LIFE HISTORY OF THE COASTAL TAILED FROG (ASCAPHUS TRUEI) ACROSS AN ELEVATIONAL GRADIENT IN NORTHERN CALIFORNIA

Abstract

The life history of a species is described in terms of its growth, longevity, and reproduction. Unsurprisingly, life history traits are known to vary in many taxa across environmental gradients. In the case of amphibians, species at high elevations and latitudes tend to have shorter breeding seasons, shorter activity periods, longer larval periods, reach sexual maturity at older ages, and produce fewer and larger clutches per year. The Coastal Tailed Frog (Ascaphus truei) is an ideal species for the study of geographic variation in life history because it ranges across most of the Pacific Northwest from northern California into British Columbia, and along its range it varies geographically in larval period and morphology. During a California Department of Fish and Wildlife restoration project in the Trinity Alps Wilderness, I had incidental captures of Coastal Tailed Frog larvae and adults. To date, no population across the species' range has been described above 2000m. These populations in the Trinity Alps range from 150m to over 2100m in elevation, and those that are in the higher part of the range are likely living at the species' maximum elevational limit. iii In this study, I examined size, growth, larval period, size at sexual maturity, and longevity of A. truei across populations along an elevational gradient in the Klamath Mountains of northern California. I calculated growth rates and movement by individually marking tadpoles and post-metamorphic frogs with visual implant elastomer (VIE), then tracking them from May through October of 2018. I described the length of the larval period using length-density histograms to visualize larval cohorts, I determined size at sexual maturity using secondary sexual characteristics of post-metamorphic frogs, and I determined longevity using skeletochronology. I found that the larval period of A. truei in the Klamath Mountains of northern California ranges from two years in low and mid-elevations, to at least three years in high elevations. I also found decreased body size and increased growth rates of tadpoles with increasing elevation. Post-metamorphic frogs grew at similar rates as previously described coastal California populations. There was high site fidelity and significantly greater movement during the months of June and August in post-metamorphic animals. Frogs in the high elevations are capable of great longevity, with a maximum observed age estimated at eight years post-metamorphosis. The high elevation populations described here have the longest larval period documented in California. This study also provides the first field estimates of larval growth rates and the first longevity estimates of post metamorphic frogs in California. Future laboratory experiments will be necessary to separate phenotypic plasticity of life history traits from true genetic differences between A. truei populations in the Klamath Mountains of northern California, as potential explanations for the variation seen.

Full text

LIFE HISTORY OF THE COASTAL TAILED FROG (ASCAPHUS TRUEI) ACROSS
AN ELEVATIONAL GRADIENT IN NORTHERN CALIFORNIA
By
Adrian Daniel Macedo
A Thesis Presented to
The Faculty of Humboldt State University
In Partial Fulfillment of the Requirements for the Degree
Master of Science in Biology
Committee Membership
Dr. John O. Reiss, Committee Chair
Dr. Daniel C. Barton, Committee Member
Dr. Karen L. Pope, Committee Member
Dr. Sharyn B. Marks, Committee Member
Dr. Erik Jules, Program Graduate Coordinator
December 2019

ii
ABSTRACT
LIFE HISTORY OF THE COASTAL TAILED FROG (ASCAPHUS TRUEI) ACROSS
AN ELEVATIONAL GRADIENT IN NORTHERN CALIFORNIA
Adrian D. Macedo
The life history of a species is described in terms of its growth, longevity, and
reproduction. Unsurprisingly, life history traits are known to vary in many taxa across
environmental gradients. In the case of amphibians, species at high elevations and
latitudes tend to have shorter breeding seasons, shorter activity periods, longer larval
periods, reach sexual maturity at older ages, and produce fewer and larger clutches per
year.
The Coastal Tailed Frog (Ascaphus truei) is an ideal species for the study of
geographic variation in life history because it ranges across most of the Pacific Northwest
from northern California into British Columbia, and along its range it varies
geographically in larval period and morphology. During a California Department of Fish
and Wildlife restoration project in the Trinity Alps Wilderness, I had incidental captures
of Coastal Tailed Frog larvae and adults. To date, no population across the species’ range
has been described above 2000m. These populations in the Trinity Alps range from 150m
to over 2100m in elevation, and those that are in the higher part of the range are likely
living at the species’ maximum elevational limit.

iii
In this study, I examined size, growth, larval period, size at sexual maturity, and
longevity of A. truei across populations along an elevational gradient in the Klamath
Mountains of northern California. I calculated growth rates and movement by
individually marking tadpoles and post-metamorphic frogs with visual implant elastomer
(VIE), then tracking them from May through October of 2018. I described the length of
the larval period using length-density histograms to visualize larval cohorts, I determined
size at sexual maturity using secondary sexual characteristics of post-metamorphic frogs,
and I determined longevity using skeletochronology.
I found that the larval period of A. truei in the Klamath Mountains of northern
California ranges from two years in low and mid-elevations, to at least three years in high
elevations. I also found decreased body size and increased growth rates of tadpoles with
increasing elevation. Post-metamorphic frogs grew at similar rates as previously
described coastal California populations. There was high site fidelity and significantly
greater movement during the months of June and August in post-metamorphic animals.
Frogs in the high elevations are capable of great longevity, with a maximum observed
age estimated at eight years post-metamorphosis.
The high elevation populations described here have the longest larval period
documented in California. This study also provides the first field estimates of larval
growth rates and the first longevity estimates of post metamorphic frogs in California.
Future laboratory experiments will be necessary to separate phenotypic plasticity of life
history traits from true genetic differences between A. truei populations in the Klamath
Mountains of northern California, as potential explanations for the variation seen.

iv
ACKNOWLEDGEMENTS
I would like to first and foremost thank my parents: Richard and Suzanna
Macedo. I was fortunate enough to have been raised by two excellent scientists and
naturalists, my father a biologist for the California Department of Fish and Wildlife
(CDFW), my mother a high school science teacher. I also had the privilege to have been
raised in the mountains of northern California and had a perennial creek and forested
country behind my house that would inspire me to dedicate my life to the understanding
and preservation of nature. At an early age, I would spend hours in the forest catching
frogs, salamanders, picking flowers, and I would bring them to my parents. My parents to
this day continue to support and nurture my passion for the natural world, and I owe them
the greatest acknowledgements in that regard.
I would like to acknowledge Justin Garwood who first hired me as a technician in
the backcountry of the Trinity Alps Wilderness to remove non-native fish and study
amphibians and reptiles. It was there where I started catching Coastal Tailed Frogs
(Ascaphus truei), and it was Justin Garwood who encouraged me to pursue their study.
He also provided me with skeletochronology slides from the East van Matre Creek and
some data from the Canyon Creek A. truei populations.
I would like to thank Justin Demianew—a fellow alpsman and invaluable
colleague in the roughest field conditions and the most complicated statistical analyses.
In the field with him by my side I felt like no snowstorm, lack of funding, flood events,

v
or forest fire could stop us from collecting data and producing novel scientific work. He
aided me immensely and is an excellent teacher.
I must not forget to acknowledge Mary Carlquist, my field technician, for her
bravery and toughness in the field. She was able to help me collect data day and night for
70 days in the wilderness, she learned how to drive stick shift in one single afternoon, and
she was able bounce back to work after serious injury. Mary was one of those rare
assistants who, along with giving me a hand, also thought deeply about the purpose of the
study and always had good thoughts and questions.
To all the undergraduate assistants in the laboratory: Julie Trejo, Daisy Ceja,
Ryan Aberg, Robyn Botsch, Syndey Gerstenberg, Bailey Andrews, Monica Jarquin, and
Mario Vasquez. I would like to thank you for your dedication and company. As well as
others whom helped me in the field: Chelsea Stewart, Ethan Snee, Shannon Hedge, James
Bettaso, Kyle Orr, and Adam Mohr.
To the thank the folks in the Dr. John Reiss/Dr. Mihai Tomescu lab: Kelly Pfeiler,
Kyle Orr, Jaclyn Patmore, Megan Nibbelink, Shayda Abidi, and Dr. Allison Bronson for
support in the forms of good company in the lab, taco Tuesdays, and listening to my
complaints about R and scientific writing. I would also like to thank some of my other
graduate student colleagues who supported me friends for the past two years such as Skye
Salanek, Ashley Abitz, and Jason Holmes.
I would like to thank my funders for financial support: CSU’s Research,
Scholarship, and Creative Activities Program, CDFW State Wildlife Grants, and Biology
Graduate Student Association. I would like to thank Susan Wright in the HSU Biological

vi
Sciences Stockroom, David Baston in the CNRS CORE, and Colin Wingfield in the
Wildlife Stockroom for material support.
I would like to thank my graduate advisor Dr. John Reiss, who accepted my
proposal for this project, met with me countless times, and read and edited countless
drafts of writing and presentations all with a smile and friendly face. John’s kindness
towards me and help was a breath of fresh air. I would like to thank Dr. Karen Kiemnec-
Tyburczy for assistance and funding to do the A. truei genetic work, and Dr. R. Bruce
Bury for advice and guidance. Lastly, I would like to thank my committee members: Dr.
Karen Pope, Dr. Sharyn Marks, and Dr. Daniel Barton for their valuable insight and
helping me improve my scientific writing.

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TABLE OF CONTENTS
ABSTRACT ................................................................................................................... ii
ACKNOWLEDGEMENTS ........................................................................................... iv
TABLE OF CONTENTS .............................................................................................. vii
LIST OF TABLES ..........................................................................................................x
LIST OF FIGURES ....................................................................................................... xi
LIST OF APPENDICES .............................................................................................. xiii
CHAPTER ONE: LARVAL LIFE HISTORY OF THE COASTAL TAILED FROG
(ASCAPHUS TRUEI) ACROSS AN ELEVATIONAL GRADIENT IN NORTHERN
CALIFORNIA .................................................................................................................1
Introduction .................................................................................................................1
Research objectives and predictions .........................................................................5
Importance and need ................................................................................................8
Methods.......................................................................................................................8
Study area ................................................................................................................8
Site selection ............................................................................................................9
Stream sampling design ......................................................................................... 10
Larval capture and immobilization ......................................................................... 11
Measurements ........................................................................................................ 12
Immobilization and marking .................................................................................. 12
Field collection ...................................................................................................... 14
Statistical methods ................................................................................................. 15
Results ....................................................................................................................... 16

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Growth/developmental rates and body size of tadpoles........................................... 16
Size cohorts and length of larval period .................................................................. 20
Bergmann’s rule..................................................................................................... 23
Discussion ................................................................................................................. 24
Growth................................................................................................................... 24
Larval period ......................................................................................................... 26
Bergmann’s rule..................................................................................................... 29
Genetic differences between populations ................................................................ 30
Management implications ...................................................................................... 30
CHAPTER TWO: LIFE HISTORY OF POST-METAMORPHIC COASTAL TAILED
FROGS (ASCAPHUS TRUEI) IN THE TRINITY ALPS WILDERNESS OF
NORTHERN CALIFORNIA ..................................................................................... 32
Introduction ............................................................................................................... 32
Research Objectives and Predictions ...................................................................... 35
Importance and need .............................................................................................. 36
Methods..................................................................................................................... 36
Study area .............................................................................................................. 36
Post-metamorphic frog capture .............................................................................. 36
Measurements ........................................................................................................ 37
Marking ................................................................................................................. 38
Field collection and skeletochronology .................................................................. 38
Statistical methods ................................................................................................. 39
Results ....................................................................................................................... 41
Growth rates of post-metamorphic frogs ................................................................ 41

ix
Adult and immature age classes ............................................................................. 42
Size at sexual maturity ........................................................................................... 42
Timing of breeding and oviposition ........................................................................ 48
Longevity............................................................................................................... 48
Site fidelity and movement..................................................................................... 51
Discussion ................................................................................................................. 55
Growth and development ....................................................................................... 55
Body size of sexually mature animals..................................................................... 56
Timing of copulation and oviposition ..................................................................... 58
Site fidelity and movement..................................................................................... 58
Longevity............................................................................................................... 59
Management implications ...................................................................................... 60
REFERENCES .............................................................................................................. 62
APPENDICES ............................................................................................................... 67

x
LIST OF TABLES
Table 1. Capture mark-recapture (CMR) and ancillary (non-CMR) study sites across an
elevational gradient in the Trinity Alps, CA, USA ......................................................... 11
Table 2. Growth rates of A. truei tadpoles from May 2018 to August 2018 across an
elevation gradient in northern California, USA. ............................................................. 17
Table 3. GLM model of the influence of body length, sex, and their interaction on sexual
maturity of Ascaphus truei in northern California, USA. ................................................ 44
Table 4. Mean snout-vent length (SVL) of post-metamorphic frogs in the Trinity Alps of
northern California,USA. ............................................................................................... 45
Table 5. Longitudinal (upstream/downstream) movement distances of the Coastal Tailed
Frog populations of the Trinity Alps Wilderness of California, USA. ............................ 52

xi
LIST OF FIGURES
Figure 1. Alternative predictions of the relationship of body size to elevation of Coastal
Tailed Frogs in northern California, USA. .......................................................................7
Figure 2. Capture mark-recapture (CMR) and ancillary (non-CMR) study sites across
low, mid, and high elevations within the Trinity River watershed in northern California.
...................................................................................................................................... 10
Figure 3. Coastal Tailed Frog tadpole with visual implant elastomer (VIE) marking. .... 14
Figure 4. Growth rate of tadpoles across an elevational gradient in northern California,
USA. Model variance is reported in standard error. ........................................................ 18
Figure 5. Growth rates of tadpoles across the months of June (left), July (middle) and
August (right) of the survey season between elevation categories in northern California.
The horizonal lines represent the medians and the lower and upper hinges correspond to
the first and third quartiles (the 25th and 75th percentiles). The whiskers extend from the
hinge to the largest value (upper) and smallest value (lower) no further than 1.5 times the
interquartile range. ......................................................................................................... 19
Figure 6. Density plots of total length histograms of Coastal Tailed Frog tadpoles from
the high elevations of northern California. Once a tadpole began metamorphosis it was
removed......................................................................................................................... 21
Figure 7. Density plots of total length histograms of Coastal Tailed Frog tadpoles from
the mid elevations of northern California. Once a tadpole began metamorphosis it was
removed......................................................................................................................... 22
Figure 8. Density plots of total length histograms of Coastal Tailed Frog tadpoles from
the low elevations of northern California. Once a tadpole began metamorphosis it was
removed......................................................................................................................... 23
Figure 9. Body size of A. truei tadpoles at developmental stage 37 across elevation in
inland northern California, USA. The horizontal lines represent the median, and the lower
and upper hinges correspond to the first and third quartiles (the 25th and 75th
percentiles). The whiskers extend from the hinge to the largest value (upper) and smallest
value (lower) no further than 1.5 times the interquartile range........................................ 24
Figure 10. Density histogram of Coastal Tailed Frog tadpole total lengths between Siligo
and East van Matre Creek from the Trinity Alps of northern California. ........................ 28

xii
Figure 11. Snout-to-vent length frequency histograms for mature (in dark gray and
dashed borders) and immature (in white and solid borders) male and female Coastal
Tailed Frogs from the Trinity Alps Wilderness of northern California, USA. Bins are
1mm wide. ..................................................................................................................... 43
Figure 12. Binomial logistic regression of body size at sexual maturity of A. truei in
northern California, USA. Plot of least squares regression on body length. .................... 44
Figure 13. Body length of mature (right) vs immature (left) females (F) and males (M)
Coastal Tailed Frogs in northern California, USA. The horizonal lines represent the
medians and the lower and upper hinges correspond to the first and third quartiles (the
25th and 75th percentiles). The whiskers extend from the hinge to the largest value
(upper) and smallest value (lower) no further than 1.5 times the interquartile range. ...... 46
Figure 14. Body length of mature Ascaphus truei across elevation in northern California,
USA. The horizonal lines represent the medians and the lower and upper hinges
correspond to the first and third quartiles (the 25th and 75th percentiles). The whiskers
extend from the hinge to the largest value (upper) and smallest value (lower) no further
than 1.5 times the interquartile range. ............................................................................ 47
Figure 15. Median age (post-metamorphosis) of A. truei across elevation categories in
northern California, USA. Sample size: high elevation: 38, mid elevation: four, low
elevation: two. The horizontal lines represent the medians and the lower and upper hinges
correspond to the first and third quartiles (the 25th and 75th percentiles). The whiskers
extend from the hinge to the largest value (upper) and smallest value (lower) no further
than 1.5 times the interquartile range. ............................................................................ 49
Figure 16. Phalanx section showing two LAGs from a post-metamorphic A. truei from a
low elevation population site in northern California, USA. Scale bar = 200um. ............. 50
Figure 17. Phalanx section showing five LAGs from a post-metamorphic A. truei from a
high elevation population site in northern California, USA. Scale bar = 100um. ............ 51
Figure 18. Distribution of longitudinal (upstream>0.0, downstream<0.0) movement
distances relative to each capture of Coastal Tailed Frogs in the Trinity Alps Wilderness
of northern California, USA. .......................................................................................... 53
Figure 19. Linear movement distance (m) of Coastal Tailed Frogs across the months of
the survey season in the Trinity Alps Wilderness of northern California, USA. .............. 54

xiii
LIST OF APPENDICES
Appendix A. Stages of development for Coastal Tailed Frog (Ascaphus truei) tadpoles,
adapted from Gosner (1960), Brown (1990), Brown (1989), and John Reiss
(unpublished). This table was design for use in the field on hatched tadpoles, therefore it
does not include stages below 26. .................................................................................. 67
Appendix B. Larval Coastal Tailed Frog specimens collected from inland California and
vouchered in the Humboldt State Vertebrate Museum (HSUMV). ................................. 72
Appendix C. Post-metamorphic Coastal Tailed Frog Voucher Specimens at the
Humboldt State Vertebrate Museum (HSUVM). ............................................................ 74
Appendix D. Protocol developed to prepare slides of Coastal Tailed Frogs for estimating
ages using skeletochronology. ....................................................................................... 75
Appendix E. Study site photos of low elevation habitat composed of a dense canopy of
late seral Douglas-fir (Pseudotsuga menziesii), Big-leaf Maple (Acer macrophylum), Red
Alder (Alnus rubrus), and Tan Oak(Notholithocarpus densiflorus) in northern California.
...................................................................................................................................... 78
Appendix F. Study site photos of mid-elevation site habitat composed of a dense canopy
of late seral Douglas-fir (Pseudotsuga menziesii), Big-leaf Maple Maple (Acer
macrophylum), Red Alder (Alnus rubrus), and Tan Oak (Notholithocarpus densiflorus) in
northern California, USA. .............................................................................................. 79
Appendix G. Site photos of high elevation habitat composed of very little canopy with a
few scattered tree species, including Jeffrey Pine (Pinus jefferi), Western White Pine
(Pinus monticola), and Foxtail Pine (Pinus balfouriana) in northern California, USA. ... 80
Appendix H. Total length frequency histograms of Coastal Tailed Frogs across low
elevation study sites and dates they were surveyed......................................................... 81
Appendix I. Total length frequency histograms of Coastal Tailed Frogs across mid-
elevation study sites and dates they were surveyed......................................................... 82
Appendix J. Total length frequency histograms of Coastal Tailed Frogs across high
elevation study sites and dates they were surveyed......................................................... 83
Appendix K. Developmental Stage frequency histograms of Coastal Tailed Frogs across
Elevation Category and dates they were surveyed. ......................................................... 84

1
CHAPTER ONE: LARVAL LIFE HISTORY OF THE COASTAL TAILED FROG
(ASCAPHUS TRUEI) ACROSS AN ELEVATIONAL GRADIENT IN NORTHERN
CALIFORNIA
Introduction
The life history of a species is described in terms of its growth, longevity, and
reproduction. Unsurprisingly, life history traits are known to vary in many taxa across
environmental gradients (Begon et al. 1990). For example, one well known
ecogeographical rule is Bergmann’s rule, which states that within endothermic
vertebrates, there is a general intra- and interspecific trend towards larger body size in
cooler environments (Bergmann 1847). Amphibian adults, although ectotherms, also
follow Bergmann’s rule, with 67% of species overall, and 62% of anuran species, having
larger body size at high latitudes or elevations (Ashton 2002). Intraspecifically,
amphibian populations at high elevations and latitudes tend to have shorter breeding
seasons, shorter activity periods, longer larval periods, reach sexual maturity later, and
produce fewer and larger clutches per year relative to those at lower elevations (Baraquet
et al., Bears et al. 2009, Lindgren and Laurila 2005, Miaud et al. 1999, Moore 1949, and
Morrison and Hero 2003). Here I will focus on the larval phase of the life cycle; in
particular, larval period and larval size variation.
Such variation has been documented in a number of species. For example, Green
Frog (Rana clamitans) larvae in montane populations overwinter twice before completing

2
metamorphosis, while lowland populations metamorphose within the same year (Berven
et al. 1979). Interestingly, with respect to Bergmann’s rule, larvae of Western Spotted
Frogs (Rana pretiosa) have a pattern that contrasts with that described for other species:
larvae are significantly larger in lowland populations than in highland populations; this
pattern is thought to be due to genetic differences between the populations (Licht 1975).
Phenotypic plasticity in growth and development rates may occur in response to
differing environmental conditions. Variation in growth and development at the
proximate level can lead to differences in life history traits like longevity and maturity
across latitudinal and elevational gradients (Lindgren and Laurila 2005, Licht 1975).
Reznick (1990) developed two models to understand the proximate origins for
variation in age and length at maturity of guppies (Poecilia reticulata). The first model
used constant values for the body length that initiates maturation and the time interval
between initiation and completion of maturation. In this way, the model assumes that
development (size, growth rate, minimum size to initiate maturation, and maximum size
at metamorphosis) does not respond to variation in the environment, meaning there is no
plasticity in their development across different environmental conditions. The second
model allowed for plasticity in development in response to variation in the environment.
Using these two models, Reznick (1990), found that guppies in favorable
conditions (more food) could grow faster and initiate the process of maturation earlier or
at a larger size. In unfavorable conditions (less food), individuals grow slower and reach
maturity later, or at a smaller size. This suggested that growth rates are plastic in guppies;
however, guppies were not flexible in the amount of time it takes to complete maturation

3
once the process was initiated (Reznick 1990). These findings suggest that flexible
growth rates could produce variation in body size and time to reach maturity in guppies
(Reznick 1990).
These same models were applied to the post-metamorphic Common Frog (Rana
esculenta) with similar results – in environments with a shorter activity period, post-
metamorphic frogs grew more slowly and reached sexual maturity later. However, adult
Common Frogs at high elevations eventually attained a greater final length (Miaud et al.
1999). This was because they also had greater longevity than individuals in populations
with a longer activity period (Miaud et al. 1999).
By contrast, when comparing populations from high and low latitudes using a
common garden experiment, individuals collected from higher latitudes actually had
increased growth rates compared to individuals from lower latitudes (Lindgren and
Laurila 2005). This is an example of countergradient variation with respect to growth
rates. Countergradient variation is a geographical pattern of genotypes (with respect to
environments) in which genetic influences on a trait oppose environmental influences,
thereby minimizing phenotypic change along the gradient (Conover et al. 1995). This
increase in growth was not due to a high food intake rate, but higher growth efficiency in
high latitude populations (Lindgren and Laurila 2005). Increased growth rates in high
elevation populations have also been described for Western Spotted Frogs, Rana pretiosa
(Licht 1975). However, in natural environments, countergradient variation means that
environmental conditions can dampen or mask phenotypic variation such as higher
growth efficiency in high elevations, so that observed growth rates may look the same or

4
appear lower in high elevations. While these lasting effects have been documented for
post-metamorphic anurans, they remain undocumented for larvae. Measuring variation in
growth and development rates in larvae can help explain why life history traits such as
larval size and larval period differ among populations.
The Coastal Tailed Frog (Ascaphus truei) is an ideal species for the study of
geographic and elevational variation in life history, because it ranges across most of the
Pacific Northwest from northern California into British Columbia, and from near sea
level to high elevations in interior mountains. Along that range, A. truei varies widely in
larval period (Bury and Adams 1999, Metter 1967, and Wallace and Diller 1998), with a
range from one to at least three years across a south-north latitudinal gradient (Bury and
Adams 1999). Populations in the mountains of northwestern Washington are thought to
have a four-year larval period (Brown 1990).
Little is known with regards to growth rates of Coastal Tailed Frogs across their
range. Studies assessing growth rates of tadpoles (using body mass measurements) in
coastal British Columbia, Canada suggested that decreased light, nutrients, and increased
consumer density all had a negative influence on growth (Kiffney and Richardson 2001,
Mallory and Richardson 2005). No studies to date have calculated growth rates for
tadpoles in California.
During the summers of 2016-2018, I worked for the California Department of
Fish and Wildlife (CDFW) on a restoration project in the Trinity Alps Wilderness and
encountered Coastal Tailed Frogs while removing fish from a stream at 2,100m elevation.
In California, A. truei are commonly found in headwater streams of coastal forests, and

5
no population across the species’ entire range has been described above ~2,000m (Bury
1968). I was immediately intrigued by this high elevation population and sought to learn
about how the species survives in the vastly different conditions of coastal and mountain
streams. Coastal Tailed Frogs are cold, headwater stream specialists, yet appear to be
elevational generalists. The discovery of these populations extends the range from the
coast to the slopes of the highest peaks in the Klamath-Siskiyou Mountains. Coastal
Tailed Frogs have clearly been able to find the specific habitats to meet their unique life
history needs at a range of elevations. However, the broad climatic differences across
elevations (e.g., snow at the high elevations) likely influence life history traits such as
size, growth rates, longevity, age at sexual maturity and length of larval development
period. Here I examine these for larval animals; I report on post-metamorphic animals in
Chapter Two, below.
Research objectives and predictions
My objective was to describe whether life history traits of larval A. truei in
northern California differ across an elevational gradient, and if larval A. truei follow
Bergmann’s rule along the same gradient. My predictions were as follows:
Growth and development of larvae
I proposed three alternative predictions for the variation of growth of A. truei tadpoles
across an elevational gradient:
1. No difference: Growth rates do not differ across elevations.

6
2. Growth rates of tadpoles are greater in populations at higher elevations, as
reported for Rana pretiosa (Licht 1975) and R. temporaria (Lindgren and Laurila
2005).
3. Growth rates are lower in higher elevation populations, as reported for Poecilia
reticulata (Reznick 1990), Ascaphus truei (Kiffney and Richardson 2001, Mallory
and Richardson 2005) and Rana temporaria (Miaud et al. 1999).
Length of the larval period
I proposed two alternative predictions for the length of the larval period across an
elevational gradient:
1. Null hypothesis: Age at metamorphosis is the same across an elevational gradient
in northern California and is two years (the maximum described currently for
California) (Bury and Adams 1999 and Wallace and Diller 1998).
2. Age at metamorphosis is one to two years in lowland populations, as was
described in coastal California (Wallace and Diller 1998) and up to four years in
higher elevations, as was described for populations in the mountains of
northwestern Washington (Brown 1990).
Bergmann’s rule
I developed three alternative predictions for larvae with regards to Bergmann’s rule
(Figure1):
1. There is no difference in body size (at a given developmental stage) across an
elevational gradient.

7
2. Larval body size (at a given developmental stage) increases along an increasing
elevational gradient.
3. Tadpoles (at a given developmental stage) are smaller in higher elevation
populations, as described for Western Spotted Frogs (Licht 1975).
Figure 1. Alternative predictions of the relationship of body size to elevation of Coastal
Tailed Frogs in northern California, USA.

8
Importance and need
Most of the research on Coastal Tailed Frogs in California has focused on how the
species may respond to human impact and climate change and has been conducted in
coastal areas. Only a few studies consider the life history variation across California
(Bury and Adams 1999, and Wallace and Diller 1998). The Coastal Tailed Frog is a
Species of Special Concern in California, more information is needed regarding
population dynamics, and life history studies could aid in determining which life history
stages limit population growth (Thomson et al. 2016). This chapter describes new
localities and information on life history traits of larval coastal tailed frog populations
that will help inform how populations can respond to disturbance and climate change
based on their life history strategy and plasticity in strategy.
Methods
Study area
This study was conducted along an elevational gradient from the upper reaches of
the Trinity River in Siskiyou and Trinity Counties, CA (Decimal Degrees, WGS84:
40.929834, -122.889931) down to the lower reaches of the Trinity River, Humboldt
County, CA (Decimal Degrees, WGS84: 40.89425, -123.69389, Figure 2) with elevations
ranging from ~150 m to ~2100 m.

9
All the study sites lie within the Klamath Mountain bioregion, which ranges
from northern California to south-eastern Oregon (Skinner et al. 2006). In California, this
bioregion lies between the northern Californian coast on the west and the southern
Cascades range to the east and contains the Klamath and Trinity River systems.
The climate is Mediterranean, and is characterized by wet, cool winters and dry,
warm summers. The proximity to the Pacific Ocean causes a moisture and temperature
gradient that leads to characteristic patterns of precipitation via orographic effects.
Average precipitation over the entire region is 101.2 cm annually with most rainfall from
October to April (Skinner et al. 2006). The summer months are typically warm and dry,
and infrequent precipitation events during this time of year occur in the form of
thunderstorms (Ferlatte 1974). The average April 1st snowpack is 259 cm at an elevation
of 2,042 m (Skinner et al. 2006), and in the highest elevations 5m of snow was measured
in mid-May of 2017 (personal observation). Snowpack does not persist more than a
month in the summer at low and mid-elevation sites and habitat is usually clear of snow
by May (personal observation). Summer air temperatures at the lower elevations sites
frequently reach 38°C or more, whereas temperatures in the high elevations sites rarely
reach 32°C.
Site selection
I selected four high elevation study sites (~1500m to ~2100m), seven mid-
elevation sites (~600 to ~1500m), and five low elevation sites (~150m to ~600m) (Table
1). Sites were selected from a combination of previous explorations, discussions with
herpetologists, and locations reported from museum collections. High elevation sites

10
were all in the Trinity Alps Wilderness and included the Echo Lake, Siligo Creek, and
Canyon Creek Basins, while the mid- and low-elevation sites extended along the lower
Trinity River watershed, near the towns of Helena, Burnt Ranch, and Willow Creek,
(Figure 2; Table 1; for site photos, see Appendices E-G).
Figure 2. Capture mark-recapture (CMR) and ancillary (non-CMR) study sites across
low, mid, and high elevations within the Trinity River watershed in northern California.
Stream sampling design
At ten of the study sites, I conducted a minimum of one survey to collect data on
larval period and age structure. I also selected two high, two mid, and two low elevation
streams for a rigorous capture-mark-recapture (CMR) study to examine growth rates
(Table 1). All CMR sites were sampled every other week from May 2018-August 2018.
Each stream was surveyed along a 200m stretch or more.
Burnt Ranch
Helena

11
Table 1. Capture mark-recapture (CMR) and ancillary (non-CMR) study sites across an
elevational gradient in the Trinity Alps, CA, USA
CMR Site Names
Elevation
(m)
Elevation
Category
UTM Easting
(WGS 84)
UTM Northing
(WGS 84)
Boise Creek
244
Low
444527
4531930
East Willow Creek
399
Low
440409
4528759
Headwaters of Big Creek
1009
Mid
471706
4524290
East Fork Big Creek
1082
Mid
470372
4522006
East van Matre Creek
2040
High
509101
4529635
Silago Creek
2076
High
508940
4531005
Non-CMR Site Names
Elevation
(m)
Elevation
Category
UTM Easting
(WGS 84)
UTM Northing
(WGS 84)
Lower Cedar Creek
152
Low
449840
4539431
Kirkham Creek
591
Low
443631
4536574
Little Bidden Creek
610
Low
461357
4515026
Rebekah's Creek
930
Mid
490063
4510414
Upper Cedar Creek
994
Mid
459495
4531987
East Horse Linto
1339
Mid
463995
4539212
East Stuart Fork
1361
Mid
515316
4530237
Trinity Alps Creek
1487
Mid
509946
4525589
Deep Creek
1969
High
509189
4529848
Canyon Creek
2131
High
496607
4537647
Larval capture and immobilization
I captured tadpoles using a combination of backpack electrofishing with a long-
handled dip net and rubble-rousing (light touch). I used rubble-rousing when the
backpack electrofisher batteries died or when stream connectivity was too low. I set
backpack electrofishers at 300-500V with low pulse set channel at three pulses per
second at 25% duty cycle, and a high pulse set at 30 pulses per second at 23% duty cycle.
I adjusted the voltage and pulse up or down as necessitated by stream conductivity.
Rubble-rousing in sensitive areas may alter stream bed conditions, so I minimized this by

12
using the light touch approach and spacing surveys at least two weeks apart. Once I
captured an animal or animals, they were transferred to plastic bags with cool stream
water and the site of capture was recorded.
Measurements
For larvae, I made the following measurements:
Snout-vent length (SVL): Measured from the tip of the snout to the posterior end
of the anal flap.
Total length: from the tip of snout to tip of tail.
Developmental stage: I scored the developmental stage with a staging table
(modified from Gosner 1960) for Ascaphus, modified for use in the field by John
Reiss and me (Appendix A). I measured hind-limb bud length through the
tadpole’s anal flap when hindlimb buds were visible. Because some stages were
difficult to distinguish in the field, I grouped them into a series of stages; for
example, between stages 28 and 34 toe differentiation and development is
obscured by the anal flap, but the length of the limb bud can be measured.
Therefore, any tadpole that had a limb bud length less than 0.7mm I reported as
stage 28-33, and any tadpole with limb bud greater than 0.7mm I reported as stage
34-36 (Appendix A). This inability to distinguish stages 29 through 33 is reflected
as a gap in the developmental stage histograms (Appendix K).
Immobilization and marking
I anesthetized tadpoles in the field with MS-222 (300 mg/L in stream water,
buffered to pH 7.0 with 600mg/L of baking soda) to immobilize them prior to marking

13
and measurement. I used the same solution for all larvae from the same stream. One at a
time, I placed individuals in the solution, and they were removed once they were
determined to be unresponsive (i.e., failed to exhibit a righting reflex). Once removed, I
rinsed them with stream water to prevent overdose. I followed anesthetizing protocols
used previously for Coastal Tailed Frogs (Chelgren and Adams 2017).
I marked tadpoles using visual implant elastomer (VIE) (Northwest Marine
Technology Inc. Seattle, Washington). I marked each animal for individual recognition
using a combination of four possible colors (yellow, orange, red, and blue). I made the
marks just under the skin, 3-5mm long and sometimes smaller for very small individuals.
I viewed marks with a UV light in a darkened area to enhance visibility. Tadpoles were
marked on the tail in upper and lower (i.e., dorsal and ventral to the midline, respectively)
and front and back positions (the front position being directly posterior to the vent, the
back position being posterior to the mark closer to the vent; Figure 3). Marking methods
followed Chelgren and Adams (2017) and 625 individual marking codes were generated
using SalaMarker (MacNeil et al. 2011).

14
Field collection
Because there are no museum collections of Coastal Tailed Frogs from any
population over 2000m elevation, I collected 36 larvae that were either found dead, found
inside snakes, or died incidentally from anesthesia/handling, and eight larvae and one
sub-adult from newly described high elevation sites. Collections were vouchered at the
HSU Vertebrate Museum (Appendix B).
Figure 3. Coastal Tailed Frog tadpole with visual implant elastomer (VIE)
marking.

15
Statistical methods
I performed all analyses in the statistical program R (Version 3.2.2, R Core
Development Team 2017).
Growth rate
I estimated growth by subtracting the body size of the last capture of each
individual animal by the body size of the first capture of each individual animal, this was
compared to the number of days between captures. I compared the relationship between
growth and number of days between captures across elevation categories using a linear
mixed effects regression with elevation category as a fixed additive effect and each
individual as a random effect. Growth rates were calculated by dividing the amount of
growth by the number of days between captures and were compared across the survey
months using non-parametric Kruskal-Wallis and Conover-Iman tests (Dinno 2017).
Number of larval cohorts and age at metamorphosis
I visually inspected density frequency histograms of total length vs. frequency of
capture for each site. Since tail resorption during metamorphosis may confound size-class
analysis, I defined metamorphosing tadpoles as having emerged forelimbs
(developmental stage 41 and later) and excluded them so they would not create false
peaks or confound existing peaks. Peaks within histograms were considered distinct age
classes and the metamorphosing age class was considered a distinct separate age class.
This has been the method used to estimate larval period in the literature to date (Brown
1990, Bury and Adams 1999, Metter 1968, Wallace and Diller 1999). I also compared the
cohorts across the survey season at sites if I completed repeated surveys. This allowed me

16
to see how the tadpole cohorts change across the season and determine timing of
metamorphosis. I also made histograms of developmental stage following the same
protocol as above. This aided in defining tadpole cohorts.
Bergmann’s rule
I compared body size (SVL) of tadpoles across elevation categories, using the
body size of tadpoles of development stage 37, and analyzed differences using a non-
parametric Kruskal-Wallis and Conover-Iman tests (Dinno 2017).
Results
In total, I took 2,280 measurements of tadpoles across a total of 16 sites (Figure 3)
over 70 days from May-August 2018. One thousand two hundred and thirty larvae were
individually marked, 210 (17%) of which were recaptured at least once: 161 were caught
twice, 37 caught three times, 11 caught four times, and one caught five times. The total
number of tadpoles captured was similar across elevation categories: 408 individual
tadpoles were caught in the low elevations, 484 in the mid elevations, and 338 in the high
elevations. Tadpoles were found in all the ancillary sites, but only Upper Cedar Creek,
East Stuart Fork, Kirkham Creek, and Little Bidden Creek had over 10 individuals
captured.
Growth/developmental rates and body size of tadpoles
Growth rates were significantly higher at high elevations compared to low
elevations (Std. Error=0.29, D. F.=159, P=<0.01) and mid elevations (Std. Error=0.26, D.
F.=159, P=<0.04) (Figure 4 and Table 2). Even though there was an observable

17
difference, there was no significant difference between the growth rates of low and mid
elevations (Std. Error=0.26, D. F.=159, P=0.34). There was no significant increase of
growth rates between the months of July and August in low elevations (W=0.99, D. F.=1,
P=0.16), and sample sizes were not sufficient for growth rates to be compared between
the months of May and June. In mid elevation sites growth rates increased significantly
across the months of the season (W=15.44, D. F.=2, P=<0.01). Lastly, an opposite pattern
was observed in the high elevations, in which growth rates decreased across the months
of the growing season, however there was no significant difference between the months
(W=4.31, D. F. =3, P=0.23) (Figure 5).
Table 2. Growth rates of A. truei tadpoles from May 2018 to August 2018 across an
elevation gradient in northern California, USA.
Elevation
Categories
Mean Body Growth (mm/day) Standard Error
Low
0.06
0.01
Mid
0.08
0.02
High
0.11
0.02

18
Figure 4. Growth rate of tadpoles across an elevational gradient in northern California,
USA. Model variance is reported in standard error.
Elevation
Category

19
Figure 5. Growth rates of tadpoles across the months of June (left), July (middle) and
August (right) of the survey season between elevation categories in northern California.
The horizonal lines represent the medians and the lower and upper hinges correspond to

20
the first and third quartiles (the 25th and 75th percentiles). The whiskers extend from the
hinge to the largest value (upper) and smallest value (lower) no further than 1.5 times the
interquartile range.
Size cohorts and length of larval period
At the beginning of the summer, populations from the high elevations showed two
main peaks of total length and developmental stage, followed by a third smaller peak
(Figure 6). These peaks correspond to three larval cohorts. Samples from low elevations
and mid elevations showed a single main size peak, followed by a second smaller peak
(Figure 7 and 8). These peaks correspond to two larval cohorts. Later in the summer, the
largest cohort begins to metamorphose and shrink. They were removed to avoid false
peaks. Density histograms of development stage across the season as well as ancillary
sites that were only sampled once gave further support for a larval period of two years in
low and mid elevations and three years in high elevations (Appendix G through K).

21
Figure 6. Density plots of total length histograms of Coastal Tailed Frog tadpoles from
the
high elevations of northern California. Once a tadpole began metamorphosis it was
removed.
Total Length (mm)
East van Matre
Creek
Siligo Creek

22
East
Willow
Creek
Boise
Creek
East Fork Big
Creek
Headwaters of
Big Creek
Figure 7. Density plots of total length histograms of Coastal Tailed Frog
tadpoles from the mid elevations of northern California. Once a tadpole began
metamorphosis it was removed.
Total Length (mm)

23
Bergmann’s rule
Tadpoles at developmental stage 37 were larger in low elevation sites compared
to mid (t=7.04, D. F=1, P=<0.01) and high elevation sites (t=6.75, D. F.=1, P=<0.01)
(Figure 9). There was no significant difference in body size of tadpoles between mid and
high elevation sites.
Figure 8. Density plots of total length histograms of Coastal Tailed Frog tadpoles from
the low elevations of northern California. Once a tadpole began metamorphosis it was
removed.
Boise
Creek
East
Willow
Creek

24
Figure 9. Body size of A. truei tadpoles at developmental stage 37 across elevation in
inland northern California, USA. The horizontal lines represent the median, and the lower
and upper hinges correspond to the first and third quartiles (the 25th and 75th
percentiles). The whiskers extend from the hinge to the largest value (upper) and smallest
value (lower) no further than 1.5 times the interquartile range.
Discussion
Growth
It is advantageous for species to have phenotypic plasticity in growth so that they
may respond to differing environmental conditions (Berven 1979, Conover et al. 1995).
This study describes increased rates of growth present in tadpole populations at higher

25
elevations compared to lower elevations. This contrasts with the general trend seen in
other amphibians in which growth and development rates are lower in high elevations
and latitudes (Morrison and Hero 2003). This may be a case of countergradient variation,
in which high elevation tadpoles grow faster to compensate for the differences in
environmental conditions, in particular because of a reduced growing season. As
described in the introduction, frogs at high latitudes can have higher growth rates due to
higher growth efficiency (Lindgren and Laurila 2005). Increased growth rates in high
elevation populations have also been described for Western Spotted Frogs (Licht 1975).
Countergradient variation is often missed because genotypic traits such as faster intrinsic
growth can be masked by environmental conditions. One would expect that cooler
temperatures experienced in high elevations would mask the presence of increased
growth rates. This may be a case when growth rates are able to overcompensate for
temperature effects, such that we can observe faster growth in cooler high elevation
environments.
My results also suggest that growth rates in the high elevations decrease across
the months of the growing season, while the opposite was observed in the mid elevations.
This may be because of the prolonged period of inactivity during the winter that tadpoles
face in high elevations. It is possible that in high elevations tadpoles are putting less
energy into body growth, but rather focusing on improving body condition prior to the
winter. On the other hand, mid and low elevation tadpoles can potentially grow year-
round, this has been described in post-metamorphic A. truei in coastal California
(Burkholder and Diller 2007). In colder regions, this has been described in populations of

26
post-metamorphic Asiatic Grass Frogs (Rana chensinensis) in which frogs put on more
liver and fat mass than growth in colder climates (Chen et al. 2011).
Larval period
The amount of time it takes from hatching to transformation from a tadpole to a
post-metamorphic frog (larval period) is an important aspect of an anuran’s life history. A
multi-year larval period among anurans is rare, and within the species that do have one,
there is often intraspecific variation with respect to larval period (Bury and Adams 1999,
Berven 1979, Morrison and Hero 2003). This intraspecific variation in larval period
within anuran species is likely in response to environmental conditions. In the case of the
larval period of A. truei in northern California, the present study shows there is clear
variation in the larval period across an elevational gradient. This study extends the
previously known maximum larval period in California from one-two years on the coast
(Wallace and Diller 1998) to three years in the Trinity Alps. The population previously
known with the longest larval period is from the mountains of northwestern Washington,
where a four-year larval period has been reported (Brown 1990). If it is real, this
difference between populations in Washington and California could likely be explained
by the decreased activity period and increased snowpack generally associated with higher
latitudes. Even though the populations studied in California were at a higher elevation
(2100m in California compared to 1500m in Washington), the combination of high
elevation and high latitude in Washington may even further limit activity and growth.
However, some caveats must be considered when estimating the length of the
larval period of overwintering populations of tadpoles based on a single season’s sample.

27
First, and importantly, combining samples from different streams can yield false evidence
of distinct cohorts. This occurs in the early summer samples of the East van Matre and
Siligo Creek tadpoles if they are combined into one histogram (Figure 10). This is due to
the difference in size range within a cohort between the two sites; the third cohort in
Siligo Creek is much larger than the third cohort of East van Matre. When the two sites
are combined, the larger cohort from Siligo Creek appears to be a separate, fourth cohort
(Figure 10).

28
Second, the timing of sampling is important. In late summer in the mountains of
Washington, it has been proposed that five larval age classes are present: hatchling
tadpoles (cohort-0) within the gravels at the nest site, one-year, two-year, three-year, and
metamorphosing tadpoles (cohort-4) (Brown 1990). By October, in the high country of
Washington, cohort four has transformed into young of the year frogs, and three cohorts
of tadpoles are left to overwinter (Brown 1990).
Figure 10. Density histogram of Coastal Tailed Frog tadpole total lengths between
Siligo and East van Matre Creek from the Trinity Alps of northern California.

29
In the high Trinity Alps of northern California, during the early summer (May
through early June) three cohorts of tadpoles can be observed (Figure 6). All three
cohorts were observed throughout the rest of June and July, until August, when cohort-3
transformed into young of the year frogs (Figure 6). This leaves only two cohorts of
tadpoles to overwinter in the stream, compared to what appear to be three cohorts in
mountains of Washington (Brown 1990).
If managers are measuring abundances of tadpoles or attempting to define
tadpole cohorts, the best time to do this is between May and August. If samples are taken
during October, surveyors will only find two cohorts in the high elevation streams, and
one cohort in the mid and low elevation streams.
Third, when looking at total length histograms to determine larval period it is
important to treat metamorphosing tadpoles separately. Since tadpoles shrink as their tails
are resorbed, false peaks can be made up entirely of metamorphosing individuals.
Therefore, developmental stage in conjunction with total length is helpful when
determining tadpole cohorts.
Bergmann’s rule
I found bigger larvae at the low elevation sites compared to high and mid
elevation sites, after accounting for developmental stage. Even though the pattern
appears opposite to Bergmann’s Rule, I considered a possible hypothesis: A. truei
tadpoles have a general trend of being bigger at low elevations due to the expectation that
the shortened growing season at higher elevations affect the ability of tadpoles to reach a
larger size than tadpoles in low elevations. Although I found that populations in higher

30
elevations experience higher growth rates and have an added year of growth compared to
lower elevation populations, these factors do not appear to compensate for a shorter
activity period and allow for larger body size in high elevations. Interestingly, larger
larvae in lower elevations have also been described among populations of the Western
Spotted Frog (Licht 1975).
Genetic differences between populations
Lastly, some preliminary data suggest that two different mitochondrial DNA
based clades may exist across my study sites (personal communication: Bruce Bury,
April 2018). The first clade encompasses my mid and low elevation sites, and the second
encompasses my high elevation sites. Therefore, the life history and size differences
between low and high elevation sites may be due to both genetic and environmental
reasons, and the potential countergradient patterns of growth may be due to phenotypic or
genetic plasticity among populations in my study area. Further studies are needed to
distinguish phenotypic plasticity from genetic differences in these populations.
Management implications
This chapter describes populations of larval A. truei that exist in the highest
known elevations for the species. Now that these new localities have been described, they
can be considered in wilderness management. An extended larval period and delayed age
at sexual maturity may make high elevation populations more susceptible to land use and
climate changes. Land use in the high elevations in the Trinity Alps is generally limited
to recreation, but climate change may have an impact in the long term. The climate
changes in the high elevations is predicted to include more precipitation in the form of

31
rain instead of snow (Snyder et al. 2004). This may affect recruitment because larvae stay
in these steep, fast-moving streams for at least three years, it puts them more at risk of
being crushed by rolling rocks or washed downstream in flood events or vulnerable to
drying streams if summer snowpack is reduced. In general, mid and low elevations
experience much more land use activity in terms of timber harvest, road building, etc..
There are also much higher stream and air temperatures in mid and low elevations than in
high elevations. Increasing temperatures due to climate change can also raise the
temperature of streams and reduce stream flow to potentially dangerous levels.
Quantifying life history traits provides key demographic parameters for Coastal Tailed
Frogs, which can have important implications regarding conservation and management of
this species.

32
CHAPTER TWO: LIFE HISTORY OF POST-METAMORPHIC COASTAL TAILED
FROGS (ASCAPHUS TRUEI) IN THE TRINITY ALPS WILDERNESS OF
NORTHERN CALIFORNIA
Introduction
As noted in Chapter One, life history traits are known to vary in many taxa across
environmental gradients (Begon et al. 1990). For example, amphibians tend to follow
Bergmann’s rule, with larger adults at high elevations and latitudes (Ashton 2002).
Intraspecifically, amphibian populations at high elevations and latitudes tend to have
shorter breeding seasons, shorter activity periods, longer larval periods, reach sexual
maturity later, and produce fewer and larger clutches per year relative to those at lower
elevations (Baraquet et al., Bears et al. 2009, Lindgren and Laurila 2005, Miaud et al.
1999, Moore 1949, and Morrison and Hero 2003). The Green Frog (Rana clamitans) has
a two-month breeding season in montane populations, compared to a five-month breeding
season in lowland populations (Berven et al. 1979). Elevational variation in life history is
also seen in the Argentinian anuran, the Córdoba Tree Frog (Boana cordobae). Across an
elevational gradient ranging from 800-2,400m, males at higher elevations were larger and
had greater longevity than those at lower elevations (Baraquet et al. 2018). In the
Common Frog (Rana temporaria), mean adult body length, age at maturity, and
longevity are increased at high elevations (Miaud et al. 1999).

33
Phenotypic plasticity in growth and development rates may occur in response to
differing environmental conditions. Variation in growth and development at the
proximate level can lead to differences in life history traits like longevity and maturity
across latitudinal and elevational gradients (Lindgren and Laurila 2005, Licht 1975). The
theoretical models developed by Reznick (1990) to understand the proximate origins for
variation in age and length at maturity of guppies (Poecilia reticulata) were discussed in
Chapter One, Reznick (1990), predicted that guppies in favorable conditions (more food)
would grow faster and initiate the process of maturation earlier or at a larger size. In
unfavorable conditions (less food), individuals grow slower and reach maturity later, or at
a smaller size. These same models were applied to the Common Frog (Rana temporaria)
with similar results – in environments with a shorter activity period, post-metamorphic
frogs grew slower and reached sexual maturity later. However, adult Common Frogs at
high elevations eventually attained a greater final length (Miaud et al. 1999). This was
because they also had greater longevity than individuals in populations with a longer
activity period. Since both guppies and amphibians are iteroparous and continue to grow
after maturity, individuals in populations with greater longevity were able to grow larger
after maturity (Miaud et al. 1999).
The Coastal Tailed Frog (Ascaphus truei) is an ideal species for the study of
geographic and elevational variation in life history, because it ranges across most of the
Pacific Northwest from northern California into British Columbia, and from near sea
level to interior mountains.

34
Timing of oviposition and breeding are important components of life history, yet
research on Ascaphus is difficult since egg masses are rarely found and copulation is
rarely observed in the field (Adams 1993, Bury et al. 2001, Karraker et al. 2006, Metter
1967, Palmeri-Miles et al. 2010). Males develop darkened cornified nuptial pads prior to
mating and these usually reach full development around mid-August to early September
(Metter 1964). Copulation is thought to occur in early fall, about ten months prior to the
laying of eggs in late June to late August (Karraker et al. 2006). However, Metter (1964)
suggested that individuals in coastal A. truei populations might breed every year, whereas
those in inland populations might lay eggs only every other year. This would be due to
shorter activity periods in inland populations, which might decrease the time available for
eggs to develop within the oviducts, and therefore delay oviposition to the following
year. But this is just a hypothesis. To date, nothing is known about oviposition timing and
breeding phenology across an elevational gradient in California, and any descriptions of
variation in breeding season and oviposition timing will be valuable.
Little is known with regards to age at sexual maturity, growth rates,
developmental rates, and longevity of Coastal Tailed Frogs across their range. In
California, Burkholder and Diller (2007) used growth curves to predicted age at sexual
maturity for six coastal populations in Humboldt County, CA and found that females
reached sexual maturity at approximately two-and-a-half years of age and males at one-
and-a-half years. They also found that growth rates of post-metamorphic frogs were
maximized in summer and were consistent between sexes. No other studies have
published post-metamorphic growth rates for the species.

35
Mark-recapture studies in Oregon, Montana, and British Columbia have examined
movement and site fidelity (Landreth and Ferguson 1967, Daugherty and Sheldon 1982b,
Wahbe et al. 2004). However only one study has described site fidelity of tailed frogs in
coastal California (Burkholder and Diller 2007), and site fidelity has not been compared
to inland or high elevation populations in California.
Longevity of Coastal Tailed Frogs is not known, but in its sister species, the
Rocky Mountain Tailed Frog (Ascaphus montanus), the oldest animal was estimated to
be at least 14 years old, based on mark-recapture data (Daugherty and Sheldon 1982).
Thus, there is ample opportunity to contribute to our knowledge of the life history of A.
truei by documenting variation in post-metamorphic traits across an elevational gradient
in California
Research objectives and predictions
My objective was to describe the following life history traits of post-metamorphic
A. truei in the Trinity Alps Wilderness of northern California:
1. Growth rates of post-metamorphic frogs
2. Size range of adult and immature age classes
3. Age and size at sexual maturity
4. Timing of breeding
5. Site fidelity
6. Longevity

36
Importance and need
Focusing on data from larval populations limits our understanding of the ecology
and life history of this species, especially regarding its sensitivity to land use and climate
change. Vital demographic parameters of tailed frog populations in the high elevations of
California are unknown. Examining populations of post-metamorphic frogs in the high
elevations of California will improve our understanding on how populations may respond
to environmental changes.
Methods
Study area
This study was conducted along an elevational gradient from the upper reaches of
the Trinity River in Trinity County, CA down to the lower reaches of the Trinity River,
Humboldt County, CA, with elevations ranging from ~150 m to ~2100 m. Details of the
study sites used have been given in Chapter One, above.
Post-metamorphic frog capture
I primarily used night surveys to locate and capture post-metamorphic frogs. I
started surveys at the bottom of the reach and walked in an upstream direction; I located
frogs with a headlamp and captured them by hand. I placed captured frogs in plastic bags
with cool stream water; the bags were numbered to correspond with a numbered flag
placed at each site of capture. In each stream, I measured a linear transect as close to
within the stream as possible. Each time a frog was captured I measured the distance
from the start of the reach to the capture location to measure linear within-stream

37
movement of individuals. After I completed the survey, all bags were collected, and I
measured each frog and released it at its site of capture. I measured post-metamorphic
frogs within the bag, then sanitized my hands with hand sanitizer, took the frog out of the
bag and marked and toe clipped quickly without the use of anesthesia. I took toe samples
from mid and low elevation frogs to estimate age and longevity.
Reproductive condition and development
I classified males as being in breeding condition while they had fully cornified
and dark black nuptial tubercles on the palm, forearm, and chin.
I classified females as gravid when a mass of bright yellow eggs was visible
through the transparent abdominal wall and their fingertips were black. Females develop
black fingertips prior to oviposition to aid in digging of nest sites (Daugherty and
Sheldon 1982a). I classified females as spent when no eggs were visible, but black
fingertips were present and abdominal skin was loose. I assumed females were not laying
that year when small eggs were present, and they had white fingertips. I assumed females
with no eggs and white fingertips were immature.
Measurements
Snout-vent length: From the tip of the snout to the posterior end of the cloaca.
Mass: same protocol as for larvae (Chapter One).
I made all the measurements with dial calipers to the nearest 0.1mm and I measured mass
with a 25g Pesola scale.

38
Marking
I marked frogs using visual implant elastomer (VIE) (Northwest Marine
Technology Inc. Seattle, Washington). I marked each animal for individual recognition
using a combination of four possible colors (yellow, orange, red, and blue). I made the
marks just under the skin and they were about 3-5mm long and sometimes smaller for
very small individuals. I viewed marks with a UV light in a darkened area to enhance
visibility. Post-metamorphic frogs were marked in the hands and feet. This yielded four
different body locations using four different colors (same as for tadpoles, as detailed in
Chapter One). Marking methods followed Chelgren and Adams (2017) and 625
individual marking codes were generated using SalaMarker (MacNeil et al. 2011).
Field collection and skeletochronology
I collected a total of 96 bone tissue samples (all the phalanges of the longest toe
on one hind foot) across the study area. Fifty samples had already been collected by
California Department of Fish and Wildlife biologists in the high elevation sites prior to
the study, and prepared by Matson’s Laboratories, Inc. (MT, USA). I collected samples
from mid and low elevations myself and prepared them in the laboratory at Humboldt
State University (HSU). I decalcified phalangeal bone samples in RDO Rapid
decalcifying solution (Apex Engineering Products Corporation, IL) for 2-3 hours, then
embedded in ParaPlast. I made ten micrometer thick sections using a rotary microtome
and stained them in either Ehrlich’s hematoxylin for 16 minutes or Toluidine Blue
(2g/100ml) for 2 minutes. I estimated longevity and age at sexual maturity using
skeletochronology. Skeletochronological analysis involves counting lines of arrested

39
growth (LAG)s in cross sections of bone tissue samples. Each LAG corresponds to each
winter period of hibernation (Castanet and Smirina 1990). Counts of LAGs were made by
at minimum two independent observers. When there were disagreements between
observers, both viewed the sections again and agreed upon final estimates of post-
metamorphic age. I added the skeletochronology estimate to the larval period estimate
(see statistical methods) to get the complete age estimate. In some cases I could estimate
age at sexual maturity as the point at which LAGs became closer together, due to a
slowing of growth as more resources are dedicated to reproduction, this has been
described in other skeletochronology work on amphibians (Guarino et al. 2003; Sinsch
2015). For my detailed skeletochronology protocol, see Appendix C.
Statistical methods
I performed all analyses in the statistical program R (Version 3.2.2, R Core
Development Team 2017).
Growth rate
I estimated growth by subtracting the body size of the last capture of each
individual animal by the body size of the first capture of each individual animal, this was
compared to the number of days between captures. I compared the relationship between
growth and number of days between captures using a linear mixed effects regression with
each individual as random effects and sex and maturity as interactive additive effects.

40
Adult and immature age classes of frogs
I visually compared age classes of frogs by making snout-vent length frequency
histograms in conjunction with reproductive male and female frogs (those exhibiting
secondary sexual characteristics).
Size and age of mature frogs
First, I compared body size of mature frogs between males and females using a
non-parametric Welsh’s T-test. Then, I modeled the differences between females and
males in size at reproductive maturity using a binomial general linear model with sex as a
covariate. To compare body size for mature frogs across elevation categories, I used a
non-parametric Kruskal-Wallis tests and Conover-Iman tests (Dinno 2017).
Site fidelity
I calculated movements by subtracting the location (linear distance from start of
survey reach) at the last capture from the location (linear distance from the start of the
survey reach) at the first capture. I visually assessed distribution of linear distance moved
of A. truei in histograms, compared the absolute value of linear distance moved between
frogs of different sex, maturity, and upstream vs downstream using non-parametric
Welsh’s T-tests, and compared across months of the survey season using a non-
parametric Kruskal-Wallis and Conover-Iman tests (Dinno 2017).

41
Results
In total, I took 517 measurements of post-metamorphic frogs across a total of 16
sites (Chapter One: Figure 3) over 70 days from May-August 2018. One hundred and
fifteen were individually marked: 66 were caught twice, 21 were caught three times, 14
were caught four times, five were caught five times, and one was caught six times.
However, the majority of post-metamorphic frog captures were at high elevations.
Fourteen were caught in low elevations, 32 in the mid elevations, and 110 in the high
elevations. Sixty-three were recaptured in high elevations; only three individuals were
ever recaptured in the mid elevations, and none were ever recaptured at low elevations.
At the high elevation sites, 50 frogs were sampled for skeletochronology and 38 provided
reliable age estimates; at mid elevation sites, 32 frogs were sampled and four provided
reliable age estimates; and at low elevations 14 frogs were sampled and two produced
reliable age estimates.
Growth rates of post-metamorphic frogs
There was no significant difference in growth rates between males and females
(χ2 =0.01, D. F.=1.00, P= 0.94) nor between mature and immature frogs (χ2 =0.04, D.
F.=1, P=0.84). There was no significant difference between growth rates between the
months of the season (χ2 =2.39, D. F. =2, P=0.30). Overall, post-metamorphic frog
growth rates were estimated to be 0.037mm per day (Std. Error= 0.12) in the Trinity Alps
of northern California.

42
Adult and immature age classes
The SVL’s of the smallest reproductive frogs (based on secondary sexual
characteristics) was about 30mm for males and 34mm for females. The size class
distribution plots further supported these cut off values (Figure 11). I could not
distinguish between subadult and juvenile frogs within the pre-reproductive category for
males, however, there were distinct peaks for females, possibly corresponding to distinct
cohorts. Juvenile females had a maximum size of about 28mm and subadult females had
a maximum size of about 33mm (Figure 12).
Size at sexual maturity
Males and females reach sexual maturity at the same size – 50% are mature at
about 33mm (Figure 12 and Table 4). Overall, they start out with immature males being
significantly larger than immature females (t=2.16, D. F.=91.94, P=0.03) and females
achieve significantly greater size as mature frogs (Figure 13 and Table 5) (t=9.67, D.
F.=135.3 P<0.00).

43
Figure 11. Snout-to-vent length frequency histograms for mature (in dark gray and
dashed borders) and immature (in white and solid borders) male and female Coastal
Tailed Frogs from the Trinity Alps Wilderness of northern California, USA. Bins are
1mm wide.

44
Figure 12. Binomial logistic regression of body size at sexual maturity of A. truei in
northern California, USA. Plot of least squares regression on body length.
Table 3. GLM model of the influence of body length, sex, and their interaction on sexual
maturity of Ascaphus truei in northern California, USA.
Parameters
P-value
Body Length
2.0x10-16
Sex
0.09
Body Length:Sex
0.08

45
Table 4. Mean snout-vent length (SVL) of post-metamorphic frogs in the Trinity Alps of
northern California,USA.
Immature
Mean SVL
Standard Error
Males
30.70
0.30
Females
29.45
0.52
Mature
Mean SVL
Standard Error
Males
34.32
0.42
Females
39.89
0.36
Body size of mature animals was significantly smaller in high elevations
compared to mid elevations (t=2.74, D. F. =1, P=0.0034) (Figure 14). Body size of
mature animals did not significantly differ between low and mid elevations (t=0.63, D.
F.=1, P=0.26) and between low and high elevations (t=0.95, D. F.=1, P=0.17).

46
Figure 13. Body length of mature (right) vs immature (left) females (F) and males (M)
Coastal Tailed Frogs in northern California, USA. The horizonal lines represent the
medians and the lower and upper hinges correspond to the first and third quartiles (the
25th and 75th percentiles). The whiskers extend from the hinge to the largest value
(upper) and smallest value (lower) no further than 1.5 times the interquartile range.

47
Figure 14. Body length of mature Ascaphus truei across elevation in northern California,
USA. The horizonal lines represent the medians and the lower and upper hinges
correspond to the first and third quartiles (the 25th and 75th percentiles). The whiskers
extend from the hinge to the largest value (upper) and smallest value (lower) no further
than 1.5 times the interquartile range.
of Mature Frogs

48
Timing of breeding and oviposition
Males in the high elevation sites were first seen forming tubercles on their
forearms on June 29th, and tubercles were fully formed in all mature males by July 27th.
No egg masses were ever found in the stream beds during my study. However, my
colleague and I found three A. truei eggs in the stomach of an Oregon Aquatic
Gartersnake (Thamnophis atratus hydrophilus) during late July-early August 2016. I also
have data on recaptures of a single female that lacked visible eggs, after having
previously been observed as gravid. Based on this observation, oviposition must have
happened sometime between July 28th and August 8th of 2018 in the high elevation.
Interestingly, no females with large eggs or egg masses were found during the summer of
2017.
Longevity
Mean age of post-metamorphic animals was significantly higher in high elevation
populations compared to mid-elevation populations (t = 3.74, p = 0.0003) and low
elevations (t=2.711, p=0.0049) (Figure 15, 16, and 17). The oldest individual captured in
mid elevation populations was three years post-metamorphosis, whereas the oldest
individual in high elevations was eight years post-metamorphosis. However, the sample
size for high elevations was much higher than for mid and low elevations.

49
Figure 15. Median age (post-metamorphosis) of A. truei across elevation categories in
northern California, USA. Sample size: high elevation: 38, mid elevation: four, low
elevation: two. The horizontal lines represent the medians and the lower and upper hinges
correspond to the first and third quartiles (the 25th and 75th percentiles). The whiskers
extend from the hinge to the largest value (upper) and smallest value (lower) no further
than 1.5 times the interquartile range.
Elevation Category

50
Figure 16. Phalanx section showing two LAGs from a post-metamorphic A. truei from a
low elevation population site in northern California, USA. Scale bar = 200um.

51
Figure 17. Phalanx section showing five LAGs from a post-metamorphic A. truei from a
high elevation population site in northern California, USA. Scale bar = 100um.
Site fidelity and movement
Overall, Coastal Tailed Frogs from the Trinity Alps exhibit high site fidelity to a
specific area in the stream channel (n=73, mean=12.4m, Std. Error=1.29). Both upstream
and downstream movements were minimal (Table 6). Histograms of linear movements
showed a range of 0-67.7m with most ranging from 0-20m (Figure 19). There were no
significant differences between linear movement distances between males or females, nor
between immature and mature frogs (Figure 18). There was, however, a significant drop

52
of movement distances during the month of July between the months of the survey season
(W2=41.9, D. F.=2, P=<0.01) (Figure 19). There was also significant upstream movement
during the month of June compared to July (t=7.92, D. F.=1, P<0.01) and significant
downstream movement during the month of August compared to July (t=4.60, D. F.=1,
P<0.01) (Figure 20). There were no significant differences between movement of males
and females nor mature and immature frogs between the survey months.
Table 5. Longitudinal (upstream/downstream) movement distances of the Coastal Tailed
Frog populations of the Trinity Alps Wilderness of California, USA.
Adult Females
Mean Movement (m)
Standard Error
n
Upstream
13.9
3.32
19
Downstream
12.4
2.75
20
Immature Females
Mean Movement (m)
Standard Error
n
Upstream
6.23
1.58
10
Downstream
11.1
3.76
10
Adult Males
Mean Movement (m)
Standard Error
n
Upstream
11.1
2.9
17
Downstream
12.2
4.06
12
Immature Males
Mean Movement (m)
Standard Error
n
Upstream
14.9
4.58
16
Downstream
16.9
5.61
11

53
Figure 18. Distribution of longitudinal (upstream>0.0, downstream<0.0) movement
distances relative to each capture of Coastal Tailed Frogs in the Trinity Alps Wilderness
of northern California, USA.

54
Figure 19. Linear movement distance (m) of Coastal Tailed Frogs across the months of
the survey season in the Trinity Alps Wilderness of northern California, USA.

55
Discussion
Growth and development
It is advantageous for species to have phenotypic plasticity in growth and
development rates so that they may respond to differing environmental conditions
(Berven 1979, Conover et al. 1995). Variation in growth and development can lead to the
differences between life history traits such as longevity and maturity across latitudinal
and elevational gradients (Lindgren and Laurila 2005, Licht 1975). This study describes
mean growth rates of 0.037mm per day (1.10mm per month) for both males and females
in inland populations of A. truei in California. The only other published growth rates
estimates for post-metamorphic frogs in California are from populations on the coast,
which had summer growth rates of just slightly higher for males (1.42mm/month) and for
females (1.5mm/month) greater than my estimates from the Trinity Alps (Burkholder and
Diller 2007). This suggests during the summer growth rates are similar yet slightly slower
among inland high elevation and low coastal populations of A. truei in California.
However, one important consideration is that during the winter and much of spring, the
high elevations are covered by snowpack, during which time the growth of high elevation
frogs is likely arrested. However, frogs in coastal populations can continue to grow at
approximately 0.89 mm/month through the winter (Burkholder and Diller 2007). This
suggests that overall annual growth is greater in coastal populations compared to inland
high elevation populations.

56
The relative consistency of observed growth rate does contrast with the general
trend seen in other amphibians in which growth were slower in high elevations and
latitudes (Morrison and Hero 2003).
Body size of sexually mature animals
The effects of shortened activity periods and lower temperatures on growth at
high elevations can influence body size and age at maturity as well as sex individuals.
This chapter describes growth rates to be similar to those in coastal populations, therefore
I expected body sizes across elevations to also be similar (Burkholder and Diller 2007).
In this species and most anurans, females tend to be larger than males and I expected the
same for be true for populations in the Trinity Alps (Burkholder and Diller 2007). In
general, studies have found that sexually mature frogs tend to be larger in high elevations
(Morrison and Hero 2003), therefore I expected mature frogs in the Trinity Alps to also
be larger than lowland populations.
My results show that size of sexually mature A. truei varies across elevations;
mature frogs at high elevations were significantly smaller than those at mid-elevation.
However, with only six mature frogs measured at low elevations, I could not determine
whether these were generally larger than mid-elevation frogs. Comparing to coastal
California, adult males had an average SVL of 36.7mm (Burkholder and Diller 2007),
whereas adult males in the Trinity Alps averaged 34.3mm in SVL. On the coast of
California adult females averaged 44.4mm SVL whereas adult females in the Trinity
Alps averaged 40.0mm SVL. The same pattern occurs with immature frogs from the

57
coast having a larger average SVL than immature frogs of both sexes from the Trinity
Alps.
My findings contrast with Bergmann’s rule, and with data from the European
Common Frog (Rana temporaria; Miaud et al. 1999) and other amphibians (Morrison
and Hero 2003). Coastal populations have been documented to grow year-round
(Burkholder and Diller 2007). It appears frogs in coastal areas can reach larger sizes than
inland Trinity Alps populations.
My results show that sexually mature males were on average 3.7 mm smaller than
mature females, similar to the populations examined by Burkholder and Diller (2007) on
the northern California coast. However, interestingly, in the Trinity Alps, immature males
were on average 0.45 mm larger than immature females. This is opposite to what was
described in populations on the coast of California and opposite to what I found for
mature frogs in the Trinity Alps. When I modeled size at sexual maturity between the
sexes a similar pattern emerged, with immature males being larger than immature
females, but after about 50% of frogs are mature at a size of 33mm, females begin to be
larger than males and this difference becomes greater as the proportion of mature frogs
increases. This suggests that sexual maturity is strongly associated with size (even though
adults are smaller at higher elevations), which may be useful to managers and researchers
when secondary sexual characteristics may not be visible and they need to determine
maturity.

58
Timing of copulation and oviposition
Timing of breeding and oviposition is important in studying the life history of a
species, and little is known with respect to variation across the environmental gradients.
My results suggest that copulation occurs in late July in the Trinity Alps, which is earlier
than it was thought to occur by Metter (1967) and Karraker et al. (2006), who described
copulation occurring in August and September in populations in Oregon and Washington.
However, Sever and others (2001), reported mating during May in the North Fork of Mad
River, Humboldt Co., California, and Noble and Putnam (1931) observed copulation
from June 12 to July 6 in the Olympic Mountains, Washington. Therefore, there appears
to be a high degree of variation in this regard. In this study, small sample sizes in mid and
low elevation did not allow me to address the question of whether breeding season
differed across an elevational gradient in California.
By contrast, my findings on oviposition timing in the high elevations of northern
California were similar to what is described in the literature for other populations (July
24th) (Karraker et al. 2006). Determining if populations or individuals breed every other
year in the high elevation was difficult, however the lack of females with large eggs
captured in 2017, and the instances of egg predation in 2016, and oviposition in 2018
suggests this could be a possibility.
Site fidelity and movement
Post-metamorphic tailed frogs have been reported to exhibit site fidelity, with
juveniles being the main dispersers (Daugherty and Sheldon 1982b, Wahbe et. al. 2004).
In my study area tailed frogs also exhibited high site fidelity. However, there were no

59
significant differences between the sexes nor between immature and adult frogs.
Movement distances in populations in coastal California mostly ranged from 0-30m
(Burkholder and Diller 2007) whereas in the Trinity Alps mostly ranged from 0-20m. The
biggest difference was an average of 28.0m movement downstream of mature females on
the coast, compared to an average of only 12.4m of movement in the Trinity Alps. It was
interesting to find significant upstream movement in June and significant downstream
movement in August, and a significant lack of movement in July. This increase of
upstream movement in June could be individuals trying to feed or establish home ranges
after emerging from 6-7 months of cold winter conditions. Others have also described
strong upstream movements and hypothesized this is because headwaters provide more
food (Hayes et al. 2004). I documented breeding to be potentially occurring in the sites in
late July and August as well as oviposition occurring this time. Perhaps the increase in
movement distances downstream in August is associated with breeding and/or
oviposition. Further studies over broader timescales need to be conducted.
Longevity
Longevity is an important life history and demographic trait that is notoriously
difficult to estimate. Skeletochronology allows for the estimating of post-metamorphic
age from lines of arrested growth in bone tissues. This study describes the potential for
the use of the this technique on the species and the potential for greater longevity in
populations of high elevations. This may be a response to an overall slower life history
pattern in high elevation Trinity Alps populations. The data however are limited due to

60
low sample sizes. Further research needs to be done in perfecting and validating
skeletochronology techniques for this species.
Management implications
Focusing on data from larval populations limits our understanding of the ecology
and life history of this species, especially in regard to its sensitivity to land use and
climate change. Vital demographic parameters of tailed frog populations in the high
elevations of California described in this study, particularly information on site fidelity,
movement, age at sexual maturity, breeding and oviposition timing, and longevity, are all
important in managing populations. Examining populations of post-metamorphic frogs in
the high elevations of California will improve our understanding on how populations may
respond to environmental changes.
. This study describes populations of A. truei that exist in the highest known
elevations for the species, and that now that these localities have been described, they can
be considered in wilderness management. An extended larval period (described in
Chapter one) and delayed age at sexual maturity may make high elevation populations
more susceptible to land use and climate changes. Land use in the high elevations is
limited to recreation, but climate change is predicted to include precipitation in the form
of rain instead of snow. This study reports more robust adult populations in high
elevations. The low captures of adults in mid and low elevations may signal significant
declines in these areas, but also may a reflection of reduced capture probability, since
larval numbers in the same streams are comparable (see Chapter One). In general, mid
and low elevations experience much more land use activity in terms of timber harvest,

61
road building, etc.. There are also much higher stream and air temperatures in mid and
low elevations than in high elevations. More research is needed to assess potential
population declines and reasons for declines in mid and low elevations and possible
refugia in high elevations in northern California.

62
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67
APPENDICES
Appendix A. Stages of development for Coastal Tailed Frog (Ascaphus truei) tadpoles,
adapted from Gosner (1960), Brown (1990), Brown (1989), and John Reiss
(unpublished). This table was design for use in the field on hatched tadpoles, therefore it
does not include stages below 26.
Stage
Initial criterion used
here for Ascaphus
Gosner (1960) initial criterion
# of days
since
hatching
(and
figures)
from
Brown
(1990)
and
Brown
(1989)
26
Labial tooth formula is
1/3; the tooth row in
the upper labium is
continuous and in the
lower labium, the first
and second tooth rows
are divided medially
while the third is
continuous. TL:18mm
View Hind limb diagram in #28
57 days
27
Dental ridges continue
to develop: single
ridge of the upper
labium is continuous
and consists of a
closely set double row
of teeth, and the
second and third ridges
are continuous and
each remains as a
single row
View Hind limb diagram in #28
66 days

68
Stage
Initial criterion used
here for Ascaphus
Gosner (1960) initial criterion
# of days
since
hatching
(and
figures)
from
Brown
(1990)
and
Brown
(1989)
28-30
Tadpole with well-
developed suctorial
mouth, median
opercular opening
behind mouth, full
dark coloration, and
hind limb bud 0.5-
0.7mm and completely
covered by anal fold.
87 days

69
Stage
Initial criterion used
here for Ascaphus
Gosner (1960) initial criterion
# of days
since
hatching
(and
figures)
from
Brown
(1990)
and
Brown
(1989)
31-33
Same as toe
development diagram
from Gosner (1960)

70
Stage
Initial criterion used
here for Ascaphus
Gosner (1960) initial criterion
# of days
since
hatching
(and
figures)
from
Brown
(1990)
and
Brown
(1989)
34
Tadpole with the
appearance of ankle
constriction and toes
and ranging from 0.7-
2.1mm long. Hind-
limbs still hidden by
the anal flap. Size of
the oral disk increases
but spiracle remains
0.3mm in width.
35-36
Unidentifiable on live
specimens in the field
View Gosner 1960 diagram in stage 31
37
Tadpole with five toes
distinct
Five toes distinct
38
Tadpole with toes
protruding beyond the
anal flap. Oral disk
enlarges in length and
width, and the spiracle
enlarges to width of
0.9mm
Metatarsal tubercle formed
38, 36
(Plate 1d,
1e)
39
Tadpole with ankle
beyond anal flap
Pigment free areas indicate site of
subarticular tubercle formation
40
Tadpole with knee
beyond anal flap.
Spiracle enlarges to a
width of 1.3mm and
some may show
fingertips at the
spiracle opening
Subarticular tubercles formed; cloacal
tail piece present
39, 40
(Plate Iib,
If)

71
Stage
Initial criterion used
here for Ascaphus
Gosner (1960) initial criterion
# of days
since
hatching
(and
figures)
from
Brown
(1990)
and
Brown
(1989)
41
Tadpole with
fingertips visible in
spiracle
Skin over forelimbs thin & transparent,
larval mouthparts begin to break down;
cloacal tail piece lost
41 (Plate
Ig, Ih)
42
Tadpole with
forelimbs emerged
Forelimbs emerge; labial denticles and
horny beak lost
42 (Plate
Iic)
43A
Transforming tadpole
with horny denticles
and beak lost, oral disc
retained
Angle of mouth between nostril and
midpoint of eye
43B
Transforming tadpole
with oral disc reduced
to upper and lower
“lips”
43 (Plate
Iid)
44
Transforming tadpole
with upper and lower
lips fused smoothly to
head
Angle of mouth between midpoint and
back of eye
45A
Transforming tadpole
with angle of mouth
behind posterior
border of eye
Angle of mouth behind posterior
border of eye; tail reduced to stub
45 (Plate
Iie)
45B
Metamorph frog with
tail reduced to stub
46
YOY frog with tail
stub gone
Tail stub gone
46 (Plate
Iif)

72
Appendix B. Larval Coastal Tailed Frog specimens collected from inland California and
vouchered in the Humboldt State Vertebrate Museum (HSUMV).
HSU
VM #
Site Name (refer to
Chapter One for specific
locations)
Developmental
Stage
(Appendix A)
Comments
3014
Headwaters of Big Creek
28
died during anesthesia
3013
Headwaters of Big Creek
40
found dead
3012
East van Matre Creek
40
died during anesthesia
3011
Siligo Creek
28
died during anesthesia
3015
East Fork Big Creek
34
died during anesthesia
3019
East Fork Big Creek
40
died during anesthesia
3020
Siligo Creek
37
died during anesthesia
3022
East Fork Big Creek
37
died during anesthesia
3023
Headwaters of Big Creek
37
died during anesthesia
3024
Headwaters of Big Creek
34
died during anesthesia
3025
Headwaters of Big Creek
34
died during anesthesia
3026
Headwaters of Big Creek
37
died during anesthesia
3027
Headwaters of Big Creek
43A
died during anesthesia
3028
Headwaters of Big Creek
34
died during anesthesia
3029
Headwaters of Big Creek
34
died during anesthesia
3030
Headwaters of Big Creek
34
died during anesthesia
3031
Headwaters of Big Creek
34
died during anesthesia
3032
Headwaters of Big Creek
37
died during anesthesia
3033
Headwaters of Big Creek
34
died during anesthesia
3034
Headwaters of Big Creek
34
died during anesthesia
3035
Headwaters of Big Creek
34
died during anesthesia
3036
Headwaters of Big Creek
34
died during anesthesia
3037
Headwaters of Big Creek
37
died during anesthesia
3038
Headwaters of Big Creek
34
died during anesthesia
3039
Headwaters of Big Creek
34
died during anesthesia
3040
Headwaters of Big Creek
37
died during anesthesia
3041
Headwaters of Big Creek
34
died during anesthesia
3051
East Fork Big Creek
37
died during anesthesia
3052
Headwaters of Big Creek
37
died during anesthesia
3053
Headwaters of Big Creek
38
died during anesthesia
3054
Siligo Creek
34
died during anesthesia

73
HSU
VM #
Site Name (refer to
Chapter One for specific
locations)
Developmental
Stage
(Appendix A)
Comments
3055
East van Matre Creek
34
Eaten by a Thamnophis
atratus hydrophilus
3057
East van Matre Creek
37
Eaten by a Thamnophis
atratus hydrophilus
3058
East van Matre Creek
34
Eaten by a Thamnophis
atratus hydrophilus
3059
East van Matre Creek
34
Eaten bya Thamnophis atratus
hydrophilus
3060
East van Matre Creek
34
Eaten by a Thamnophis
atratus hydrophilus
3016
Siligo Creek
34
Specifically collected for
voucher
3017
Siligo Creek
38
Specifically collected for
voucher
3018
Siligo Creek
34
Specifically collected for
voucher
3061
Siligo Creek
34
Specifically collected for
voucher
3062
Siligo Creek
37
Specifically collected for
voucher
3063
Siligo Creek
38
Specifically collected for
voucher
3064
Siligo Creek
37
Specifically collected for
voucher
3065
Siligo Creek
34
Specifically collected for
voucher

74
Appendix C. Post-metamorphic Coastal Tailed Frog Voucher Specimens at the
Humboldt State Vertebrate Museum (HSUVM).
HSUVM
#
Site Name
UTM
Easting(WGS 84)
UTM Northing
(WGS 84)
Comments
3021
Siligo
Creek
508940
4531005
immature, sex
unknown

75
Appendix D. Protocol developed to prepare slides of Coastal Tailed Frogs for estimating
ages using skeletochronology.
Skeletochronology Protocol
Dissecting out Bone:
1. Get equipment: 70% Ethanol, flame, dissection tray, paper towels, scalpel, two
forceps, glass screw top container, masking tape, and permanent marker.
2. Between each sample, sanitize tools (forceps and scalpel) by dipping in 70%
Ethanol and holding over flame until all Ethanol is burned off place them on paper
towel (get a new paper towel between each sample)
3. Using sanitized forceps, remove the toe sample from the labeled centrifuge tube and
place it on the paper towel. Set aside original centrifuge tube.
4. Using a pair of tweezers hold the toe bone, gently peel off the skin trying to get the
largest pieces possible. Sometimes you may need to make an incision using a scalpel
down the side of the toe, taking care to not cut into the bone.
5. Put tissue back in original centrifuge tube and copy the exact label from the toes
previous container on the new glass screw top container using a fine tipped sharpie.
Place the bone in the glass screw top container and fill with 70% Ethanol to fully
cover the bone.
Decalcification of Bone:
1. Tap water for 1 hour (for pieces 1/2cm diameter or less)
2. Decalcify in RDO rapid decalcifier for 2-3 hours (do not leave for too long!;
Solution is corrosive)
3. Rinse with water for 2-3 hours
4. 50% Ethanol for 1 hour
5. Store in 70% Ethanol
Embedding (either in Autotechnicron or by hand):
1. 95% Ethanol for 1 hour
2. 100% Ethanol for 1 hour
3. 100% Ethanol for 1 hour
4. Toluene for 1 hour
5. Toluene for 1 hour
6. 50% Toluene/50% Paraffin for 1 hour inside Paraffin Oven
7. Paraffin for 1 hour inside Paraffin Oven
8. Paraffin for 1 hour inside Paraffin Oven

76
9. Label and Embed in Molten Paraffin in aluminum foil mold for 24 hours in
refrigerator
Sectioning:
1. Remove embedded specimen from mold, and trim off excess paraffin with a razor.
2. Fix specimen to wooden block
3. Adjust angle of razor to 0
4. Make 10um sections in microtome
Fixing Sections to Slides:
1. Take slide without cover slip and lightly smear with Haupt’s Solution
2. Place sections onto slide
3. Float with 3% Formalin
4. Place on warming plate for 24 hours
Staining Slides:
1. Xylene for 2 minutes
2. Xylene for 2 minutes
3. 100% Ethanol for 2 minutes
4. 95% Ethanol for 2 minutes
5. 70% Ethanol for 2 minutes
6. Water for 2 minutes
7. Hematoxylin (Erlich’s or Delafield’s) for 16 minutes for Toluidine Blue use 2
minutes
8. Running Water for 4 minutes
9. Ammonia for 2 minutes
10. Water for 2 minutes
12. 70% Ethanol for 2 minutes for Toluidine Blue just drip once
13. 95% Ethanol for 2 minutes for Toluidine Blue just drip once
14. 100% Ethanol for 2 minutes for Toluidine Blue just drip once
15. 100% Ethanol for 2 minutes
16. 50% Ethanol/50% Xylene for 2 minutes
17. Xylene for 2 minutes
18. Xylene for 2 minutes
Mounting Slides
1. Take slide and add a few drops of Permount

77
2. Using coverslide forceps, place a 24x60 coverslip over slide.
3. Adjust coverslip and remove bubbles
4. Set slides out to dry for 1 week.
Viewing Slides:
1. On skeletochronology data sheet, write down the Unique ID and Collection Dates
prior to viewing slides.
2. On each slide there are 4 Unique IDs labeled on the left, and 4 phalange sections
corresponding with each Unique ID (Figure 1).
3. View each slide using a microscope starting at 10x magnification, and ending at 40x
magnification. Long bone sections can be reported at 10x magnification.
4. Indicate which sections you will be reporting in the Sections Used column.
5. Indicate which area of the section you used by imagining the section as a clock
(Figure 2)
6. Count each line of arrested growth (LAG) (Figure 3 and 4)
7. Two independent observers report their estimates, then agree on the final estimate of
age.
8. Report endosteal resorption and other comments in the notes section.

78
Appendix E. Study site photos of low elevation habitat composed of a dense canopy of
late seral Douglas-fir (Pseudotsuga menziesii), Big-leaf Maple (Acer macrophylum), Red
Alder (Alnus rubrus), and Tan Oak(Notholithocarpus densiflorus) in northern California.

79
Appendix F. Study site photos of mid-elevation site habitat composed of a dense canopy
of late seral Douglas-fir (Pseudotsuga menziesii), Big-leaf Maple Maple (Acer
macrophylum), Red Alder (Alnus rubrus), and Tan Oak (Notholithocarpus densiflorus) in
northern California, USA.

80
Appendix G. Site photos of high elevation habitat composed of very little canopy with a
few scattered tree species, including Jeffrey Pine (Pinus jefferi), Western White Pine
(Pinus monticola), and Foxtail Pine (Pinus balfouriana) in northern California, USA.

81
Appendix H. Total length frequency histograms of Coastal Tailed Frogs across low
elevation study sites and dates they were surveyed.

82
Appendix I. Total length frequency histograms of Coastal Tailed Frogs across mid-
elevation study sites and dates they were surveyed.

83
Appendix J. Total length frequency histograms of Coastal Tailed Frogs across high
elevation study sites and dates they were surveyed.

84
Appendix K. Developmental Stage frequency histograms of Coastal Tailed Frogs across
Elevation Category and dates they were surveyed.