SAGU01145 19-Mar-10 RACA NEGATIVE 0 2010 Spring Cebedilla 528943 3567760 2718 A 50 F 205.0 120 0 SAGU01156 19-Mar-10 RACA NEGATIVE 0 2010 Spring Cebedilla 528943 3567760 2718 A 50 F 325.0 155 0 SAGU01131 19-Mar-10 RACA NEGATIVE 0 2010 Spring Cebedilla 528943 3567760 2718 A 50 M 145.0 110 0 SAGU01143 19-Mar-10 RACA NEGATIVE 0 2010 Spring Cebedilla 528943 3567760 2718 J 50 U 12.5 43 0 SAGU01972 8-Jul-10 RACA NEGATIVE 0 2010 Summer Cebedilla 528943 3567760 2718 A 82 F 220.0 155 0 SAGU01856 8-Jul-10 RACA NEGATIVE 0 2010 Summer Cebedilla 528943 3567760 2718 A 82 F 240.0 146 0 SAGU02031 8-Jul-10 RACA NEGATIVE 0 2010 Summer Cebedilla 528943 3567760 2718 A 82 M 250.0 146 0 SAGU01721 8-Jul-10 RACA NEGATIVE 0 2010 Summer Cebedilla 528943 3567760 2718 J 82 U 12.5 50 0 SAGU01965 8-Jul-10 RACA NEGATIVE 0 2010 Summer Cebedilla 528943 3567760 2718 J 82 U 17.5 62 0 SAGU02084 8-Jul-10 RACA NEGATIVE 0 2010 Summer Cebedilla 528943 3567760 2718 J 82 U 12.0 53 0 SAGU01849 8-Jul-10 RACA NEGATIVE 0 2010 Summer Cebedilla 528943 3567760 2718 T 82 U SAGU01788 8-Jul-10 RACA NEGATIVE 0 2010 Summer Cebedilla 528943 3567760 2718 T 82 U SAGU01730 8-Jul-10 RACA NEGATIVE 0 2010 Summer Cebedilla 528943 3567760 2718 T 82 U SAGU01640 8-Jul-10 RACA NEGATIVE 0 2010 Summer Cebedilla 528943 3567760 2718 T 82 U SAGU01606 8-Jul-10 RACA NEGATIVE 0 2010 Summer Cebedilla 528943 3567760 2718 T 82 U SAGU01853 8-Jul-10 RACA NEGATIVE 0 2010 Summer Cebedilla 528943 3567760 2718 T 82 U SAGU01742 8-Jul-10 RACA NEGATIVE 0 2010 Summer Cebedilla 528943 3567760 2718 T 82 U SAGU01875 8-Jul-10 RACA NEGATIVE 0 2010 Summer Cebedilla 528943 3567760 2718 T 82 U SAGU01814 8-Jul-10 RACA NEGATIVE 0 2010 Summer Cebedilla 528943 3567760 2718 T 82 U SAGU01736 8-Jul-10 RACA NEGATIVE 0 2010 Summer Cebedilla 528943 3567760 2718 T 82 U SAGU02041 8-Jul-10 RACA POSITIVE 10 2010 Summer Cebedilla 528943 3567760 2718 T 82 U SAGU00293 18-Mar-09 RACA NEGATIVE 0 2009 Spring Cebedilla 528956 3567648 2683 A 70 M 280.0 132 0

SAGU00386 18-Mar-09 RACA NEGATIVE 0 2009 Spring Cebedilla 528956 3567648 2683 A 70 M 280.0 132 0 157 SAGU01256 26-Jun-09 RACA NEGATIVE 0 2009 Summer Cebedilla 528943 3567760 2718 A 85 F 295.0 169 0 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU00613 26-Jun-09 RACA NEGATIVE 0 2009 Summer Cebedilla 528943 3567760 2718 A 85 F 235.0 133 0 SAGU01251 26-Jun-09 RACA NEGATIVE 0 2009 Summer Cebedilla 528943 3567760 2718 A 85 F 117.0 105 0 SAGU01255 26-Jun-09 RACA NEGATIVE 0 2009 Summer Cebedilla 528943 3567760 2718 A 85 F 290.0 167 0 SAGU01270 26-Jun-09 RACA NEGATIVE 0 2009 Summer Cebedilla 528943 3567760 2718 A 85 M 115.0 102 0 SAGU01252 26-Jun-09 RACA NEGATIVE 0 2009 Summer Cebedilla 528943 3567760 2718 A 85 M 280.0 145 0 SAGU01272 26-Jun-09 RACA NEGATIVE 0 2009 Summer Cebedilla 528943 3567760 2718 A 85 M 360.0 147 0 SAGU01266 26-Jun-09 RACA NEGATIVE 0 2009 Summer Cebedilla 528943 3567760 2718 J 85 F 40.0 74 0 SAGU00628 26-Jun-09 RACA NEGATIVE 0 2009 Summer Cebedilla 528943 3567760 2718 J 85 M 50.0 75 0 SAGU01260 26-Jun-09 RACA NEGATIVE 0 2009 Summer Cebedilla 528943 3567760 2718 J 85 U 20.0 61 0 SAGU01162 17-Oct-09 RACA NEGATIVE 0 2009 Fall Columbus Park 497022 3571717 2233 A 78 F 30.0 63 0 SAGU01174 17-Oct-09 RACA POSITIVE 1 2009 Fall Columbus Park 497022 3571717 2233 A 78 F 31.5 67 0 SAGU01172 17-Oct-09 RACA POSITIVE 25 2009 Fall Columbus Park 497022 3571717 2233 A 78 F 31.0 61 0 SAGU01164 17-Oct-09 RACA NEGATIVE 0 2009 Fall Columbus Park 497022 3571717 2233 A 78 F 416.0 151 0 SAGU01165 17-Oct-09 RACA NEGATIVE 0 2009 Fall Columbus Park 497022 3571717 2233 J 78 U 8.0 41 0 SAGU01169 17-Oct-09 RACA NEGATIVE 0 2009 Fall Columbus Park 497022 3571717 2233 J 78 U 6.0 43 0 SAGU01177 17-Oct-09 RACA NEGATIVE 0 2009 Fall Columbus Park 497022 3571717 2233 J 78 U 5.0 33 0 SAGU01183 17-Oct-09 RACA NEGATIVE 0 2009 Fall Columbus Park 497022 3571717 2233 J 78 U 3.0 31 0 SAGU01367 17-Oct-09 RACA NEGATIVE 0 2009 Fall Columbus Park 497022 3571717 2233 T 78 U SAGU01289 17-Oct-09 RACA NEGATIVE 0 2009 Fall Columbus Park 497022 3571717 2233 T 78 U SAGU01284 17-Oct-09 RACA NEGATIVE 0 2009 Fall Columbus Park 497022 3571717 2233 T 78 U SAGU01354 17-Oct-09 RACA NEGATIVE 0 2009 Fall Columbus Park 497022 3571717 2233 T 78 U SAGU01290 17-Oct-09 RACA NEGATIVE 0 2009 Fall Columbus Park 497022 3571717 2233 T 78 U SAGU01366 17-Oct-09 RACA NEGATIVE 0 2009 Fall Columbus Park 497022 3571717 2233 T 78 U SAGU01328 17-Oct-09 RACA NEGATIVE 0 2009 Fall Columbus Park 497022 3571717 2233 T 78 U SAGU01356 17-Oct-09 RACA NEGATIVE 0 2009 Fall Columbus Park 497022 3571717 2233 T 78 U SAGU01355 17-Oct-09 RACA NEGATIVE 0 2009 Fall Columbus Park 497022 3571717 2233 T 78 U SAGU01286 17-Oct-09 RACA NEGATIVE 0 2009 Fall Columbus Park 497022 3571717 2233 T 78 U SAGU01410 27-Mar-10 RACA NEGATIVE 0 2010 Spring Columbus Park 496792 3571699 2258 A 60 M 255.0 130 0 SAGU01876 17-Jul-10 RACA NEGATIVE 0 2010 Summer Columbus Park 496792 3571699 2258 A 85 F 235.0 146 0 SAGU01969 17-Jul-10 RACA NEGATIVE 0 2010 Summer Columbus Park 496792 3571699 2258 J 85 U 17.5 55 0 SAGU01914 17-Jul-10 RACA NEGATIVE 0 2010 Summer Columbus Park 496792 3571699 2258 J 85 U 21.5 63 0 SAGU01920 17-Jul-10 RACA NEGATIVE 0 2010 Summer Columbus Park 496792 3571699 2258 J 85 U 8.0 42 0 SAGU01942 17-Jul-10 RACA NEGATIVE 0 2010 Summer Columbus Park 496792 3571699 2258 J 85 U 11.5 45 0 SAGU01915 17-Jul-10 RACA NEGATIVE 0 2010 Summer Columbus Park 496792 3571699 2258 J 85 U 17.5 50 0 SAGU01910 17-Jul-10 RACA NEGATIVE 0 2010 Summer Columbus Park 496792 3571699 2258 J 85 U 13.0 47 0 SAGU01923 17-Jul-10 RACA NEGATIVE 0 2010 Summer Columbus Park 496792 3571699 2258 J 85 U 15.0 45 0 SAGU01950 17-Jul-10 RACA NEGATIVE 0 2010 Summer Columbus Park 496792 3571699 2258 J 85 U 10.5 46 0 SAGU01982 17-Jul-10 RACA NEGATIVE 0 2010 Summer Columbus Park 496792 3571699 2258 J 85 U 9.5 45 0 SAGU01976 17-Jul-10 RACA NEGATIVE 0 2010 Summer Columbus Park 496792 3571699 2258 J 85 U 12.5 49 0

SAGU01953 17-Jul-10 RACA NEGATIVE 0 2010 Summer Columbus Park 496792 3571699 2258 J 85 U 20.5 58 0 158 SAGU01956 17-Jul-10 RACA NEGATIVE 0 2010 Summer Columbus Park 496792 3571699 2258 T 85 U 0 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU01928 17-Jul-10 RACA NEGATIVE 0 2010 Summer Columbus Park 496792 3571699 2258 T 85 U 0 SAGU01927 17-Jul-10 RACA NEGATIVE 0 2010 Summer Columbus Park 496792 3571699 2258 T 85 U 0 SAGU01907 17-Jul-10 RACA NEGATIVE 0 2010 Summer Columbus Park 496792 3571699 2258 T 85 U 0 SAGU01926 17-Jul-10 RACA NEGATIVE 0 2010 Summer Columbus Park 496792 3571699 2258 T 85 U 0 SAGU01970 17-Jul-10 RACA NEGATIVE 0 2010 Summer Columbus Park 496792 3571699 2258 T 85 U 0 SAGU01954 17-Jul-10 RACA NEGATIVE 0 2010 Summer Columbus Park 496792 3571699 2258 T 85 U 0 SAGU01924 17-Jul-10 RACA NEGATIVE 0 2010 Summer Columbus Park 496792 3571699 2258 T 85 U 0 SAGU01906 17-Jul-10 RACA NEGATIVE 0 2010 Summer Columbus Park 496792 3571699 2258 T 85 U 0 SAGU01932 17-Jul-10 RACA NEGATIVE 0 2010 Summer Columbus Park 496792 3571699 2258 T 85 U 0 SAGU01952 17-Jul-10 RACA NEGATIVE 0 2010 Summer Columbus Park 496792 3571699 2258 T 85 U 0 SAGU00756 3-Jul-09 RACA NEGATIVE 0 2009 Summer Columbus Park 496792 3571699 2258 A 90 F 30.0 70 0 SAGU00767 3-Jul-09 RACA NEGATIVE 0 2009 Summer Columbus Park 496792 3571699 2258 A 90 F 27.0 65 0 SAGU00780 3-Jul-09 RACA NEGATIVE 0 2009 Summer Columbus Park 496792 3571699 2258 A 90 F 37.0 73 0 SAGU00776 3-Jul-09 RACA NEGATIVE 0 2009 Summer Columbus Park 496792 3571699 2258 J 90 U 14.0 52 0 SAGU00777 3-Jul-09 RACA NEGATIVE 0 2009 Summer Columbus Park 496792 3571699 2258 J 90 U 11.5 54 0 SAGU00758 3-Jul-09 RACA NEGATIVE 0 2009 Summer Columbus Park 496792 3571699 2258 J 90 U 7.0 42 0 SAGU00752 3-Jul-09 RACA NEGATIVE 0 2009 Summer Columbus Park 496792 3571699 2258 J 90 U 7.5 44 0 SAGU00778 3-Jul-09 RACA NEGATIVE 0 2009 Summer Columbus Park 496792 3571699 2258 J 90 U 9.5 44 0 SAGU00757 3-Jul-09 RACA NEGATIVE 0 2009 Summer Columbus Park 496792 3571699 2258 J 90 U 28.0 65 0 SAGU00760 3-Jul-09 RACA NEGATIVE 0 2009 Summer Columbus Park 496792 3571699 2258 J 90 U 5.0 38 0 SAGU00301 18-Mar-09 RACA POSITIVE 26 2009 Spring Crosby 526477 3567242 2637 A 60 M 80.0 90 0 SAGU00095 18-Mar-09 RACA POSITIVE 324 2009 Spring Crosby 526477 3567242 2637 A 60 F 65.0 89 0 SAGU00172 18-Mar-09 RACA POSITIVE 46,428 2009 Spring Crosby 526477 3567242 2637 A 60 F 65.0 89 0 SAGU00320 18-Mar-09 RACA POSITIVE 7,399 2009 Spring Crosby 526477 3567242 2637 A 60 F 180.0 125 0 SAGU00258 18-Mar-09 RACA POSITIVE 15,498 2009 Spring Crosby 526477 3567242 2637 A 60 F 170.0 115 0 SAGU00033 18-Mar-09 RACA POSITIVE 37,854 2009 Spring Crosby 526477 3567242 2637 A 60 F 110.0 105 0 SAGU00424 18-Mar-09 RACA POSITIVE 621 2009 Spring Crosby 526477 3567242 2637 J 60 F 10.0 54 0 SAGU00171 18-Mar-09 RACA POSITIVE 4,834 2009 Spring Crosby 526477 3567242 2637 J 60 F 20.0 63 0 SAGU00300 19-Jun-09 RACA POSITIVE 166 2009 Summer Crosby 526483 3567236 2681 A 69 M 40.0 73 0 SAGU00530 19-Jun-09 RACA POSITIVE 251 2009 Summer Crosby 526483 3567236 2681 A 69 M 35.0 78 0 SAGU00714 19-Jun-09 RACA POSITIVE 1,522 2009 Summer Crosby 526483 3567236 2681 A 69 F 37.0 70 0 SAGU00793 19-Jun-09 RACA POSITIVE 2,738 2009 Summer Crosby 526483 3567236 2681 A 69 M 55.0 76 0 SAGU00070 19-Jun-09 RACA POSITIVE 3,959 2009 Summer Crosby 526483 3567236 2681 A 69 F 25.0 63 0 SAGU00023 19-Jun-09 RACA POSITIVE 8,564 2009 Summer Crosby 526483 3567236 2681 A 69 M 50.0 82 0 SAGU00522 19-Jun-09 RACA POSITIVE 21,568 2009 Summer Crosby 526483 3567236 2681 A 69 F 50.0 84 0 SAGU00787 19-Jun-09 RACA POSITIVE 440 2009 Summer Crosby 526483 3567236 2681 A 69 F 47.0 81 0 SAGU00018 19-Jun-09 RACA POSITIVE 991 2009 Summer Crosby 526483 3567236 2681 A 69 M 200.0 119 0 SAGU00701 19-Jun-09 RACA POSITIVE 1,118 2009 Summer Crosby 526483 3567236 2681 A 69 F 67.0 82 0 SAGU00457 19-Jun-09 RACA POSITIVE 1,410 2009 Summer Crosby 526483 3567236 2681 A 69 M 70.0 83 0

SAGU00708 19-Jun-09 RACA POSITIVE 1,793 2009 Summer Crosby 526483 3567236 2681 A 69 F 57.0 83 0 159 SAGU00801 19-Jun-09 RACA POSITIVE 2,623 2009 Summer Crosby 526483 3567236 2681 A 69 M 117.0 94 0 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU00087 19-Jun-09 RACA POSITIVE 6,521 2009 Summer Crosby 526483 3567236 2681 A 69 F 105.0 104 0 SAGU00525 19-Jun-09 RACA POSITIVE 14,422 2009 Summer Crosby 526483 3567236 2681 A 69 M 120.0 107 0 SAGU00012 19-Jun-09 RACA POSITIVE 33,573 2009 Summer Crosby 526483 3567236 2681 A 69 F 70.0 92 0 SAGU00513 19-Jun-09 RACA POSITIVE 42,285 2009 Summer Crosby 526483 3567236 2681 A 69 M 47.0 72 0 SAGU00693 19-Jun-09 RACA POSITIVE 600 2009 Summer Crosby 526483 3567236 2681 J 69 F 30.0 68 0 SAGU00791 19-Jun-09 RACA POSITIVE 5,872 2009 Summer Crosby 526483 3567236 2681 J 69 F 20.0 61 0 SAGU00021 19-Jun-09 RACA POSITIVE 5,929 2009 Summer Crosby 526483 3567236 2681 J 69 F 27.0 64 0 SAGU00853 19-Jun-09 RACA POSITIVE 45,156 2009 Summer Crosby 526483 3567236 2681 J 69 U 17.0 58 0 SAGU01309 6-Nov-09 RACA POSITIVE 2,507 2009 Fall Crosby 526483 3567236 2681 A 60 F 23.0 65 0 SAGU01461 6-Nov-09 RACA POSITIVE 6,978 2009 Fall Crosby 526483 3567236 2681 A 60 M 95.0 98 0 SAGU01217 6-Nov-09 RACA POSITIVE 8,800 2009 Fall Crosby 526483 3567236 2681 A 60 F 35.0 77 0 SAGU01233 6-Nov-09 RACA POSITIVE 34 2009 Fall Crosby 526483 3567236 2681 J 60 F 14.5 57 0 SAGU01081 6-Nov-09 RACA POSITIVE 88 2009 Fall Crosby 526483 3567236 2681 J 60 U 13.0 52 0 SAGU01216 6-Nov-09 RACA POSITIVE 140 2009 Fall Crosby 526483 3567236 2681 J 60 U 18.5 52 0 SAGU01304 6-Nov-09 RACA POSITIVE 228 2009 Fall Crosby 526483 3567236 2681 J 60 U 14.0 52 0 SAGU01214 6-Nov-09 RACA POSITIVE 430 2009 Fall Crosby 526483 3567236 2681 J 60 U 11.0 47 0 SAGU01443 6-Nov-09 RACA POSITIVE 594 2009 Fall Crosby 526483 3567236 2681 J 60 U 15.0 51 0 SAGU01288 6-Nov-09 RACA POSITIVE 726 2009 Fall Crosby 526483 3567236 2681 J 60 U 12.5 49 0 SAGU01452 6-Nov-09 RACA POSITIVE 1,083 2009 Fall Crosby 526483 3567236 2681 J 60 U 14.0 53 0 SAGU01203 6-Nov-09 RACA POSITIVE 1,299 2009 Fall Crosby 526483 3567236 2681 J 60 U 9.5 47 0 SAGU00568 6-Nov-09 RACA POSITIVE 1,617 2009 Fall Crosby 526483 3567236 2681 J 60 U 17.5 55 0 SAGU01446 6-Nov-09 RACA POSITIVE 2,262 2009 Fall Crosby 526483 3567236 2681 J 60 U 16.5 52 0 SAGU01455 6-Nov-09 RACA POSITIVE 5,755 2009 Fall Crosby 526483 3567236 2681 J 60 U 20.0 62 0 SAGU01209 6-Nov-09 RACA POSITIVE 155,996 2009 Fall Crosby 526483 3567236 2681 J 60 U 16.0 53 0 CHY00094 7-Nov-09 RACA POSITIVE 2,279 2009 Fall Crosby 526483 3567236 2681 T 60 U SAGU01149 18-Mar-10 RACA POSITIVE 101 2010 Spring Crosby 526483 3567236 2681 A 50 M 97.0 102 0 SAGU01141 18-Mar-10 RACA POSITIVE 414 2010 Spring Crosby 526483 3567236 2681 A 50 F 105.0 95 0 SAGU01138 18-Mar-10 RACA POSITIVE 18,756 2010 Spring Crosby 526483 3567236 2681 A 50 M 95.0 90 0 SAGU01142 18-Mar-10 RACA POSITIVE 298 2010 Spring Crosby 526483 3567236 2681 A 50 M 235.0 130 0 SAGU01137 18-Mar-10 RACA POSITIVE 21 2010 Spring Crosby 526483 3567236 2681 J 50 U 16.5 47 0 SAGU01147 18-Mar-10 RACA POSITIVE 73 2010 Spring Crosby 526483 3567236 2681 J 50 U 20.0 53 0 SAGU01133 18-Mar-10 RACA POSITIVE 90 2010 Spring Crosby 526483 3567236 2681 J 50 U 12.5 46 0 SAGU01146 18-Mar-10 RACA POSITIVE 123 2010 Spring Crosby 526483 3567236 2681 J 50 U 17.5 54 0 SAGU01155 18-Mar-10 RACA POSITIVE 369 2010 Spring Crosby 526483 3567236 2681 J 50 U 18.5 55 0 SAGU01152 18-Mar-10 RACA POSITIVE 602 2010 Spring Crosby 526483 3567236 2681 J 50 U 17.5 50 0 SAGU01136 18-Mar-10 RACA POSITIVE 12,094 2010 Spring Crosby 526483 3567236 2681 J 50 U 16.0 52 0 SAGU01159 18-Mar-10 RACA POSITIVE 46,381 2010 Spring Crosby 526483 3567236 2681 J 50 U 12.5 46 0 SAGU01715 15-Jul-10 RACA NEGATIVE 0 2010 Summer Crosby 526483 3567236 2681 A 77 F 172.5 116 0 SAGU01879 15-Jul-10 RACA NEGATIVE 0 2010 Summer Crosby 526483 3567236 2681 A 77 M 156.5 123 0

SAGU01747 15-Jul-10 RACA NEGATIVE 0 2010 Summer Crosby 526483 3567236 2681 A 77 M 147.5 115 0 160 SAGU02096 15-Jul-10 RACA NEGATIVE 0 2010 Summer Crosby 526483 3567236 2681 A 77 U 82.0 87 0 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU02092 15-Jul-10 RACA POSITIVE 8 2010 Summer Crosby 526483 3567236 2681 A 77 U 68.5 96 0 SAGU01722 15-Jul-10 RACA POSITIVE 21 2010 Summer Crosby 526483 3567236 2681 A 77 F 87.5 105 0 SAGU01716 15-Jul-10 RACA POSITIVE 51 2010 Summer Crosby 526483 3567236 2681 A 77 U 117.0 105 0 SAGU01586 15-Jul-10 RACA POSITIVE 56 2010 Summer Crosby 526483 3567236 2681 A 77 M 200.5 124 0 SAGU01701 15-Jul-10 RACA POSITIVE 86 2010 Summer Crosby 526483 3567236 2681 A 77 F 120.0 115 0 SAGU01811 15-Jul-10 RACA POSITIVE 517 2010 Summer Crosby 526483 3567236 2681 A 77 F 97.5 122 0 SAGU01753 15-Jul-10 RACA POSITIVE 640 2010 Summer Crosby 526483 3567236 2681 A 77 F 169.0 110 0 SAGU01873 15-Jul-10 RACA POSITIVE 2,124 2010 Summer Crosby 526483 3567236 2681 A 77 U 75.5 109 0 SAGU01898 15-Jul-10 RACA NEGATIVE 0 2010 Summer Crosby 526483 3567236 2681 A 77 M 350.0 165 0 SAGU01623 15-Jul-10 RACA POSITIVE 11 2010 Summer Crosby 526483 3567236 2681 A 77 M 310.0 150 0 SAGU02009 15-Jul-10 RACA POSITIVE 15 2010 Summer Crosby 526483 3567236 2681 A 77 M 175.0 157 0 SAGU01605 15-Jul-10 RACA POSITIVE 39 2010 Summer Crosby 526483 3567236 2681 A 77 F 210.0 125 0 SAGU01797 15-Jul-10 RACA POSITIVE 42 2010 Summer Crosby 526483 3567236 2681 A 77 F 250.0 160 0 SAGU01732 15-Jul-10 RACA POSITIVE 54 2010 Summer Crosby 526483 3567236 2681 A 77 M 200.0 130 0 SAGU02091 15-Jul-10 RACA POSITIVE 58 2010 Summer Crosby 526483 3567236 2681 A 77 M 185.0 112 0 SAGU01685 15-Jul-10 RACA POSITIVE 155 2010 Summer Crosby 526483 3567236 2681 A 77 F 237.5 151 0 SAGU01731 15-Jul-10 RACA POSITIVE 165 2010 Summer Crosby 526483 3567236 2681 A 77 M 300.0 154 0 SAGU01866 15-Jul-10 RACA POSITIVE 512 2010 Summer Crosby 526483 3567236 2681 A 77 M 192.5 180 0 SAGU01593 15-Jul-10 RACA POSITIVE 827 2010 Summer Crosby 526483 3567236 2681 A 77 M 305.0 172 0 SAGU01650 15-Jul-10 RACA POSITIVE 11,007 2010 Summer Crosby 526483 3567236 2681 A 77 F 186.5 141 0 SAGU00832 13-Jul-09 RACA NEGATIVE 0 2009 Summer Ft Lowell Park 511955 3569297 2444 A 91 F 260.0 134 0 SAGU00814 16-Jul-09 RACA NEGATIVE 0 2009 Summer Ft Lowell Park 512263 3569342 2417 A 87 M 280.0 150 0 SAGU00015 16-Jul-09 RACA NEGATIVE 0 2009 Summer Ft Lowell Park 512263 3569342 2417 T 87 U SAGU00815 16-Jul-09 RACA NEGATIVE 0 2009 Summer Ft Lowell Park 512263 3569342 2417 T 87 U SAGU00817 16-Jul-09 RACA NEGATIVE 0 2009 Summer Ft Lowell Park 512263 3569342 2417 T 87 U SAGU00821 16-Jul-09 RACA NEGATIVE 0 2009 Summer Ft Lowell Park 512263 3569342 2417 T 87 U SAGU00822 16-Jul-09 RACA NEGATIVE 0 2009 Summer Ft Lowell Park 512263 3569342 2417 T 87 U SAGU00823 16-Jul-09 RACA NEGATIVE 0 2009 Summer Ft Lowell Park 512263 3569342 2417 T 87 U SAGU00824 16-Jul-09 RACA NEGATIVE 0 2009 Summer Ft Lowell Park 512263 3569342 2417 T 87 U SAGU00825 16-Jul-09 RACA NEGATIVE 0 2009 Summer Ft Lowell Park 512263 3569342 2417 T 87 U SAGU00826 16-Jul-09 RACA NEGATIVE 0 2009 Summer Ft Lowell Park 512263 3569342 2417 T 87 U SAGU00829 16-Jul-09 RACA NEGATIVE 0 2009 Summer Ft Lowell Park 512263 3569342 2417 T 87 U SAGU00837 16-Jul-09 RACA NEGATIVE 0 2009 Summer Ft Lowell Park 512263 3569342 2417 T 87 U SAGU00833 16-Jul-09 RACA POSITIVE 22 2009 Summer Ft Lowell Park 512263 3569342 2417 T 87 U SAGU01394 20-Mar-10 RACA POSITIVE 14 2010 Spring Ft Lowell Park 512263 3569342 2417 A 53 F 300.0 139 0 SAGU01403 20-Mar-10 RACA POSITIVE 205 2010 Spring Ft Lowell Park 512263 3569342 2417 A 53 F 205.0 128 0 SAGU01135 20-Mar-10 RACA POSITIVE 262 2010 Spring Ft Lowell Park 512263 3569342 2417 A 53 M 350.0 150 0 SAGU01381 20-Mar-10 RACA POSITIVE 400 2010 Spring Ft Lowell Park 512263 3569342 2417 A 53 M 350.0 150 0 SAGU01153 20-Mar-10 RACA POSITIVE 1,128 2010 Spring Ft Lowell Park 512263 3569342 2417 A 53 F 275.0 125 0

SAGU01401 20-Mar-10 RACA POSITIVE 1,429 2010 Spring Ft Lowell Park 512263 3569342 2417 A 53 F 145.0 125 0 161 SAGU01405 20-Mar-10 RACA POSITIVE 2,144 2010 Spring Ft Lowell Park 512263 3569342 2417 A 53 F 275.0 135 0 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU01408 20-Mar-10 RACA POSITIVE 19,444 2010 Spring Ft Lowell Park 512263 3569342 2417 A 53 F 275.0 135 0 SAGU01734 6-Jul-10 RACA NEGATIVE 0 2010 Summer Ft Lowell Park 512263 3569342 2417 A 80 F 340.0 153 0 SAGU01964 6-Jul-10 RACA POSITIVE 59 2010 Summer Ft Lowell Park 512263 3569342 2417 A 80 M 270.0 148 0 SAGU01943 6-Jul-10 RACA POSITIVE 102 2010 Summer Ft Lowell Park 512263 3569342 2417 A 80 F 200.0 132 0 SAGU01279 28-Jun-09 RACA POSITIVE 190 2009 Summer Kennedy Park 499319 3560515 2435 A 88 F 51.0 82 0 SAGU01278 28-Jun-09 RACA NEGATIVE 0 2009 Summer Kennedy Park 499319 3560515 2435 J 88 F 20.0 62 0 SAGU01257 28-Jun-09 RACA NEGATIVE 0 2009 Summer Kennedy Park 499319 3560515 2435 J 88 U 5.5 43 0 SAGU01258 28-Jun-09 RACA NEGATIVE 0 2009 Summer Kennedy Park 499319 3560515 2435 J 88 U 8.5 47 0 SAGU01262 28-Jun-09 RACA NEGATIVE 0 2009 Summer Kennedy Park 499319 3560515 2435 J 88 U 6.0 42 0 SAGU01263 28-Jun-09 RACA NEGATIVE 0 2009 Summer Kennedy Park 499319 3560515 2435 J 88 U 7.0 45 0 SAGU01274 28-Jun-09 RACA NEGATIVE 0 2009 Summer Kennedy Park 499319 3560515 2435 J 88 U 8.0 49 0 SAGU01276 28-Jun-09 RACA NEGATIVE 0 2009 Summer Kennedy Park 499319 3560515 2435 J 88 U 7.5 45 0 SAGU01280 28-Jun-09 RACA NEGATIVE 0 2009 Summer Kennedy Park 499319 3560515 2435 J 88 U 7.0 43 0 SAGU01277 28-Jun-09 RACA POSITIVE 2 2009 Summer Kennedy Park 499319 3560515 2435 J 88 F 18.5 62 0 SAGU00561 10-Oct-09 RACA NEGATIVE 0 2009 Fall Kennedy Park 499317 3560523 2435 A 76 F 25.0 68 0 SAGU00562 10-Oct-09 RACA NEGATIVE 0 2009 Fall Kennedy Park 499317 3560523 2435 A 76 F 31.0 70 0 SAGU00632 10-Oct-09 RACA NEGATIVE 0 2009 Fall Kennedy Park 499317 3560523 2435 A 76 M 50.0 77 0 SAGU00635 10-Oct-09 RACA NEGATIVE 0 2009 Fall Kennedy Park 499317 3560523 2435 J 76 F 17.0 61 0 SAGU01729 1-Jul-10 RACA NEGATIVE 0 2010 Summer Kennedy Park 499317 3560523 2435 A 81 U 50.0 93 0 SAGU00472 30-Jun-09 RACA NEGATIVE 0 2009 Summer TVGR 529239 3567209 2696 A 84 F 25.5 68 0 SAGU00458 30-Jun-09 RACA NEGATIVE 0 2009 Summer TVGR 529239 3567209 2696 A 84 F 15.5 58 0 SAGU00447 30-Jun-09 RACA NEGATIVE 0 2009 Summer TVGR 529239 3567209 2696 A 84 M 240.0 134 0 SAGU00232 30-Jun-09 RACA NEGATIVE 0 2009 Summer TVGR 529239 3567209 2696 J 84 U 8.0 47 0 SAGU00451 30-Jun-09 RACA NEGATIVE 0 2009 Summer TVGR 529239 3567209 2696 J 84 U 5.5 40 0 SAGU01029 30-Jun-09 RACA NEGATIVE 0 2009 Summer TVGR 529239 3567209 2696 J 84 U 4.5 38 0 SAGU00569 30-Jun-09 RACA NEGATIVE 0 2009 Summer TVGR 529239 3567209 2696 J 84 U 5.0 36 0 SAGU00570 30-Jun-09 RACA NEGATIVE 0 2009 Summer TVGR 529239 3567209 2696 J 84 U 6.5 46 0 SAGU00448 30-Jun-09 RACA NEGATIVE 0 2009 Summer TVGR 529239 3567209 2696 J 84 U 10.0 51 0 SAGU00275 30-Jun-09 RACA NEGATIVE 0 2009 Summer TVGR 529239 3567209 2696 J 84 U 5.5 41 0 SAGU00606 30-Jun-09 RACA NEGATIVE 0 2009 Summer TVGR 529239 3567209 2696 T 84 U SAGU00799 30-Jun-09 RACA NEGATIVE 0 2009 Summer TVGR 529239 3567209 2696 T 84 U SAGU01253 30-Jun-09 RACA NEGATIVE 0 2009 Summer TVGR 529239 3567209 2696 T 84 U SAGU00619 30-Jun-09 RACA NEGATIVE 0 2009 Summer TVGR 529239 3567209 2696 T 84 U SAGU00615 30-Jun-09 RACA POSITIVE 2,405 2009 Summer TVGR 529239 3567209 2696 T 84 U SAGU01275 30-Jun-09 RACA POSITIVE 2,545 2009 Summer TVGR 529239 3567209 2696 T 84 U SAGU00618 30-Jun-09 RACA POSITIVE 5,237 2009 Summer TVGR 529239 3567209 2696 T 84 U SAGU00604 30-Jun-09 RACA POSITIVE 8,016 2009 Summer TVGR 529239 3567209 2696 T 84 U SAGU00319 30-Jun-09 RACA POSITIVE 9,115 2009 Summer TVGR 529239 3567209 2696 T 84 U SAGU00092 30-Jun-09 RACA POSITIVE 22,865 2009 Summer TVGR 529239 3567209 2696 T 84 U

SAGU00603 30-Jun-09 RACA POSITIVE 24,254 2009 Summer TVGR 529239 3567209 2696 T 84 U 162 SAGU01462 5-Nov-09 RACA NEGATIVE 0 2009 Fall TVGR 529239 3567209 2696 A 66 F 17.5 57 0 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU01312 5-Nov-09 RACA NEGATIVE 0 2009 Fall TVGR 529239 3567209 2696 A 66 F 22.5 59 0 SAGU01297 5-Nov-09 RACA POSITIVE 214 2009 Fall TVGR 529239 3567209 2696 A 66 F 18.5 58 0 SAGU01291 5-Nov-09 RACA NEGATIVE 0 2009 Fall TVGR 529239 3567209 2696 A 66 F 98.0 112 0 SAGU01298 5-Nov-09 RACA NEGATIVE 0 2009 Fall TVGR 529239 3567209 2696 A 66 F 318.0 137 0 SAGU01306 5-Nov-09 RACA NEGATIVE 0 2009 Fall TVGR 529239 3567209 2696 A 66 M 156.0 156 0 SAGU01301 5-Nov-09 RACA NEGATIVE 0 2009 Fall TVGR 529239 3567209 2696 J 66 U 11.0 47 0 SAGU01308 5-Nov-09 RACA NEGATIVE 0 2009 Fall TVGR 529239 3567209 2696 J 66 U 18.5 42 0 SAGU01311 5-Nov-09 RACA NEGATIVE 0 2009 Fall TVGR 529239 3567209 2696 J 66 U 17.0 39 0 SAGU01307 5-Nov-09 RACA NEGATIVE 0 2009 Fall TVGR 529239 3567209 2696 J 66 U 13.0 49 0 SAGU01319 5-Nov-09 RACA NEGATIVE 0 2009 Fall TVGR 529239 3567209 2696 J 66 U 8.0 41 0 SAGU01318 5-Nov-09 RACA NEGATIVE 0 2009 Fall TVGR 529239 3567209 2696 J 66 U 8.5 43 0 SAGU01320 5-Nov-09 RACA POSITIVE 3 2009 Fall TVGR 529239 3567209 2696 J 66 U 8.0 40 0 SAGU01463 5-Nov-09 RACA POSITIVE 9 2009 Fall TVGR 529239 3567209 2696 J 66 U 17.5 42 0 SAGU01917 16-Jul-10 RACA NEGATIVE 0 2010 Summer TVGR 529239 3567209 2696 A 80 F 190.0 155 0 SAGU01798 16-Jul-10 RACA POSITIVE 17 2010 Summer TVGR 529239 3567209 2696 A 80 F 157.5 120 0 SAGU01767 16-Jul-10 RACA NEGATIVE 0 2010 Summer TVGR 529239 3567209 2696 J 80 U 19.5 59 0 SAGU01717 16-Jul-10 RACA NEGATIVE 0 2010 Summer TVGR 529239 3567209 2696 J 80 U 26.0 65 0 SAGU01817 16-Jul-10 RACA NEGATIVE 0 2010 Summer TVGR 529239 3567209 2696 J 80 U 20.0 53 0 SAGU01691 16-Jul-10 RACA NEGATIVE 0 2010 Summer TVGR 529239 3567209 2696 T 80 U SAGU01724 16-Jul-10 RACA NEGATIVE 0 2010 Summer TVGR 529239 3567209 2696 T 80 U SAGU01880 16-Jul-10 RACA NEGATIVE 0 2010 Summer TVGR 529239 3567209 2696 T 80 U SAGU02018 16-Jul-10 RACA NEGATIVE 0 2010 Summer TVGR 529239 3567209 2696 T 80 U SAGU01810 16-Jul-10 RACA NEGATIVE 0 2010 Summer TVGR 529239 3567209 2696 T 80 U SAGU01944 16-Jul-10 RACA NEGATIVE 0 2010 Summer TVGR 529239 3567209 2696 T 80 U SAGU01802 16-Jul-10 RACA NEGATIVE 0 2010 Summer TVGR 529239 3567209 2696 T 80 U SAGU01854 16-Jul-10 RACA NEGATIVE 0 2010 Summer TVGR 529239 3567209 2696 T 80 U SAGU01744 16-Jul-10 RACA NEGATIVE 0 2010 Summer TVGR 529239 3567209 2696 T 80 U SAGU01646 16-Jul-10 RACA NEGATIVE 0 2010 Summer TVGR 529239 3567209 2696 T 80 U SAGU02040 16-Jul-10 RACA NEGATIVE 0 2010 Summer TVGR 529239 3567209 2696 T 80 U SAGU01786 16-Jul-10 RACA NEGATIVE 0 2010 Summer TVGR 529239 3567209 2696 T 80 U SAGU01872 16-Jul-10 RACA NEGATIVE 0 2010 Summer TVGR 529239 3567209 2696 T 80 U SAGU01858 16-Jul-10 RACA NEGATIVE 0 2010 Summer TVGR 529239 3567209 2696 T 80 U SAGU01778 16-Jul-10 RACA NEGATIVE 0 2010 Summer TVGR 529239 3567209 2696 T 80 U SAGU01860 16-Jul-10 RACA NEGATIVE 0 2010 Summer TVGR 529239 3567209 2696 T 80 U SAGU01848 16-Jul-10 RACA NEGATIVE 0 2010 Summer TVGR 529239 3567209 2696 T 80 U SAGU01939 16-Jul-10 RACA NEGATIVE 0 2010 Summer TVGR 529239 3567209 2696 T 80 U SAGU01727 16-Jul-10 RACA NEGATIVE 0 2010 Summer TVGR 529239 3567209 2696 T 80 U SAGU01930 16-Jul-10 RACA NEGATIVE 0 2010 Summer TVGR 529239 3567209 2696 T 80 U SAGU00343 6-Oct-08 RAYA POSITIVE 1,049 2008 Fall Chimenea 538690 3561186 5384 A 73 F 29.0 60 150

SAGU00228 12-Oct-08 RAYA NEGATIVE 0 2008 Fall Chimenea 536814 3557155 3352 A 70 F 80.0 83 4 163 SAGU00105 12-Oct-08 RAYA NEGATIVE 0 2008 Fall Chimenea 536814 3557155 3352 A 70 M 33.0 67 0 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU00110 12-Oct-08 RAYA NEGATIVE 0 2008 Fall Chimenea 536814 3557155 3352 A 70 M 29.5 62 7 SAGU00194 12-Oct-08 RAYA NEGATIVE 0 2008 Fall Chimenea 536870 3557425 3391 A 72 M 37.0 68 20 SAGU00168 12-Oct-08 RAYA POSITIVE 14 2008 Fall Chimenea 536814 3557155 3352 A 70 M 33.0 65 2 SAGU00217 12-Oct-08 RAYA POSITIVE 19 2008 Fall Chimenea 536870 3557425 3391 A 72 F 42.0 78 12 SAGU00213 12-Oct-08 RAYA POSITIVE 30 2008 Fall Chimenea 536870 3557425 3391 A 72 M 31.0 69 8 SAGU00350 12-Oct-08 RAYA POSITIVE 31 2008 Fall Chimenea 536814 3557155 3352 A 70 F 74.0 81 4 SAGU00229 12-Oct-08 RAYA POSITIVE 8,870 2008 Fall Chimenea 536877 3557491 3402 A 76 M 29.0 58 20 SAGU00193 12-Oct-08 RAYA POSITIVE 17,887 2008 Fall Chimenea 536924 3557681 3444 A 74 M 31.0 67 30 SAGU00103 12-Oct-08 RAYA POSITIVE 74,480 2008 Fall Chimenea 536917 3557639 3454 A 77 M 30.5 64 25 SAGU00240 12-Oct-08 RAYA POSITIVE 76,003 2008 Fall Chimenea 536924 3557681 3444 A 74 M 34.0 63 5 SAGU00164 12-Oct-08 RAYA POSITIVE 116,747 2008 Fall Chimenea 536877 3557491 3402 A 76 M 27.0 63 12 SAGU00102 12-Oct-08 RAYA POSITIVE 122,560 2008 Fall Chimenea 536924 3557681 3444 A 74 F 63.0 78 6 SAGU00107 12-Oct-08 RAYA POSITIVE 307,884 2008 Fall Chimenea 536917 3557639 3454 A 77 F 75.0 80 25 SAGU00090 27-Oct-08 RAYA POSITIVE 5,515 2008 Fall Chimenea 537029 3557789 3508 A 68 F 91.0 83 100 SAGU00082 27-Oct-08 RAYA POSITIVE 15,320 2008 Fall Chimenea 537029 3557789 3508 A 68 M 33.0 54 50 SAGU00084 27-Oct-08 RAYA POSITIVE 6,628 2008 Fall Chimenea 537029 3557789 3508 J 68 M 23.0 58 3 SAGU00081 31-Oct-08 RAYA POSITIVE 6,137 2008 Fall Chimenea 537038 3557841 3553 A 72 M 23.5 54 15 SAGU00061 31-Oct-08 RAYA POSITIVE 43,197 2008 Fall Chimenea 537038 3557841 3553 A 72 M 29.0 60 50 SAGU00078 31-Oct-08 RAYA POSITIVE 922 2008 Fall Chimenea 537038 3557841 3553 A 72 F 75.0 83 25 SAGU00080 31-Oct-08 RAYA POSITIVE 6,213 2008 Fall Chimenea 536915 3557568 3441 A N/A M 33.0 59 20 SAGU00088 31-Oct-08 RAYA POSITIVE 20,855 2008 Fall Chimenea 537038 3557841 3553 A 72 M 35.5 64 40 SAGU00083 31-Oct-08 RAYA POSITIVE 27,616 2008 Fall Chimenea 536925 3557648 3468 A 72 M 30.5 54 10 SAGU00064 31-Oct-08 RAYA POSITIVE 45,101 2008 Fall Chimenea 537038 3557841 3553 A 72 M 34.0 63 15 SAGU00075 31-Oct-08 RAYA POSITIVE 52,203 2008 Fall Chimenea 536915 3557568 3441 A N/A M 30.0 57 20 SAGU00065 31-Oct-08 RAYA POSITIVE 63,171 2008 Fall Chimenea 536892 3557505 3426 A 80 M 34.0 60 3 SAGU00067 31-Oct-08 RAYA POSITIVE 66,254 2008 Fall Chimenea 537038 3557841 3553 A 72 F 61.0 77 8 SAGU00074 31-Oct-08 RAYA POSITIVE 70,749 2008 Fall Chimenea 537038 3557841 3553 A 72 M 32.0 59 8 SAGU00027 25-Jan-09 RAYA POSITIVE 891 2008 Winter Chimenea 536872 3557494 3404 A 60 F 44.0 68 1 SAGU00286 25-Jan-09 RAYA POSITIVE 667,675 2008 Winter Chimenea 536921 3557672 3445 A 60 M 32.0 57 50 SAGU00420 16-Mar-09 RAYA POSITIVE 118,313 2009 Spring Chimenea 536871 3557421 3363 J 61 M 30.0 58 5 SAGU00996 8-Jun-09 RAYA POSITIVE 2,869 2009 Summer Chimenea 536934 3557675 3423 A 73 M 34.0 65 40 SAGU00961 8-Jun-09 RAYA NEGATIVE 0 2009 Summer Chimenea 536893 3557503 3423 J 81 U 3.5 32 0 SAGU00974 8-Jun-09 RAYA NEGATIVE 0 2009 Summer Chimenea 536893 3557503 3423 J 81 U 5.0 35 0 SAGU00978 8-Jun-09 RAYA NEGATIVE 0 2009 Summer Chimenea 536893 3557503 3423 J 81 U 3.0 33 0 SAGU01012 8-Jun-09 RAYA POSITIVE 4 2009 Summer Chimenea 536919 3557594 3417 J 82 U 5.0 36 0 SAGU00970 8-Jun-09 RAYA POSITIVE 6 2009 Summer Chimenea 536893 3557503 3423 J 81 U 6.0 39 1 SAGU00981 8-Jun-09 RAYA POSITIVE 14 2009 Summer Chimenea 536893 3557503 3423 J 81 U 4.5 36 0 SAGU01015 8-Jun-09 RAYA POSITIVE 37 2009 Summer Chimenea 536934 3557675 3423 J 73 U 5.0 35 0 SAGU00963 8-Jun-09 RAYA POSITIVE 44 2009 Summer Chimenea 536808 3557160 3343 J 70 U 11.0 48 3

SAGU00992 8-Jun-09 RAYA POSITIVE 75 2009 Summer Chimenea 536934 3557675 3423 J 73 U 6.0 37 0 164 SAGU00975 8-Jun-09 RAYA POSITIVE 174 2009 Summer Chimenea 536893 3557503 3423 J 81 U 5.5 38 0 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU00984 8-Jun-09 RAYA POSITIVE 243 2009 Summer Chimenea 536808 3557160 3343 J 70 U 7.0 39 1 SAGU00967 8-Jun-09 RAYA POSITIVE 254 2009 Summer Chimenea 536808 3557160 3343 J 70 U 7.0 40 0 SAGU00983 8-Jun-09 RAYA POSITIVE 418 2009 Summer Chimenea 536808 3557160 3343 J 70 U 8.5 44 0 SAGU01018 8-Jun-09 RAYA POSITIVE 847 2009 Summer Chimenea 536934 3557675 3423 J 73 U 5.0 30 0 SAGU00976 8-Jun-09 RAYA POSITIVE 1,057 2009 Summer Chimenea 536900 3556955 3314 J U 0 SAGU01000 8-Jun-09 RAYA POSITIVE 1,076 2009 Summer Chimenea 536934 3557675 3423 J 73 U 5.0 34 0 SAGU00962 8-Jun-09 RAYA POSITIVE 3,556 2009 Summer Chimenea 536808 3557160 3343 J 70 U 3.5 32 0 SAGU00997 9-Jun-09 RAYA POSITIVE 30 2009 Summer Chimenea 536906 3556954 3314 A 71 M 35.0 62 10 SAGU01025 9-Jun-09 RAYA POSITIVE 1 2009 Summer Chimenea 537020 3557774 3485 J 85 U 6.0 40 0 SAGU01037 9-Jun-09 RAYA POSITIVE 3 2009 Summer Chimenea 537020 3557774 3485 J 85 U 2.0 26 0 SAGU00994 9-Jun-09 RAYA POSITIVE 98 2009 Summer Chimenea 536861 3557443 3444 J 78 U 4.0 35 0 SAGU00995 9-Jun-09 RAYA POSITIVE 144 2009 Summer Chimenea 536906 3556954 3314 J 71 U 4.0 35 0 SAGU01016 9-Jun-09 RAYA POSITIVE 464 2009 Summer Chimenea 536906 3556954 3314 J 71 U 4.0 35 0 SAGU01007 9-Jun-09 RAYA POSITIVE 609 2009 Summer Chimenea 536906 3556954 3314 J 71 U 4.0 34 0 SAGU00999 9-Jun-09 RAYA POSITIVE 827 2009 Summer Chimenea 536861 3557443 3444 J 78 U 5.0 36 0 SAGU00993 9-Jun-09 RAYA POSITIVE 1,119 2009 Summer Chimenea 536906 3556954 3314 J 71 U 5.0 35 0 SAGU01017 9-Jun-09 RAYA POSITIVE 1,510 2009 Summer Chimenea 536906 3556954 3314 J 71 U 4.0 36 0 SAGU01047 9-Jun-09 RAYA POSITIVE 2,530 2009 Summer Chimenea 537020 3557774 3485 J 85 U 6.0 40 0 SAGU01013 9-Jun-09 RAYA POSITIVE 6,617 2009 Summer Chimenea 536861 3557443 3444 J 78 U 6.5 40 0 SAGU01009 9-Jun-09 RAYA POSITIVE 6,867 2009 Summer Chimenea 536906 3556954 3314 J 71 U 4.0 35 0 SAGU00998 9-Jun-09 RAYA POSITIVE 16,087 2009 Summer Chimenea 536861 3557443 3444 J 78 U 6.5 39 0 SAGU01036 9-Jun-09 RAYA POSITIVE 34,842 2009 Summer Chimenea 537020 3557774 3485 J 85 U 5.0 36 0 SAGU01001 9-Jun-09 RAYA NEGATIVE 0 2009 Summer Chimenea 536886 3557491 3407 T 75 U 3.0 SAGU01049 9-Jun-09 RAYA NEGATIVE 0 2009 Summer Chimenea 536886 3557491 3407 T 75 U 2.0 SAGU01026 9-Jun-09 RAYA NEGATIVE 0 2009 Summer Chimenea 536886 3557491 3407 T 75 U 1.5 SAGU01046 9-Jun-09 RAYA NEGATIVE 0 2009 Summer Chimenea 536886 3557491 3407 T 75 U 1.0 SAGU01027 9-Jun-09 RAYA NEGATIVE 0 2009 Summer Chimenea 536886 3557491 3407 T 75 U 1.5 SAGU01043 9-Jun-09 RAYA NEGATIVE 0 2009 Summer Chimenea 536886 3557491 3407 T 75 U 2.0 SAGU01050 9-Jun-09 RAYA NEGATIVE 0 2009 Summer Chimenea 536886 3557491 3407 T 75 U 1.0 SAGU01041 9-Jun-09 RAYA NEGATIVE 0 2009 Summer Chimenea 536886 3557491 3407 T 75 U 1.5 SAGU01038 9-Jun-09 RAYA POSITIVE 1,143 2009 Summer Chimenea 536886 3557491 3407 T 75 U 2.0 SAGU01040 9-Jun-09 RAYA POSITIVE 238,867 2009 Summer Chimenea 536886 3557491 3407 T 75 U 2.0 SAGU01073 2-Oct-09 RAYA POSITIVE 202 2009 Fall Chimenea 537099 3559823 4321 A 76 F 21.0 61 20 SAGU01061 2-Oct-09 RAYA POSITIVE 1,040 2009 Fall Chimenea 537099 3559823 4321 A 76 M 16.0 52 10 SAGU01439 2-Oct-09 RAYA POSITIVE 1,290 2009 Fall Chimenea 537099 3559823 4321 A 76 F 84.0 81 20 SAGU01440 2-Oct-09 RAYA POSITIVE 8,901 2009 Fall Chimenea 537099 3559823 4321 A 76 F 23.0 60 20 SAGU01110 5-Oct-09 RAYA POSITIVE 3 2009 Fall Chimenea 536936 3557673 3473 A 80 F 13.0 47 5 SAGU01114 5-Oct-09 RAYA POSITIVE 5 2009 Fall Chimenea 536936 3557673 3473 A 80 M 14.0 47 30 SAGU01113 5-Oct-09 RAYA POSITIVE 247 2009 Fall Chimenea 536936 3557673 3473 A 80 M 13.5 49 1

SAGU01130 5-Oct-09 RAYA POSITIVE 15,075 2009 Fall Chimenea 536936 3557673 3473 A 80 M 15.5 51 50 165 SAGU01128 5-Oct-09 RAYA POSITIVE 6 2009 Fall Chimenea 536936 3557673 3473 A 80 M 22.0 54 10 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU01104 5-Oct-09 RAYA POSITIVE 7 2009 Fall Chimenea 536936 3557673 3473 A 80 M 17.5 53 3 SAGU01115 5-Oct-09 RAYA POSITIVE 11 2009 Fall Chimenea 536936 3557673 3473 A 80 M 32.5 61 10 SAGU01118 5-Oct-09 RAYA POSITIVE 26 2009 Fall Chimenea 536936 3557673 3473 A 80 M 36.5 68 20 SAGU01120 5-Oct-09 RAYA POSITIVE 131 2009 Fall Chimenea 536936 3557673 3473 A 80 F 20.5 55 10 SAGU01121 5-Oct-09 RAYA POSITIVE 21,979 2009 Fall Chimenea 537038 3557795 3540 A 76 M 18.0 53 2 SAGU00754 5-Oct-09 RAYA POSITIVE 5,252 2009 Fall Chimenea 537040 3557865 3640 J 72 M 10.0 46 5 SAGU00751 10-Oct-09 RAYA POSITIVE 1 2009 Fall Chimenea 536909 3557519 3414 A 70 M 14.5 49 5 SAGU01123 10-Oct-09 RAYA POSITIVE 7 2009 Fall Chimenea 536909 3557519 3414 A 70 M 12.0 47 4 SAGU00567 10-Oct-09 RAYA POSITIVE 43 2009 Fall Chimenea 536909 3557519 3414 A 70 M 13.5 46 0 SAGU01107 10-Oct-09 RAYA POSITIVE 67 2009 Fall Chimenea 536909 3557519 3414 A 70 M 17.5 50 6 SAGU01268 10-Oct-09 RAYA POSITIVE 695 2009 Fall Chimenea 536909 3557519 3414 A 70 F 11.0 44 2 SAGU01106 10-Oct-09 RAYA POSITIVE 16,532 2009 Fall Chimenea 536909 3557519 3414 A 70 M 17.5 53 4 SAGU00753 10-Oct-09 RAYA POSITIVE 17,755 2009 Fall Chimenea 536909 3557519 3414 A 70 M 15.0 47 0 SAGU01267 10-Oct-09 RAYA POSITIVE 55,707 2009 Fall Chimenea 536909 3557519 3414 A 70 M 10.5 44 8 SAGU00813 10-Oct-09 RAYA POSITIVE 8 2009 Fall Chimenea 536909 3557519 3414 A 70 M 22.5 53 30 SAGU01122 10-Oct-09 RAYA POSITIVE 11 2009 Fall Chimenea 536909 3557519 3414 A 70 M 18.0 51 1 SAGU01374 10-Oct-09 RAYA POSITIVE 23 2009 Fall Chimenea 536909 3557519 3414 A 70 M 13.5 48 3 SAGU01373 10-Oct-09 RAYA POSITIVE 37 2009 Fall Chimenea 536909 3557519 3414 A 70 F 54.0 81 10 SAGU00819 10-Oct-09 RAYA POSITIVE 563 2009 Fall Chimenea 536909 3557519 3414 A 70 F 27.0 59 3 SAGU01378 10-Oct-09 RAYA POSITIVE 855 2009 Fall Chimenea 536909 3557519 3414 A 70 M 28.0 53 5 SAGU00634 10-Oct-09 RAYA POSITIVE 1,943 2009 Fall Chimenea 536909 3557519 3414 A 70 F 77.0 81 20 SAGU01116 10-Oct-09 RAYA POSITIVE 5,026 2009 Fall Chimenea 536909 3557519 3414 A 70 M 22.0 53 50 SAGU01103 10-Oct-09 RAYA POSITIVE 18,156 2009 Fall Chimenea 536909 3557519 3414 A 70 M 15.5 48 6 SAGU01124 10-Oct-09 RAYA POSITIVE 133 2009 Fall Chimenea 536909 3557519 3414 J 70 M 10.0 42 1 SAGU01293 10-Jan-10 RAYA POSITIVE 12,264 2009 Winter Chimenea 536900 3557528 3419 A 60 F 58 SAGU00930 10-Jan-10 RAYA POSITIVE 64,965 2009 Winter Chimenea 536928 3557691 3446 A F 73.0 87 10 SAGU00927 10-Jan-10 RAYA POSITIVE 149,182 2009 Winter Chimenea 536920 3557553 3414 A 60 M 57 4 SAGU02095 10-Jun-10 RAYA NEGATIVE 0 2010 Summer Chimenea 536901 3557513 3432 J 78 U 7.5 37 0 SAGU02054 10-Jun-10 RAYA NEGATIVE 0 2010 Summer Chimenea 536901 3557513 3432 J 78 U 5.5 34 0 SAGU02067 10-Jun-10 RAYA NEGATIVE 0 2010 Summer Chimenea 536879 3557493 3430 J 85 U 6.5 35 1 SAGU02032 10-Jun-10 RAYA NEGATIVE 0 2010 Summer Chimenea 536879 3557493 3430 J 85 U 4.5 33 0 SAGU01647 10-Jun-10 RAYA POSITIVE 12 2010 Summer Chimenea 536901 3557513 3432 J 78 U 7.0 38 1 SAGU02029 10-Jun-10 RAYA POSITIVE 41 2010 Summer Chimenea 536874 3557425 3428 J 74 U 6.5 32 0 SAGU02011 10-Jun-10 RAYA POSITIVE 65 2010 Summer Chimenea 536874 3557425 3428 J 74 U 6.5 34 0 SAGU02049 13-Jul-10 RAYA NEGATIVE 0 2010 Summer Chimenea 536909 3557519 3414 J 82 U 6.0 33 0 SAGU01862 13-Jul-10 RAYA NEGATIVE 0 2010 Summer Chimenea 536909 3557519 3414 J 82 U 7.5 35 0 SAGU02034 13-Jul-10 RAYA NEGATIVE 0 2010 Summer Chimenea 536909 3557519 3414 J 82 U 8.0 35 0 SAGU02050 13-Jul-10 RAYA NEGATIVE 0 2010 Summer Chimenea 536909 3557519 3414 J 82 U 5.5 35 0 SAGU00163 8-Oct-08 RAYA NEGATIVE 0 2008 Fall Tanque Verde 538896 3568552 4268 A 81 M 70.5 82 100

SAGU00326 8-Oct-08 RAYA NEGATIVE 0 2008 Fall Tanque Verde 538896 3568552 4268 A 81 M 70.5 82 100 166 SAGU00292 8-Oct-08 RAYA POSITIVE 73 2008 Fall Tanque Verde 538067 3569196 3884 A 53 F 61.0 83 50 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU00411 17-Oct-08 RAYA POSITIVE 3 2008 Fall Tanque Verde 534131 3569561 3403 A 76 M 23.0 60 15 SAGU00288 17-Oct-08 RAYA POSITIVE 16,109 2008 Fall Tanque Verde 534131 3569561 3403 A 76 F 24.0 58 3 SAGU00475 17-Oct-08 RAYA POSITIVE 32,943 2008 Fall Tanque Verde 533620 3569280 3371 A 70 M 27.0 68 50 SAGU00303 17-Oct-08 RAYA POSITIVE 160,282 2008 Fall Tanque Verde 532908 3569016 3191 A 62 M 30.0 65 50 SAGU00434 17-Oct-08 RAYA POSITIVE 3,734 2008 Fall Tanque Verde 534131 3569561 3403 A 76 F 55.0 79 15 SAGU00290 17-Oct-08 RAYA POSITIVE 18,531 2008 Fall Tanque Verde 533142 3569098 3292 A 76 F 48.0 80 40 SAGU00233 17-Oct-08 RAYA POSITIVE 21,728 2008 Fall Tanque Verde 534131 3569561 3403 A 76 F 28.0 65 60 SAGU00443 17-Oct-08 RAYA POSITIVE 29,648 2008 Fall Tanque Verde 533142 3569098 3292 A 76 M 28.5 60 25 SAGU00464 17-Oct-08 RAYA POSITIVE 63,244 2008 Fall Tanque Verde 534131 3569561 3403 A 76 M 29.0 64 5 SAGU00456 17-Oct-08 RAYA POSITIVE 72,717 2008 Fall Tanque Verde 534131 3569561 3403 A 76 F 64.0 87 30 SAGU00482 17-Oct-08 RAYA POSITIVE 86,141 2008 Fall Tanque Verde 533620 3569280 3371 A 70 F 53.0 78 40 SAGU00391 17-Oct-08 RAYA POSITIVE 110,025 2008 Fall Tanque Verde 534131 3569561 3403 A 76 M 30.0 66 30 SAGU00388 17-Oct-08 RAYA POSITIVE 143,645 2008 Fall Tanque Verde 532961 3569004 3257 A 60 F 65.0 79 40 SAGU00426 17-Oct-08 RAYA POSITIVE 192,338 2008 Fall Tanque Verde 533620 3569280 3371 A 70 F 61.0 79 30 SAGU00387 18-Oct-08 RAYA POSITIVE 78,112 2008 Fall Tanque Verde 535602 3569761 3575 A 78 M 31.0 61 90 SAGU00283 18-Oct-08 RAYA POSITIVE 249,092 2008 Fall Tanque Verde 535602 3569761 3575 A 78 M 33.0 66 60 SAGU00312 18-Oct-08 RAYA POSITIVE 2,079 2008 Fall Tanque Verde 535418 3569792 3563 A 70 F 69.0 75 25 SAGU00274 18-Oct-08 RAYA POSITIVE 13,433 2008 Fall Tanque Verde 535602 3569761 3575 A 78 F 67.5 82 90 SAGU00460 18-Oct-08 RAYA POSITIVE 33,371 2008 Fall Tanque Verde 534870 3569546 3477 A 68 F 66.0 85 30 SAGU00462 18-Oct-08 RAYA POSITIVE 71,364 2008 Fall Tanque Verde 535466 3569792 3565 A 72 F 54.0 78 80 SAGU00237 18-Oct-08 RAYA POSITIVE 106,638 2008 Fall Tanque Verde 535602 3569761 3575 A 78 F 46.0 75 20 SAGU00297 18-Oct-08 RAYA POSITIVE 118,912 2008 Fall Tanque Verde 535091 3569611 3491 A 71 F 49.5 69 50 SAGU00165 18-Oct-08 RAYA POSITIVE 123,842 2008 Fall Tanque Verde 534870 3569546 3477 A 68 F 64.0 80 50 SAGU00130 18-Oct-08 RAYA POSITIVE 161,719 2008 Fall Tanque Verde 535602 3569761 3575 A 78 F 48.0 78 80 SAGU00400 18-Oct-08 RAYA POSITIVE 289,640 2008 Fall Tanque Verde 534870 3569546 3477 A 68 M 35.0 64 10 SAGU00189 18-Oct-08 RAYA POSITIVE 451,161 2008 Fall Tanque Verde 535091 3569611 3491 A 71 M 30.5 64 50 SAGU00230 18-Oct-08 RAYA POSITIVE 667,285 2008 Fall Tanque Verde 534870 3569546 3477 A 68 M 37.0 67 30 SAGU00295 18-Oct-08 RAYA POSITIVE 792,477 2008 Fall Tanque Verde 534870 3569546 3477 A 68 F 42.0 69 20 SAGU00435 18-Oct-08 RAYA POSITIVE 869,461 2008 Fall Tanque Verde 534870 3569546 3477 A 68 M 25.0 62 5 SAGU00221 18-Oct-08 RAYA POSITIVE 1,526,741 2008 Fall Tanque Verde 534870 3569546 3477 A 68 F 56.0 76 1 SAGU00869 16-Jun-09 RAYA POSITIVE 76 2009 Summer Tanque Verde 532915 3569014 3340 J 85 U 7.0 38 0 SAGU00865 16-Jun-09 RAYA POSITIVE 90 2009 Summer Tanque Verde 533476 3569104 3288 J 75 U 7.5 38 0 SAGU00860 16-Jun-09 RAYA POSITIVE 134 2009 Summer Tanque Verde 533473 3569088 3320 J 70 U 4.0 32 0 SAGU00847 16-Jun-09 RAYA POSITIVE 525 2009 Summer Tanque Verde 534174 3569533 3395 J 84 U 7.0 38 0 SAGU00868 16-Jun-09 RAYA POSITIVE 755 2009 Summer Tanque Verde 533149 3569100 3251 J 90 U 6.0 38 0 SAGU00867 16-Jun-09 RAYA POSITIVE 2,882 2009 Summer Tanque Verde 533149 3569100 3251 J 90 U 5.5 38 5 SAGU00841 16-Jun-09 RAYA POSITIVE 4,744 2009 Summer Tanque Verde 533149 3569100 3251 J 90 U 7.0 41 5 SAGU00842 16-Jun-09 RAYA POSITIVE 9,931 2009 Summer Tanque Verde 533259 3569094 3275 J 79 U 5.0 36 3 SAGU00870 16-Jun-09 RAYA POSITIVE 16,899 2009 Summer Tanque Verde 533473 3569088 3320 J 70 U 5.0 38 0

SAGU00843 16-Jun-09 RAYA POSITIVE 20,232 2009 Summer Tanque Verde 533157 3569098 J 78 U 7.0 40 20 167 SAGU00855 16-Jun-09 RAYA POSITIVE 41,707 2009 Summer Tanque Verde 534193 3569525 3357 J 83 U 5.5 37 0 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU00854 16-Jun-09 RAYA POSITIVE 46,225 2009 Summer Tanque Verde 533473 3569088 3320 J 70 U 6.0 39 0 SAGU00861 16-Jun-09 RAYA POSITIVE 81,291 2009 Summer Tanque Verde 532915 3569014 3340 J 85 U 8.5 45 1 SAGU00844 16-Jun-09 RAYA POSITIVE 112,976 2009 Summer Tanque Verde 533259 3569094 3275 J 79 U 5.0 38 5 SAGU00848 16-Jun-09 RAYA POSITIVE 23,504 2009 Summer Tanque Verde 534193 3569525 3357 T 83 U 6.0 SAGU00856 16-Jun-09 RAYA POSITIVE 43,221 2009 Summer Tanque Verde 534193 3569525 3357 T 83 U 8.0 SAGU00802 17-Jun-09 RAYA POSITIVE 2,102 2009 Summer Tanque Verde 534672 3569573 3484 J 76 U 8.0 43 0 SAGU00586 17-Jun-09 RAYA POSITIVE 9,217 2009 Summer Tanque Verde 534672 3569573 3484 J 76 U 9.5 46 10 SAGU00588 17-Jun-09 RAYA POSITIVE 13,553 2009 Summer Tanque Verde 534672 3569573 3484 J 76 U 8.5 43 0 SAGU00578 17-Jun-09 RAYA POSITIVE 17,583 2009 Summer Tanque Verde 534672 3569573 3484 J 76 U 8.0 40 0 SAGU00806 17-Jun-09 RAYA POSITIVE 20,845 2009 Summer Tanque Verde 534672 3569573 3484 J 76 U 7.5 43 1 SAGU00851 17-Jun-09 RAYA POSITIVE 22,635 2009 Summer Tanque Verde 534672 3569573 3484 J 76 U 7.5 39 8 SAGU00592 17-Jun-09 RAYA POSITIVE 107,105 2009 Summer Tanque Verde 534672 3569573 3484 J 76 U 8.0 41 0 SAGU00596 17-Jun-09 RAYA POSITIVE 119,933 2009 Summer Tanque Verde 534672 3569573 3484 J 76 U 7.5 41 5 SAGU00804 17-Jun-09 RAYA NEGATIVE 0 2009 Summer Tanque Verde 534875 3569543 3477 T 85 U N/A SAGU00858 17-Jun-09 RAYA NEGATIVE 0 2009 Summer Tanque Verde 534875 3569543 3477 T 85 U 12.0 SAGU00792 17-Jun-09 RAYA POSITIVE 448 2009 Summer Tanque Verde 534875 3569543 3477 T 85 U 15.0 SAGU00788 17-Jun-09 RAYA POSITIVE 469 2009 Summer Tanque Verde 534875 3569543 3477 T 85 U 15.0 SAGU00808 17-Jun-09 RAYA POSITIVE 37,715 2009 Summer Tanque Verde 534875 3569543 3477 T 85 U 10.0 SAGU00816 22-Jun-09 RAYA POSITIVE 486,913 2009 Summer Tanque Verde 539602 3568638 4409 A 80 M 30.5 58 30 SAGU00835 22-Jun-09 RAYA POSITIVE 1,470,188 2009 Summer Tanque Verde 539602 3568638 4409 A 80 M 25.5 58 30 SAGU00906 7-Apr-10 RAYA NEGATIVE 0 2010 Spring Tanque Verde 534211 3569510 3424 A 61 M 28.5 56 10 SAGU01588 8-Jun-10 RAYA POSITIVE 1,167 2010 Summer Tanque Verde 534679 3569564 3439 T 80 U 0 SAGU01283 20-Oct-09 RAYA NEGATIVE 0 2009 Fall Tanque Verde 534166 3569556 3456 A 76 M 15.0 53 10 SAGU01460 20-Oct-09 RAYA POSITIVE 630 2009 Fall Tanque Verde 532924 3569016 3234 A 73 M 25.0 62 2 SAGU00638 20-Oct-09 RAYA POSITIVE 122,059 2009 Fall Tanque Verde 533401 3569215 3456 A 70 F 35.0 62 30 SAGU01207 26-Oct-09 RAYA POSITIVE 905 2009 Fall Tanque Verde 534166 3569556 3456 A 70 M 21.5 54 0 SAGU01212 26-Oct-09 RAYA POSITIVE 1,070 2009 Fall Tanque Verde 534166 3569556 3456 A 70 M 25.0 53 10 SAGU01193 26-Oct-09 RAYA POSITIVE 38,679 2009 Fall Tanque Verde 534652 3569600 3508 A 63 M 20.0 58 10 SAGU01219 26-Oct-09 RAYA POSITIVE 109,738 2009 Fall Tanque Verde 534652 3569600 3508 A 63 M 26.0 60 10 SAGU00147 1-Oct-08 RAYA NEGATIVE 0 2008 Fall Wildhorse 530225 3563487 3346 A 82 M 44.5 75 25 SAGU00493 1-Oct-08 RAYA NEGATIVE 0 2008 Fall Wildhorse 530225 3563487 3346 A 82 M 27.0 63 4 SAGU00251 1-Oct-08 RAYA NEGATIVE 0 2008 Fall Wildhorse 529888 3563725 3200 A 72 F 95.0 90 N/A SAGU00255 1-Oct-08 RAYA NEGATIVE 0 2008 Fall Wildhorse 529888 3563725 3200 A 72 M 76.0 82 30 SAGU00253 1-Oct-08 RAYA NEGATIVE 0 2008 Fall Wildhorse 529888 3563725 3200 A 72 M 40.5 68 150 SAGU00190 1-Oct-08 RAYA NEGATIVE 0 2008 Fall Wildhorse 529879 3563665 3188 A 80 M 72.0 88 30 SAGU00123 1-Oct-08 RAYA NEGATIVE 0 2008 Fall Wildhorse 530091 3563480 3283 A 75 M 44.0 80 30 SAGU00126 1-Oct-08 RAYA NEGATIVE 0 2008 Fall Wildhorse 530140 3563430 3309 A 85 M 58.0 84 20 SAGU00184 1-Oct-08 RAYA NEGATIVE 0 2008 Fall Wildhorse 530128 3563429 3290 A 83 M 70.0 93 50 SAGU00257 1-Oct-08 RAYA POSITIVE 5 2008 Fall Wildhorse 529737 3563871 3112 A 72 M 80.0 86 25

SAGU00259 1-Oct-08 RAYA POSITIVE 6 2008 Fall Wildhorse 529770 3563843 3136 A 71 M 84.0 90 10 168 SAGU00134 1-Oct-08 RAYA POSITIVE 7 2008 Fall Wildhorse 530091 3563480 3283 A 75 M 66.0 83 30 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU00182 1-Oct-08 RAYA NEGATIVE 0 2008 Fall Wildhorse 529836 3563790 3166 J 79 U 11.2 52 6 SAGU00112 1-Oct-08 RAYA POSITIVE 5 2008 Fall Wildhorse 529710 3563881 3105 J 66 U 10.5 52 25 SAGU00187 1-Oct-08 RAYA POSITIVE 13 2008 Fall Wildhorse 529992 3563599 3199 J 81 M 15.0 82 6 SAGU00179 11-Oct-08 RAYA NEGATIVE 0 2008 Fall Wildhorse 529073 3564232 2882 A 74,71,70 F 10.0 49 7 SAGU00177 11-Oct-08 RAYA NEGATIVE 0 2008 Fall Wildhorse 529073 3564232 2882 A 74,71,70 M 16.0 57 5 SAGU00160 11-Oct-08 RAYA NEGATIVE 0 2008 Fall Wildhorse 529073 3564232 2882 A 74,71,70 M 15.0 55 1 SAGU00178 11-Oct-08 RAYA NEGATIVE 0 2008 Fall Wildhorse 529073 3564232 2882 A 74,71,70 M 18.5 56 25 SAGU00180 11-Oct-08 RAYA NEGATIVE 0 2008 Fall Wildhorse 529452 3564069 3005 A 78 M 20.0 58 20 SAGU00152 11-Oct-08 RAYA NEGATIVE 0 2008 Fall Wildhorse 529452 3564069 3005 A 78 M 17.0 62 7 SAGU00153 11-Oct-08 RAYA NEGATIVE 0 2008 Fall Wildhorse 529459 3564068 3049 A 75 M 15.0 54 3 SAGU00176 11-Oct-08 RAYA NEGATIVE 0 2008 Fall Wildhorse 529073 3564232 2882 A 74,71,70 F 48.0 78 8 SAGU00155 11-Oct-08 RAYA NEGATIVE 0 2008 Fall Wildhorse 529073 3564232 2882 A 74,71,70 F 28.0 69 5 SAGU00173 11-Oct-08 RAYA NEGATIVE 0 2008 Fall Wildhorse 529073 3564232 2882 A 74,71,70 F 69.0 82 15 SAGU00151 11-Oct-08 RAYA NEGATIVE 0 2008 Fall Wildhorse 529526 3563878 3082 A 78 M 34.0 67 16 SAGU00156 11-Oct-08 RAYA NEGATIVE 0 2008 Fall Wildhorse 529478 3564033 3035 J 78 M 16.0 57 6 SAGU00474 22-Oct-08 RAYA NEGATIVE 0 2008 Fall Wildhorse 531243 3563340 4041 A 62 F 68.0 72 125 SAGU00465 22-Oct-08 RAYA NEGATIVE 0 2008 Fall Wildhorse 530756 3563403 3694 A 68 F 42.0 68 60 SAGU00437 22-Oct-08 RAYA NEGATIVE 0 2008 Fall Wildhorse 530644 3563348 3518 A 59 F 40.0 64 20 SAGU00423 22-Oct-08 RAYA NEGATIVE 0 2008 Fall Wildhorse 530644 3563348 3518 J 59 M 25.0 61 15 SAGU00014 5-Feb-09 RAYA POSITIVE 116 2008 Winter Wildhorse 529732 3563874 3115 J 62 M 15.0 48 2 SAGU00538 20-Mar-09 RAYA NEGATIVE 0 2009 Spring Wildhorse 529744 3563857 3120 A 78 M 16.5 52 10 SAGU00508 20-Mar-09 RAYA NEGATIVE 0 2009 Spring Wildhorse 529807 3563823 3162 A 75 M 17.5 54 8 SAGU00517 20-Mar-09 RAYA NEGATIVE 0 2009 Spring Wildhorse 529877 3563730 3189 A 70 F 69.0 85 40 SAGU00549 20-Mar-09 RAYA NEGATIVE 0 2009 Spring Wildhorse 529405 3564094 2980 A 72 F 35.0 68 10 SAGU00541 20-Mar-09 RAYA NEGATIVE 0 2009 Spring Wildhorse 529481 3564022 A 72 F 75.0 80 7 SAGU00553 20-Mar-09 RAYA NEGATIVE 0 2009 Spring Wildhorse 529568 3563868 A 72 F 31.0 71 15 SAGU00527 20-Mar-09 RAYA NEGATIVE 0 2009 Spring Wildhorse 529807 3563823 3162 A 75 F 66.0 82 15 SAGU00519 20-Mar-09 RAYA NEGATIVE 0 2009 Spring Wildhorse 529877 3563730 3189 A 70 M 34.0 62 60 SAGU00537 20-Mar-09 RAYA NEGATIVE 0 2009 Spring Wildhorse 529723 3563877 3104 A 76 M 27.0 64 40 SAGU00543 20-Mar-09 RAYA NEGATIVE 0 2009 Spring Wildhorse 529070 3564236 2865 J 64 M 17.0 56 5 SAGU00546 20-Mar-09 RAYA NEGATIVE 0 2009 Spring Wildhorse 529070 3564236 2865 J 64 M 14.0 46 8 SAGU00547 20-Mar-09 RAYA NEGATIVE 0 2009 Spring Wildhorse 529568 3563868 J 72 M 16.0 54 8 SAGU00559 20-Mar-09 RAYA NEGATIVE 0 2009 Spring Wildhorse 529723 3563877 3104 J 76 M 17.0 53 15 SAGU00510 20-Mar-09 RAYA NEGATIVE 0 2009 Spring Wildhorse 529877 3563730 3189 J 70 U 5.0 35 0 SAGU00558 4-Apr-09 RAYA NEGATIVE 0 2009 Spring Wildhorse 530162 3563442 3291 A 66 M 17.0 54 7 SAGU00010 4-Apr-09 RAYA NEGATIVE 0 2009 Spring Wildhorse 530186 3563472 3307 A 63 F 45.0 69 30 SAGU00539 4-Apr-09 RAYA NEGATIVE 0 2009 Spring Wildhorse 530165 3563454 A 62 M 25.0 62 100 SAGU00556 4-Apr-09 RAYA NEGATIVE 0 2009 Spring Wildhorse 530176 3563457 3298 A 68 M 22.0 62 60 SAGU00471 4-Apr-09 RAYA NEGATIVE 0 2009 Spring Wildhorse 529880 3563727 3188 A 66 M 31.5 62 50

SAGU00690 28-May-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 530632 3563351 3653 A 81 M 23.0 61 40 169 SAGU00670 28-May-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 530219 3563482 3332 A 90 M 20.0 50 2 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU00664 28-May-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 530632 3563351 3653 A 81 F 40.0 71 12 SAGU00672 28-May-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 530219 3563482 3332 A 90 M 34.0 60 10 SAGU00683 29-May-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 530182 3563469 3314 A 79 F 16.0 52 1 SAGU00663 29-May-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 530182 3563469 3314 A 79 M 17.0 55 25 SAGU00685 29-May-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 530160 3563438 3305 A 85 F 51.0 80 12 SAGU00712 29-May-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 530160 3563438 3305 A 85 F 68.0 85 10 SAGU00665 29-May-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 530182 3563469 3314 A 79 F 24.0 63 20 SAGU00678 29-May-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 530182 3563469 3314 A 79 M 25.0 58 60 SAGU00667 29-May-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 530182 3563469 3314 A 79 M 24.0 62 20 SAGU00662 29-May-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 530182 3563469 3314 J 79 U 6.5 38 0 SAGU00686 29-May-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 530182 3563469 3314 J 79 U 5.0 34 0 SAGU00676 29-May-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 530182 3563469 3314 J 79 U 6.0 33 0 SAGU00675 29-May-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 530182 3563469 3314 J 79 U 7.0 36 0 SAGU00691 29-May-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 530160 3563438 3305 J 85 U 3.5 31 0 SAGU00694 29-May-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 530160 3563438 3305 J 85 U 6.5 38 0 SAGU00692 29-May-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 530160 3563438 3305 J 85 U 4.0 34 0 SAGU00695 29-May-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 530160 3563438 3305 J 85 U 4.5 35 0 SAGU00697 29-May-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 530160 3563438 3305 J 85 U 4.5 32 0 SAGU00710 11-Jun-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 529792 3563838 3127 A 78 F 20.0 54 10 SAGU00715 11-Jun-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 529407 3564103 3005 A 79 F 15.0 55 5 SAGU00717 11-Jun-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 529888 3563730 3188 A 78 F 30.0 63 40 SAGU00002 11-Jun-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 529888 3563730 3188 A 78 M 20.0 57 5 SAGU00702 11-Jun-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 529888 3563730 3188 A 78 M 19.0 57 20 SAGU00720 11-Jun-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 529407 3564103 3005 A 79 M 20.0 55 10 SAGU00698 11-Jun-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 529888 3563730 3188 A 78 F 72.0 79 50 SAGU00706 11-Jun-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 529888 3563730 3188 A 78 F 63.0 80 40 SAGU00719 11-Jun-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 529407 3564103 3005 A 79 F 67.0 85 10 SAGU00026 11-Jun-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 529407 3564103 3005 A 79 F 68.0 83 5 SAGU00718 11-Jun-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 529077 3564237 2867 A 90 F 43.0 72 5 SAGU00009 11-Jun-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 529465 3564079 3033 A 79 M 38.5 71 12 SAGU00699 11-Jun-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 529792 3563838 3127 J 78 U 4.0 39 0 SAGU00007 11-Jun-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 529792 3563838 3127 J 78 U 3.0 34 0 SAGU00713 11-Jun-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 529792 3563838 3127 J 78 U 5.0 39 1 SAGU00932 26-Jun-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 529816 3563815 3170 T 84 U SAGU00942 26-Jun-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 529816 3563815 3170 T 84 U SAGU01044 26-Jun-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 529816 3563815 3170 T 84 U SAGU00551 26-Jun-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 529816 3563815 3170 T 84 U SAGU00489 26-Jun-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 529816 3563815 3170 T 84 U SAGU00944 26-Jun-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 529816 3563815 3170 T 84 U

SAGU01048 26-Jun-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 529816 3563815 3170 T 84 U 170 SAGU01024 26-Jun-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 529816 3563815 3170 T 84 U Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU00183 26-Jun-09 RAYA NEGATIVE 0 2009 Summer Wildhorse 529816 3563815 3170 T 84 U SAGU00952 26-Jun-09 RAYA POSITIVE 1,017 2009 Summer Wildhorse 529816 3563815 3170 T 84 U SAGU01321 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529879 3563717 3203 A 80 F 12.0 44 15 SAGU01323 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529879 3563717 3203 A 80 M 12.0 46 5 SAGU01341 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529879 3563717 3203 A 80 M 13.0 46 15 SAGU01127 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529459 3564060 3061 A 72 F 65.0 80 10 SAGU00641 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529801 3563833 3145 A 75 F 40.0 72 15 SAGU00643 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529801 3563833 3145 A 75 F 42.0 73 15 SAGU00645 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529801 3563833 3145 A 75 F 43.0 77 10 SAGU00655 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529801 3563833 3145 A 75 F 21.0 58 5 SAGU01091 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529801 3563833 3145 A 75 F 32.0 66 20 SAGU01372 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529801 3563833 3145 A 75 F 48.0 72 5 SAGU01082 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529801 3563833 3145 A 75 F 28.0 60 15 SAGU01337 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529879 3563717 3203 A 80 F 36.0 67 10 SAGU01339 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529879 3563717 3203 A 80 F 18.5 56 10 SAGU01333 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529879 3563717 3203 A 80 F 62.0 83 20 SAGU01332 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529879 3563717 3203 A 80 F 64.5 70 20 SAGU01331 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529879 3563717 3203 A 80 F 20.0 54 5 SAGU01334 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529879 3563717 3203 A 80 F 45.0 76 15 SAGU01350 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529459 3564060 3061 A 72 M 18.0 53 15 SAGU01264 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529801 3563833 3145 A 75 M 18.0 51 10 SAGU01093 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529801 3563833 3145 A 75 M 13.0 50 10 SAGU01092 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529801 3563833 3145 A 75 M 30.0 57 15 SAGU00565 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529801 3563833 3145 A 75 M 25.0 57 10 SAGU00657 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529801 3563833 3145 A 75 M 25.0 55 10 SAGU00652 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529801 3563833 3145 A 75 M 14.0 49 15 SAGU01089 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529801 3563833 3145 A 75 M 35.0 60 10 SAGU01096 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529801 3563833 3145 A 75 M 28.0 58 25 SAGU01347 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529879 3563717 3203 A 80 M 23.0 56 3 SAGU01348 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529879 3563717 3203 A 80 M 17.0 50 15 SAGU01322 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529879 3563717 3203 A 80 M 26.0 53 10 SAGU01338 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529879 3563717 3203 A 80 M 25.0 56 10 SAGU00639 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529801 3563833 3145 J 75 U 4.0 32 2 SAGU01329 14-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 529879 3563717 3203 J 80 U 5.0 35 10 SAGU00646 19-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 530769 3563410 3717 A 75 F 13.0 47 15 SAGU01359 19-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 530167 3563446 3336 A 82 M 10.0 46 15 SAGU00649 19-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 531292 3563346 4065 A 70 F 71.0 82 50 SAGU01099 19-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 530769 3563410 3717 A 75 F 17.0 53 15 SAGU00840 19-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 530545 3563377 3482 A 80 F 51.0 70 10

SAGU01085 19-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 530167 3563446 3336 A 82 F 21.0 56 20 171 SAGU01231 19-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 530103 3563460 3281 A 75 F 25.0 59 10 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU01248 19-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 530103 3563460 3281 A 75 F 24.0 57 5 SAGU01370 19-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 531115 3563321 3977 A 80 M 14.0 51 10 SAGU01088 19-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 530545 3563377 3482 A 80 M 18.0 52 15 SAGU01238 19-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 530103 3563460 3281 A 75 M 19.0 50 15 SAGU01239 19-Oct-09 RAYA NEGATIVE 0 2009 Fall Wildhorse 530103 3563460 3281 A 75 M 23.0 57 2 SAGU01249 4-Jan-10 RAYA POSITIVE 41 2009 Winter Wildhorse 529803 3563825 3140 A 58 F 53.5 77 10 SAGU01134 18-Mar-10 RAYA NEGATIVE 0 2010 Spring Wildhorse 529891 3563728 3172 A F 60.0 79 10 SAGU01157 18-Mar-10 RAYA NEGATIVE 0 2010 Spring Wildhorse 530086 3563480 3273 A M 23.5 53 8 SAGU01132 18-Mar-10 RAYA NEGATIVE 0 2010 Spring Wildhorse 529887 3563733 3188 A M 31.5 60 8 SAGU01687 29-May-10 RAYA NEGATIVE 0 2010 Summer Wildhorse 529887 3563720 3169 A 70 F 41.5 72 10 SAGU01738 29-May-10 RAYA NEGATIVE 0 2010 Summer Wildhorse 529887 3563720 3169 A 70 F 71.5 82 12 SAGU01723 29-May-10 RAYA NEGATIVE 0 2010 Summer Wildhorse 530135 3563428 3337 A 76 F 45.5 70 2 SAGU01718 29-May-10 RAYA NEGATIVE 0 2010 Summer Wildhorse 530103 3563478 3270 A 64 M 46.5 68 25 SAGU01838 29-May-10 RAYA NEGATIVE 0 2010 Summer Wildhorse 529467 3564082 3028 A 78 M 40.5 68 5 SAGU01881 29-May-10 RAYA NEGATIVE 0 2010 Summer Wildhorse 529887 3563720 3169 T 70 U SAGU01883 29-May-10 RAYA NEGATIVE 0 2010 Summer Wildhorse 529887 3563720 3169 T 70 U SAGU01818 29-May-10 RAYA NEGATIVE 0 2010 Summer Wildhorse 529887 3563720 3169 T 70 U SAGU01819 29-May-10 RAYA NEGATIVE 0 2010 Summer Wildhorse 529887 3563720 3169 T 70 U SAGU01870 29-May-10 RAYA NEGATIVE 0 2010 Summer Wildhorse 529887 3563720 3169 T 70 U SAGU01766 29-May-10 RAYA NEGATIVE 0 2010 Summer Wildhorse 529887 3563720 3169 T 70 U SAGU01980 29-May-10 RAYA NEGATIVE 0 2010 Summer Wildhorse 529887 3563720 3169 T 70 U SAGU01683 29-May-10 RAYA NEGATIVE 0 2010 Summer Wildhorse 529887 3563720 3169 T 70 U SAGU01749 29-May-10 RAYA NEGATIVE 0 2010 Summer Wildhorse 529887 3563720 3169 T 70 U SAGU01782 29-May-10 RAYA NEGATIVE 0 2010 Summer Wildhorse 529887 3563720 3169 T 70 U CHY00748 12-Jul-10 RAYA NEGATIVE 0 2010 Summer Wildhorse 529878 3563716 3169 J U 40 0 CHY00745 12-Jul-10 RAYA NEGATIVE 0 2010 Summer Wildhorse 530160 3563437 3330 J U 32 0 CHY00746 12-Jul-10 RAYA NEGATIVE 0 2010 Summer Wildhorse 530160 3563437 3330 J U 35 0 SAGU00440 22-Oct-08 RAYA POSITIVE 38 2008 Fall Berghausen 524786 3565393 2747 J 66 M 21.5 55 0 CHY02843 17-Oct-09 RAYA NEGATIVE 0 2009 Fall Beryl Pond 500167 3561567 2384 J U CHY02842 17-Oct-09 RAYA NEGATIVE 0 2009 Fall Beryl Pond 500167 3561567 2384 J U CHY02844 17-Oct-09 RAYA NEGATIVE 0 2009 Fall Beryl Pond 500167 3561567 2384 J U SAGU01599 08-Jun-10 RAYA NEGATIVE 0 2010 Summer Beryl Pond 500167 3561567 2384 A 80 F 31.5 69 0 SAGU01781 08-Jun-10 RAYA NEGATIVE 0 2010 Summer Beryl Pond 500167 3561567 2384 J 80 U 7.5 40 0 CHY00375 14-Jun-09 RAYA NEGATIVE 0 2009 Summer Campus Ag Farm 505852 3571359 2323 J U CHY00376 14-Jun-09 RAYA POSITIVE 3,155 2009 Summer Campus Ag Farm 505852 3571359 2323 J U SAGU01033 10-Jun-09 RAYA POSITIVE 112,550 2009 Summer Campus Ag Farm 505852 3571359 2323 T U SAGU01039 10-Jun-09 RAYA POSITIVE 119,209 2009 Summer Campus Ag Farm 505852 3571359 2323 T U SAGU00272 10-Jun-09 RAYA POSITIVE 122,316 2009 Summer Campus Ag Farm 505852 3571359 2323 T U SAGU01022 10-Jun-09 RAYA POSITIVE 146,675 2009 Summer Campus Ag Farm 505852 3571359 2323 T U

SAGU01045 10-Jun-09 RAYA POSITIVE 153,466 2009 Summer Campus Ag Farm 505852 3571359 2323 T U 172 SAGU00555 10-Jun-09 RAYA POSITIVE 163,815 2009 Summer Campus Ag Farm 505852 3571359 2323 T U Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU00444 10-Jun-09 RAYA POSITIVE 256,876 2009 Summer Campus Ag Farm 505852 3571359 2323 T U SAGU01023 10-Jun-09 RAYA POSITIVE 259,685 2009 Summer Campus Ag Farm 505852 3571359 2323 T U SAGU00542 10-Jun-09 RAYA POSITIVE 331,554 2009 Summer Campus Ag Farm 505852 3571359 2323 T U SAGU00094 10-Jun-09 RAYA POSITIVE 344,893 2009 Summer Campus Ag Farm 505852 3571359 2323 T U SAGU01035 10-Jun-09 RAYA POSITIVE 590,210 2009 Summer Campus Ag Farm 505852 3571359 2323 T U SAGU00564 13-Oct-09 RAYA NEGATIVE 0 2009 Fall Campus Ag Farm 505852 3571359 2323 J 75 M 6.5 38 0 SAGU01254 13-Oct-09 RAYA NEGATIVE 0 2009 Fall Campus Ag Farm 505852 3571359 2323 J 75 U 6.5 37 0 SAGU01094 13-Oct-09 RAYA NEGATIVE 0 2009 Fall Campus Ag Farm 505852 3571359 2323 J 75 M 11 47 0 SAGU00450 22-Oct-08 RAYA POSITIVE 207,177 2008 Fall Caruso 524827 3565480 2726 J 70 F 22 63 0 SAGU00089 29-Oct-08 RAYA POSITIVE 1,006,288 2008 Fall Caruso 524831 3565471 2732 J 72 M 14 54 1 SAGU01302 21-Oct-09 RAYA POSITIVE 2,664 2009 Fall Caruso 524827 3565480 2726 J 72 F 12 46 0 SAGU01300 21-Oct-09 RAYA POSITIVE 6,914 2009 Fall Caruso 524827 3565480 2726 J 72 F 14 48 0 SAGU01608 10-Jun-10 RAYA NEGATIVE 0 2010 Summer Caruso 524827 3565480 2726 J 80 U 9 45 0 SAGU02035 10-Jun-10 RAYA NEGATIVE 0 2010 Summer Caruso 524827 3565480 2726 J 80 U 7 37 0 SAGU02039 10-Jun-10 RAYA NEGATIVE 0 2010 Summer Caruso 524827 3565480 2726 J 80 U 6.5 32 0 SAGU02068 10-Jun-10 RAYA NEGATIVE 0 2010 Summer Caruso 524827 3565480 2726 J 80 U 6.5 36 0 SAGU02066 10-Jun-10 RAYA POSITIVE 2 2010 Summer Caruso 524827 3565480 2726 J 80 U 9 38 0 SAGU02055 10-Jun-10 RAYA POSITIVE 4 2010 Summer Caruso 524827 3565480 2726 J 80 F 15 47 0 SAGU02063 10-Jun-10 RAYA POSITIVE 6 2010 Summer Caruso 524827 3565480 2726 J 80 F 10.5 46 0 SAGU02047 10-Jun-10 RAYA POSITIVE 7 2010 Summer Caruso 524827 3565480 2726 J 80 U 9.5 43 0 SAGU02058 10-Jun-10 RAYA POSITIVE 13 2010 Summer Caruso 524827 3565480 2726 J 80 U 5.5 37 0 SAGU02030 10-Jun-10 RAYA POSITIVE 26 2010 Summer Caruso 524827 3565480 2726 J 80 M 11.5 47 0 SAGU02070 10-Jun-10 RAYA POSITIVE 31 2010 Summer Caruso 524827 3565480 2726 J 80 U 5.5 34 0 SAGU02061 10-Jun-10 RAYA POSITIVE 35 2010 Summer Caruso 524827 3565480 2726 J 80 U 7.5 39 0 SAGU02062 10-Jun-10 RAYA POSITIVE 39 2010 Summer Caruso 524827 3565480 2726 J 80 U 7.5 39 0 SAGU02025 10-Jun-10 RAYA POSITIVE 3,472 2010 Summer Caruso 524827 3565480 2726 J 80 U 7.5 39 0 Warren Frog 01-Sep-08 RAYA POSITIVE 2,066 2008 Fall Davison 521921 3561788 2898 U U SAGU00467 30-Dec-08 RAYA POSITIVE 919,309 2008 Winter Davison 521921 3561788 2898 A 55 M 23 64 0 SAGU00393 30-Dec-08 RAYA POSITIVE 2,031,708 2008 Winter Davison 521921 3561788 2898 A 55 U 28 60 0 SAGU00008 14-Jan-09 RAYA POSITIVE 308,877 2009 Winter Davison 521921 3561788 2898 A 60 F SAGU00433 14-Jan-09 RAYA POSITIVE 494,892 2009 Winter Davison 521921 3561788 2898 U 60 U SAGU00028 14-Jan-09 RAYA POSITIVE 4,140,300 2009 Winter Davison 521921 3561788 2898 U 60 U SAGU00790 02-Jul-09 RAYA POSITIVE 1,651 2009 Summer Davison 521950 3561765 2932 J 82 U 5.5 38 0 SAGU01179 16-Oct-09 RAYA POSITIVE 129 2009 Fall Davison 521918 3561790 2890 J 75 U 3 23 0 SAGU01189 16-Oct-09 RAYA POSITIVE 2,802 2009 Fall Davison 521918 3561790 2890 J 75 M 9 42 0 SAGU01166 16-Oct-09 RAYA POSITIVE 3,182 2009 Fall Davison 521918 3561790 2890 J 80 U 3 33 0 SAGU01168 16-Oct-09 RAYA POSITIVE 3,703 2009 Fall Davison 521918 3561790 2890 J 75 F 15 53 0 SAGU01368 17-Oct-09 RAYA POSITIVE 5,013 2009 Fall Davison 521918 3561790 2890 J 75 U 4 32 0 SAGU01175 16-Oct-09 RAYA POSITIVE 5,393 2009 Fall Davison 521918 3561790 2890 J 75 U 3 29 1

SAGU01173 16-Oct-09 RAYA POSITIVE 10,866 2009 Fall Davison 521918 3561790 2890 J 75 U 5.5 37 0 173 SAGU01416 18-Oct-09 RAYA POSITIVE 12,541 2009 Fall Davison 521918 3561790 2890 J 79 U 3.5 31 0 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU01360 18-Oct-09 RAYA POSITIVE 14,368 2009 Fall Davison 521918 3561790 2890 J 79 U 4 29 0 SAGU01180 16-Oct-09 RAYA POSITIVE 21,158 2009 Fall Davison 521918 3561790 2890 J 75 M 6.5 35 0 SAGU01184 16-Oct-09 RAYA POSITIVE 32,427 2009 Fall Davison 521918 3561790 2890 J 75 M 10.5 44 0 SAGU01364 17-Oct-09 RAYA POSITIVE 54,632 2009 Fall Davison 521918 3561790 2890 J 75 U 5 34 0 SAGU01170 16-Oct-09 RAYA POSITIVE 189,247 2009 Fall Davison 521918 3561790 2890 J 75 M 11 45 0 SAGU01357 17-Oct-09 RAYA POSITIVE 241,504 2009 Fall Davison 521918 3561790 2890 J 75 M 8 44 0 SAGU00660 18-Oct-09 RAYA POSITIVE 260,961 2009 Fall Davison 521918 3561790 2890 J 79 U 3 26 0 SAGU01161 16-Oct-09 RAYA POSITIVE 619,632 2009 Fall Davison 521918 3561790 2890 J 80 U 4.5 36 0 SAGU01449 02-Nov-09 RAYA POSITIVE 2,580,090 2009 Fall Davison 521950 3561765 2932 J M 15.5 36 0 SAGU00911 26-Dec-09 RAYA POSITIVE 2,449 2009 Winter Davison 521950 3561765 2932 A 50 F 37 63 0 SAGU00910 09-Jan-10 RAYA POSITIVE 3,248 2010 Winter Davison 521921 3561788 2898 J 55 U 30 0 SAGU00919 09-Jan-10 RAYA POSITIVE 260,463 2010 Winter Davison 521921 3561788 2898 J U SAGU01947 01-Jul-10 RAYA POSITIVE 14 2010 Summer Davison 521921 3561788 2898 J U SAGU01689 01-Jul-10 RAYA POSITIVE 46 2010 Summer Davison 521921 3561788 2898 J U SAGU00454 22-Oct-08 RAYA POSITIVE 1,761 2008 Fall Delaney 525733 3565208 2734 J 67 M 8.5 47 1 SAGU00146 22-Oct-08 RAYA POSITIVE 15,495 2008 Fall Hernbrode 521921 3561788 2898 J 64 M 18.5 53 0 SAGU00401 22-Oct-08 RAYA POSITIVE 45,834 2008 Fall Hernbrode 521921 3561788 2898 J 64 M 19 55 0 SAGU00001 10-Dec-08RAYA - deaPOSITIVE 841 2008 Winter Hernbrode 521921 3561788 2898 A M 25 65 SAGU00019 10-Dec-08RAYA - deaPOSITIVE 4,207 2008 Winter Hernbrode 521921 3561788 2898 A F 15 55 SAGU00016 10-Dec-08RAYA - deaPOSITIVE 302,967 2008 Winter Hernbrode 521921 3561788 2898 A F 25 56 SAGU00004 10-Dec-08RAYA - deaPOSITIVE 1,311,276 2008 Winter Hernbrode 521921 3561788 2898 A M 18.8 57 CHY00092 10-Dec-08YA - alive/dPOSITIVE 4,237,330 2008 Winter Hernbrode 521921 3561788 2898 A M 18 61 SAGU00135 18-Dec-08 RAYA POSITIVE 35,629 2008 Winter Hernbrode 521921 3561788 2898 A M 23.5 50 SAGU00431 18-Dec-08 RAYA POSITIVE 1,366,734 2008 Winter Hernbrode 521921 3561788 2898 A M 30.5 59.5 SAGU00416 30-Dec-08 RAYA POSITIVE 452,744 2008 Winter Hernbrode 521921 3561788 2898 A 55 M 59 0 SAGU00425 30-Dec-08 RAYA POSITIVE 637,572 2008 Winter Hernbrode 521921 3561788 2898 A 55 U 25 52 0 SAGU00478 30-Dec-08 RAYA POSITIVE 1,080,001 2008 Winter Hernbrode 521921 3561788 2898 A 55 M 30 61 0 SAGU00442 30-Dec-08 RAYA POSITIVE 1,411,645 2008 Winter Hernbrode 521921 3561788 2898 A 55 M 26.5 58 0 SAGU00488 14-Jan-09 RAYA POSITIVE 53,852 2009 Winter Hernbrode 521921 3561788 2898 U 60 U SAGU00025 14-Jan-09 RAYA POSITIVE 1,365,857 2009 Winter Hernbrode 521921 3561788 2898 U 60 U SAGU00783 02-Jul-09 RAYA POSITIVE 956 2009 Summer Hernbrode 521950 3561765 2932 A 88 M 28 60 0 SAGU00807 02-Jul-09 RAYA POSITIVE 1,706 2009 Summer Hernbrode 521950 3561765 2932 J 88 U 2.5 32 0 SAGU00810 02-Jul-09 RAYA POSITIVE 3,358 2009 Summer Hernbrode 521950 3561765 2932 J 88 U 6.5 37 0 SAGU00794 01-Sep-09 RAYA NEGATIVE 0 2009 Fall Hernbrode 521950 3561765 2932 A F 49 0 SAGU01178 16-Oct-09 RAYA POSITIVE 1,257 2009 Fall Hernbrode 521918 3561790 2890 J 75 U 5 37 0 SAGU01181 16-Oct-09 RAYA POSITIVE 1,669 2009 Fall Hernbrode 521918 3561790 2890 A 75 M 24 58 0 SAGU01084 02-Nov-09 RAYA POSITIVE 2,243 2009 Fall Hernbrode 521950 3561765 2932 J 70 U 28 SAGU01185 18-Oct-09 RAYA POSITIVE 2,798 2009 Fall Hernbrode 521918 3561790 2890 J 79 M 6.5 37 0 SAGU00566 18-Oct-09 RAYA POSITIVE 3,629 2009 Fall Hernbrode 521918 3561790 2890 A 79 M 17 46 0

SAGU01353 17-Oct-09 RAYA POSITIVE 4,867 2009 Fall Hernbrode 521918 3561790 2890 J 78 F 12 46 0 174 SAGU01163 16-Oct-09 RAYA POSITIVE 5,656 2009 Fall Hernbrode 521918 3561790 2890 J 75 F 12.5 47 0 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU01182 16-Oct-09 RAYA POSITIVE 8,479 2009 Fall Hernbrode 521918 3561790 2890 J 75 U 2.5 29 0 SAGU01187 16-Oct-09 RAYA POSITIVE 8,832 2009 Fall Hernbrode 521918 3561790 2890 J 75 U 3 27 0 SAGU01363 18-Oct-09 RAYA POSITIVE 9,774 2009 Fall Hernbrode 521918 3561790 2890 J 79 U 3 31 0 SAGU01176 16-Oct-09 RAYA POSITIVE 13,797 2009 Fall Hernbrode 521918 3561790 2890 J 75 M 11 45 0 SAGU01188 16-Oct-09 RAYA POSITIVE 54,606 2009 Fall Hernbrode 521918 3561790 2890 J 75 U 2 26 0 SAGU01171 16-Oct-09 RAYA POSITIVE 83,915 2009 Fall Hernbrode 521918 3561790 2890 J 75 U 3.5 30 0 SAGU00656 18-Oct-09 RAYA POSITIVE 337,831 2009 Fall Hernbrode 521918 3561790 2890 J 79 U 3 26 0 SAGU01315 12-Dec-09 RAYA POSITIVE 11,498,309 2009 Winter Hernbrode 521950 3561765 2932 J M 45 SAGU00929 05-Jan-10 RAYA POSITIVE 28,715 2010 Winter Hernbrode 521921 3561788 2898 T 60 U SAGU01240 19-Feb-10 RAYA POSITIVE 24 2010 Winter Hernbrode 521950 3561765 U U 51 SAGU00427 01-Jul-09 RAYA NEGATIVE 0 2009 Summer Judy Fraser's 500423 3561991 2399 A 82 F 70 50.5 0 SAGU00093 01-Jul-09 RAYA NEGATIVE 0 2009 Summer Judy Fraser's 500423 3561991 2399 A 82 F 28 64 0 SAGU00459 01-Jul-09 RAYA NEGATIVE 0 2009 Summer Judy Fraser's 500423 3561991 2399 A 82 F 35 67 0 SAGU00622 01-Jul-09 RAYA NEGATIVE 0 2009 Summer Judy Fraser's 500268 3561973 2406 J 85 U 2 27 0 SAGU00625 01-Jul-09 RAYA NEGATIVE 0 2009 Summer Judy Fraser's 500423 3561991 2399 J 82 U 17.5 54 0 SAGU01215 04-Nov-09 RAYA NEGATIVE 0 2009 Fall Judy Fraser's 500423 3561991 2399 A 72 F 58 82 0 SAGU01213 04-Nov-09 RAYA NEGATIVE 0 2009 Fall Judy Fraser's 500423 3561991 2399 A 72 M 36 64 0 SAGU01201 04-Nov-09 RAYA NEGATIVE 0 2009 Fall Judy Fraser's 500423 3561991 2399 J F 19 51 0 SAGU01226 04-Nov-09 RAYA POSITIVE 0 2009 Fall Judy Fraser's 500423 3561991 2399 A 72 F 58 72 0 SAGU01196 04-Nov-09 RAYA POSITIVE 5 2009 Fall Judy Fraser's 500423 3561991 2399 J 67 M 16.5 48 0 SAGU01454 04-Nov-09 RAYA POSITIVE 7 2009 Fall Judy Fraser's 500423 3561991 2399 A 72 F 64 79 0 SAGU01653 17-May-10 RAYA NEGATIVE 0 2010 Summer Judy Fraser's 500423 3561991 2399 A F 67 0 SAGU01934 09-Jun-10 RAYA NEGATIVE 0 2010 Summer Judy Fraser's 500423 3561991 2399 A 80 F 48.5 74 0 SAGU01774 09-Jun-10 RAYA NEGATIVE 0 2010 Summer Judy Fraser's 500423 3561991 2399 A 80 F 46 78 0 SAGU01903 09-Jun-10 RAYA NEGATIVE 0 2010 Summer Judy Fraser's 500423 3561991 2399 A 80 F 40.5 76 0 SAGU02099 09-Jun-10 RAYA NEGATIVE 0 2010 Summer Judy Fraser's 500423 3561991 2399 A 80 F 43.5 74 0 SAGU01948 09-Jun-10 RAYA NEGATIVE 0 2010 Summer Judy Fraser's 500423 3561991 2399 A 80 F 48.5 70 0 SAGU01570 09-Jun-10 RAYA NEGATIVE 0 2010 Summer Judy Fraser's 500423 3561991 2399 A 80 F 41.5 72 0 SAGU01783 09-Jun-10 RAYA NEGATIVE 0 2010 Summer Judy Fraser's 500423 3561991 2399 J 80 F 19.5 55 0 SAGU02097 09-Jun-10 RAYA NEGATIVE 0 2010 Summer Judy Fraser's 500423 3561991 2399 J 80 F 13 49 0 SAGU01589 09-Jun-10 RAYA NEGATIVE 0 2010 Summer Judy Fraser's 500423 3561991 2399 J 80 F 15.5 52 0 SAGU01702 09-Jun-10 RAYA NEGATIVE 0 2010 Summer Judy Fraser's 500423 3561991 2399 J 80 F 9.5 44 0 SAGU01728 09-Jun-10 RAYA NEGATIVE 0 2010 Summer Judy Fraser's 500423 3561991 2399 J 80 F 17.5 50 0 SAGU01789 09-Jun-10 RAYA NEGATIVE 0 2010 Summer Judy Fraser's 500423 3561991 2399 J 80 M 13.5 47 0 SAGU01625 11-Jun-10 RAYA NEGATIVE 0 2010 Summer Olais J 80 U 7.5 34 0 SAGU01639 11-Jun-10 RAYA NEGATIVE 0 2010 Summer Olais J 80 U 8 37 0 SAGU01936 11-Jun-10 RAYA NEGATIVE 0 2010 Summer Olais J 80 U 7.5 40 0 SAGU01632 11-Jun-10 RAYA NEGATIVE 0 2010 Summer Olais J 80 U 11.5 44 0 SAGU01595 11-Jun-10 RAYA NEGATIVE 0 2010 Summer Olais J 80 U 9.5 40 0

SAGU02089 11-Jun-10 RAYA NEGATIVE 0 2010 Summer Olais J 80 U 12 49 0 175 SAGU00280 22-Oct-08 RAYA POSITIVE 581 2008 Fall Reifschneider 525399 3566027 2693 U 68 F 149 118 0 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU00396 22-Oct-08 RAYA POSITIVE 1,846 2008 Fall Roscoe 524955 3565613 2725 J 70 M 11.5 45 0 SAGU00468 22-Oct-08 RAYA POSITIVE 5,140 2008 Fall Roscoe 524955 3565613 2725 J 70 M 9.5 46 0 SAGU00408 22-Oct-08 RAYA POSITIVE 7,887 2008 Fall Roscoe 524955 3565613 2725 J 70 U 6.5 40.5 0 SAGU00469 22-Oct-08 RAYA POSITIVE 10,276 2008 Fall Roscoe 524955 3565613 2725 J 70 M 10 44 0 SAGU00404 22-Oct-08 RAYA POSITIVE 13,156 2008 Fall Roscoe 524955 3565613 2725 J 70 F 13 48.5 0 SAGU00461 22-Oct-08 RAYA POSITIVE 16,959 2008 Fall Roscoe 524955 3565613 2725 J 70 M 11.5 48 0 SAGU00486 22-Oct-08 RAYA POSITIVE 17,937 2008 Fall Roscoe 524955 3565613 2725 J 70 M 12.5 49 0 SAGU00383 22-Oct-08 RAYA POSITIVE 19,898 2008 Fall Roscoe 524955 3565613 2725 J 70 M 9.5 43 0 SAGU00271 22-Oct-08 RAYA POSITIVE 30,736 2008 Fall Roscoe 524955 3565613 2725 J 70 F 10.5 47 0 SAGU00455 22-Oct-08 RAYA POSITIVE 97,892 2008 Fall Roscoe 524955 3565613 2725 J 70 M 10 48 0 SAGU00418 22-Oct-08 RAYA POSITIVE 1,482,726 2008 Fall Roscoe 524955 3565613 2725 J 70 F 10 47 0 SAGU01314 21-Oct-09 RAYA POSITIVE 136 2009 Fall Roscoe 524955 3565613 2725 J 70 M 13 46 0 SAGU01303 21-Oct-09 RAYA POSITIVE 4,733 2009 Fall Roscoe 524955 3565613 2725 A 70 F 22 60 0 CHY02192 04-Sep-08 RAYA NEGATIVE 0 2008 Fall Schwalbe 523973 3559254 2988 A F CHY02198 04-Sep-08 RAYA NEGATIVE 0 2008 Fall Schwalbe 523973 3559254 2988 A M CHY02190 04-Sep-08 RAYA NEGATIVE 0 2008 Fall Schwalbe 523973 3559254 2988 A M CHY02195 04-Sep-08 RAYA NEGATIVE 0 2008 Fall Schwalbe 523973 3559254 2988 A M CHY02189 04-Sep-08 RAYA NEGATIVE 0 2008 Fall Schwalbe 523973 3559254 2988 A M CHY02188 04-Sep-08 RAYA NEGATIVE 0 2008 Fall Schwalbe 523973 3559254 2988 J U CHY02185 04-Sep-08 RAYA NEGATIVE 0 2008 Fall Schwalbe 523973 3559254 2988 J U SAGU00167 29-Sep-08 RAYA NEGATIVE 0 2008 Fall Schwalbe 523973 3559254 2988 A 70 F 34 72 SAGU00161 29-Sep-08 RAYA NEGATIVE 0 2008 Fall Schwalbe 523973 3559254 2988 A 70 F 60 84 SAGU00120 29-Sep-08 RAYA NEGATIVE 0 2008 Fall Schwalbe 523973 3559254 2988 A 70 F 62 89 SAGU00170 29-Sep-08 RAYA NEGATIVE 0 2008 Fall Schwalbe 523973 3559254 2988 A 70 F 76 86 SAGU00115 29-Sep-08 RAYA NEGATIVE 0 2008 Fall Schwalbe 523973 3559254 2988 A 70 U 33 65 SAGU00111 29-Sep-08 RAYA NEGATIVE 0 2008 Fall Schwalbe 523973 3559254 2988 J 70 U 25 58 SAGU00162 29-Sep-08 RAYA NEGATIVE 0 2008 Fall Schwalbe 523973 3559254 2988 J 70 U 22 74 SAGU00166 29-Sep-08 RAYA NEGATIVE 0 2008 Fall Schwalbe 523973 3559254 2988 J 70 U 22 53 SAGU00114 29-Sep-08 RAYA NEGATIVE 0 2008 Fall Schwalbe 523973 3559254 2988 J 70 U 20 59 SAGU00119 29-Sep-08 RAYA NEGATIVE 0 2008 Fall Schwalbe 523973 3559254 2988 J 70 U 28 63 SAGU00955 18-Jun-09 RAYA NEGATIVE 0 2009 Summer Schwalbe 523973 3559259 3016 A 80 F 31 61 0 SAGU00239 18-Jun-09 RAYA NEGATIVE 0 2009 Summer Schwalbe 523973 3559259 3016 A 80 F 17.5 54 0 SAGU00099 18-Jun-09 RAYA NEGATIVE 0 2009 Summer Schwalbe 523973 3559259 3016 A 80 F 44.5 74 0 SAGU01042 18-Jun-09 RAYA NEGATIVE 0 2009 Summer Schwalbe 523973 3559259 3016 A 80 F 41.5 69 0 SAGU00091 18-Jun-09 RAYA NEGATIVE 0 2009 Summer Schwalbe 523973 3559259 3016 A 80 M 20 55 0 SAGU00951 18-Jun-09 RAYA NEGATIVE 0 2009 Summer Schwalbe 523973 3559259 3016 A 80 M 19.5 56 0 SAGU00314 18-Jun-09 RAYA NEGATIVE 0 2009 Summer Schwalbe 523973 3559259 3016 A 80 M 26 61 0 SAGU00410 18-Jun-09 RAYA NEGATIVE 0 2009 Summer Schwalbe 523973 3559259 3016 A 80 M 21 58 1 SAGU00145 18-Jun-09 RAYA NEGATIVE 0 2009 Summer Schwalbe 523973 3559259 3016 A 80 M 21 56 0

SAGU00700 18-Jun-09 RAYA NEGATIVE 0 2009 Summer Schwalbe 523973 3559259 3016 A 80 M 22.5 56 0 176 SAGU01034 18-Jun-09 RAYA NEGATIVE 0 2009 Summer Schwalbe 523973 3559259 3016 A 80 M 26.5 55 0 Water Number UTM UTM Elevation Life Weight SVL Mite Frog ID Date Species Result Year Season Location Temp Sex Zoospores (easting) (northing) (ft) Stage (g) (mm) Load (oF) SAGU00409 18-Jun-09 RAYA NEGATIVE 0 2009 Summer Schwalbe 523973 3559259 3016 A 80 M 22.5 55 0 SAGU00096 18-Jun-09 RAYA NEGATIVE 0 2009 Summer Schwalbe 523973 3559259 3016 J 80 F 15.5 51 0 SAGU01028 18-Jun-09 RAYA NEGATIVE 0 2009 Summer Schwalbe 523973 3559259 3016 J 80 M 20.5 54 0 SAGU01445 03-Nov-09 RAYA NEGATIVE 0 2009 Fall Schwalbe 523975 3559253 3016 A 69 F 50 75 0 SAGU01224 03-Nov-09 RAYA NEGATIVE 0 2009 Fall Schwalbe 523975 3559253 3016 J 69 U 3.5 23 0 SAGU01451 03-Nov-09 RAYA NEGATIVE 0 2009 Fall Schwalbe 523975 3559253 3016 J 69 U 3 26 0 SAGU01200 03-Nov-09 RAYA POSITIVE 4 2009 Fall Schwalbe 523975 3559253 3016 A 69 M 29.5 55 0 SAGU01457 03-Nov-09 RAYA POSITIVE 6 2009 Fall Schwalbe 523975 3559253 3016 A 69 F 49 75 0 SAGU01465 03-Nov-09 RAYA POSITIVE 9 2009 Fall Schwalbe 523975 3559253 3016 J 69 U 4 28 0 SAGU01458 03-Nov-09 RAYA POSITIVE 12 2009 Fall Schwalbe 523975 3559253 3016 A 69 F 21.5 58 0 SAGU01195 03-Nov-09 RAYA POSITIVE 13 2009 Fall Schwalbe 523975 3559253 3016 A 69 M 24.5 57 0 SAGU01459 03-Nov-09 RAYA POSITIVE 18 2009 Fall Schwalbe 523975 3559253 3016 A 69 F 24 56 0 SAGU01222 03-Nov-09 RAYA POSITIVE 96 2009 Fall Schwalbe 523975 3559253 3016 A 69 M 28 59 0 SAGU01466 03-Nov-09 RAYA POSITIVE 134 2009 Fall Schwalbe 523975 3559253 3016 A 69 M 23.5 57 0 SAGU01326 19-Dec-09 RAYA NEGATIVE 0 2009 Winter Schwalbe 523975 3559253 3016 J M SAGU01680 04-Jun-10 RAYA NEGATIVE 0 2010 Summer Schwalbe 523973 3559259 2988 A 80 F 44.5 76 0 SAGU01973 04-Jun-10 RAYA NEGATIVE 0 2010 Summer Schwalbe 523973 3559259 2988 A 80 F 61 78 0 SAGU01776 04-Jun-10 RAYA NEGATIVE 0 2010 Summer Schwalbe 523973 3559259 2988 A 80 M 19 61 0 SAGU01908 04-Jun-10 RAYA NEGATIVE 0 2010 Summer Schwalbe 523973 3559259 2988 A 80 M 21.5 53 0 SAGU01677 04-Jun-10 RAYA NEGATIVE 0 2010 Summer Schwalbe 523973 3559259 2988 A 80 M 25.5 52 0 SAGU01821 04-Jun-10 RAYA NEGATIVE 0 2010 Summer Schwalbe 523973 3559259 2988 A 80 M 23 56 0 SAGU01867 04-Jun-10 RAYA NEGATIVE 0 2010 Summer Schwalbe 523973 3559259 2988 A 80 M 28.5 62 0 SAGU01918 04-Jun-10 RAYA NEGATIVE 0 2010 Summer Schwalbe 523973 3559259 2988 A 80 M 28 57 0 SAGU02065 04-Jun-10 RAYA NEGATIVE 0 2010 Summer Schwalbe 523973 3559259 2988 J 80 U 5 27 0 SAGU01792 04-Jun-10 RAYA POSITIVE 3 2010 Summer Schwalbe 523973 3559259 2988 A 80 F 25 53 0 177 APPENDIX B: ALL WATER FILTER SAMPLES COLLECTED AT PONDS FROM THE RINCON MOUNTAINS AND TUCSON BASIN

Water Water ID Date Number Zoospores Result Location UTM (easting) UTM (northing) Elevation Temp Berg 1 29-Oct-08 0 NEGATIVE Berghausen 524786 3565393 2747 70 SAGU01663 10-Jun-10 97 POSITIVE Berghausen 524786 3565393 2747 SAGU01521 21-Oct-09 0 NEGATIVE Berghausen 524786 3565393 2747 70 SAGU01506 01-Jul-09 0 NEGATIVE Berghausen 524786 3565393 2747 88 SAGU01532 08-Jun-10 0 NEGATIVE Beryl Pond 500167 3561567 2384 80 SAGU01514 06-Jul-09 0 NEGATIVE Beryl Pond 500167 3561567 2384 SAGU01556 6-Jun-10 0 NEGATIVE Campus Ag Farm 505852 3571359 2323 79 SAGU01556 06-Jun-10 0 NEGATIVE Campus Ag Farm 505852 3571359 2323 79 SAGU01507 13-Oct-09 0 NEGATIVE Campus Ag Farm 505852 3571359 2323 75 SAGU01507 13-Oct-09 0 NEGATIVE Campus Ag Farm 505852 3571359 2323 75 SAGU01656 10-Jun-10 9,659 POSITIVE Caruso 524827 3565480 2726 80 SAGU01522 21-Oct-09 0 NEGATIVE Caruso 524827 3565480 2726 72 SAGU01505 01-Jul-09 0 NEGATIVE Caruso 524827 3565480 2726 86 Carusoe 1 29-Oct-08 74 POSITIVE Caruso 524831 3565471 2732 72 SAGU01508 15-Oct-09 0 NEGATIVE Cebadilla 528955 3567668 2691 71 CE031910 19-Mar-10 0 NEGATIVE Cebadilla 528943 3567760 2718 50 7/8/10Cebedilla 8-Jul-10 0 NEGATIVE Cebadilla 528943 3567760 2718 82 SAGU01497 26-Jun-09 0 NEGATIVE Cebadilla 528943 3567760 2718 85 CH 3402 12-Oct-08 0 NEGATIVE Chimenea 536877 3557491 3402 76 CH 8140 4-Oct-08 0 NEGATIVE Chimenea 542598 3564136 8140 57 CH 7580 4-Oct-08 0 NEGATIVE Chimenea 542047 3562890 7580 CH 7088 5-Oct-08 0 NEGATIVE Chimenea 541566 3562376 7088 58 CH 6509 5-Oct-08 0 NEGATIVE Chimenea 540726 3562100 6509 62 CH 5888 6-Oct-08 0 NEGATIVE Chimenea 539653 3561663 5888 62 CH 5571 6-Oct-08 0 NEGATIVE Chimenea 539173 3561445 5571 67 CH 3938 10-Oct-08 0 NEGATIVE Chimenea 537180 3558598 3938 69 CH 4420 10-Oct-08 0 NEGATIVE Chimenea 537084 3559946 4420 79 SAGU01543 1-Oct-09 0 NEGATIVE Chimenea 539429 3561593 5845 69 SAGU01542 1-Oct-09 0 NEGATIVE Chimenea 538635 3561111 5389 80 SAGU01545 2-Oct-09 0 NEGATIVE Chimenea 537596 3560329 5028 70 178 SAGU01516 5-Oct-09 0 NEGATIVE Chimenea 537040 3557865 3640 72 Water Water ID Date Number Zoospores Result Location UTM (easting) UTM (northing) Elevation Temp SAGU01501 10-Oct-09 0 NEGATIVE Chimenea 536909 3557519 3414 70 SAGU01474 2-Jun-09 0 NEGATIVE Chimenea 541853 3562637 7380 58 SAGU01475 2-Jun-09 0 NEGATIVE Chimenea 541379 3562369 6942 70 SAGU01476 2-Jun-09 0 NEGATIVE Chimenea 540665 3562048 6529 78 SAGU01477 2-Jun-09 0 NEGATIVE Chimenea 542039 3563518 7924 68 SAGU01478 3-Jun-09 0 NEGATIVE Chimenea 539701 3561649 5916 63 SAGU01479 3-Jun-09 0 NEGATIVE Chimenea 538834 3561379 5482 81 SAGU01480 8-Jun-09 0 NEGATIVE Chimenea 536893 3557503 3423 81 SAGU01484 15-Jun-09 0 NEGATIVE Chimenea 537579 3560322 5010 70 SAGU01485 15-Jun-09 0 NEGATIVE Chimenea 537089 3559933 4407 82 SAGU01486 15-Jun-09 0 NEGATIVE Chimenea 537148 3558639 3978 80 SAGU01541 30-Sep-09 0 NEGATIVE Chimenea 541854 3562651 7367 62 SAGU01498 30-Sep-09 0 NEGATIVE Chimenea 540730 3562090 6554 72 SAGU01531 10-Jun-10 0 NEGATIVE Chimenea 536901 3557513 3432 78 SAGU01535 10-Jun-10 0 NEGATIVE Chimenea 537037 3557858 3600 78 6/15/10CH7400 15-Jun-10 0 NEGATIVE Chimenea 541860 3562691 7400 52 6/15/10CH6923 15-Jun-10 0 NEGATIVE Chimenea 541412 3562384 6923 78 6/15/10CH6542 15-Jun-10 0 NEGATIVE Chimenea 540719 3562104 6542 76 6/16/10CH5738 16-Jun-10 0 NEGATIVE Chimenea 539379 3561509 5738 73 6/16/10CH5384 16-Jun-10 0 NEGATIVE Chimenea 538638 3561124 5384 73 6/16/10CH5030 17-Jun-10 0 NEGATIVE Chimenea 537601 3560340 5030 80 6/17/10CH4461 17-Jun-10 0 NEGATIVE Chimenea 537090 3559920 4461 80 CH071310 13-Jul-10 0 NEGATIVE Chimenea 536909 3557519 3414 82 SAGU01544 1-Oct-09 5 POSITIVE Chimenea 542049 3563526 7972 61 SAGU01546 2-Oct-09 1 POSITIVE Chimenea 537081 3559959 4495 79 SAGU01473 9-Jun-09 87 POSITIVE Chimenea 537038 3557851 3546 76 1/10/10CH3419 10-Jan-10 127 POSITIVE Chimenea 536900 3557528 3419 60 3/17/10CH3432 16-Mar-10 28 POSITIVE Chimenea 536913 3557511 3432 40 6/16/10CH7926 16-Jun-10 227 POSITIVE Chimenea 542040 3563520 7926 61 SAGU01560 30-Sep-09 0 NEGATIVE Chimenea 541382 3562404 6969 67 SAGU01551 17-Oct-09 0 NEGATIVE Columbus Park 497022 3571717 2233 78 SAGU01513 3-Jul-09 0 NEGATIVE Columbus Park 486792 3571699 2258 90 CO032110 27-Mar-10 0 NEGATIVE Columbus Park 496792 3571699 2258 60

SAGU01670 17-Jul-10 0 NEGATIVE Columbus Park 496792 3571699 2258 85 179 SAGU01529 6-Nov-09 0 NEGATIVE Crosby 526483 3567236 2681 60 Water Water ID Date Number Zoospores Result Location UTM (easting) UTM (northing) Elevation Temp SAGU01492 19-Jun-09 0 NEGATIVE Crosby 526483 3567236 2681 69 7/15/10Sky 15-Jul-10 0 NEGATIVE Crosby 526483 3567236 2681 77 CRO31810 18-Mar-10 6 POSITIVE Crosby 526483 3567236 2681 50 SAGU01512 02-Jul-09 0 NEGATIVE Davison 521950 3561765 2932 82 SAGU01657 10-Jun-10 6 POSITIVE Davison 521921 3561788 2898 SAGU01509 17-Oct-09 55 POSITIVE Davison 521918 3561790 2890 75 SAGU01523 21-Oct-09 0 NEGATIVE Delaney 525733 3565208 2734 72 SAGU01659 10-Jun-10 0 NEGATIVE Delaney 525733 3565208 2734 79 SAGU01502 01-Jul-09 0 NEGATIVE Delaney 525733 3565208 2734 84 Delaney 1 29-Oct-08 0 NEGATIVE Delaney 525742 3565227 2758 70 SAGU01520 9-Oct-09 0 NEGATIVE Ft Lowell Park 512020 3569323 2465 72 SAGU01515 7-Jul-09 0 NEGATIVE Ft Lowell Park 511955 3569297 2444 SAGU01499 16-Jul-09 0 NEGATIVE Ft Lowell Park 512263 3569342 2417 87 FTL032010 20-Mar-10 0 NEGATIVE Ft Lowell Park 512263 3569342 2417 53 7/6/10FtLowell 6-Jul-10 0 NEGATIVE Ft Lowell Park 512263 3569342 2417 80 SAGU01658 10-Jun-10 85 POSITIVE Hernbrode 521921 3561788 2898 SAGU01510 17-Oct-09 11 POSITIVE Hernbrode 521918 3561790 2890 78 Hern 1 29-Oct-08 2 POSITIVE Hernbrode 521921 3561788 2898 70 Hern 2 29-Oct-08 0 NEGATIVE Hernbrode 521921 3561788 2898 63 SAGU01511 02-Jul-09 0 NEGATIVE Hernbrode 521950 3561765 2932 88 SAGU01500 02-Sep-09 0 NEGATIVE Hernbrode 521950 3561765 2932 Hern 3 30-Dec-08 511 POSITIVE Hernbrode 521921 3561788 2898 55 SAGU01490 01-Jul-09 0 NEGATIVE Judy Fraser's 500423 3561991 2399 82 SAGU01550 04-Nov-09 0 NEGATIVE Judy Fraser's 500423 3561991 2399 SAGU01558 09-Jun-10 0 NEGATIVE Judy Fraser's 500423 3561991 2399 78 SAGU01488 28-Jun-09 0 NEGATIVE Kennedy Park 499319 3560515 2435 88 SAGU01502 10-Oct-09 19 POSITIVE Kennedy Park 499317 3560523 2435 76 SAGU01660 11-Jun-10 0 NEGATIVE Olais 80 Karen 1 29-Oct-08 0 NEGATIVE Reifschneider 525399 3566027 2693 72 SAGU01524 21-Oct-09 0 NEGATIVE Reifschneider 525399 3566027 2693 70 SAGU01664 10-Jun-10 0 NEGATIVE Reifschneider 525399 3566027 2693 80 SAGU01503 01-Jul-09 0 NEGATIVE Reifschneider 525399 3566027 2693 85 SAGU01525 21-Oct-09 0 NEGATIVE Roscoe 524955 3565613 2725 70

SAGU01662 10-Jun-10 0 NEGATIVE Roscoe 524955 3565613 2725 79 180 SAGU01504 01-Jul-09 0 NEGATIVE Roscoe 524955 3565613 2725 84 Water Water ID Date Number Zoospores Result Location UTM (easting) UTM (northing) Elevation Temp Roscoe 1 29-Oct-08 499 POSITIVE Roscoe 524955 3565613 2725 70 SAGU01533 04-Jun-10 0 NEGATIVE Schwalbe 523973 3559259 2988 80 SAGU01501 01-Jul-09 0 NEGATIVE Schwalbe 523973 3559259 3016 84 Cecil 1 29-Oct-08 0 NEGATIVE Schwalbe 523971 3559252 3013 63 SAGU01549 03-Nov-09 0 NEGATIVE Schwalbe 523975 3559253 3016 69 SAGU01507 01-Jul-09 0 NEGATIVE Schwalbe 523973 3559259 3016 84 Cecil 2 29-Oct-08 0 NEGATIVE Schwalbe 523971 3559252 3013 63 TV 4551 3-Nov-08 0 NEGATIVE Tanque Verde 539979 3568602 4551 70 TV 4004 8-Oct-08 0 NEGATIVE Tanque Verde 538110 3569124 4004 69 TV 3191 17-Oct-08 0 NEGATIVE Tanque Verde 532908 3569016 3191 62 TV 3477 18-Oct-08 0 NEGATIVE Tanque Verde 534870 3569546 3477 68 TV 6800 25-Oct-08 0 NEGATIVE Tanque Verde 542167 3566048 6800 48 TV 4997 3-Nov-08 0 NEGATIVE Tanque Verde 540576 3567704 4997 SAGU01547 6-Oct-09 0 NEGATIVE Tanque Verde 538106 3569121 4012 75 SAGU01518 7-Oct-09 0 NEGATIVE Tanque Verde 539688 3568600 4462 70 SAGU01519 7-Oct-09 0 NEGATIVE Tanque Verde 540979 3568324 4893 65 SAGU01487 16-Jun-09 0 NEGATIVE Tanque Verde 532906 3564020 3214 73 SAGU01493 22-Jun-09 0 NEGATIVE Tanque Verde 538116 3569138 4026 89 SAGU01494 22-Jun-09 0 NEGATIVE Tanque Verde 539677 3568593 4450 88 TV3468 5-Jan-10 0 NEGATIVE Tanque Verde 534877 3569542 3468 48 SAGU01665 7-Apr-10 0 NEGATIVE Tanque Verde 533149 3569093 3317 51 SAGU01540 8-Jun-10 0 NEGATIVE Tanque Verde 532920 3569019 3360 70 SAGU01536 8-Jun-10 0 NEGATIVE Tanque Verde 534385 3569453 3395 82 SAGU01530 8-Jun-10 0 NEGATIVE Tanque Verde 534885 3569544 3485 82 SAGU01491 17-Jun-09 38 POSITIVE Tanque Verde 534672 3569573 3484 76 SAGU01548 20-Oct-09 0 NEGATIVE Tanque Verde 532924 3569016 3234 73 SAGU01527 26-Oct-09 0 NEGATIVE Tanque Verde 534878 3569542 3523 70 SAGU01496 23-Jun-09 0 NEGATIVE Tanque Verde 540823 3568348 4825 91 SAGU01539 2-Jun-10 0 NEGATIVE Tanque Verde 538111 3569116 3983 68 SAGU01554 3-Jun-10 0 NEGATIVE Tanque Verde 539697 3568599 4484 84 SAGU01534 3-Jun-10 0 NEGATIVE Tanque Verde 540341 3568256 4712 69 SAGU01526 26-Oct-09 188 POSITIVE Tanque Verde 534176 3569533 3362 63 SAGU01528 5-Nov-09 0 NEGATIVE TVGR 529239 3567209 2696 66

SAGU01489 30-Jun-09 0 NEGATIVE TVGR 529239 3567209 2696 84 181 7/16/10TVGR 16-Jul-10 0 NEGATIVE TVGR 529239 3567209 2696 80 Water Water ID Date Number Zoospores Result Location UTM (easting) UTM (northing) Elevation Temp WH 3309 1-Oct-08 0 NEGATIVE Wildhorse 530140 3563430 3309 85 WH 5132 20-Oct-08 0 NEGATIVE Wildhorse 534187 3564189 5132 59 WH 4992 20-Oct-08 0 NEGATIVE Wildhorse 533281 3563577 4992 WH 4074 22-Oct-08 0 NEGATIVE Wildhorse 531316 3563358 4074 63 SAGU01503 12-Oct-09 0 NEGATIVE Wildhorse 531858 3563466 4331 70 SAGU01504 12-Oct-09 0 NEGATIVE Wildhorse 533281 3563576 5029 60 SAGU01506 14-Oct-09 0 NEGATIVE Wildhorse 529459 3564060 3061 72 SAGU01553 19-Oct-09 0 NEGATIVE Wildhorse 530103 3563460 3281 75 SAGU01472 28-May-09 0 NEGATIVE Wildhorse 531259 3563340 4036 80 SAGU01471 29-May-09 0 NEGATIVE Wildhorse 530212 3563476 3333 83 SAGU01481 10-Jun-09 0 NEGATIVE Wildhorse 533644 3563062 5579 82 SAGU01482 10-Jun-09 0 NEGATIVE Wildhorse 533279 3563577 5039 SAGU01483 11-Jun-09 0 NEGATIVE Wildhorse 529407 3564103 3005 79 WH3353 4-Jan-10 0 NEGATIVE Wildhorse 530173 3563444 3329 64 SAGU01661 18-Mar-10 0 NEGATIVE Wildhorse 529887 3563733 3188 SAGU01508 29-May-10 0 NEGATIVE Wildhorse 529080 3564239 2899 90 SAGU01537 1-Jun-10 0 NEGATIVE Wildhorse 531683 3563343 4202 69 SAGU01557 1-Jun-10 0 NEGATIVE Wildhorse 530204 3563482 3373 82 SAGU01555 7-Jun-10 0 NEGATIVE Wildhorse 533633 3563048 5570 80 SAGU01559 7-Jun-10 0 NEGATIVE Wildhorse 533168 3563635 4910 80 WH 2882 11-Oct-08 6 POSITIVE Wildhorse 529073 3564232 2882 71 WH 5646 20-Oct-08 1 POSITIVE Wildhorse 533655 3563047 5646 60 SAGU01505 12-Oct-09 562 POSITIVE Wildhorse 533623 3563055 5593 60 SAGU01552 19-Oct-09 10 POSITIVE Wildhorse 531210 3563331 4030 73 182 APPENDIX C: LOCATIONS WHERE BOTH FROGS WERE SWABBED AND WATER FILTRATION SAMPLES WERE COLLECTED - Frog Water Mean Frog Min Frog Max Frog Zoospores L Location Species Sampled # Frogs Result Result Zoospores Zoospores Zoospores 1 Wildhorse HYAR 3 - + 0 0 0 4,495 Chimenea HYAR 8 - + 0 0 0 6 Wildhorse HYAR 5 + + 8,834 152 18,576 12 Chimenea HYAR 8 + - 11,251 0 61,735 0 Chimenea HYAR 4 + - 9,904 1 31,434 0 Wildhorse HYAR 10 + - 8,489 13 77,317 0 Chimenea HYAR 5 + - 1,925 6 5,468 0 Chimenea HYAR 1 + - 503 503 503 0 Chimenea HYAR 11 + - 463 0 2,869 0 Chimenea HYAR 13 + - 54 0 460 0 Chimenea HYAR 2 + - 32 0 63 0 Wildhorse HYAR 4 + - 28 0 71 0 Chimenea HYAR 7 + - 19 0 134 0 Chimenea HYAR 1 + - 3 3 3 0 Chimenea HYAR 8 + - 2 0 16 0 Wildhorse HYAR 15 + - 2 0 25 0 Chimenea HYAR 8 - - 0 0 0 0 Chimenea HYAR 6 - - 0 0 0 0 Chimenea HYAR 9 - - 0 0 0 0 Chimenea HYAR 6 - - 0 0 0 0 Chimenea HYAR 2 - - 0 0 0 0 Chimenea HYAR 1 - - 0 0 0 0 Chimenea HYAR 10 - - 0 0 0 0 183

- Frog Water Mean Frog Min Frog Max Frog Zoospores L Location Species Sampled # Frogs Result Result Zoospores Zoospores Zoospores 1 Chimenea HYAR 4 - - 0 0 0 0 Chimenea HYAR 7 - - 0 0 0 0 Chimenea HYAR 8 - - 0 0 0 0 Chimenea HYAR 6 - - 0 0 0 0 Chimenea HYAR 4 - - 0 0 0 0 Chimenea HYAR 18 - - 0 0 0 0 Chimenea HYAR 8 - - 0 0 0 0 Chimenea HYAR 8 - - 0 0 0 0 Chimenea HYAR 8 - - 0 0 0 0 Chimenea HYAR 12 - - 0 0 0 0 Chimenea HYAR 9 - - 0 0 0 0 Chimenea HYAR 1 - - 0 0 0 0 Tanque Verde HYAR 1 - - 0 0 0 0 Tanque Verde HYAR 8 - - 0 0 0 0 Wildhorse HYAR and RAYA 5 - + 0 0 0 82 Wildhorse HYAR and RAYA 7 - + 0 0 0 51 Tanque Verde HYAR and RAYA 26 + + 12,077 0 119,933 302 Tanque Verde HYAR and RAYA 15 + - 293,407 1,251 1,526,741 0 Tanque Verde HYAR and RAYA 1 + - 160,282 160,282 160,282 0 Chimenea HYAR and RAYA 2 + - 62,808 8,870 116,747 0 Tanque Verde HYAR and RAYA 17 + - 10,781 0 109,738 0 Chimenea HYAR and RAYA 26 + - 4,522 0 55,707 0 Chimenea HYAR and RAYA 3 + - 1,751 0 5,252 0 Tanque Verde HYAR and RAYA 11 + - 71 0 630 0

Tanque Verde HYAR and RAYA 8 + - 16 0 58 0 184

- Frog Water Mean Frog Min Frog Max Frog Zoospores L Location Species Sampled # Frogs Result Result Zoospores Zoospores Zoospores 1 Tanque Verde HYAR and RAYA 8 + - 12 0 99 0 Tanque Verde HYAR and RAYA 10 + - 10 0 62 0 Chimenea HYAR and RAYA 7 + - 2 0 12 0 Tanque Verde HYAR and RAYA 3 + - 1 0 4 0 Tanque Verde HYAR and RAYA 26 + - 1 0 21 0 Wildhorse HYAR and RAYA 12 + - 1 0 12 0 Wildhorse HYAR and RAYA 16 + - 1 0 6 0 Wildhorse HYAR and RAYA 8 + - 0 0 4 0 Chimenea HYAR and RAYA 4 - - 0 0 0 0 Chimenea HYAR and RAYA 1 - - 0 0 0 0 Tanque Verde HYAR and RAYA 6 - - 0 0 0 0 Tanque Verde HYAR and RAYA 1 - - 0 0 0 0 Tanque Verde HYAR and RAYA 10 - - 0 0 0 0 Tanque Verde HYAR and RAYA 8 - - 0 0 0 0 Tanque Verde HYAR and RAYA 8 - - 0 0 0 0 Tanque Verde HYAR and RAYA 8 - - 0 0 0 0 Tanque Verde HYAR and RAYA 1 - - 0 0 0 0 Wildhorse HYAR and RAYA 8 - - 0 0 0 0 Wildhorse HYAR and RAYA 4 - - 0 0 0 0 Wildhorse HYAR and RAYA 1 - - 0 0 0 0 Wildhorse HYAR and RAYA 4 - - 0 0 0 0 Wildhorse HYAR and RAYA 2 - - 0 0 0 0 Kennedy Park RACA 4 - + 0 0 0 151 Kennedy Park RACA 1 - + 0 0 0 82

Crosby RACA 12 + + 6,610 21 46,381 47 185

- Frog Water Mean Frog Min Frog Max Frog Zoospores L Location Species Sampled # Frogs Result Result Zoospores Zoospores Zoospores 1 Crosby RACA 17 + - 11,225 34 155,996 0 Crosby RACA 21 + - 9,595 166 45,156 0 TVGR RACA 21 + - 3,545 0 24,254 0 Ft. Lowell Park RACA 8 + - 3,128 14 19,444 0 Crosby RACA 24 + - 683 0 11,007 0 Ft. Lowell Park RACA 3 + - 54 0 102 0 Kennedy Park RACA 10 + - 19 0 190 0 TVGR RACA 14 + - 16 0 214 0 Cebedilla RACA 13 + - 3 0 21 0 Ft. Lowell Park RACA 13 + - 2 0 22 0 Columbus Park RACA 18 + - 1 0 25 0 TVGR RACA 25 + - 1 0 17 0 Cebedilla RACA 17 + - 1 0 10 0 Cebedilla RACA 10 - - 0 0 0 0 Cebedilla RACA 5 - - 0 0 0 0 Columbus Park RACA 1 - - 0 0 0 0 Columbus Park RACA 10 - - 0 0 0 0 Columbus Park RACA 23 - - 0 0 0 0 Ft. Lowell Park RACA 1 - - 0 0 0 0 Campus Ag Farm RACA & RAYA 11 + - 44,591 0 201,684 0 Campus Ag Farm RACA & RAYA 16 + - 26 12 41 0 Chimenea RAYA 4 + + 406,306 70,780 969,050 1,818 Chimenea RAYA 1 + + 41,615 41,615 41,615 223 Chimenea RAYA 1 + + 12,264 12,264 12,264 1,020

Chimenea RAYA 8 + + 1 0 5 694 186

- Frog Water Mean Frog Min Frog Max Frog Zoospores L Location Species Sampled # Frogs Result Result Zoospores Zoospores Zoospores 1 Caruso RAYA 1 + + 1,006,288 1,006,288 593 Davison RAYA 3 + + 100,383 5,013 241,504 441 Hernbrode RAYA 1 + + 4,867 4,867 90 Caruso RAYA 14 + + 363 2 3,472 77,268 Davison RAYA 1 + - 1,651 1,651 0 Hernbrode RAYA 3 + - 2,007 956 3,358 0 Caruso RAYA 2 + - 4,789 2,664 6,914 0 Roscoe RAYA 2 + - 2,435 136 4,733 0 Schwalbe RAYA 11 + - 37 4 134 0 Judy Fraser's RAYA 6 + - 4 1 7 0 Schwalbe RAYA 10 + - 3 3 0 Hernbrode RAYA 1 - - 0 0 0 Campus Ag Farm RAYA 3 - - 0 0 0 Beryl Pond RAYA 2 - - 0 0 0 Judy Fraser's RAYA 12 - - 0 0 0 Judy Fraser's RAYA 5 - - 0 0 0 Olais RAYA 6 - - 0 0 0 187 APPENDIX D: ADDITIONAL FIGURES FOR CHAPTER 1

(a) (b)

Figure 1.1 Prevalence (a) and intensity (b) of Batrachochytrium dendrobatidis infection across species, all life stages, locations, seasons, and elevations combined. HYAR = Hyla arenicolor, RACA = Rana catesbeiana, RAYA = Rana yavapaiensis. Error bars represent 95% confidence intervals.

188 (a) (b)

Figure 1.2 Prevalence (a) and intensity (b) of Batrachochytrium dendrobatidis infection across life stages and species, all drainages, seasons, and elevations combined. HYAR = Hyla arenicolor, RACA = Rana catesbeiana, RAYA = Rana yavapaiensis. Error bars represent 95% confidence intervals.

189 (a) (b)

Figure 1.3 Prevalence (a) and intensity (b) of Batrachochytrium dendrobatidis from Hyla arenicolor across locations, all life stages, elevations and seasons combined. Error bars represent 95% confidence intervals.

190 (a) (b)

Figure 1.4 Prevalence (a) and intensity (b) of Batrachochytrium dendrobatidis from Hyla arenicolor across life stages and locations from all elevations and seasons. Error bars represent 95% confidence intervals.

191 (a) (b)

Figure 1.5 Prevalence (a) and intensity (b) of Batrachochytrium dendrobatidis from Hyla arenicolor across seasons, all life stages, locations, and elevations combined. Error bars represent 95% confidence intervals.

192 (a) (b)

Figure 1.6 Prevalence (a) and intensity (b) of Batrachochytrium dendrobatidis from Hyla arenicolor across seasons and locations, all life stages and elevations combined. No H. arenicolor were swabbed from Chimenea Canyon and none tested positive from Tanque Verde during the winter. Error bars represent 95% confidence intervals.

193 (a) (b)

Figure 1.7 Prevalence (a) and intensity (b) of Batrachochytrium dendrobatidis from Rana yavapaiensis across locations, all life stages, elevations and seasons combined. Error bars represent 95% confidence intervals.

194 (a) (b)

Figure 1.8 Prevalence (a) and intensity (b) of Batrachochytrium dendrobatidis from Rana yavapaiensis across life stages and locations from all elevations and seasons. Error bars represent 95% confidence intervals.

195 (a) (b)

Figure 1.9 Prevalence (a) and intensity (b) of Batrachochytrium dendrobatidis from Rana yavapaiensis across seasons, all life stages, locations, and elevations combined. One R. yavapaiensis adult tested positive in the spring. Error bars represent 95% confidence intervals.

196 (a) (b)

Figure 1.10 Prevalence (a) and intensity (b) of Batrachochytrium dendrobatidis from Rana yavapaiensis across seasons and locations, all life stages and elevations combined. One R. yavapaiensis was swabbed and tested positive from Chimenea in the spring, one tested positive from Wildhorse in the summer, none were swabbed from Tanque Verde Canyon in the winter or spring, and none tested positive from Wildhorse Canyon during the spring. Error bars represent 95% confidence intervals. 197 (a) (b)

Figure 1.11 Prevalence (a) and intensity (b) of Batrachochytrium dendrobatidis from Rana catesbeiana across ponds throughout the Tucson Basin, Arizona, USA, all life stages combined. No error bars were calculated for Columbus and Kennedy Parks, which both had two positive frogs. TVGR = Tanque Verde Guest Ranch. Error bars represent 95% confidence intervals.

198

Figure 1.12 Prevalence of Batrachochytrium dendrobatidis from Rana catesbeiana across life stages and ponds throughout the Tucson Basin, Arizona, USA. TVGR = Tanque Verde Guest Ranch. No tadpoles from Kennedy Park or juveniles from Ft. Lowell Park were swabbed; one tadpole was swabbed at Crosby pond and one adult was swabbed at Campus Ag; values with no error bars represent locations where <3 individuals of that life stage tested positive. Error bars represent 95% confidence

intervals. 199

Figure 1.13 Intensity of Batrachochytrium dendrobatidis from Rana catesbeiana across life stages and ponds throughout the Tucson Basin, Arizona, USA. No tadpoles from Kennedy Park or juveniles from Ft. Lowell Park were swabbed; no tadpoles or juveniles from Columbus Park tested positive; values with no error bars represent locations where <3 individuals of that life stage tested positive. Error bars represent 95% confidence intervals. 200 APPENDIX E: STATISTICAL TABLES FOR CHAPTER 1

The following tables relate one of two things: 1. The odds of species/life stages/seasons in the columns on the left testing positive for Bd as compared to species/life stages/seasons in the row on the top a. In Table 1.1a this would be described as: i. The odds of HYAR testing positive for Bd were 0.87 less than RACA and 1.18 less than RAYA ii. The odds of RACA testing positive for Bd were 0.31 less than RAYA b. These data describe the statistical differences between values on prevalence figures of the same species/life stages/seasons 2. The multiplicative differences of Bd infection loads (in zoospores) between species/life stages/seasons in the columns on the left and species/life stages/seasons in the row on the top a. In Table 1.1b this would be described as i. Of the frogs that were positive for Bd, infection loads in HYAR were 3.11 times less than RACA and 8.99 times less than RAYA ii. Of the frogs that were positive for Bd, infection loads in RACA were 2.89 times less than RAYA. b. These data describe the statistical differences between values on infection intensity figures of the same species/life stages/season • For all tables, HYAR = Hyla arenicolor, RACA = Rana catesbeiana, RAYA = Rana yavapaiensis . P-values are in parentheses; grey shaded values differ significantly (p ≤ 0.05).

201

Table 1.1 Odds of testing positive for Bd (a) and multiplicative differences of Bd infection loads (b) from three species of frogs in three canyons in the Rincon Mountains, Arizona and the surrounding Tucson Basin, Arizona, all life stages, locations, seasons, and elevations combined.

(a) HYAR RACA RAYA (b) HYAR RACA RAYA -0.87 -1.18 -3.11 -8.99 HYAR x (<0.0001) (<0.0001) HYAR x (0.0022) (<0.0001) RACA x -0.31 (0.0051) RACA x -2.89 (0.0052) RAYA x RAYA x

202 Table 1.2 Odds of testing positive for Bd (a) and multiplicative differences of Bd infection loads (b) across life stages of three species of frogs in three canyons in the Rincon Mountains, Arizona and the surrounding Tucson Basin, Arizona, all locations, seasons, and elevations combined.

(a) Tadpoles Metamorphs Adults RAYA RACA HYAR RAYA RACA HYAR RAYA RACA HYAR 0.03 1.20 -1.74 -0.33 0.48 -1.21 -1.27 0.44 RAYA x (0.2533) (0.0263) (<0.0001) (0.1540) (0.1700) (0.0154) (0.0120) (0.1846) -1.78 Tadpoles 1.17 -0.36 0.45 -1.24 -1.31 0.41 RACA x (0.0235) (<0.0001) (0.1173) (0.1582) (0.0077) (0.0063) (0.1748) -2.95 -1.54 -0.72 -2.41 -2.48 -0.77 HYAR x (<0.0001) (0.0014) (0.1267) (<0.0001) (<0.0001) (0.1055) 1.41 2.23 0.53 0.47 2.18 RAYA x (<.0001) (<0.0001) (0.0770) (0.1038) (<0.0001) 0.82 -0.88 -0.94 0.77 Metamorphs RACA x (0.0298) (0.0198) (0.0157) (0.0298) -1.69 -1.76 -0.05 HYAR x (<0.0001) (0.0001) (0.1907) -0.07 1.65 RAYA x (0.1082) (<0.0001) Adults 1.71 RACA x (<0.0001) HYAR x

203 Table 1.2 (cont.) Odds of testing positive for Bd (a) and multiplicative differences of Bd infection loads (b) across life stages of three species of frogs in three canyons in the Rincon Mountains, Arizona and the surrounding Tucson Basin, Arizona, all locations, seasons, and elevations combined.

(b) Tadpoles Metamorphs Adults RAYA RACA HYAR RAYA RACA HYAR RAYA RACA HYAR 1.62 234.42 8.71 6.92 100.00 1.23 7.59 53.7 RAYA x (0.7283) (0.0063) (0.0465) (0.1220) (0.0010) (0.8555) (0.0735) (0.0004) 144.54 5.37 4.27 61.66 -1.32 4.68 33.11 Tadpoles RACA x (0.0032) (0.1068) (0.2228) (0.0029) (0.8015) (0.1482) (0.0015) -26.92 -33.88 -2.34 -190.55 -30.9 -4.37 HYAR x (0.0386) (0.0325) (0.6372) (0.0013) (0.0286) (0.3538) -1.26 11.48 -7.08 -1.15 6.17 RAYA x (0.7720) (0.0185) (0.0024) (0.8287) (0.0027) 14.45 -5.62 1.1 7.76 Metamorphs RACA x (0.0188) (0.0262) (0.8931) (0.0078) -81.28 -13.18 -1.86 HYAR x (<0.0001) (0.0171) (0.4806) 6.17 43.65 RAYA x (0.009) (<0.0001) Adults 7.08 RACA x (0.0037) HYAR x

204

Table 1.3 Odds of testing positive for Bd (a) and multiplicative differences of Bd infection loads (b) from Hyla arenicolor across three canyons in the Rincon Mountains, Arizona, USA, all life stages, elevations and seasons combined. Tanque Tanque (a) Chimenea Verde Wildhorse (b) Chimenea Verde Wildhorse -0.20 0.60 1.28 9.89 Chimenea x (0.0451) (0.0030) Chimenea x (0.6807) (0.0054) Tanque 0.40 7.75 Verde x (0.0142) Tanque Verde x (0.0059)

205 Table 1.4 Odds of testing positive for Bd (a) and multiplicative differences of Bd infection loads (b) from Hyla arenicolor across life stages and locations, all elevations and seasons combined. No Hyla arenicolor tadpoles tested positive from Chimenea or Wildhorse, so no comparisons were made involving these variables (indicated by a diagonal line).

(a) Tadpoles Juveniles Adults CH TV WH CH TV WH CH TV WH CH x 0.58 0.73 -2.00 -0.14 0.00 0.84 Tadpoles TV X (0.3042) (0.2968) (0.0274) (0.2813) (0.3002) (0.1194) WH X 0.15 -2.58 -0.72 -0.58 0.26 CH X (0.5238) (0.0036) (0.0562) (0.1020) (0.2753) -2.73 -0.87 -0.73 0.11 Juveniles TV X (0.0039) (0.0738) (0.1107) (0.3773) 2.00 2.84 WH X 1.86 (0.0129) (0.0079) (0.0003) 0.15 0.98 CH X (0.0813) (0.0001) Adults 0.84 TV X (0.0002) WH X

206 Table 1.4 (cont.) Odds of testing positive for Bd (a) and multiplicative differences of Bd infection loads (b) from Hyla arenicolor across life stages and locations, all elevations and seasons combined. No Hyla arenicolor tadpoles tested positive from Chimenea or Wildhorse, so no comparisons were made involving these variables (indicated by a diagonal line).

(b) Tadpoles Juveniles Adults CH TV WH CH TV WH CH TV WH CH x -29.75 Tadpoles -26.79 6.33 -4.08 -13.64 -1.17 TV X (0.1410) (0.1560) (0.3765) (0.4007) (0.1168) (0.9271) WH X -1.11 169.74 6.57 1.96 22.84 CH X (0.9639) (0.0099) (0.2315) (0.6667) (0.0588) Juveniles 188.49 7.29 2.18 25.37 TV X (0.0130) (0.2872) (0.6592) (0.0784) -25.85 -86.39 -7.43 WH X (0.0200) (0.0008) (0.1433) -3.34 3.48 CH X (0.0991) (0.1261) Adults 11.63 TV X (0.0015) WH X

207 Table 1.5 Odds of testing positive for Bd (a) and multiplicative differences of Bd infection loads (b) from Hyla arenicolor across seasons, all life stages, locations, and elevations combined. (a) Fall Winter Spring Summer (b) Fall Winter Spring Summer -1.54 -0.99 0.14 0.56 -0.2 -0.05 Fall x (0.0155) (<0.0001) (0.0468) Fall x (0.4178) (0.4825) (0.8320) 0.55 1.68 -0.76 -0.61 Winter x (0.2393) (0.0098) Winter x (0.2860) (0.3797) 1.13 0.15 Spring x (<0.0001) Spring x (0.6251) Summer x Summer x

208

Table 1.6 Odds of testing positive for Batrachochytrium dendrobatidis in Hyla arenicolor across seasons and locations, all life stages and elevations combined.

Fall Winter Tanque Tanque Chimenea Verde Wildhorse Chimenea Verde Wildhorse -3.00 -1.86 -0.24 -3.31 Fall Chimenea x (<0.0001) (<0.0001) (1.0000) (0.0002) 1.18 2.81 -0.32 Tanque Verde x (<0.0001) (0.0055) (0.4774) 1.65 -1.47 Wildhorse x (0.1073) (0.0515) Winter Chimenea x -3.75 Tanque Verde x (0.0171) Wildhorse x

209 Table 1.6 (cont.) Odds of testing positive for Batrachochytrium dendrobatidis in Hyla arenicolor across seasons and locations, all life stages and elevations combined.

Spring Summer Tanque Tanque Chimenea Verde Wildhorse Chimenea Verde Wildhorse -5.30 -2.37 -0.89 -2.57 -0.94 -1.27 Fall Chimenea (<0.0001) (<0.0001) (0.2247) (<0.0001) (0.0896) (0.0386) -2.29 0.65 2.16 2.11 1.77 Tanque Verde (<0.0001) (0.0330) (<0.0001) 0.50 (0.0499) (<0.0001) (<0.0001) -3.44 -0.50 -0.65 0.95 0.61 Wildhorse (<0.0001) (0.0844) 0.99 (0.0541) (0.0291) (0.0243) (0.1021) Winter Chimenea -5.68 -2.77 -1.31 -2.88 -1.36 -1.68 Tanque Verde (<0.0001) (0.0546) (1.0000) (0.0524) (0.8933) (0.4618) -1.35 1.59 3.04 2.70 Wildhorse (0.5680) (0.0714) 3.09 (0.0019) 1.48 (0.1289) (0.0016) (0.0055) 3.49 4.99 3.36 4.94 4.60 Spring Chimenea x (<0.0001) (<0.0001) (<0.0001) (<0.0001) (<0.0001) -0.07 1.51 1.17 Tanque Verde x 1.56 (0.0082) (0.2491) (0.0039) (0.0231) -1.65 -0.07 -0.41 Wildhorse x (0.0117) (0.5521) (0.4289) 1.69 1.34 Summer Chimenea x (0.0004) (0.0061) -0.35 Tanque Verde x (0.3245)

Wildhorse x 210

Table 1.7 Multiplicative differences of Batrachochytrium dendrobatidis infection loads from Hyla arenicolor across seasons and locations, all life stages and elevations combined. Differences Locations within a season with a diagonal line instead of values indicate either H. arenicolor tested positive or were swabbed at that location in that season.

Fall Winter Tanque Tanque Chimenea Verde Wildhorse Chimenea Verde Wildhorse -83.80 -14.41 Fall Chimenea x (<0.0001) (0.0304) -2.26 (0.5939) 5.82 37.10 Tanque Verde x (0.0757) (0.0081) Wildhorse x 6.38 (0.2435) Winter Chimenea x Tanque Verde x Wildhorse x

211 Table 1.7 (cont.) Multiplicative differences of Batrachochytrium dendrobatidis infection loads from Hyla arenicolor across seasons and locations, all life stages and elevations combined. Differences Locations within a season with a diagonal line instead of values indicate either H. arenicolor tested positive or were swabbed at that location in that season.

Spring Summer Tanque Chimenea Tanque Verde Wildhorse Chimenea Verde Wildhorse -673.21 -73.79 1.18 -224.06 -6.99 Fall Chimenea (<0.0001) (0.0005) (0.9385) (<0.0001) (0.2409) -4.06 (0.4344) -8.03 99.30 -2.67 11.98 20.65 Tanque Verde (0.0297) 1.14 (0.9046) (0.0308) (0.2633) (0.1129) (0.0784) -46.73 17.07 -15.55 Wildhorse (0.0046) -5.12 (0.2505) (0.2200) (0.0344) 2.06 (0.6865) 3.55 (0.4962) Winter Chimenea Tanque Verde -298.02 -32.67 2.68 -99.19 -3.10 Wildhorse (0.0003) (0.0368) (0.6884) (0.0040) (0.5846) -1.80 (0.7931) 797.66 96.25 165.90 Spring Chimenea x 9.12 (0.0217) (0.0013) 3.00 (0.1737) (0.0037) (0.0040) 87.43 -3.04 10.55 18.18 Tanque Verde x (0.0347) (0.2180) (0.1436) (0.0989) -265.48 -8.29 Wildhorse x (0.0060) (0.3873) -4.81 (0.5315) 32.04 55.22 Summer Chimenea x (0.0191) (0.0094) Tanque Verde x 1.72 (0.7894) Wildhorse x

212 Table 1.8 Odds of testing positive for Bd (a) and multiplicative differences of Bd infection loads (b) from Rana yavapaiensis across three canyons in the Rincon Mountains, Arizona, USA, all life stages, elevations and seasons combined. Tanque Tanque (a) Chimenea Verde Wildhorse (b) Chimenea Verde Wildhorse x 0.02 5.35 x -16.99 300.48 Chimenea (0.3585) (<0.0001) Chimenea (<0.0001) (<0.0001) Tanque 5.32 Tanque 5,104.03 Verde x (<0.0001) Verde x (<0.0001)

213 Table 1.9 Odds of testing positive for Bd (a) and multiplicative differences of Bd infection loads (b) from Rana yavapaiensis across life stages and locations, all elevations and seasons combined. (a) Tadpoles Metamorphs Adults CH TV WH CH TV WH CH TV WH -3.28 1.51 -2.62 -5.71 0.90 -3.69 -3.30 2.09 CH x (0.0012) (0.2365) (0.0003) (<0.0001) (0.4301) (<0.0001) (<0.0001) (0.0521) 4.80 0.66 -2.43 4.18 -0.41 -0.02 5.37 Tadpoles TV x (<0.0001) (0.3229) (0.1190) (<0.0001) (0.7618) (1.0000) (<0.0001) -4.13 -7.22 -0.61 -5.20 -4.81 0.057 WH x (<0.0001) (<0.0001) (0.6103) (<0.0001) (<0.0001) (1.0000) -3.09 3.52 -1.07 -0.68 4.71 CH x (0.0017) (<0.0001) (0.0456) (0.1572) (<0.0001) 6.61 2.02 2.41 7.79 Metamorphs TV x (<0.0001) (0.1338) (0.0642) (<0.0001) -4.59 -4.20 1.19 WH x (<0.0001) (<0.0001) (0.1374) 0.39 5.78 CH x (0.3320) (<0.0001) Adults 5.38 TV x (<0.0001) WH x

214 Table 1.9 (cont.) Odds of testing positive for Bd (a) and multiplicative differences of Bd infection loads (b) from Rana yavapaiensis across life stages and locations, all elevations and seasons combined. (b) Tadpoles Metamorphs Adults CH TV WH CH TV WH CH TV WH 1.59 6.74 49.41 1.82 8938.57 23.5 1.52 7861.46 CH x (0.8340) (0.5590) (0.0445) (0.7600) (0.0008) (0.1456) (0.8469) (0.0005) 4.25 31.17 1.15 5638.66 14.83 -1.04 4959.19 Tadpoles TV x (0.6140) (0.0056) (0.9126) (0.0001) (0.0858) (0.9791) (<0.0001) 7.33 -3.70 1325.65 3.49 -4.43 1165.91 WH x (0.4643) (0.6300) (0.0288) (0.6668) (0.6094) (0.0267) -27.14 180.91 -2.10 -32.50 159.11 CH x (<0.0001) (0.0072) (0.5012) (0.0022) (0.0035) 4910.21 12.91 -1.20 4318.52 Metamorphs TV x (<0.0001) (0.0406) (0.8868) (<0.0001) -380.30 -5879.03 -1.14 WH x (0.0004) (<0.0001) (0.9494) -15.46 334.48 CH x (<0.0001) (<0.0001) Adults 5170.60 TV x (<0.0001) WH x

215 Table 1.10 Odds of testing positive for Bd (a) and multiplicative differences of Bd infection loads (b) from Rana yavapaiensis across seasons, all life stages, locations, and elevations combined. (a) Fall Winter Spring Summer (b) Fall Winter Spring Summer -2.44 3.02 0.50 -0.29 -1.57 0.34 Fall x (0.0151) (<0.0001) (0.0046) Fall x (0.6209) (0.2987) (0.1492) 5.46 2.94 -1.28 0.64 Winter x (<0.0001) (0.0025) Winter x (0.4258) (0.2888) -2.52 1.92 Spring x (<0.0001) Spring x (0.2067) Summer x Summer x

216 Table 1.11 Odds of testing positive for Batrachochytrium dendrobatidis in Rana yavapaiensis across seasons and locations, all life stages and elevations combined. Locations within a season with a diagonal line instead of values indicate either no R. yavapaiensis were swabbed or <2 frogs tested positive at that location in that season.

Fall Winter Tanque Chimenea Verde Wildhorse Chimenea Tanque Verde Wildhorse 0.99 5.27 2.32 3.15 Fall Chimenea x (0.1288) (<0.0001) (0.1771) (0.0810) 4.56 1.60 2.43 Tanque Verde x (<0.0001) (0.4109) (0.1912) -3.05 -2.25 Wildhorse x (0.0018) (0.0523) 1.82 Winter Chimenea x (1.0000) Tanque Verde x Wildhorse x

217 Table 1.11 (continued) Odds of testing positive for Batrachochytrium dendrobatidis in Rana yavapaiensis across seasons and locations, all life stages and elevations combined. Locations within a season with a diagonal line instead of values indicate either no R. yavapaiensis were swabbed or <2 frogs tested positive at that location in that season.

Spring Summer Tanque Tanque Chimenea Verde Wildhorse Chimenea Verde Wildhorse 5.81 1.64 -0.20 6.01 Fall Chimenea (<0.0001) (0.0166) (1.0000) (<0.0001) 5.09 0.93 -0.91 5.29 Tanque Verde (<0.0001) (0.1731) (0.4281) (<0.0001) 0.41 -3.73 -5.57 0.61 Wildhorse (0.5998) (<0.0001) (<0.0001) (0.4034) 4.46 0.35 -1.46 4.64 Winter Chimenea (0.0011) (1.0000) (1.0000) (0.0006) Tanque Verde 3.23 -0.77 -2.58 3.36 Wildhorse (0.0642) (1.0000) (1.0000) (0.0532) Spring Chimenea x Tanque Verde x -5.02 -6.85 -0.75 Wildhorse x (<0.0001) (<0.0001) (1.0000) -1.75 4.65 Summer Chimenea x (0.0173) (<0.0001) 7.54 Tanque Verde x (<0.0001) Wildhorse x

218 Table 1.12 Multiplicative differences of Batrachochytrium dendrobatidis infection loads in Rana yavapaiensis across seasons and locations, all life stages and elevations combined. Locations within a season with a diagonal line instead of values indicate either no R. yavapaiensis were swabbed or <2 frogs tested positive at that location in that season. Fall Winter Tanque Tanque Chimenea Verde Wildhorse Chimenea Verde Wildhorse -8.69 724.28 -10.08 35.72 Fall Chimenea x (0.001) (<0.0001) (0.1759) (0.0788) 6292.09 310.33 Tanque Verde x (<0.0001) -1.16 (0.9301) (0.0053) -7303.87 -20.28 Wildhorse x (<0.0001) (0.2022) 360.23 Winter Chimenea x (0.0178) Tanque Verde x Wildhorse x

219 Table 1.12 (continued) Multiplicative differences of Batrachochytrium dendrobatidis infection loads in Rana yavapaiensis across seasons and locations, all life stages and elevations combined. Locations within a season with a diagonal line instead of values indicate either no R. yavapaiensis were swabbed or <2 frogs tested positive at that location in that season. Spring Summer Tanque Chimenea Verde Wildhorse Chimenea Tanque Verde Wildhorse Fall Chimenea -1.08 (0.9169) -51.6 (<0.0001) Tanque Verde 8.01 (0.0399) -5.94 (0.0878) -785.22 -37375.76 Wildhorse (<0.0001) (<0.0001) Winter Chimenea 9.3 (0.2271) -5.12 (0.3924) Tanque Verde Wildhorse -38.73 (0.0899) -1843.4 (0.0008) Spring Chimenea x Tanque Verde x Wildhorse x Summer Chimenea x -47.6 (<0.0001) Tanque Verde x Wildhorse x

220 Table 1.13 Odds of testing positive for Bd (a) and multiplicative differences of Bd infection loads (b) from Rana catesbeiana across ponds throughout the Tucson Basin, Arizona, USA, all life stages combined. Campus Ag Ft. Lowell Kennedy (a) Farm Cebadilla Columbus Crosby Park Park TVGR Campus Ag 3.07 3.50 -1.87 1.85 2.19 Farm x (0.0004) (0.0002) (1.0000) (0.0283) 2.39 (0.0063) (0.0034) 0.44 -4.95 -1.21 -0.67 -0.87 Cebadilla x (0.4752) (<0.0001) (0.1069) (0.4238) (0.0962) -5.38 -1.65 -1.11 -1.31 Columbus x (<0.0001) (0.0674) (0.2762) (0.0439) 3.73 4.27 4.06 Crosby x (0.0001) (<0.0001) (<0.0001) Ft. Lowell 0.34 Park x 0.54 (0.4748) (0.3694) -0.20 Kennedy Park x (0.4562) TVGR x

221 Table 1.13 (continued) Odds of testing positive for Bd (a) and multiplicative differences of Bd infection loads (b) from Rana catesbeiana across ponds throughout the Tucson Basin, Arizona, USA, all life stages combined. Campus Ft Lowell Kennedy (b) Ag Farm Cebadilla Columbus Crosby Park Park TVGR Campus Ag 446.68 2398.83 7.24 602.56 12.30 Farm x (<0.0001) (<0.0001) (0.0006) 8.91 (0.0160) (0.0004) (0.0055) -61.66 -50.12 1.35 -36.31 Cebadilla x 5.25 (0.4458) (0.0039) (0.0143) (0.8883) (0.0223) -323.59 -263.03 -3.89 -194.98 Columbus x (0.0010) (0.0036) (0.5689) (0.0047) 83.18 Crosby x 1.23 (0.7913) (0.0110) 1.70 (0.5036) 67.61 Ft Lowell Park x (0.0265) 1.38 (0.7744) -50.12 Kennedy Park x (0.0349) TVGR x

222 Table 1.14 Odds of testing positive for Batrachochytrium dendrobatidis in Rana catesbeiana across life stages and ponds throughout the Tucson Basin, Arizona, USA. Tadpoles Campus Ag Cebedilla Columbus Ft Lowell TVGR Campus Ag X 1.62 (0.0258) 2.87 (<0.0001) 2.77 (0.0002) 1.74 (0.0010) Cebedilla X 0.69 (1.0000) 0.56 (1.0000) -0.43 (0.5853) Tadpoles Columbus X -1.24 (0.8365) -2.24 (0.0432) Ft Lowell X -1.67 (0.0494)

Metamorphs Campus Ag Cebedilla Columbus Crosby Kennedy TVGR Campus 3.39 Ag -0.63 (0.6628) 2.29 (0.0010) (<0.0001) -3.33 (0.0008) 2.5 (0.0009) 2.29 (0.0002) Cebedilla -2.87 (0.0328) 0.09 (1.0000) 1.22 (0.7527) -5.58 (<0.0001) 0.29 (1.0000) 0.08 (1.0000) -1.69 -1.51 Tadpoles Columbus -4.6 (0.0010) (0.3387) -0.6 (1.0000) -7.3 (<0.0001) (0.5111) -1.7 (0.2504) -1.12 -0.85 Ft Lowell -4.12 (0.0019) (0.4353) 0.01 (1.0000) -6.81 (<0.0001) (0.6002) -1.1 (0.3070) TVGR -2.59 (0.0210) 0.39 (0.4623) 1.51 (0.0568) -5.3 (<0.0001) 0.62 (0.3476) 0.39 (0.3649) Campus Ag X 2.54 (0.0645) 3.63 (0.0111) -3.04 (0.1702) 2.72 (0.0576) 2.54 (0.0561) -0.35 -0.57 Cebedilla X 0.53 (1.0000) -6.21 (<0.0001) (1.0000) (0.6713) -2.01 -2.24 Metamorphs Columbus X -7.92 (<0.0001) (0.2131) (0.0892) 5.67 5.49 Crosby X (<0.0001) (<0.0001) -0.84 223 Kennedy X (0.4752)

Table 1.14 (cont.) Odds of testing positive for Batrachochytrium dendrobatidis in Rana catesbeiana across life stages and ponds throughout the Tucson Basin, Arizona, USA. Adults Campus Ag Cebedilla Columbus Crosby Ft Lowell Kennedy TVGR 3.33 1.56 -1.41 -0.04 1.02 Campus Ag (<0.0001) (0.0288) (0.0101) (0.5609) (0.223) 1.57 (0.019) 1.13 -0.66 -3.65 -2.24 -1.2 -0.65 Cebedilla (0.8337) (0.8893) (<0.0001) (0.0348) (0.5308) (0.8108) -2.43 -5.38 -4.01 -2.95 -2.41 Tadpoles Columbus -0.66 (1) (0.0797) (<0.0001) (0.0007) (0.0562) (0.0735) -1.86 -4.89 -3.49 -2.42 -1.86 Ft Lowell -0.04 (1) (0.1013) (<0.0001) (0.0008) (0.0756) (0.096) 1.45 -0.36 -3.36 -1.97 -0.91 -0.35 TVGR (0.0597) (0.5178) (<0.0001) (0.0142) (0.4355) (0.4896) 3.57 1.81 1.3 1.83 Campus Ag (0.0109) (0.2176) -1.12 (1) 0.23 (1) (0.5888) (0.1945) 0.52 -1.3 -4.29 -2.89 -1.85 -1.3 Cebedilla (1.000) (0.2506) (<0.0001) (0.0032) (0.1754) (0.2423) -1.19 -2.99 -5.99 -4.61 -3.54 -2.98 Columbus (0.9312) (0.024) (<0.0001) (<0.0001) (0.0207) (0.0221) Metamorphs 6.55 4.77 1.84 4.25 4.79 Crosby (<0.0001) (<0.0001) (0.1577) 3.2 (0.0167) (0.0008) (<0.0001) 0.23 -1.59 -4.61 -3.22 -2.15 -1.59 Kennedy (1.000) (0.1722) (<0.0001) (0.0025) (0.1215) (0.1639) 0.68 -1.12 -4.11 -2.73 -1.66 -1.11 TVGR (0.5837) (0.2539) (<0.0001) (0.0037) (0.1804) (0.2384)

224 Table 1.14 (cont.) Odds of testing positive for Batrachochytrium dendrobatidis in Rana catesbeiana across life stages and ponds throughout the Tucson Basin, Arizona, USA. Adults Campus

Ag Cebedilla Columbus Crosby Ft Lowell Kennedy TVGR Campus Ag X -2.47 -5.52 -3.07 -2.51 Cebedilla X (0.0250) (<0.0001) -0.36 (1) (0.0255) (0.0267) -3.41 -2.01 -0.97 -0.42 Columbus X (<0.0001) (0.0389) (0.5751) (0.774) Adults 1.36 2.41 2.96 Crosby X (0.1112) (0.0122) (<0.0001) 0.8 1.35 Ft Lowell X (0.6882) (0.1814) Kennedy X -0.05 (1) TVGR X

225 Table 1.15 Multiplicative differences of Batrachochytrium dendrobatidis infection loads in Rana catesbeiana across life stages and ponds throughout the Tucson Basin, Arizona, USA. Locations within a life stage with a diagonal line instead of values indicate <3 frogs tested positive at that location in that life stage. Tadpoles Campus Ag Cebedilla Columbus Ft Lowell TVGR Campus Ag X 85.59 (0.0023) 196.67 (0.0004) 6.81 (0.0187) Cebedilla X -2.3 (0.7243) -63.9 (0.0414) Tadpoles Columbus X -316.44 Ft Lowell X (0.0043)

Metamorphs Campus Ag Cebedilla Columbus Crosby Kennedy TVGR Campus 311.08 155.48 27.59 685.96 779.21 Ag (<0.0001) (0.0014) (<0.0001) (<0.0001) (<0.0001) Cebedilla -1.41 (0.8811) -2.82 (0.6680) -15.9 (0.1601) 1.49 (0.8668) 1.78 (0.8045)

Tadpoles Columbus -13.74 -77.41 Ft Lowell -6.87 (0.4027) (0.2797) (0.0281) -2.98 (0.6464) -2.74 (0.6644) 25.25 116.56 126.53 TVGR 50.51 (0.0091) (0.0547) 4.48 (0.1077) (0.0030) (0.0015) Campus -54.83 Ag X -9.43 (0.2366) (0.0048) -3.27 (0.5438) -1.88 (0.7179) -64.57 Cebedilla X (0.0318) -3.85 (0.5707) -2.22 (0.7192) Metamorphs Columbus X Crosby X 19 (0.0403) 27.94 (0.0066)

-29.52 226 Kennedy X (0.1467)

Table 1.15 (cont.) Multiplicative differences of Batrachochytrium dendrobatidis infection loads in Rana catesbeiana across life stages and ponds throughout the Tucson Basin, Arizona, USA. Locations within a life stage with a diagonal line instead of values indicate <3 frogs tested positive at that location in that life stage. Adults Campus Ag Cebedilla Columbus Crosby Ft Lowell Kennedy TVGR Campus 216.96 144.55 828.13 22.49 21.32 62.56 85.86 Ag (0.0003) (0.0016) (<0.0001) (<0.0001) (0.0003) (0.0044) (0.0005) -2.09 -3.03 1.89 -19.51 -20.49 -7.13 Cebedilla (0.7555) (0.6459) (0.7845) (0.1203) (0.1314) (0.4017) -5.06 (0.4645) Tadpoles Columbus -9.54 -14.78 -2.58 -94.95 -100.58 -33.55 -25.13 Ft Lowell (0.3368) (0.2663) (0.6837) (0.0179) (0.0243) (0.1318) (0.1496) 36.37 23.47 134.47 3.65 3.45 10.34 13.80 TVGR (0.0222) (0.0601) (0.0014) (0.1034) (0.2427) (0.1311) (0.0617) Campus -9.12 -10.15 -1.77 -68.25 -71.17 -27.61 -18.95 Ag (0.2517) (0.2215) (0.7442) (0.0042) (0.0062) (0.0833) (0.0904) -10.74 -11.95 -2.09 -80.38 -83.82 -32.51 -22.32 Cebedilla (0.3121) (0.2849) (0.7401) (0.0268) (0.0302) (0.1352) (0.1557) Columbus Metamorphs 6.49 5.18 29.70 -1.27 -1.33 Crosby (0.1846) (0.2477) (0.0058) (0.6541) (0.6965) 2.03 (0.6106) 2.89 (0.3602) -102.70 -159.15 -27.78 -1022.57 -1083.20 -361.37 -270.69 Kennedy (0.0498) (0.0375) (0.1544) (0.0004) (0.0007) (0.0122) (0.0130) -36.14 -40.22 -7.02 -270.57 -282.15 -109.44 -75.12 TVGR (0.0639) (0.0523) (0.2669) (0.0002) (0.0003) (0.0148) (0.0135)

227 Table 1.15 (cont.) Multiplicative differences of Batrachochytrium dendrobatidis infection loads in Rana catesbeiana across life stages and ponds throughout the Tucson Basin, Arizona, USA. Locations within a life stage with a diagonal line instead of values indicate <3 frogs tested positive at that location in that life stage. Adults Campus

Ag Cebedilla Columbus Crosby Ft Lowell Kennedy TVGR Campus -17.81 -3.11 -114.45 -121.23 -40.44 -30.30 Ag X (0.2347) (0.6258) (0.0138) (0.0192) (0.1126) (0.1275) -1.80 -69.47 -72.44 -28.10 -19.29 Cebedilla X (0.7903) (0.0321) (0.036) (0.1522) (0.1759) -296.45 -309.13 -119.9 -82.31 Adults Columbus X (0.0001) (0.0003) (0.0130) (0.0117) -1.06 Crosby X (0.9345) 2.71 (0.4697) 3.75 (0.2622) Ft Lowell X 2.63 (0.5278) 3.76 (0.3165) Kennedy X -2.84 (0.6393) TVGR X

228 229

APPENDIX F: BACKYARD POND INFORMATION

Home Owner Location House # Baker Near Kennedy Park 1 Fraser Near Kennedy Park 2 Campus Ag Farm Campus Ag Farm 3 Reifschneider Notch Neighborhood 4 Caruso Notch Neighborhood 5 Roscoe Notch Neighborhood 6 Delaney Notch Neighborhood 7 Berghausen Notch Neighborhood 8 Davison Houghton and Old Spanish Trail 9 Hernbrode Houghton and Old Spanish Trail 10 Olais Old Spanish Trail and Escalante 11 Schwalbe Old Spanish Trail and Escalante 12